Malformations of the Nervous System Handbook of Clinical Neurology Series

Foreword The period of intrauterine and neonatal brain development is crucial for everything that we will become and ca...

Author: Christopher P. Cannon

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Foreword

The period of intrauterine and neonatal brain development is crucial for everything that we will become and can accomplish in the rest of our life. In that short period of time the outline of the human brain develops into a tremendously complex organ consisting of 100 billion neurons, each making between 1000 and 100 000 contacts with other particular groups of neurons by means of some 100 000 km of nerve fibers. Each group of neurons has to be born at the right moment, migrate to the site where they differentiate and make their specific contacts in a limited critical period of brain development in order to function later in a normal way. Building such a complex structure as the brain in such a brief period is certainly a most demanding task for nature. It is in fact a wonder that it does not often end with catastrophic failures in one of the numerous exactly timed and extremely complex processes; instead it mostly results in a healthy baby with good potential for the rest of its existence. The present volume of the Handbook of Clinical Neurology, edited by Harvey Sarnat and Paolo Curatolo, deals with those children in whom brain development has resulted in a malformation of the central nervous system. This field has recently gained exciting new insights, for instance from molecular genetics, which are integrated in this volume. Section I of this volume follows the new integrative classification and deals with midline hypoplasias, disorders of segmentation of the neural tube, hamartomatous disorders of cellular lineage, disorders of radial neuroblast migration and cerebral cortical architecture and other dysgeneses. Section II describes the different clinical manifestations of CNS malformations, followed by sections on diagnostic methods, management and treatment. The editors of this volume of the third series of the Handbook of Clinical Neurology are to be congratulated in bringing together a wide range of internationally acknowledged experts to describe the new developments and their clinical implications. We are grateful to the authors, whose excellent chapters are the basis for this valuable volume. In addition we are, of course, also grateful to the team at Elsevier – and in particular to Ms Lynn Watt and Mr Michael Parkinson in Edinburgh – for their expert assistance in every stage of the development and production of this volume. Michael J. Aminoff François Boller Dick F. Swaab

Preface

Embryology is the essence of understanding development. Development is the essence of pediatric neurology. Training programs in our discipline, both in Europe and in North America, as well as in Latin America and Australia, presently provide our residents with only a weak foundation in neuroembryology or ontogeny that is, at best, disjointed as isolated topics in molecular genetics, neuroimaging and neuropathology, without systematic integration of these individual perspectives to enable conceptualization of the mechanisms of the developmental processes. Each perspective has much to contribute and complements the others but none alone can impart a full comprehension of normal embryological and fetal development, much less address abnormal development and its clinical manifestations. In this volume we have attempted to provide such an integrated approach. One of us previously made such an incipient attempt [Sarnat HB (1992). Cerebral Dysgenesis. Embryology and Clinical Expression. Oxford University Press, New York], but that monograph was written just at the end of the era of classic embryology and as the new era of molecular genetic programming was being initiated. The many modern advances in neuroimaging, clinical neurophysiology and neuropathology using immunohistochemistry were also in more juvenile stages of development in 1992. Molecular genetics has already contributed much to our knowledge of the genetic etiologies of many malformations of the nervous system but just knowing the names and loci of particular genes associated with particular disturbances in developmental processes, such as neuroblast migration, is not the full picture of their influence upon development that direct tissue examination with modern techniques can complement. The demonstration by neuroimaging of the neonatal, and indeed the intrauterine fetal, brain may reveal many pathological findings diagnostic of certain malformations, hydrocephalus and other conditions but this gross cerebral anatomy and its distortions do not show the microscopic features of cellular architecture, as in cortical lamination and heterotopia in neuroblast migratory disorders, or of abnormal cellular lineage and growth, as in tuberous sclerosis and hemimegalencephaly. Neither gross nor microscopic anatomy provides physiological data of synaptic function that identify epilepsy as a symptom in many malformations, as does the electroencephalogram. The need for integration of these individually important disciplines becomes increasingly obvious to provide better care for individuals affected by malformations of the nervous system. One of us tried to encapsulate, in a single text, modern knowledge ranging from molecular genetics to clinical presentations in a single disease [Curatolo P, Ed. (2003). Tuberous Sclerosis Complex: from Science to Clinical Phenotypes. Mac Keith Press, London]. This present volume is a single source that encompasses the various aspects of cerebral malformations in an integrated fashion. Finally, we appeal to our readers to provide thorough data about their own cases of malformation that might further elucidate mechanisms and etiologies. This can be accomplished by good neuroimaging. Recent advances in MRI techniques and analysis, including volumetric morphology, spectroscopy and functional neuroimaging, extract more information and may better characterize in vivo malformations of the central nervous system. Neurophysiology, including recent advances in EEG monitoring, also offers important contributions. The EEG is valuable in assessing electrocerebral maturation and in identifying clinically silent paroxysmal foci, even in the absence of seizures. Genetic studies are essential to confirm diagnoses suggested by clinical features and imaging patterns. Postmortem examination in those patients who do not survive despite the best of care adds a dimension not duplicated by any of the clinical techniques during life. Autopsy rates have fallen in recent years, in large part driven by fiscal restraints imposed by hospital business administrators who have little knowledge of or interest in academics or even in patient care, entrepreneurs for whom profit is the goal and investment in knowledge means little. We must not lose our identity and dignity as professionals and physicians with a more noble agenda. Harvey B. Sarnat, MD Paolo Curatolo, MD

List of contributors

C. Arpino Tor Vergata University of Rome, Rome, Italy

L. Genitori Ospedale Pediatrico Meyer, Florence, Italy

K.M. Barlow University of Calgary and Alberta Children’s Hospital, Calgary, Alberta, Canada

F. Giordano Ospedale Pediatrico Meyer, Florence, Italy

P.G. Barth Emma Childrens Hospital/Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands G. Battaglia Neurological Institute C. Besta, Milan, Italy E. Boltshauser University Children’s Hospital, Zurich, Switzerland R. Bombardieri Tor Vergata University of Rome, Rome, Italy M.H. Carstens Cardinal Glennon Children’s Hospital, Saint Louis University, Saint Louis, MO, USA C. Cerminara Tor Vergata University of Rome, Rome, Italy S. Cianfarani Tor Vergata University of Rome, Rome, Italy P. Curatolo Tor Vergata University of Rome, Rome, Italy G. di Pietro Ospedale Pediatrico Meyer, Florence, Italy

J.G. Gleeson University of California San Diego, La Jolla, CA, USA W.D. Graf Children’s Mercy Hospitals and Clinics and University of Missouri, Kansas City, MO, USA T. Granata Neurological Institute C. Besta, Milan, Italy J.S. Hahn Stanford University School of Medicine and the Lucile Packard Childrens Hospital, Stanford, CA, USA L.D. Hamiwka University of Calgary and Alberta Children’s Hospital, Calgary, Alberta, Canada P. Humphreys Children’s Hospital of Eastern Ontario, Ottawa, Ontario, Canada K. Imai The Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada A.M. Innes University of Calgary and Alberta Children’s Hospital, Calgary, Alberta, Canada

P.A. Donati Ospedale Pediatrico Meyer, Florence, Italy

R. Leventer Royal Children’s Hospital, Parkville, Victoria, Australia

L. Flores-Sarnat Alberta Children’s Hospital, Calgary, Alberta, Canada

J.K. Mah University of Calgary and Alberta Children’s Hospital, Calgary, Alberta, Canada

xii

LIST OF CONTRIBUTORS

F. Mussa Ospedale Pediatrico Meyer, Florence, Italy G. Oliveri Ospedale Le Scotte, Siena, Italy H. Otsubo The Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada I. Pascual-Castroviejo University Hospital La Paz, Madrid, Spain J.D. Pinter University of California Davis Medical Center, Sacramento, CA, USA M. Sanzo Ospedale Pediatrico Meyer, Florence, Italy Luigi Sardo Ospedale Pediatrico Meyer, Florence, Italy

J.R Siebert Children’s Hospital and Regional Medical Center and University of Washington, Seattle, WA, USA B. Talim Hacettepe University Children’s Hospital, Ankara, Turkey T. Tanaka Graduate School of Medicine and Faculty of Medicine, University of Tokyo, Tokyo, Japan H. Topalog˘lu Hacettepe University Children’s Hospital, Ankara, Turkey I.E.B. Tuxhorn Epilepsy Center in the Neurology Institute, Cleveland, OH, USA A. Volzone Tor Vergata University of Rome, Rome, Italy

B. Spacca Ospedale Pediatrico Meyer, Florence, Italy

E.C. Wirrell University of Calgary and Alberta Children’s Hospital, Calgary, Alberta, Canada

H.B. Sarnat University of Calgary and Alberta Children’s Hospital, Calgary, Alberta, Canada

F. Woermann Epilepsy Center Bethel, Evangelisches Krankenhaus BielefeldCampus – Mara Hospital, Bielefeld, Germany

Contents

Foreword Preface List of contributors

vii ix xi

SECTION I. Specific malformations using the new integrative classification Revised classification 1. Axes and gradients of the neural tube and gradients for a morphological molecular genetic classification of nervous system malformations Laura Flores-Sarnat and Harvey B. Sarnat (Calgary, Canada)

3

Midline hypoplasias 2. Holoprosencephaly Jin. S. Hahn (Stanford, CA, USA)

13

3. Septo-optic-pituitary dysplasia Peter Humphreys (Ottawa, Canada)

39

4. Rhombencephalosynapsis Peter G. Barth (Amsterdam, Netherlands)

53

5. Embryology and malformations of the forebrain commissures Harvey B. Sarnat (Calgary, Canada)

67

Disorders of segmentation of the neural tube 6. Disorders of segmentation of the neural tube: Chiari malformations Harvey B. Sarnat (Calgary, Canada)

89

7. Disorders of segmentation of the neural tube: agenesis of selective neuromeres Harvey B. Sarnat (Calgary, Canada)

105

8. Cerebellar hypoplasias Eugen Boltshauser (Zurich, Switzerland)

115

Hamartomatous disorders of cellular lineage 9. Tuberous sclerosis Paolo Curatolo and Roberta Bombardieri (Rome, Italy)

129

xiv

CONTENTS

10. Hemimegalencephaly syndrome Laura Flores-Sarnat (Calgary, Canada)

153

Disorders of radial neuroblast migration and cerebral cortical architecture 11. Periventricular nodular heterotopia Giorgio Battaglia and Tiziana Granata (Milan, Italy)

177

12. Subcortical laminar (band) heterotopia Teruyuki Tanaka and Joseph G. Gleeson (Tokyo, Japan and La Jolla, CA, USA)

191

13. Lissencephaly type I Richard Leventer (Parkville, Australia)

205

14. Lissencephaly type II Haluk Topalog˘lu and Beril Talim (Ankara, Turkey)

219

15. Schizencephaly Tiziana Granata and Giorgio Battaglia (Milan, Italy)

235

Disorders of neural crest induction of non-neural tissues 16. Neural tube programming and the pathogenesis of craniofacial clefts, part I: the neuromeric organization of the head and neck Michael H. Carstens (St Louis, MO, USA)

247

17. Neural tube programming and the pathogenesis of craniofacial clefts, part II: mesenchyme, pharyngeal arches, developmental fields; and the assembly of the human face Michael H. Carstens (St Louis, MO, USA)

277

18. The oral–facial–digital syndromes Joseph R. Siebert (Seattle, WA, USA)

335

Other dysgeneses 19. Congential vascular malformations in childhood Ignacio Pascual-Castroviejo (Madrid, Spain)

347

20. Acquired, induced and secondary malformations of the developing central nervous system Harvey B. Sarnat (Calgary, Canada)

371

SECTION II. Comparative manifestations of central nervous system malformations 21. Epilepsy in patients with cerebral malformations Lorie D. Hamiwka and Elaine C. Wirrell (Calgary, Canada)

383

22. Neuromuscular disorders associated with cerebral malformations Jean K. Mah (Calgary, Canada)

403

23. Neuroendocrine complications of central nervous system malformations Stefano Cianfarani (Rome, Italy)

433

24. Cerebral dysgeneses associated with chromosomal disorders Joseph D. Pinter (Sacramento, CA, USA)

451

CONTENTS 25. Cerebral dysgeneses secondary to metabolic diseases in fetal life William D. Graf (Kansas City, MO, USA)

xv 459

SECTION III. Diagnostic methods 26. Imaging malformations of cortical development Ingrid E.B. Tuxhorn and Friedrich Woermann (Bielefeld, Germany) 27. Clinical neurophysiology of cortical malformations: magnetoencephalography and electroencephalography Hiroshi Otsubo and Katsumi Imai (Toronto, Canada)

479

503

28. Molecular genetic testing and genetic counseling A. Micheil Innes (Calgary, Canada)

517

29. Embryology and neuropathological examination of central nervous system malformations Harvey B. Sarnat (Calgary, Canada)

533

SECTION IV. Management of central nervous system malformations 30. Medical treatment in children with central nervous system malformations Paolo Curatolo, Roberta Bombardieri and Caterina Cerminara (Rome, Italy)

557

31. Surgical treatment of central nervous system malformations Lorenzo Genitori, Pier Arturo Donati, Flavio Giordano, Massimiliano Sanzo, Federico Mussa, Luigi Sardo, Barbara Spacca, Giovanni di Pietro and Giuseppe Oliveri (Florence, Italy)

569

32. Neurorehabilitation of children with cerebral palsy Karen Maria Barlow (Calgary, Canada)

591

33. Educational, cognitive, behavioral and language development issues Carla Arpino, Anna Volzone and Paolo Curatolo (Rome, Italy)

611

Index

627

Color plate section

645

Section I Specific malformations using the new integrative classification

Handbook of Clinical Neurology, Vol. 87 (3rd series) Malformations of the Nervous System H. B. Sarnat, P. Curatolo, Editors # 2008 Elsevier B. V. All rights reserved

Revised classification Chapter 1

Axes and gradients of the neural tube for a morphological/ molecular genetic classification of nervous system malformations LAURA FLORES-SARNAT*,1 AND HARVEY B. SARNAT2 *

Alberta Children’s Hospital, Division of Pediatric Neurology, Calgary, Alberta, Canada;

2

University of Calgary Faculty of Medicine and Alberta Children’s Hospital, Calgary, Alberta, Canada

1.1. Introduction and historical note Classification is among the most primordial elements of human thought. From the time that the neonate begins to distinguish self from the rest of the world, s/he has already initiated the first process of classification. In later infancy, it is extended to distinguish living from nonliving objects, people from other living things, familiar from unfamiliar and, much later, reality from fantasy. Classification is how we organize our thoughts to put things into perspective and to compare new with previous experiences. Even individuals with mental retardation are able to classify the things and events in their lives, although this skill may be delayed, as with other aspects of their development. Inability to compare and classify leads to confusion, poor perspective, impaired judgment, and may even be a contributory factor in psychosis and schizophrenia. The classification of nervous system malformations has been the obsession of many investigators in this field for more than a century and continues to be a prominent part of the discussion of any article describing abnormal development. Until the past couple of decades, nearly all the focus was upon descriptive morphogenesis and this focus continues to be prominent in publications by anatomically oriented specialists such as neurosurgeons and neuroradiologists (Barkovich, 2001). Describing abnormal morphology in the context of embryology was a major advance (Sarnat, 1997),

because disturbances in ontogenesis are an important basis for anatomically abnormally formed brains as an end-result examined either by imaging, neuropathology or, ideally, both. Contributions by neuropathology to microscopic dysgeneses and classification of malformations provides another perspective of normal and abnormal neural tissue organization and cytological abnormalities. An example of a microscopic dysgenesis is individual heterotopic neurons in white matter that appear isolated by histological stains, but with special immunocytochemical techniques their synaptic connections with overlying cortex can be demonstrated and thus they might contribute to epilepsy. Another example is a small zone of disoriented and displaced neurons within the cortex, associated with focally disrupted lamination and abnormal synaptic circuitry. The application of modern techniques of histochemistry and immunocytochemistry has enabled the identification of specific types of cell involved, synaptic organization and their state of maturation compared with normal controls, another form of classification. The introduction of molecular genetics in the 1980s and the rapid expansion of this new discipline to understanding the genetic programming of development, the relations between genes simultaneously or sequentially expressed and the recognition of the specific genetic mutations and deletions in many cerebral malformations has been a truly revolutionary advance in both neuroembryology and in providing an insight

*Correspondence to: Laura Flores-Sarnat MD, Alberta Children’s Hospital, Division of Paediatric Neurology, 2888 Shaganappi Trail NW, Calgary, Alberta T3B 6A8, Canada. E-mail: [email protected], Tel: þ1-403-955-3013, Fax: þ1-403-955-2922.

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L. FLORES-SARNAT AND H. B. SARNAT

not previously possible to understand mechanisms of dysgenesis and classify malformations. These data are far from complete at this time but enough information has now been documented that molecular genetics can be used in refining morphological schemes of abnormal development of the nervous system. Whereas some geneticists in particular suggest replacing all morphological classifications with genetic schemes, this approach is as limited as the pure morphological one that fails to provide an etiological basis for dysgeneses. Pure genetic schemes also would be of little practical value to physicians, who must make a clinical diagnosis and confirm it by anatomical imaging, provide treatment, prognosis and genetic counseling of the family because the phenotype/genotype correlation often is highly variable and not all details of even the genetic factors are known. In some cases, an apparent genetic defect may be secondary, as downregulation of an important gene in a cascade in which the primary mutation is another gene underexpressed earlier in the cascade, or the apparent overexpression of normal antagonistic genes, or heterotropism, with nonuniform genetic defects in different parts of the tissue or among different cells at the same site. Because of the limitations of both pure morphological and pure genetic approaches to the classification of nervous system malformations, we have proposed a combined scheme that integrates the two and enables the recognition of gradients of genetic expression, whether or not the specific defective gene is known (Sarnat, 2000; Sarnat and Flores-Sarnat, 2001a, 2002, 2004). It further provides the flexibility needed for future revision and for the inclusion of nongenetic developmental defects acquired during fetal and early postnatal development. This classification has undergone several revisions and this present chapter offers yet another update to incorporate new data and new understanding of some cerebral dysgeneses, in particular those associated with neural crest defects and neurocutaneous syndromes (Flores-Sarnat and Sarnat, 2006). Several ‘regional’ classifications have been elaborated by various authors, limited to malformations in particular parts of the brain, such as dysgeneses of the posterior fossa, the corpus callosum or the cerebral cortex. These schemes were designed for special conditions, symptoms or signs, such as ataxia or epilepsy, but, however well intentioned these highly focused classifications are, all special-purpose regional classifications fall short in the context of portraying the overall picture involving the entire neuraxis. Many disturbances of neuroblast migration produce abnormal gyration of the cerebral cortex but also involve migratory abnormalities in the cerebellum and sometimes the brainstem, and these features are not consid-

ered by ‘regional’ schemes that focus entirely on the cerebrum. An example is the Walker–Warburg syndrome, in which migratory disturbances are seen from the rhombic lip of His to the pontine and inferior olivary nuclei as well as to the cerebral cortex, and the cerebellar cortex and nuclei also have focal dysplasias. Even the epilepsies for which these schemes are principally designed may have important subcortical generators that contribute to the seizure disorder. In this same regard, schemes limited to the central nervous system also are too restrictive. Neural crest originates within the central nervous system but forms the entire peripheral nervous system and also induces many mesodermal structures not of neuroectodermal origin. Many neurocutaneous syndromes may be explained as neurocristopathies. Purely genetic schemes of classification provide a limited value in disorders in which a similar anatomical malformation may be due to several different genetic mutations. An example is holoprosencephaly (see Ch. 2). Another example is the Joubert syndrome, with multiple genetic associations (Valente et al., 2005). Other examples include the listing of genetic etiologies of microcephaly, neuroblast migratory disorders to the cerebral cortex and the genetically determined epilepsies. These schemes have merit for special purposes but cannot be universally applied because they are not integrated with morphology and do not emphasize variants due to gradients along the axes. One must also exercise caution that the strong human urge to categorize and classify does not impede, rather than facilitate, biological concepts. We mentally divide the body into systems for convenience, to better comprehend their functions. But these various systems are so interrelated, including – and perhaps especially – during development, that scientifically they cannot be segregated in isolation from one another. This ontogenesis of the brain is intimately associated with its arterial supply, parenchymal microcirculation, perfusion and oxygenation. The importance of relating these two systems, as an example, was articulated in 1976 by the German embryologist Erich Blechschmidt, whose monograph was recently updated and translated to English (Blechschmidt, 2004): Concepts such as body ‘systems’ (e.g. the cardiovascular system, the nervous system, etc.) are quite artificial; their only use is for the convenience of dividing the subject matter into sections or chapters. Body systems do not exist in reality. . .. The body functions as a whole and it is only as a whole that we should attempt to comprehend it.

AXES AND GRADIENTS OF THE NEURAL TUBE

5

Nevertheless, body systems do exist and every disorder of the digestive system does not necessarily affect the nervous system unless secondary to chronic absorptive and nutritional factors. The 19th century ‘fusion concept’ of the interdependence of all body systems may, at times, be too extreme a view to be applicable in all situations. On the other hand, cutaneous lesions often are good clinical markers of developmental abnormalities in the nervous system, as with some neurocutaneous syndromes. Yet another aspect to be considered is the process of induction, not only induction of the neural tube by extraneural tissues but also induction of extraneural tissues by the neural tube to produce, for example, associated craniofacial dysmorphisms (see Induction, below). Finally, one must consider that some malformations may not be of genetic origin and may be secondary to acquired defects in the developing brain, as might be induced by congenital infections or by ischemic episodes in the fetus that cause infarcts in the white matter that interrupt radial glial fibers that guide migratory neuroblasts. Any reliable classification scheme of dysgeneses of the nervous system must have flexibility to change and to incorporate nongenetic malformations. 1.1.1. Axes of the neural tube and gradients of genetic expression Bilateral symmetry evolved early in phylogeny and was recently demonstrated in microfossils of some of the earliest multicellular organisms to develop, as early as the pre-Cambrian period, about 480–500 million years ago (Bottjer, 2005). In the ontogeny of vertebrates, bilateral symmetry appears at gastrulation, in the stage of the primitive node (Hensen node) and primitive streak, at the time of appearance of the notochordal process and the neural plate. Not only is bilateral symmetry established at this time but the three fundamental axes of the body and of the future neural tube also become evident: a longitudinal axis with rostral and caudal ends (cephalization), a vertical axis with dorsal and ventral surfaces and a horizontal axis with medial (proximal) and lateral (distal) positions along this axis (Figs. 1.1 and 1.2). Structures differentiate in these three axes along a gradient: in the longitudinal axis, some structures develop only at the rostral end (head and organs of special senses) and others, only at the caudal end (tail). In the vertical axis, some structures develop only on the ventral side (most visceral organs) and others are confined to the dorsal side (spinal column; spinal cord); within the neural tube there are ventral structures (motor

Fig. 1.1. Drawing of neural tube before closure of the neuropores, to illustrate the three axes: longitudinal, vertical and horizontal. Many genes exhibit a gradient of diminishing expression along these axes and the extent of the gradient explains many malformations.

neurons) and dorsal structures (roof plate, epiphysis, collicular plate of midbrain). In the horizontal axis, some structures develop abnormally proximally, near the midline (limb girdle muscular dystrophy), whereas others develop only distally, as in the fingers and toes (syndactyly; polydactyly). These axes are evident in

6

L. FLORES-SARNAT AND H. B. SARNAT the neural tube long before its closure and all differentiating structures either are rostral or caudal, dorsal or ventral, medial or lateral. Positions of individual structures may change during the course of development, with growth and maturation, but each has its predetermined position in the neural tube. These positions at different gestational ages are predictable because each gene that programs development has a gradient of diminishing expression along the three axes as well: the expression of the gene is stronger at one end of the gradient and weakens in progressively more distant sites. Many genes have simultaneous expression and gradients in all three axes of the neural tube. The six genes recognized as defective in holoprosencephaly provide good examples of this phenomenon (Sarnat and Flores-Sarnat, 2001b). Some genes, notably the homeobox genes of neural tube segmentation, confine their expression to certain neuromeres and do not follow a gradient of diminishing expression (see Ch. 7). An example is EGR2 (homologue of Krox-20 in the mouse), which is expressed only in rhombomeres r3 and r5 and inhibits neural crest formation in those segments. A few normal embryological phenomena may appear to be disorders of segmentation but are actually physiological developmental phenomena; an example is secondary neurulation (see Ch. 7). Other genes may cause defective neuroblast migration that may or may not be expressed as a gradient. Downregulation of the EMX2 gene is responsible for schizencephaly but the normal fetal expression of EMX2 is in a rostrocaudal gradient within the forebrain, particularly the neocortex. Some genes are primarily involved with cellular lineage and many developmental defects due to faulty expression of these genes are secondary, such as the neuroblast migratory disturbances due to abnormal maturation of radial glial cells to guide the neuroblasts, as observed in tuberous sclerosis and hemimegalencephaly (Flores-Sarnat et al., 2003; also see Chs 9 and 10).

1.2. Upregulation/downregulation versus overexpression/underexpression Fig. 1.2. Drawing of the human neural tube during early fetal life, showing that the three principal axes persist and are not altered by changes in overall shape of the neural tube because of the normal formation of flexures. (Reproduced from Sarnat and Flores-Sarnat, 2004 with permission from John Wiley & Sons, Inc.)

The regulation and expression of developmental genes are not semantically identical and interchangeable (Sarnat and Menkes, 2000). The term upregulation implies overproduction of a genetic transcription product that is then overexpressed in the tissue. Downregulation is the opposite: underproduction of the genetic transcription product leads to underexpression in the tissue. Overexpression and underexpression are less specific than upregulation and downregulation. Whereas overexpression may be due to upregulation,

AXES AND GRADIENTS OF THE NEURAL TUBE it also may be due to normal amounts of transcription product, but lack of equilibrium with the product of an antagonistic gene having the opposite gradient of expression. If the antagonistic gene is downregulated, the balance is lost and the other gene becomes overexpressed without being upregulated. Thus, in the vertical axis, overexpression of a ventralizing gene may be due to either upregulation of that gene or downregulation of its antagonistic dorsalizing gene in the same vertical axis. Some genes act in a cascade, so that, if the earliest or first gene in the series is downregulated, subsequent genes in the cascade are not activated and do not become adequately expressed. The morphogenic effect of overexpression in the neural tube is hyperplasia and/or duplication of structures, following the gradient of the defective gene. The effect of underexpression, by contrast, is hypoplasia, aplasia or noncleavage in the midline.

1.3. Examples of disturbances in axes and gradients 1.3.1. Longitudinal axis With the initial formation of the neural tube, before it is completely closed, the longitudinal axis resembles a straight line. With the subsequent development of the cervical, pontine and other flexures and the differentiation of new structures in the rostral end of the neural tube in particular, the axis adapts to the new shape. The axes are, therefore, not dependent upon flexures or changing overall shape of the neural tube with subsequent development, and the longitudinal axis follows the curvature of the tube rather than remaining a straight line (Figs. 1.1 and 1.2). 1.3.1.1. Rostrocaudal gradient Holoprosencephaly provides an excellent example of this gradient. The dysgenesis always involves the forebrain with failed or incomplete cleavage of the prosencephalon, but in many cases there also is noncleavage of the thalamus, hypothalamus and other diencephalic structures, and the gradient may extend to the midbrain, with noncleavage of the superior colliculi, aqueductal atresia and continuity of the oculomotor nuclei across the midline. This gradient may be seen with any of the six identified genes that may be defective in holoprosencephaly. Anencephaly is another example, with failed cerebral cortical differentiation and additional but variable dysgenesis of deep telencephalic and diencephalic nuclei (basal ganglia, thalamus and hypothalamus). In this case, the face frequently shows midfacial hypoplasia in addition to the extensive loss of the calvarium, both craniofacial defects related to abnormal mesencephalic neural crest formation because the ros-

7

trocaudal gradient extends to the dorsal midbrain, just as in holoprosencephaly (Sarnat and Flores-Sarnat, 2002). As another example, the gene ZIC2, a mutation of which is responsible for schizencephaly, normally exhibits a rostrocaudal gradient of diminishing expression within the cerebral cortex of the mouse (Cecchi, 2000), although the lateromedial gradient in the horizontal axis also is severely affected in schizencephaly. 1.3.1.2. Caudorostral gradient Sacral agenesis is an example of defective expression in this gradient. This malformation is an absence of the caudal spinal column, ranging from an almost inconsequential agenesis of the most caudal segment to the most frequent agenesis of the entire sacrum and, in more severe cases, a deletion of lumbar and even thoracic vertebrae; this agenesis of the vertebrae follows a caudorostral gradient. The notochord is absent or defective at those levels where vertebral agenesis results from defective or absent sclerotome formation in the embryo, and segmental amyoplasia also results. Failure of normal notochordal induction of the floor plate of the neural tube leads to severe dysplasias of the spinal cord, with apparent fusion of ventral horns and a fragmented and poorly formed central canal with heterotopic ependyma but normal spinal cord at more rostral levels where the vertebrae are formed and presumably the notochord was present for normal embryonic induction (Sarnat et al., 1976; Sarnat, 1992). 1.3.2. Vertical axis 1.3.2.1. Dorsoventral gradient Many genes, particularly those of the bone morphogenic protein (BMP) family, the wingless (WNT) family, several of the paired homeobox gene (PAX) family and the zinc-finger ZIC2 gene, have a strong dorsoventral expression. Upregulation of these genes causes the formation of dorsal structures of the neural tube even in ventral regions, and hyperplasia of structures in dorsal regions. Downregulation, by contrast, allows overexpression of antagonistic ventralizing genes, with the formation of ventral structures even in dorsal regions, and aplasia or hypoplasia of dorsal structures. Several malformations involve dorsalizing genes in the vertical axis and produce dysgeneses explicable on that basis. Rhombencephalosynapsis is an aplasia of the cerebellar vermis and relatively normal formation of the cerebellar hemispheres; the medial sides of the two hemispheres are fused in the midline and occasionally mildly hypoplastic, but without intervening subarachnoid space between them as occurs in Joubert syndrome and Dandy–Walker malformation (see Ch. 4). It may be associated with septo-optic-pituitary dysplasia, and both this latter condition and

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rhombencephalosynapsis may be regarded as a defect of the dorsoventral gradient (Sarnat, 2000). In holoprosencephaly, the midbrain may show apparent ‘fusion’ of the superior colliculi and aqueductal atresia, representing not only the rostrocaudal gradient reaching the mesencephalic neuromere but also defective genetic expression in the dorsoventral gradient of the vertical axis; the ventral structures, such as the cerebral peduncles and the substantia nigra, are spared (Sarnat and Flores-Sarnat, 2001b). Upregulation of genes of the dorsoventral gradient in the spinal cord may cause duplication of the dorsal horns (Sarnat, 2000; Sarnat and Menkes, 2000). 1.3.2.2. Ventrodorsal gradient The most studied and most important of the genes that show a strong ventrodorsal gradient in normal developing brain and spinal cord is Sonic hedgehog (SHH). Of the six or more well documented genes associated with holoprosencephaly (see Ch. 2), only ZIC2 is a dorsalizing gene; the other five, including SHH and its receptor gene, Patched (PTCH), are all strong ventralizing genes, i.e. they follow a ventrodorsal gradient of expression. Recent evidence indicates that SHH reverses the polarity of its gradient rostral to the mesencephalon, however. Sacral agenesis follows a caudorostral gradient in the longitudinal axis, as discussed above, but also has a strong ventrodorsal gradient of defective genetic expression in the vertical axis because the absent or defective portion of notochord does not secrete the transcription product of SHH that is important for induction of the floor plate. The ventral horns of the dysplastic spinal cord are much more abnormal than are the dorsal horns or white matter of the dorsal columns, and the roof plate develops and forms a dorsal median septum. Upregulation in the ventrodorsal gradient of the vertical axis of the spinal cord, by contrast, from excessive SHH expression, results in duplication of the central canal and hypertrophy of the ventral horns or even diplomyelia or duplication of the spinal cord restricted to a region, such as the lumbar or cervical (Sarnat, 2000). An old terminology based upon human chauvinism renamed ventral as anterior and dorsal as posterior (e.g. anterior horn cells versus the correct ventral horn cells) because 19th century biologists considered humans to be superior, in part because of their upright posture, and that this posture merited redesignation of positional labels. Many other animals also ambulate or ambulated on two legs, including kangaroos, many primates, birds, some lizards and many dinosaurs; on the other hand, human newborns and infants during the first months of life, and also adults during sleep, do

not exhibit an upright posture. There is no justification for using different labels to designate human anatomy; nonstandardized terminology between species, including our own, leads to confusion rather than enhanced understanding. The correct terms, ventral and dorsal, denote the relative anatomical positions of structures and are independent of posture. 1.3.3. Horizontal axis 1.3.3.1. Mediolateral gradient Holoprosencephaly again provides a prototype example of a defective mediolateral gradient in the horizontal axis. Whereas in the lissencephalies the entire cerebral cortex is abnormally laminated, in holoprosencephaly the neocortex is continuous across the midline because of failure of cleavage to form the interhemispheric fissure. The frontal midline and parasagittal regions exhibit severely disrupted microscopic architecture, whereas the more lateral regions of cortex are less dysplastic and may even be normal; radial glia are fragmented and disoriented in the medial zones but span the cerebral mantle normally in the lateral regions (Sarnat and Flores-Sarnat, 2001b). The extent of the mediolateral gradient in holoprosencephaly may contribute to the degree of mental retardation, cognitive deficits, aphasias and epilepsy. Septo-optic-pituitary dysplasia is another example of midline and paramedian structures being severely altered and more lateral regions remaining unaffected, but the rostrocaudal gradient also plays a role. 1.3.3.2. Lateromedial gradient One example of this gradient is provided by pontocerebellar hypoplasia, which is really a progressive degenerative disease that begins in fetal life and extends well into the postnatal period (Barth, 1993, 2000). The cerebellar vermis remains well preserved until late stages, whereas the most lateral parts of the folia in the lateral cerebellar hemispheres are poorly laminated and show extensive loss of all types of neurons and reactive gliosis (Sarnat and Flores-Sarnat, 2001a). The dorsoventral gradient also affects the cerebellar malformation. Another example is schizencephaly, already mentioned above as a combined rostrocaudal gradient defect in the longitudinal axis and lateromedial gradient in the horizontal axis.

1.4. Induction Induction is the influence of one embryonic tissue upon another, each then diverging to develop as different mature tissues. Induction may occur between two traditional germ layers, such as the notochordal (mesoderm) induction of the floor plate of the neural tube

AXES AND GRADIENTS OF THE NEURAL TUBE (ectoderm), or it may occur within the same germ layer, as with the retina (ectoderm), inducing formation of a lens and cornea (ectoderm) instead of those tissues simply forming more epidermis. Induction may imply influences upon or within the developing neuroectoderm, as in the examples just cited, or the neural tube may induce nonneural structures, as with the induction of craniofacial development mediated by the prosencephalic and mesencephalic neural crest tissue. Defects in induction of the intercanthal ligament, from prosencephalic neural crest, may result in hypertelorism, often associated with agenesis of the corpus callosum because both this neural crest and the callosal plate originate in the lamina terminalis, or hypotelorism, as is often associated with holoprosencephaly or other craniofacial dysmorphisms (Sarnat et al., 2007).

1.5. Neurocristopathies and neurocutaneous syndromes: gradients of genetic expression affecting neural crest Neurocristopathy is a term coined by Bolande to define diseases that could be attributed to an embryonic disturbance in the formation, migration or terminal differentiation of neural crest tissue (Bolande, 1974). He originally classified the neurocristopathies as either ‘simple’ or ‘complex’: aganglionic megacolon, neurofibromatosis and neurocutaneous melanosis, but later extended his list (Bolande, 1997). Many other diseases are now recognized to be primarily of neural crest origin and can be identified with the site of formation and initial migration of neural crest in the dorsal part of the neural tube, in the prosencephalon, mesencephalon and rhombencephalon. Furthermore, some neurocristopathies, such as Waardenburg syndrome, may also be classified as neurocutaneous syndromes (Sarnat and Flores-Sarnat, 2005; Flores-Sarnat and Sarnat, 2007). These disorders may involve the peripheral nervous system and sometimes the central nervous system, as well as many nonneural neural crest derivatives, such as the craniofacial membranous bones and cartilages, nerve sheaths, blood vessels and cutaneous melanocytes. The term neurocutaneous syndromes originated with Yakovlev and Guthrie (1931).

1.6. Scheme of classification of central nervous system malformations Table 1.1 is a classification based upon our most recently published scheme (Sarnat and Flores-Sarnat, 2004) with additional modifications including, not previously published, the neurocristopathies. Items that necessarily overlap with other headings are crossreferenced. Well documented specific genes are cited

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Table 1.1 Etiological classification of human nervous system malformations as patterns of genetic expression I. Genetic mutations expressed in the primitive streak or node A. Upregulation of organizer genes 1. Duplication of neural tube 2. Conjoined twins B. Downregulation of organizer genes 1. Agenesis of neural tube II. Disorders of genetic gradients in the axes of the neural tube A. Vertical axis 1. Overexpression in the ventrodorsal gradient a. Duplication of spinal central canal b. Duplication of ventral horns of spinal cord c. Diplomyelia (and diastematomyelia?) d. Duplication of neural tube e. Ventralizing induction of somite (segmental amyoplasia) 2. Underexpression in the ventrodorsal gradient a. Fusion of ventral horns of spinal cord b. Sacral (thoraco-lumbo-sacral) agenesis c. Arrhinencephaly d. Holoprosencephaly 3. Overexpression in the dorsoventral gradient a. Duplication of dorsal horns of spinal cord b. Duplication of dorsal brainstem structures 4. Underexpression in the dorsoventral gradient a. Fusion of dorsal horns of spinal cord b. Septo-optic-pituitary dysplasia c. Rhombencephalosynapsis B. Longitudinal axis 1. Disorders in the rostrocaudal gradient a. Holoprosencephaly involving diencephalon and mesencephalon b. Anencephaly 2. Disorders in the caudorostral gradient a. Sacral agenesis C. Horizontal axis 1. Disorders in the mediolateral gradient a. Holoprosencephaly 2. Disorders in the lateromedial gradient a. Pontocerebellar hypoplasia b. Schizencephaly (EMX2) III. Disorders of segmentation of the neural tube A. Increased homeobox domains and/or ectopic expression 1. Lumbosacral meningomyelocele (?) 2. Chiari II malformation B. Decreased homeobox domains and/or neuromere deletion 1. Agenesis of mesencephalon and metencephalon (EN2) 2. Lumbosacral meningomyelocele 3. Global cerebellar aplasia or hypoplasia 4. Agenesis of basal telencephalic nuclei (EMX1?) (continued)

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Table 1.1

Table 1.1

(Continued)

(Continued)

IV. Disorders of neuroepithelial cell proliferation A. Generalized cerebral hypoplasia V. Disorders of decreased or increased apoptosis of neuroepithelial cells A. Megalencephaly (e.g. Sotos syndrome) B. Spinal muscular atrophy (SMN, NAIP) VI. Aberrations in cell lineages by genetic mutation A. Nonneoplastic 1. Striated muscle in the central nervous system 2. Dysplastic gangliocytoma of the cerebellum (Lhermitte–Duclos) 3. Tuberous sclerosis 4. Hemimegalencephaly (also VIII. Disorders of symmetry) B. Neoplastic 1. Myomedulloblastoma 2. Dysembryoplastic neuroepithelial tumors 3. Gangliogliomas and other mixed neural tumors VII. Disorders of symmetry A. Hemimegalencephaly (also see IV. Aberrations of cellular lineage) B. Hemicerebellar megalencephaly, including dysplastic gangliocytoma of cerebellum (Lhermitte–Duclos disease) VIII. Disorders of secretory molecules and genes that mediate migrations A. Neuroblast migrations 1. Initial course of neuroblast migration a. Filamin-1 (X-linked dominant periventricular nodular heterotopia) 2. Middle course of neuroblast migration a. Doublecortin (DCX; X-linked dominant subcortical laminar heterotopia or band heterotopia) b. LIS1 (type I lissencephaly or Miller–Dieker syndrome) c. Fukutin (type II lissencephaly; Fukuyama muscular dystrophy) d. Empty spiracles (EMX2; schizencephaly) e. Astrotactin 3. Late course of neuroblast migration; architecture of cortical plate a. Reelin (RLN; pachygyria and cerebellar hypoplasia) b. Disabled-1 (DAB1; also VLDL/Apoe2R?App receptor defect; downstream of RLN, EMX2 and DCX) c. L1-NCAM (X-linked hydrocephalus and pachygyria with aqueductal stenosis) B. Glioblast migration C. Focal migratory disturbances due to acquired lesions of the fetal brain IX. Disorders of secretory molecules and genes that attract or repel axonal growth cones A. Netrin downregulation

B. Keratan sulfate and other glycosaminoglycan downregulations C. S-100b protein downregulation or upregulation (?) X. Disorders of neural crest (neurocristopathies) A. Prosencephalic neural crest 1. Epidermal nevus syndrome (midline vertical facial nevus of nasal and frontal hyper- or hypopigmentation) 2. Hypertelorism, e.g. associated with agenesis of corpus callosum 3. Frontal encephaloceles through either fonticulus frontalis or foramen cecum B. Mesencephalic neural crest 1. Hypotelorism and midfacial hypoplasia, e.g. holoprosencephaly 2. Aplasia of cranial vault and midfacial hypoplasia in anencephaly C. Rhombencephalic neural crest 1. Sturge–Weber disease 2. Aganglionic megacolon (Hirschsprung’s disease) D. Neurocristopathies involving all sites of origin of neural crest (neurofibromatosis I; Waardenburg syndrome) A periodically updated list of genes documented to be associated with specific human malformations and also providing references for each genetic mutation or deletion, is published in the European Journal of Paediatric Neurology (Sarnat 2005). A list and discussion of these genes also appears in Chapter 28.

parenthetically for some malformations but this scheme is designed to show patterns of genetic expression rather than to be a list of genes with their associated dysgenesis if mutated or deleted. It must always be borne in mind that many genes act sequentially in series and that multiple genes are coexpressed at the same time, thus showing overlapping expression. Some genes are expressed early then become dormant, only to be re-expressed later for a different purpose; some genes are expressed early for differentiation and then continue to be expressed even in adult life for maintenance of identity of particular neurons (Sarnat and Menkes, 2000). These temporal factors make it difficult to develop schemes that are closely time-linked as if they were a simple sequence of one following another, as frequently is presented in traditional classifications (Sarnat, 1992; Volpe, 1995).

1.7. Conclusions Many malformations of the brain and spinal cord follow gradients of defective genetic expression along the three axes of the neural tube and these gradients explain

AXES AND GRADIENTS OF THE NEURAL TUBE many features of the malformation, regardless of whether the precise gene is identified. Some may exhibit gradients of defective genetic expression in all three axes simultaneously, exemplified by holoprosencephaly and regardless of which of the recognized genes is involved. The rostrocaudal and/or dorsoventral gradients reaching the embryonic mesencephalic neuromere may result in defective neural crest formation and migration, resulting in abnormal neural induction of craniofacial structures to produce midfacial hypoplasia and other facial dysmorphisms. These gradients of genetic expression in the axes of the neural tube should be considered when interpreting neuroimaging studies or neuropathological examinations of brains with congenital malformations, at all fetal and postnatal ages. Classifications of cerebral malformations should integrate criteria of both morphogenesis and molecular genetic programming. The patterns of faulty genetic expression along the axes may be more informative about pathogenesis than the identity of the specific genetic defect. We have here added new categories of disorders of neuroepithelial cell proliferation (global cerebral hypoplasia), neuroepithelial apoptosis and the neurocristopathies to the classification scheme we previously proposed.

References Barkovich AJ, Kuzniecky RI, Jackson GD, et al (2001). Classification system for malformations of cortical development: update 2001. Neurology 57: 2168–2178. Barth PG (1993). Pontocerebellar hypoplasia: an overview of a group of inherited neurodegenerative disorders with fetal onset. Brain Dev 6: 411–422. Barth PG (2000). Pontocerebellar hypoplasia: how many types? Eur J Paediatr Neurol 4: 161–162. Blechschmidt E (2004). The ontogenetic basis of human anatomy. A biodynamic approach to development from conception to birth. Revised, edited and translated by B Freeman. North Atlantic Books, Berkely, CA, pp. 2–3. Bolande RP (1974). The neurocristopathies. A unifying concept of disease arising in neural crest maldevelopment. Hum Pathol 5: 409–429. Bolande RP (1997). Neurocristopathy: its growth and development in 20 years. Pediatr Pathol Lab Med 17: 1–25. Bottjer DJ (2005). The early evolution of animals. Sci Am 293: 42–47. Cecchi C (2000). Emx homeogenes and mouse brain development. Trends Neurosci 23: 347–352. Flores-Sarnat L, Sarnat HB (2007). Embryology of the neurocutaneous syndromes. In: M Ruggieri, I Pascual-Castroviejo, C Di Rocco (Eds.), Neurocutaneous Disorders:

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Phakomatoses and Hamartoneoplastic Syndromes. Springer, New York, in press. Flores-Sarnat L, Sarnat HB, Da´vila-Gutie´rrez G, et al. (2003). Hemimegalencephaly: Part 2. Neuropathology suggests a disorder of cellular lineage. J Child Neurol 18: 776–785. Sarnat HB (1992). Cerebral Dysgenesis. Embryology and Clinical Expression. Oxford University Press, New York. Sarnat HB (1997). Microscopic criteria to determine gestational age of the fetal and neonatal brain. In: JH Garcıá, (Ed.), Neuropathology: The Diagnostic Approach. MosbyYearbook, St Louis, pp. 529–540. Sarnat HB (2000). The new neuroembryology: molecular genetic classification of central nervous system malformations. J Child Neurol 15: 675–687. Sarnat HB (2001). Congenital malformations of the nervous system: a neuropathological perspective. Neuroimaging Clin North Am 11: 57–77. Sarnat HB (2005). Central nervous system malformations: locations of known human mutations. Eur J Paediatr Neurol 9: 427–431. Sarnat HB, Flores-Sarnat L (2001a). What’s new in neuroembryology? A new classification of malformations of the nervous system. Integration of morphological and molecular genetic criteria. Eur J Paediatr Neurol 5: 57–64. Sarnat HB, Flores-Sarnat L (2001b). Neuropathologic research strategies in holoprosencephaly. J Child Neurol 16: 918–931. Sarnat HB, Flores-Sarnat L (2002). Molecular genetic and morphological integration in malformations of the nervous system for etiologic classification. Semin Pediatr Neurol 9: 335–344. Sarnat HB, Flores-Sarnat L (2004). Integrative classification of morphology and molecular genetics in central nervous system malformations. Am J Med Genet 126A: 386–392. Sarnat HB, Flores-Sarnat (2005). Embryology of the neural crest: its inductive role in neurocutaneous syndromes. J Child Neurol 20: 637–643. Sarnat HB, Menkes JH (2000). The new neuroembryology: how to construct a neural tube. J Child Neurol 15: 110–124. Sarnat HB, Flores-Sarnat L, Carstens MH (2007). Hypotelorism vs hypertelorism: craniofacial dysmorphisms secondary to defective induction by neural crest associated with cerebral malformations. J Child Neurol 21: in press. Sarnat HB, Case ME, Graviss R (1976). Sacral agenesis. Neurologic and neuropathologic features. Neurology 26: 1124–1129. Valente EM, Marsh SE, Louis CM, et al. (2005). A molecular classification of Joubert syndrome. Ann Neurol 58 (Suppl 9): S90. Volpe JJ (1995). Neurology of the Newborn, 3rd edn. WB Saunders, Philadelphia. Yakovlev PI, Guthrie RH (1931). Congenital ectodermoses (neurocutaneous syndromes) in epileptic patients. Arch Neurol Psychiatr 26: 1145–1194.


Midline hypoplasias Chapter 2

Holoprosencephaly JIN S. HAHN* Department of Neurology, Stanford University School of Medicine and the Lucile Packard Children’s Hospital at Stanford, Stanford, CA, USA

2.1. Introduction Holoprosencephaly (HPE) is a complex congenital brain malformation characterized by failure of the forebrain to bifurcate into two hemispheres, a process normally complete by the fifth week of gestation (Golden, 1999). HPE is the most common developmental defect of the forebrain and midface in humans and occurs in 1 in 250 pregnancies (Matsunaga and Shiota, 1977). Because only 3% of fetuses with HPE survive to delivery (Cohen, 1989b), the live birth prevalence is only about 1 in 10 000 (Croen et al., 1996; Rasmussen et al., 1996; Bullen et al., 2001). Two-thirds of affected patients have alobar HPE, the most severe form (Ming and Muenke, 1998). Modern high-resolution brain magnetic resonance imaging (MRI) has increased the identification of children with less severe forms who have gone undiagnosed in the past. Therefore, the true live birth prevalence of HPE is likely to be higher than previously estimated, and the proportion of the cases with milder subtypes appears to be increasing in modern series (Stashinko et al., 2004).

2.2. Historical background While individuals with cyclopia have been described for centuries in mythology and in the scientific literature since the late 18th century (Siebert et al., 1990), it was not until 1882 that Kundrat first described the cerebral changes of HPE, including absent olfactory nerves. Believing the absence of olfactory lobes and bulbs was the cardinal feature, he termed the condition ‘arrhinencephaly’ (Kundrat, 1882). Yakovlev recognized involvement of the entire telencephalon and called the single

telencephalic ventricle a ‘holosphere’ and the malformation ‘holotelencephaly’ (Yakovlev, 1959). DeMyer found that the thalamus and other diencephalic structures were also involved and coined the still-favored term ‘holoprosencephaly’ to indicate that the defect involved the entire prosencephalon (DeMyer and Zeman, 1963).

2.3. Definition and classification HPE has traditionally been classified according to DeMyer’s division into three grades of severity: alobar, semilobar and lobar. These classifications have been based on neuropathological series, but neuroimaging series have more recently contributed to our understanding of the neuroanatomical abnormalities in HPE. In addition to these classic forms, there is another milder subtype of HPE, called middle interhemispheric variant (MIH) or syntelencephaly (Barkovich and Quint, 1993; Simon et al., 2002). The sine qua non feature of HPE is an incomplete separation of the cerebral hemispheres. The lack of cleavage, or lack of separation, of midline structures that is the defining feature of holoprosencephaly is sometimes inappropriately referred to as ‘fusion’ of the cerebral hemispheres, which wrongly implies that the structures grew together after being initially separate. 2.3.1. Alobar holoprosencephaly In the most severe form, alobar HPE, there is complete or nearly complete lack of separation of the cerebral hemispheres with a single midline forebrain ventricle (a crescent shaped monoventricle), which often communicates with a dorsal cyst (Fig. 2.1). The cerebral

*Correspondence to: Jin S. Hahn MD, Department of Neurology, Stanford University Medical Center, 300 Pasteur Drive, Room A343, Stanford, CA 94305–5235, USA. E-mail: [email protected], Tel: þ1-650-723-6841, Fax: þ1-650-725-7459.

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Fig. 2.1. MRI of a neonate with alobar HPE. (A) Axial T2-weighted image demonstrating incomplete separation of the two hemispheres and thalami, and a large dorsal cyst (DC). (B) Coronal T2-weighted image showing a continuity of gray matter in the midline without an interhemispheric fissure. The ventricular system is composed of a single midline monoventricle (MV). (C) Sagittal T1-weighted image showing absence of corpus callosum and a monoventricle that communicates with the dorsal cyst.

holosphere usually has the appearance of a pancake-like mass of tissue in the rostralmost portion of the calvaria. The posterior aspect of the cerebrum is shaped like a horseshoe with the posterior dorsal rim composed of a thin, cyst-like membrane (Fig. 2.2) (Golden, 1999).

This membrane is the posterior roof of the monoventricle. When the dorsal cyst is smaller, the posterior dorsal rim is located more posteriorly and the holosphere may have a cup-like appearance (DeMyer, 1987). The interhemispheric fissure, falx cerebri and corpus callosum

Fig. 2.2. (A) View from the inferior aspect of a fixed brain from a 3-day old newborn (37-weeks gestational age) with alobar HPE and severe hydrocephalus, who died shortly after birth. The hemispheres are completely noncleaved, while the temporal lobes are underdeveloped but separated. The interhemispheric fissure and the olfactory tracts and bulbs are absent. Part of the right posterior cortex extends inferiorly and overlies the right cerebellar hemisphere. (B) The view from the posterior aspect of the brain shows a large sac-like structure (the monoventricle) surrounded by the thinned cortex of the holosphere. The posterior rim of the cerebrum (arrowheads) is shaped like an inverted U and remnants of a thin membrane are attached to the rim. This membrane is the posterior roof of the monoventricle. The thalamic nuclei (black arrow) appear completely nonseparated and are attached caudally to an abnormal appearing mesencephalon. Courtesy of Dr Hannes Vogel.

HOLOPROSENCEPHALY are completely absent. The basal ganglia and hypothalamic and thalamic nuclei lack separation, resulting in absence of the third ventricle (Simon and Barkovich, 2001). At times, a mass of deep gray matter is noted with poor differentiation of the different nuclei (striatum and thalamus). This mass may be attached to the holosphere by a small anterior midline hinge of tissue that lacks corticospinal, corticothalamic and thalamocortical tracts (Muenke, 1995). Olfactory bulbs and tracts are absent (Yamada et al., 2004). 2.3.2. Semilobar holoprosencephaly In semilobar HPE, the anterior hemispheres fail to separate, while some portions of the posterior hemispheres show separation (Fig. 2.3). The noncleaved frontal lobes are usually small. The frontal horns of the lateral ventricle are absent but posterior horns and trigones are present. The septum pellucidum is absent. The corpus callosum is absent anteriorly but the splenium of the corpus callosum is present. The anterior extent of the corpus callosum development correlates with the anterior extent of the interhemispheric fissure formation (Simon and Barkovich, 2001). On imaging studies, some portions of the posterior interhemispheric fissure, falx cerebri and splenium of the corpus callosum can be identified. The deep gray nuclei are incompletely separated and can usually be identified as discrete structures, usually resulting in a small third ventricle (Simon et al., 2000). The head of the caudate nuclei is often noncleaved. Dorsal cysts are sometimes seen in semilobar HPE, especially when

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there is nonseparation of the thalamic nuclei. The olfactory bulbs are either absent or hypoplastic. 2.3.3. Lobar holoprosencephaly In lobar HPE, a milder form, the cerebral hemispheres are fairly well developed and separated, while only the most rostral/ventral aspects of the neocortex are nonseparated (Fig. 2.4). Again, the corpus callosum is absent in the region affected. The posterior half of the corpus callosum (including the splenium and body) is present, although the genu may be poorly developed. Rudimentary formation of the frontal horns is usually present. The third ventricle is fully formed. The interhemispheric fissure and falx cerebri extend anteriorly beneath the frontal bones, although the most anterior aspects of the falx are hypoplastic (Simon and Barkovich, 2001). The thalamic nuclei may be fully separated, although an enlarged massa intermedia may be present. A dorsal cyst is usually absent. Some of the patients with lobar HPE may fall within the spectrum of septo-optic dysplasia (Barkovich et al., 1989). Olfactory bulbs and tracts may be present, although they are usually hypoplastic. 2.3.4. Middle interhemispheric variant (syntelencephaly) Middle interhemispheric variant (MIH) is a recently described subtype (Barkovich and Quint, 1993; Simon et al., 2002). In contrast to ‘classic’ HPE, the posterior frontal and parietal lobes fail to separate in MIH, while

Fig. 2.3. MRI of a 3-year-old patient with semilobar HPE. (A) Axial T2-weighted image showing absence of interhemispheric fissure anteriorly. The posterior hemispheres are well separated and the posterior horns of the lateral ventricles are well formed. The heads of the caudate are nonseparated and the thalami are partially separated. A small dorsal cyst is present (DC). (B) Coronal T2-weighted image of the same patient showing a monoventricle (MV) and partial nonseparation of the thalamic nuclei. (C) A sagittal T1-weighted image of a different patient with semilobar HPE demonstrates absence of the genu and body of the corpus callosum but presence of the splenium (arrowhead).

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Fig. 2.4. MRI of a 16-month-old patient with lobar HPE. (A) Axial T2-weighted image showing cerebral hemispheres that are fairly well separated both anteriorly and posteriorly. The fontal horns are poorly developed (arrowheads). (B) Coronal T1weighted image showing failure of complete separation of the frontal lobes with continuity of gray matter in the inferior frontal regions (arrowheads). (C) Sagittal T1-weighted image demonstrating that the body and splenium of the corpus callosum are present (arrowhead) but the genu is not developed.

Fig. 2.5. MRI of two patients with MIH. (A) Axial T1-weighted image showing continuity of gray matter in the posterior frontal lobes across the midline. (B) Coronal 1-weighted image (same patient as A) through incompletely separated hemispheres at the level of the body of the lateral ventricle showing continuity of the gray matter (black arrows). (C) In a different patient, a sagittal T1-weighted image through the midline shows the presence of the genu and splenium of the corpus callosum (black arrows). The body of the corpus callosum is absent in the region of nonseparated hemispheres (white arrowhead).

the poles of the frontal and occipital lobes are well separated (Fig. 2.5) (Barkovich and Quint, 1993; Simon et al., 2002). The caudate nuclei and thalami are often incompletely separated (Simon et al., 2002). The most anteroinferior portions of the prosencephalon are spared, resulting in separation of the inferior frontal lobes, lentiform nuclei, and hypothalamic nuclei. Although there is some disagreement as to whether MIH should be classified as a subtype HPE, it does have the cardinal features of HPE including incomplete separation of the cerebral hemispheres and some deep gray nuclei. More detailed characteristics of MIH are provided in the Neuroimaging section below.

2.3.5. Newer classification schema The classification of classic HPE based on the degree of hemispheric nonseparation falls within a spectrum. DeMyer (1987) brought attention to this spectrum when he stated: ‘from the holospheric brain with no hint of an interhemispheric fissure, the spectrum of the malformation extends in unbroken continuity through intermediate and minimal stages’. Neuroradiologists have found that categorizing an individual case into one of the three classic forms may be challenging (Simon and Barkovich, 2001), particularly in milder subtypes. For example, the precise distinction between lobar and semilobar HPE is difficult in some

HOLOPROSENCEPHALY cases. In addition, the deep gray nuclei, which frequently show incomplete separation in HPE, may be just as important in predicting outcome and function (Simon et al., 2000; Plawner et al., 2002). Classification scoring systems that also take into account the abnormalities of deep gray nuclei, cortical malformations, pituitary gland and other structures have been proposed and found to be of value when correlating with neurodevelopmental outcome (Simon and Barkovich, 2001; Plawner et al., 2002). Ideally, a classification scheme should incorporate genetic criteria into the morphological criteria. Genetic classification of HPE has been more difficult to accomplish because of the complex nature and heterogeneity of the disorder (see Etiology, below). Furthermore, only a small proportion (15–20%) of human HPE cases have had an identifiable mutation in genes known to cause HPE (Ming and Muenke, 2002), making a classification system based on genotype difficult at best.

2.4. Etiology The etiologies of HPE are heterogeneous and include environmental and genetic causes. At its most fundamental level, HPE may be thought of as a disorder of faulty patterns of gene expression in the early embryonic nervous system. The cardinal feature of HPE is a failure of embryonic forebrain to bifurcate into the two cerebral hemispheres. Although the developmental defect is referred to as ‘failure of cleavage’ of the forebrain, this term does not accurately reflect the abnormality. The process by which two eyes and two hemispheres are formed is more accurately one of growth, i.e. an increase in number of cells (Muenke, 1995). Abnormal patterning in early embryonic nervous system from genetic and environmental factors may perturb normal growth, either by decreasing the normal proliferation of cells or by abnormally increasing programmed cell death. These factors may lead to morphological abnormality in the brain, which seemingly appear as a ‘failure of cleavage’ or ‘fusion’ of midline structures. 2.4.1. Neurodevelopmental abnormalities in holoprosencephaly Before examining the etiologies of HPE, it is useful to discuss the proposed mechanisms of pathogenesis in HPE based on recent studies in humans and animal models. These studies have shown that the primary defect in HPE is in the vertical gradients (dorsoventral and ventrodorsal) patterning of the rostral neural tube, which occurs during the first 4 weeks of gestation

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(Golden, 1999). In normal embryonic forebrain development, a complex interaction between ventralizing signals (molecules arising from the floor plate and prechordal plate, primarily Sonic hedgehog (SHH), that act on embryonic cells to give them characteristics of ventral CNS) and dorsalizing signals (molecules arising from the roof plate that act on cells to give characteristics of dorsal CNS) modulate regional identity of tissues along the vertical axis of the neural tube (Simon and Barkovich, 2001). The gradients of ventral and dorsal signaling molecules induce a distinct combination of genes and transcription factors in populations of cells at different levels along the vertical axis. These transcription factors induce the production of other molecules that lead to cell differentiation at various levels. The presumed mechanism in HPE is thought to be the lack of production of ventralizing factors or an overproduction of dorsalizing factors, resulting in noncleavage of the midline structures and producing holoprosencephalic brains (Golden, 1999). As hypothesized by Sarnat and Flores-Sarnat (2001), gradients in other axes play a role in determining neuropathological abnormalities in HPE and can explain the wide spectrum of abnormalities. In addition to the vertical gradient, there is also a gradient in the anteroposterior (rostrocaudal) axis (Rubenstein et al., 1998). Studies indicate that the areas most consistently malformed in classic HPE (cortex, striatum and hypothalamus) belong to the same embryonic anteroposterior domain, namely the secondary prosencephalon. The more posterior structures, such as the thalamus (diencephalon) and midbrain (mesencephalon), are less frequently affected since they are more distant from the anterior pole. A mediolateral gradient in the horizontal axis also exists in HPE. Neuroimaging studies also suggest that the frequency of noncleavage in HPE depends on proximity to the midline and to the ventricular system. Structures adjacent to the midline or ventricles (such as the cingulate cortex, caudate nucleus and medial hypothalamus) are more frequently noncleaved than the structures normally located farther from the midline or ventricles (such as globus pallidus) (Simon and Barkovich, 2001). Neuropathologically, the cortical microarchitecture (lamination) and the radial glial fibers tend to be more disrupted medially in medial parts of the forebrain, while the lateral cortex is usually spared (Sarnat and Flores-Sarnat, 2001). Sonic hedgehog is important in ventral patterning and the formation of the ventrodorsal gradient. It plays a critical role in midline patterning of human embryonic forebrain and CNS development. SHH is

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expressed in the notochord, the floorplate of the neural tube, the posterior limb buds, and the gut. SHH is also expressed in prechordal plate and plays an essential role in craniofacial development including that of the eyes. Deficiencies in SHH expression in the prechordal plate thus account for the common association of craniofacial abnormalities in HPE. The genetic pathways involved in dorsal patterning and roof plate differentiation include the bone morphogenetic proteins (BMPs) (Golden, 1999) and ZIC2, which create a dorsoventral gradient. 2.4.2. Genetic causes 2.4.2.1. Chromosomal anomalies Chromosomal anomalies including trisomies, duplications, deletions and ring arrangements play an important role in HPE. About 40% of live births with HPE have a chromosomal anomaly, and trisomy 13 accounts for over half of these cases (Croen et al., 1996). Of infants born with trisomy 13, 70% have holoprosencephaly (Taylor, 1968). Trisomy 18 is the second most common chromosomal abnormality. In fetal series of HPE, the incidence of abnormal karyotype by amniocentesis ranged from 29% to 37%, with approximately 70–80% of the abnormal karyotype being trisomy 13 (Berry et al., 1990; Blaas et al., 2002). Other abnormalities of chromosome 13 (e.g. deletions and duplications of different regions on 13q and ring chromosome 13) and partial deletions of chromosome 18 have been seen in patients with HPE. Detailed listing of chromosomal anomalies associated with HPE has been provided by Muenke (1995). Marcorelles et al. (2002) also described a group of five children with the MIH type of HPE who had monosomy 13q. They postulated that haploinsufficiency of the critical area of 13q32 (which contains ZIC2 gene) was responsible for HPE. The prognosis in HPE is much worse for those with cytogenetic abnormalities. In live case series, only 2% with cytogenetic anomalies survived beyond 1 year, compared to 30–54% for those without (Croen et al., 1996; Olsen et al., 1997). 2.4.2.2. Genetic syndromes associated with holoprosencephaly Several multiple malformation syndromes have been associated with HPE, with as many as 25% of HPE cases having a recognizable monogenic syndrome (Croen et al., 1996; Olsen et al., 1997). These include pseudotrisomy 13 (Young and Madders, 1987) and the Pallister-Hall, Meckel and velocardiofacial syndromes (Siebert et al., 1990). Others are listed in Table 2.1. Approximately 5% of patients with Smith–Lemli–

Table 2.1 Genetic syndromes associated with holoprosencephaly Aicardi syndrome CHARGE association DiGeorge sequence Kallmann syndrome Lambotte syndrome Majewski syndrome

Meckel syndrome

Varadi syndrome

Pallister-Hall syndrome Pseudotrisomy 13

Velocardiofacial syndrome Walker–Warburg syndrome Zellweger syndrome

Rubinstein–Taybi syndrome Smith–Lemli–Opitz syndrome Steinfield syndrome

Opitz syndrome have HPE. Children with this syndrome have a defect in 7-dehydrocholesterol reductase, the enzyme that catalyzes the final step of cholesterol biosynthesis (Kelley et al., 1996). Defective cholesterol synthesis may have a role in the pathogenesis of HPE through the SHH signaling pathway, since cholesterol is required for activation of SHH. 2.4.2.3. Familial holoprosencephaly In addition to the association of HPE with chromosomal anomalies and monogenic syndromes, familial cases of nonsyndromic HPE with normal chromosomes have been described (Ming and Muenke, 1998). The inheritance in familial HPE may be autosomal dominant, autosomal recessive and possibly X-linked. The clinical variability within a single pedigree can be quite large, with some having severe brain malformations while others have microforms and function fairly normally. In autosomal dominant HPE, for an obligate carrier the risk of severe HPE was 16–21%, and the risk for less severe form or a microform was 13–14%. The maximum overall risk for some effect was 35% and the penetrance was, therefore, estimated to be 70% autosomal dominant HPE (Cohen, 1989a). 2.4.2.4. Gene mutations Based on nonrandom chromosomal rearrangements, at least 12 different loci on 11 different chromosomes have been implicated in HPE (Roessler and Muenke, 1998). To date mutations in eight genes have been implicated in human HPE: SHH, Patched-1 (PTCH), TGIF, TDGF1/cripto, ZIC2, SIX3, GLI2, and FAST1 (Ming and Muenke, 2002). Two of these genes (SHH and PTCH) encode members of the Sonic hedgehog signaling pathway, which regulates ventral development in both the forebrain and spinal cord.

HOLOPROSENCEPHALY 2.4.3. Sonic hedgehog pathway The first gene identified as a cause of human HPE was Sonic hedgehog (SHH) (Roessler et al., 1996), which maps to the HPE3 locus at chromosome 7q36. SHH is a potent morphogen expressed in Hensen’s node, the notochord and the ventral forebrain, and is critical for ventral patterning of the embryonic neural tube (Roelink et al., 1995). SHH is a secreted molecule that undergoes autocatalytic cleavage into amino- and carboxy-terminal domains (Lee et al., 1994). The active amino-terminal cleavage product of secreted SHH (SHH-N) covalently binds to cholesterol (Porter et al., 1996). Mice with homozygous null mutations in SHH develop brain and facial abnormalities consistent with holoprosencephaly (Chiang et al., 1996).

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Human mutations have been discovered in SHH (Roessler et al., 1996), which encodes a secreted signaling molecule localized at early stages to ventral domains in the developing neural tube and patched-1 (PTCH) (Ming et al., 2002). Haploinsufficiency of SHH is responsible for the brain and facial malformations seen in autosomal dominant HPE kindreds and in sporadic cases. Mutations in two other genes in the SHH pathways have been implicated in human HPE, PTCH and smoothened (SMO). PTCH encodes a receptor for SHH. The binding of activated SHH to PTCH in turn stops inhibition of the constitutive signaling activity of SMO, a transmembrane protein (Fig. 2.6). Therefore, the mutations that enhance PTCH activity should decrease SHH activity and result in the HPE pheno-

Fig. 2.6. Summary of Sonic hedgehog (Shh) signaling. The release and extracellular accumulation of Shh is regulated by Dispatched A (Disp). Patched (Ptc) is a transmembrane protein, which in the absence of hedgehog ligand inhibits another transmembrane protein, Smoothened (Smo). The binding of Shh to Ptc relieves this inhibition and initiates an intracellular signaling cascade, which ultimately results in activation of the GLI family of transcription factors. Su(fu), suppressor of fused; CBP, cyclic AMP-regulated element binding protein. Modified from Hayhurst and McConnell, 2003.

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type. Although mutations in PTCH have been identified in HPE (Ming et al., 2002), mutations in SMO have not yet been identified. SMO in turn causes an activation of a complex series of intracellular events that convert the GLI proteins (a family of zinc-finger transcription factors) into transcriptional activators (Hayhurst and McConnell, 2003) (Fig. 2.6). GLI proteins bind to and upregulate target genes in the WNT and BMP families, which encode dorsalizing morphogens (Ming and Muenke, 1998). GLI2 mutations, also found in human HPE, may cause defective translocation of GLI proteins to the nuclei by coexpressed ZIC proteins (Cohen, 2003). This complex signaling pathway appears critical for maintaining a balance between ventralizing and dorsalizing forces required for normal forebrain induction and subsequent regional brain development. The hedgehog signaling network and its role in holoprosencephaly has been recently reviewed in detail (Cohen, 2003; Hayhurst and McConnell, 2003). 2.4.4. Nodal/TGFb pathway Three additional HPE gene mutations implicate the Nodal signaling pathway, which plays a vital role in neural patterning. The pathway includes: transcriptional corepressor TG-interacting factor (TGIF), TDGF1/ cripto and FAST1 (Cohen, 2003). TGIF maps to chromosome 18p11.3 within the HPE4 locus (Gripp et al., 2000) and has been implicated in two important signaling pathways. First, TGIF blocks the activity of SMAD transcription factors and is activated by Nodal signaling (Gripp et al., 2000). Secondly, TGIF also appears to compete for binding to retinoid receptors and may thus act as a suppressor of gene transcription regulated by retinoic acid, which itself is an important regulatory molecule and a known teratogenic cause of HPE (Bertolino et al., 1995). TDGF1/cripto encodes a membrane-associated protein that serves as a co-receptor for Nodal signaling (de la Cruz et al., 2002). FAST1 is involved in regulatory feedback loops that control the duration and intensity of Nodal signals in early patterning (Pogoda et al., 2000).

other identified HPE genes. This gene appears to be significant in neural tube closure and differentiation of the roof plate of the developing embryo. This dorsalizing effect of ZIC2 may be the reason why most patients with ZIC2 mutations have only mild facial abnormalities (Wallis and Muenke, 2000; Dubourg et al., 2004). In a mouse model, mutation of zic-2 led to defects in neural tube closure and HPE, apparently from decreased mitosis and increased apoptosis (Nagai et al., 2000). SIX3 encodes a homeodomain transcription factor expressed in ventral forebrain (Oliver et al., 1995) and maps to the HPE2 locus on chromosome 2p21. Six3 mutant animals develop midline forebrain and eye anomalies (Wallis et al., 1999), suggesting its involvement in head midline and eye formation. 2.4.5.1. Incidence of gene mutations in HPE While progress has been made in identifying gene mutations associated with HPE, the current known mutations have been identified in only 15–20% of the HPE cases in a cohort with normal karyotypes (Ming and Muenke, 2002). Molecular screening of SHH, ZIC2, SIX3 and TGIF in patients with HPE and normal karyotype carried out in France revealed 34 heterozygous mutations out of 200 cases (88 fetuses and 112 living children) (Dubourg et al., 2004). A total of 17 had a mutation in SHH (12 familial cases and 5 sporadic cases), 7 in ZIC2, 8 in SIX3 and 2 in TGIF. This 17% incidence was much higher than the less than 5% found in a population-based molecular screening of five genes carried out in a small cohort of sporadic HPE patients in California (Nanni et al., 2000). Given the complex and multiple signaling pathways that have been implicated in HPE, the future discovery of numerous other genes is likely. SHH is the most frequently identified gene defect in HPE (Dubourg et al., 2004) and was identified in 3.4% of sporadic HPE cases (Nanni et al., 1999). In contrast, SHH mutations were detected in 10 of 27 families (37%) with autosomal dominant transmission of HPE. 2.4.6. Genotype–phenotype correlation

2.4.5. Other gene mutations The other known HPE genes do not play an obvious role in either of the above pathways. ZIC2 encodes a zinc-finger transcription factor homologous to the odd-paired gene in Drosophila (Mizugishi et al., 2001) and maps to chromosome 13q32. ZIC2 is unique among HPE genes in that it is expressed in dorsal and ventral midline regions of the telencephalon, rather than predominantly in ventral regions as

There is great phenotypic variability in HPE, even in familial forms with known mutations. In familial HPE, such as that due to SHH mutation, variable penetrance has been noted (Cohen, 1989a; Wallis and Muenke, 2000; Dubourg et al., 2004). Members of the same family with the implicated mutation may be severely affected while others with the same mutation or deletion are only mildly affected with ‘microforms’ of HPE and may be neurologically normal. These

HOLOPROSENCEPHALY microforms include microcephaly, hypotelorism, single maxillary central incisor, iris coloboma, absent frenulum and hyposmia (Muenke et al., 1994; Heussler et al., 2002). Because these individuals are still at an increased risk for having children with HPE, it is important to carefully look for these signs in family members of children with HPE. The molecular screening study from France (Dubourg et al., 2004) has led to some themes in genotype–phenotype correlations in HPE. Mutations of the SHH gene have been found predominantly in subjects with nasal aperture stenosis, cerebellar hypoplasia and pituitary gland anomalies. Ocular malformations are preferentially associated with SHH and SIX3 mutations. Unlike SHH, SIX3 and TGIF genes, ZIC2 is less likely to be implicated with severe facial malformations. The wide variability in the phenotype even in familial cases with the same mutation have led some to hypothesize a multiple hit hypothesis (Ming and Muenke, 2002), which implicates more than one gene or the interaction of a gene and an environmental factor. 2.4.7. Environmental causes Evidence from many human studies and animal models implicate multiple environmental factors in the pathogenesis of HPE (Cohen and Shiota, 2002). Prenatal exposures to a variety of toxins, medications and infections have also been reported in cases of HPE. Potential teratogens include alcohol (Croen et al., 2000), anticonvulsants (Kotzot et al., 1993; Holmes and Harvey, 1994; Rosa, 1995), retinoic acid (De Wals et al., 1991), cigarette smoking (Croen et al., 2000) and congenital cytomegalovirus infection (Byrne et al., 1987). However, the only environmental influence that has been well established is maternal diabetes (Barr et al., 1983; Martinez-Frias et al., 1998). A diabetic mother’s risk of having a child with HPE is approximately 1%, a greater than 100-fold increase over of the general population. A recent large population-based study of HPE in California also revealed that cigarette smoking is associated with a four-fold increase in risk for HPE (Croen et al., 2000). The mechanisms by which teratogens cause HPE remain unknown. Extensive animal studies have clearly shown, however, that exposure to various agents including alcohol and retinoic acid during the period of gastrulation and forebrain induction (equivalent to around the fourth week of gestation in humans) causes HPE (Siebert et al., 1991; Sulik et al., 1995; Helms et al., 1997). Some teratogens may interfere with the Sonic hedgehog signaling pathways by perturbing cholesterol biosynthesis or the ability of target tissue to sense or

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transduce the Sonic hedgehog signal (Cohen and Shiota, 2002). While relatively low doses of these teratogens by themselves may not be sufficient to cause HPE, they may act synergistically with other genetic or environmental factors to produce the HPE phenotype (Ming and Muenke, 2002). Likewise, while a single HPE gene mutation by itself may not be sufficient to produce HPE in a patient, another factor, such as teratogens, may work in concert to generate the HPE phenotype. 2.4.7.1. Cholesterol hypothesis The critical role of cholesterol in this pathway is particularly interesting when considering the possible mechanisms involved in HPE in the Smith–Lemli– Opitz syndrome. Mutations in 7-dehydrocholesterol reductase (DHCR7) have been shown to be the major defect in this syndrome (Wassif et al., 1998). Mutations in DHCR7 have been reported in fetuses with HPE, including the common mutation associated with Smith–Lemli–Opitz syndrome (IVS8–1G!C) (Nowaczyk et al., 2001) and novel missense mutation in G344D (Shim et al., 2004). These mutations in DHCR7 lead to reduction in the enzyme activity and lower cholesterol levels. This reduction in turn is thought to reduce the binding of cholesterol to the amino-terminus of SHH and thus its activation. While low cholesterol could possibly interfere with the covalent modification required for normal SHH function, the interference may occur instead at the level of signal reception (Cooper et al., 1998). Further evidence for the importance of cholesterol in brain development has come from studies showing that steroidal alkaloids, such as cyclopamine and jervine, cause HPE in several animals (Keeler, 1975; Coventry et al., 1998; Golden, 1999). Ingestion of cyclopamine, a derivative of the desert corn lily (Veratrum californicum), by pregnant sheep was noted in the 1960s to cause cyclopia in lambs (Binns et al., 1963). The mechanism by which these agents cause HPE appears to be through inhibition of target-tissue response to Shh (Cooper et al., 1998; Incardona and Roelink, 2000). Taken together, these findings provide strong evidence that teratogens and gene defects exert their effects through the same or similar molecular pathways. Maternal cholesterol levels during early gestation may underlie the wide phenotypic variability of severity of HPE. Supporting this idea, those patients with the Smith–Lemli–Opitz syndrome and HPE whose mothers had the highest cholesterol levels have been found to have the least severe HPE (Kelley et al., 1996). Rarely, first trimester exposure to cholesterol-lowering statins drugs has been associated with HPE (Edison and Muenke, 2004, 2005). As more becomes understood

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Table 2.2 Gene mutation and loci associated with holoprosencephaly

Gene SHH Patched-1 (PTCH) GLI2 TGIF TDGF1/cripto FAST1 ZIC2 SIX3

Candidate gene designation HPE3 HPE7 HPE4

HPE5 HPE2

Locus 7q36 9q22 2q24 18p11.3 3p21.31 8q24 13q32 2p21

Source: adapted from Dubourg et al., 2004.

about the molecular mechanisms, maternal cholesterol could represent an important modifiable risk factor. Evaluation of a child with HPE usually includes high-resolution chromosome studies. If the results are normal, HPE gene mutation analysis should be considered (Table 2.2). Mutational analyses for SHH, TGIF, SIX3 and ZIC2 are currently available commercially. Other genes (known and candidate) that are being tested on a research basis include PTCH, DKK1, GLI2, TDGF1 and FAST1. In certain circumstances, a genetic evaluation to assess for syndromic HPE may be warranted (Table 2.1). The parents should be examined for possible features of HPE microforms.

2.5. Neuroimaging Advances in neuroimaging have improved our understanding of the pathogenesis of HPE. Our group has published several neuroimaging studies of a large cohort of HPE patients (Simon et al., 2000, 2001, 2002; Barkovich et al., 2002a, 2002b). These studies have provided a new grading system for various components of HPE, which allowed the correlation of imaging findings and clinical characteristics (Lewis et al., 2002; Plawner et al., 2002). The studies have also led to a better understanding of the embryological derangements that lead to HPE. 2.5.1. Deep gray nuclei While the deep gray nuclear structures and diencephalon are often profoundly affected in many patients with HPE, until recently there were no formal analyses of the subcortical structures. In contrast to previous studies that indicated that the thalamus was the most affected subcortical structure (Probst, 1979; Cohen, 1989b), a

neuroimaging study of 57 classic HPE patients (43 MRI studies and 14 high-quality computed tomography (CT) studies) revealed that the hypothalamus and caudate nuclei were the most commonly nonseparated deep gray structures in HPE (Simon et al., 2000). Nearly all patients (99%) with classic HPE had some degree of hypothalamic nonseparation. The caudate nuclei were not fully separated in 96% of the cases. The thalami were the least frequently involved of the deep gray nuclei, showing nonseparation in 67%. Abnormal orientation of the long axis of the thalamus (outside of 30–45 ) was seen in 71% of the cases. In 27% of the cases, the mesencephalon showed some degree of noncleavage. This was surprising, since HPE was thought to be a ‘prosencephalic disorder’. The midbrain involvement may be seen pathologically as a failure of two distinct paired superior and inferior colliculi to form, continuity of the oculomotor nuclei across the midline, and aqueductal atresia or stenosis (Vogel et al., 1990; Sarnat and Flores-Sarnat, 2001). In 11% of the HPE cases, a single deep gray nuclear mass without discrete basal ganglia, thalami and mesencephalon was noted (Fig. 2.7). The pattern of deep gray nuclei abnormalities, in particular the universal involvement of the hypothalamus, supports the theory that a lack of induction of the most rostral aspects of the embryonic floor plate is the cause of classic HPE. 2.5.2. Midline dorsal cyst The presence of a dorsal cyst strongly correlates with the degree of nonseparation of the thalami (Simon et al., 2001; Plawner et al., 2002). It seems likely that the nonseparated thalamus physically blocks egress of cerebrospinal fluid (CSF) from the third ventricle, resulting in expansion of the posterodorsal portion of the ventricle to form the dorsal cyst. Abnormalities of the aqueduct of Sylvius, such as atresia or stenosis, that have been found in HPE (Vogel et al., 1990) may also contribute to the obstruction of CSF flow. The posterior location of most dorsal cysts is most consistent with egress of the CSF through the path of least resistance, the thin posterior wall of the third ventricle in the suprapineal recess. Supporting this theory, hydrocephalus is often noted in association with dorsal cysts (Simon and Barkovich, 2001; Plawner et al., 2002). The degree of CSF obstruction determines the size of the dorsal cyst. The gross morphological descriptions of the holosphere as pancake, cup or ball shape is merely a reflection of the degree of obstruction. Smaller dorsal cysts frequently disappear after ventriculoperitoneal shunting. Occasionally, the dorsal cyst herniates through the anterior fontanelle to form a vertex encephalocele that is unique in HPE (Sarnat and Flores-Sarnat, 2001).

HOLOPROSENCEPHALY

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Fig. 2.7. (A, B) Axial T1-weighted and (C) coronal T1-weighted MRI of a newborn with alobar HPE showing a single central deep gray nuclei mass (arrowheads in A and C) without discrete basal ganglia, thalamic nuclei and mesencephalon. The axial images (B) show that the gyri are also broad with shallow sulci.

The dorsal cyst of HPE is similar in appearance to the interhemispheric cyst associated with agenesis of the corpus callosum (Young et al., 1992). The latter is frequently misdiagnosed as HPE but is distinguished by normal cleavage of the basal forebrain structures. Differentiating these can be especially difficult when the abnormal brain anatomy is further distorted by hydrocephalus. Definitive diagnosis in these cases often requires a repeat MRI after decompression. 2.5.3. Cortical gyral abnormalities In a neuroimaging study of 96 classic HPE patients, the cortical thickness was normal in all patients and gyral/sulcal sizes were normal in 83% (Barkovich et al., 2002a). Eight patients, all with alobar HPE, had diffuse dysplastic cortex consisting of broad gyri with too few sulci (Fig. 2.7). Four patients, all with lobar HPE, had localized dysplastic cortex limited to the midline frontal cortex. Although the gyri appear broad and may appear as pachygyria, the measurements of cortical thickness are usually normal. Surprisingly only four of 96 patients with classic HPE had subcortical heterotopia, which consisted of large masses that crossed the midline in the noncleaved region anterior to the anterior termination interhemispheric fissure (Fig. 2.8). In general, when localized cortical dysplasia or subcortical heterotopia was present, they tended to be located in the midline or paramedian region, consistent with the mediolateral gradient (Sarnat, 1992). In classic HPE, sylvian fissures are displaced further anteriorly and medially as HPE became more

severe (Barkovich et al., 2002a). More severe cases were characterized by more horizontal orientation, and anterior and medial displacement of the sylvian fissures. In most severe cases, no sylvian fissure could be identified. The sylvian angle (the angle between lines drawn tangential to the sylvian fissures) correlated strongly with the severity of classic HPE (lobar, semilobar, alobar) and with the degree of abnormal frontal lobe development, being largest in the most severe and smallest in the least severe cases (Barkovich et al., 2002a). 2.5.4. White matter abnormalities Magnetic resonance imaging studies of the white matter in HPE have focused on callosal abnormalities (Simon and Barkovich, 2001; Simon et al., 2002). A recent study of white matter maturity in HPE noted a tendency for delay in myelination in classic HPE, but not in MIH (Barkovich et al., 2002b). Diffusion tensor imaging (DTI) techniques have been applied to analyze in vivo the brainstem white matter abnormality in HPE (Albayram et al., 2002). In patients with alobar HPE, the cortico-ponto-spinal tracts were absent bilaterally, confirming the findings from prior neuropathological studies (Kobori et al., 1987). In most patients with less severe types of HPE, the tracts were present bilaterally. HPE type and neurodevelopmental score correlated strongly with cortico-ponto-spinal tracts and middle cerebellar peduncle dimensions. These findings demonstrate that analysis of white matter tracts in HPE using DTI adds complementary information to traditional MRI analysis.

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Fig. 2.8. (A, B) MRI of a 19-year-old patient with lobar holoprosencephaly and subcortical heterotopia. (A) Axial T2-weighted image showing the presence of a nearly complete interhemispheric fissure (arrows) and lateral ventricles. Therefore, this patient was classified as lobar HPE. (B) Coronal spoiled gradient-recalled image showing a large infolding of dysplastic cortex (white arrowheads) in the right hemisphere that crosses the midline. This patient was severely affected and had intractable epilepsy. (C) T2-weighted coronal MRI of a 1.5-year-old patient with MIH and subcortical heterotopia consisting of large infolding dysplastic cortex (black arrowheads). This child also had severe localization-related epilepsy. (D) Coronal spoiled gradientrecalled image in a patient with semilobar HPE showing heterotopia at the roof of the monoventricle (white arrowheads).

2.5.5. Other cerebral anomalies On rare occasions, other cerebral anomalies may be associated with HPE, including schizencephaly, Dandy– Walker malformation, and various encephaloceles. A rare case of diffuse polymicrogyria in a patient with MIH has been reported (Takanashi et al., 2003). 2.5.6. Vascular anomalies The anterior circulation vasculature is often abnormal in HPE. In more severe types (alobar and semi-

lobar) of HPE, patients demonstrate a lack of formation of normal middle and anterior cerebral arteries. In these cases, a rete of vessels arises from the internal carotid artery. In less severe cases, including MIH, the arterial system is nearly normal but an azygous, or unpaired, anterior cerebral artery is nearly always noted (Simon and Barkovich, 2001). In lobar and less severe semilobar HPE, prenatal power Doppler imaging studies have shown an anomalous trajectory of the anterior cerebral artery following a course close to the frontal bone (Blin et al., 2004).

HOLOPROSENCEPHALY 2.5.7. Middle interhemispheric variant The neuroimaging features of the subtype MIH are different from classic HPE (Fig. 2.5). Unlike classic HPE, where the most severely nonseparated region of the hemispheres is the basal forebrain, the posterior frontal and parietal lobes in MIH are affected. The more anterior portion of the frontal lobes and the occipital lobes are well separated in MIH. The genu and splenium of the corpus callosum appear normally formed, but the callosal body is absent. The hypothalamus and lentiform nuclei appeared normally separated in all MIH patients but the caudate nuclei and thalami were incompletely separated in many cases (Simon et al., 2002). The sylvian fissures in most patients were oriented nearly vertically and were abnormally connected across the midline over the vertex of the brain (Simon et al., 2002). Approximately two-thirds of the MIH patients had either subcortical heterotopic gray matter or cortical dysplasia (Fig. 2.8C). As in other types of HPE, the anterior vasculature was abnormal, with an azygous anterior cerebral artery noted in all cases. The pattern of defects suggests that MIH is caused by impaired induction or differentiation of the embryonic roof plate. Studies of the homologous mouse gene zic-2 have indicated that it promotes embryonic roof plate differentiation in the dorsal midline of the neural tube after closure (Nagai et al., 2000). Roof plate specific properties such as increased apoptotic rate and decreased mitotic rate are critical to the formation of the interhemispheric fissure. Compromise of dorsal induction and roof plate differentiation, therefore, are plausible mechanisms for the formation of MIH. The recent report of an MIH patient with a ZIC2 mutation provides further evidence to support this hypothesis (Brown et al., 2001). In addition, monosomy 13q was recently discovered in five patients with MIH (Marcorelles et al., 2002). In this neuropathological series, it was postulated that the loss of function in the ZIC2 gene, which has been mapped to a critical segment of chromosome 13, is responsible for MIH. Because the dorsal structures are primarily affected in MIH, malformations due to incomplete formation of ventrally derived structures, such as hypothalamic nonseparation and midfacial hypoplasia, are less common. 2.5.8. Neuroimaging technical approaches High-resolution MRI scans that include thin-section image sequences in three orthogonal planes (axial, sagittal and coronal) are preferred. The study should also include a volumetric dataset (three-dimensional spoiled gradient-echo sequences), which displays good gray–white matter differentiation and permits refor-

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matting in other planes and volumetric analyses (Simon and Barkovich, 2001). To determine the type of HPE, careful assessment of the telencephalon is required. Close attention is paid to the presence of anterior and posterior interhemispheric fissures and the localization of nonseparation of the two hemispheres. In addition, the basal ganglia, thalamic nuclei, hypothalamus, pituitary gland and mesencephalon are analyzed systematically, as they are often involved in HPE. Neuroimaging evaluation of the brain in HPE may be difficult in young infants with microcephaly because of the small brain size and immature myelination. Follow-up imaging after a period of brain growth may be required. Difficulties in assessment also occur when hydrocephalus distorts underlying brain structures (Simon and Barkovich, 2001). Definitive diagnosis in these cases often requires repeat MRI after decompression. Ideally, a pediatric neuroradiologist with experience in brain malformations should review the imaging studies. Approximately one-fifth of the imaging studies referred to our centers for HPE fail to meet the HPE neuroimaging criteria (Stashinko et al., 2004). The ultimate diagnoses given to these studies include septo-optic dysplasia, agenesis of corpus callosum or callosal agenesis with interhemispheric cyst. The dorsal cyst of HPE is similar in appearance to the interhemispheric cyst associated with agenesis of the corpus callosum (type 1b) (Young et al., 1992; Barkovich et al., 2001). The latter is frequently misdiagnosed as HPE but is distinguished by normal cleavage of the basal forebrain structures. In the past the term ‘lobar holoprosencephaly’ has been used for another malformation characterized by complete separation of the neocortex across the midline, usually with an absent or hypoplastic corpus callosum. However, as Kobori et al. (1987) point out, classifying this group as holoprosencephaly with complete separation of the hemispheres is a contradiction of terms and the term ‘holoprosencephaly’ should be restricted to malformations with midline continuity of the neocortex. Many of the reported cases of such lobar HPE appear to represent callosal agenesis with interhemispheric cyst. Even though in some of these cases, fusion of the thalamic nuclei may be identified, in absence of some degree of neocortical nonseparation, these cases should not be classified as HPE. Nevertheless, cases with thalamic fusion may share similar pathogenetic mechanisms involved in HPE and further studies are needed to understand the boundaries and continuities of midline malformations. Using three-dimensional imaging with reconstruction, detailed topological studies have been performed

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in HPE (Takahashi et al., 2003, 2004). These studies provide complementary anatomical information that may not be apparent from conventional MR imaging. In semilobar HPE the core anomaly found was a rostrocaudally aligned midline gray matter ‘seam’ that extends from the suprachiasmatic hypothalamus to a caudally positioned diminutive body and splenium of the corpus callosum (Takahashi et al., 2003). In the rostral to caudal direction, the interhemispheric fissure also transitions from being absent to a shallow but deepening zone and finally to a zone where the fissure is at full depth. At this full-depth zone, there is continuous gray matter linkage between the neocortex of the cerebral surface and the midline gray matter seam. Caudal to the seam, the telencephalic structures are normally separated (between the right and left hemispheres) and the seam is replaced by the posterior corpus callosum. The hippocampal formation in the temporal lobes appears normal. The temporal limb of the choroid fissure is closed. In more severe types of HPE (alobar), however, the structures of the temporal axis are formed but the temporal limb of the choroids fissure is splayed open (Takahashi et al., 2004). 2.5.9. Fetal neuroimaging Prenatal ultrasounds have been used to detect the central nervous system and facial abnormalities of severe HPE as early as the first trimester (Filly et al., 1984; Nyberg et al., 1987; Tongsong et al., 1999). Failure to identify the characteristics of the developing choroid plexuses (‘butterfly sign’) during the first trimester may be a sensitive indicator of HPE (Sepulveda et al., 2004). In alobar and semilobar HPE, prenatal diagnosis can readily be made by ultrasound (Peebles, 1998). The sensitivity of ultrasonography for detection of milder forms of HPE (lobar and MIH) may be low, since in these forms the anterior and posterior interhemispheric fissures are present and the characteristic dorsal cyst of HPE is often absent. One helpful clue in lobar HPE is the anomalous trajectory of the anterior cerebral artery detected by prenatal power Doppler imaging (Blin et al., 2004). Because of noncleavage of the inferior frontal lobes in lobar HPE, the anterior cerebral artery is pushed anteriorly and distally, resulting in a thin diameter and position along the frontal skull bone. In our experience, prenatal ultrasonography has low sensitivity. Although prenatal ultrasound was performed in 93% of 104 HPE patients (weighted toward less severe type), prenatal diagnosis was made in only 22% (Stashinko et al., 2004). Fetal MRI will provide better characterization of the malformations (Sonigo et al., 1998). Modern ultrafast MRI techniques reduce movement artifacts

significantly and are ideal for fetal imaging (Fig. 2.9). Fetal MRI has been used to diagnosis various forms of HPE including alobar, semilobar, lobar (Wong et al., 2005) and middle interhemispheric variant (Pulitzer et al., 2004). Other midline anomalies, such as agenesis of corpus callosum (isolated or with interhemispheric cysts), absence of septum pellucidum, and hydrocephalus with communication of the lateral ventricles, are sometimes misdiagnosed prenatally as HPE (Malinger et al., 2005). The detection of craniofacial malformations associated with HPE on prenatal imaging often aid in the diagnosis of HPE.

2.6. Neuropathological findings Most of our understanding and classification scheme of HPE has been based on gross and histopathological architecture of the HPE brain and in vivo neuroimaging studies. However, modern neuropathological techniques, such as immunocytochemistry for cell markers, markers of apoptosis and cell cycling and in situ hybridization have been infrequently applied to the study of HPE brain tissue (Sarnat and Flores-Sarnat, 2001). Some controversy exists regarding the cytoarchitecture of the cerebral cortex. However, more recent studies indicate that, while rare cases may show migration defects, the basic neuronal migration is intact, leading to a six-layer cortex (Muenke, 1995; Golden, 1999; Judas et al., 2003). Periventricular and white matter glioneuronal heterotopia are also encountered in rare cases. The defects in cortical organization that have been observed by others may represent secondary injury to the cerebral cortex or an abnormality in connections into and out of the cerebral cortex (Golden, 1999). In a neuropathological study of a newborn with semilobar HPE and 18p deletion, the neocortex retained its basic six-layered lamination but displayed a number of intralaminar and modular architectonic disturbances (Judas et al., 2003). Rapid Golgi impregnations revealed the presence of both normal and altered types of neocortical neurons. The most prominent finding was the increased soma size, dendritic length and dendritic arborization of the holoprosencephalic layer III pyramidal neurons. The authors postulated that the fivefold dendritic overgrowth in associative cortico-cortical pyramidal neurons was paradoxically related to severely diminished cortical afferent inputs. The preservation of the basic six-layer neocortical pattern in semilobar HPE suggests there is no major alteration in the inside-out migration and laminar positioning of cortical neurons.

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Fig. 2.9. Single-shot fast spin-echo MR sequences obtained in three planes, axial (A), coronal (B) and sagittal (C), in a 33-week gestational age fetus with HPE. The images show what appear to be alobar HPE with poorly separated frontal lobes and a large monoventricle that communicates with a large dorsal cyst (DC). (D) A fetal ultrasound image showing similar morphology to the axial MR image (A). The bright signal on the right of B is due to surface coil artifact.

The pia mater overlying the cortex often shows extensive glioneuronal heterotopia that form a ‘crust’ over the surface of the brain. This layer is not usually present in midgestation fetuses with HPE but is seen in late gestation and postnatally (Golden, 1999). These heterotopia are thought to be due to radial overmigration through breaks in the pia mater and also the absence or paucity of the subpial granular layer of glial cells of Brun (Sarnat and Flores-Sarnat, 2001). The hippocampus is virtually always present, although it may show incomplete or abnormal development (Golden, 1999). The cerebellum may show various degrees of cortical dysplasia, heterotopia or both, particularly in trisomy 13 and other cytogenetic abnormalities (Golden, 1999).

2.7. Clinical manifestations Children with HPE experience numerous medical and neurological problems, including craniofacial defects,

mental retardation, epilepsy, weakness, spasticity, dystonia, choreoathetosis and endocrinopathies (Barr and Cohen, 1999; Plawner et al., 2002; Hahn and Plawner, 2004). Developmental disability affects nearly all patients with HPE. The severity of the brain malformation determines the degree of delay and neurological problems. Barr and Cohen (1999) have previously reported poor survival and performance in a large group of patients with alobar HPE. Severe cases are identified at the time of birth or, more commonly today, prenatally. Neonates manifest early signs such as apneic spells, seizures, abnormal neonatal reflexes and abnormal tone. In semilobar HPE, the detection of midline craniofacial abnormalities at birth may prompt neuroimaging studies that reveal the brain abnormalities. Affected children without such anomalies may be referred at a later age for microcephaly, developmental delay or cerebral palsy, although more than 90% of the cases are diagnosed before the age of 1 (Stashinko et al., 2004). Children with milder

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forms of HPE, such as lobar and MIH, may escape early detection, since they display mild or moderate developmental delay, learning problems or cerebral palsy at a later age. Even in these milder cases, because of improved neuroimaging the vast majority of the cases are diagnosed before 2 years of age (Stashinko et al., 2004). Studies of the clinical characteristics of all types of HPE from a prospectively collected case series from the Carter Centers for Brain Research in Holoprosencephaly and Related Malformations (a national consortium funded by a not-for-profit foundation) have contributed to our understanding of the broad spectrum of outcomes in HPE (Lewis et al., 2002; Plawner et al., 2002). These studies included 83 children (41 male and 42 female) with HPE evaluated at one of the Centers (Kennedy Krieger Institute, Texas Scottish Rite Hospital or Stanford University Medical Center) between 1998 and 2001. Just over half had semilobar and about 15% each had alobar, lobar and MIH types. The age range for each type was broad: alobar 0.1–2.6, semilobar 0.1–13.9, lobar 0.8–19, and MIH 0.5–14 years at the time of evaluation. The following summarizes some of the clinical problems and neurological disorders in our cohort of children, which presently numbers more than 120 (Lewis et al., 2002; Plawner et al., 2002; Hahn and Plawner, 2004), as well as those in the series from Barr and Cohen (1999). 2.7.1. Craniofacial anomalies It has been long recognized that patients with HPE have midline craniofacial malformation of highly variable severity (Table 2.3, Fig. 2.10). Severe abnormalities, which are associated with neonatal mortality, include cyclopia (a single central eye with a proboscis above), ethmocephaly (a median proboscis attached between two separate but severely hypoteloric eyes) and cebocephaly (hypotelorism with a single nostril and aperture). Moderate defects include cleft lip and palate. Premaxillary agenesis is one of the characteristic facial malformations in HPE and consists of a combination of median cleft of the upper lip, orbital hypotelorism, flat nasal bridge and nasal alae but no septum, and absence of the premaxillary bone and other median plane bones of the face (Fig. 2.10C) (DeMyer, 1987). Unilateral and bilateral cleft lip may also occur in HPE. In bilateral cleft lip, a median intermaxillary rudiment exists in between the clefts (Fig. 2.10D). Milder defects include hypotelorism, midfacial hypoplasia, iris colobomas, single central maxillary incisors and absence of labial frenulum (Ming and Muenke, 1998). DeMyer noted that in most cases the severity of the brain abnormality paralleled

Table 2.3 Congenital malformations associated with holoprosencephaly Region

Malformation

Head

Microcephaly Hydrocephalus Encephalocele Hypotelorism Hypertelorism Anophthalmia Microphthalmia Fused orbits Cyclopia Coloboma Epicanthal folds Ptosis Ethmocephaly Flat nose Philtrum pit Single nares (cebocephaly) Septal defect/obstruction/deviation Pyriform sinus stenosis Proboscis Premaxillary agenesis Single maxillary central incisor Fused teeth Missing teeth Cleft lip (median, unilateral, or bilateral) Cleft palate (median, unilateral, or bilateral) Spina bifida, meningomyelocele Limb reduction Digit anomalies (syndactyly, polydactyly) Club feet Supernumerary nipples Cardiac defect (Tetralogy of Fallot, truncus arteriosus, septal defects) Scoliosis Abnormal genitalia

Eye

Nose

Teeth

Lip Palate Others

Source: modified from Hahn and Plawner (2004).

that of the face in HPE, and advanced the dictum ‘the face predicts the brain’ (DeMyer et al., 1964). For example, cyclopia and ethmocephaly are always associated with alobar HPE (Muenke, 1995). With premaxillary agenesis with midline cleft, alobar HPE is usually but not invariably found. We no longer need to depend on this association, since high-resolution MRI provides neuroanatomical details of brain anomalies that ultimately predict outcome. In our cohort of 117 patients, we did not observe any patients with extremely severe abnormalities, such as cyclopia or ethmocephaly (Fig. 2.10A), most probably

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Fig. 2.10. Spectrum of craniofacial malformations in HPE. (A) Cyclopia with a proboscis attached above. Courtesy of Dr Hannes Vogel. (B) Cebocephaly in a 2-month-old infant girl with alobar HPE and severe microcephaly. The infant had frequent breathing problems and episodic hypoxemia due to stenotic single nasal aperture. The eyes have been pixilated in this figure. (C) Premaxillary agenesis with median cleft in a 7-month-old boy with alobar HPE and microcephaly. The characteristic facial malformation consists of a combination of median cleft of the upper lip, orbital hypotelorism, absent nasal bridge and flat nasal alae. (D) Bilateral cleft lip and cleft palate in a 7-month-old boy with semilobar HPE and severe hydrocephalus. The clefting is worse on the right. The midline tissue present in between the clefts represents median intermaxillary rudiment. (E) Orbital hypotelorism in a 16-month-old girl with semilobar HPE. Mild microcephaly was also present. (F) Single maxillary central incisor (arrow) in a 14-month-old boy with lobar HPE.

because these malformations are associated with early neonatal death. Cebocephaly was seen in one patient (Fig. 2.10B). Severe defects, i.e. premaxillary agenesis (midline cleft, hypotelorism and flat nose) occurred in 15%. Moderate defects, including unilateral or bilateral clefts, midface hypoplasia and moderate hypotelorism occurred in 15%. Mild malformations, including mild hypotelorism (Fig. 2.10E) and single maxillary central incisor (Fig. 2.10F), were seen in 41%. A total of 27% of our patient cohort had no facial dysmorphisms. In our clinical studies, we found a correlation between the severity of the facial malformations and the grade of HPE (Lewis et al., 2002; Plawner et al., 2002). Thus the more severe the facial malformation the more likely it was that the child would have a severe type of HPE. However, there were many exceptions (Plawner et al., 2002). For example, three of 21 alobar patients had no discernible facial dysmorphism and two of 17 lobar HPE patients had premaxillary agenesis with median cleft. Craniofacial malformations in patients with MIH were either absent or usual-

ly mild, often manifesting as hypertelorism (Lewis et al., 2002; Simon et al., 2002). The degree of midfacial hypoplasia has been hypothesized to depend on the rostrocaudal gradient of patterning defects (Sarnat and Flores-Sarnat, 2001). Since the midbrain is the most rostral structure from which neural crest tissue forms and migrates, if the gradient extends as far caudally as the midbrain it may interfere with midfacial development. Supporting this hypothesis, in our cohort of HPE patients (92 in whom the neuroimaging of the mesencephalon could be assessed adequately), the severity of craniofacial malformation correlated with the grade of mesencephalic nonseparation (unpublished data from the Carter Centers HPE Database). Patients born with extremely severe craniofacial malformations often die during infancy. Those with less severe malformations, such as cleft palates often have feeding or airway difficulties. Surgical repair of the cleft is often performed if the infant survives beyond infancy.

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Holoprosencephaly has also been associated with congenital malformations outside of the other head and brain regions, particularly if the HPE occurs as part of a chromosomal disorder. Some of these are listed in Table 2.3. 2.7.2. Developmental dysfunction and behavior Severe to profound developmental delay and mental retardation are common in more severe forms of HPE (Barr and Cohen, 1999). Alobar HPE patients have previously been described as not being aware of their environment or showing evidence of useful vision or hearing (DeMyer and Zeman, 1963; DeMyer, 1987). However, our experience has shown that patients with HPE, including many with alobar HPE, do interact with their environment and family, as well as responding to visual and auditory stimuli. They develop the ability to recognize certain voices and noises and react to those sounds in a reproducible manner (Barr and Cohen, 1999). Those without severe ocular anomalies will develop the ability to focus on faces, track objects and respond to facial expressions. These developmental milestones are usually delayed. The developmental outcomes and prognosis in HPE have been derived from series composed primarily of alobar HPE (the most severe form) (DeMyer, 1987; Barr and Cohen, 1999). Until recently, there were no large series examining the outcomes in less severe forms of HPE (such as semilobar and lobar). Recent studies have shown that the neurodevelopmental function is more favorable in these patients (Lewis et al., 2002; Plawner et al., 2002). These studies have found an inverse correlation between the grade of classic HPE and developmental functions, including mobility, hand/arm function and expressive language (Plawner et al., 2002). As shown previously, alobar HPE patients were severely affected and made minimal developmental progress. None of the alobar HPE patients were able to walk, use their hand/arm to reach and attain objects, or utter single words. Vocalizations were severely limited and were often hoarse or ‘barking’ in nature (Barr and Cohen, 1999). Only four of 30 patients with semilobar HPE had normal or mildly abnormal hand/arm function and only two could speak in multiword sentences. In contrast, approximately half of the lobar HPE patients were able to walk independently or with assistance, use their hands/arms normally or with mild dysfunction and speak single words or multiword sentences. The developmental functions of the MIH group were comparable to the lobar HPE group in terms of mobility but somewhat better in hand/arm function and speech (Lewis et al., 2002). A total of 40% were

able to ambulate with or without support, 73% were able to use their hands/arms with only mild dysfunction and 75% were able to utter single words or speak in multiword sentences. A neuropsychological study of nine patients with HPE (16 months to 17 years) suggested a pattern of relative strengths in receptive language and socialization skills, and weaknesses in visual reasoning and nonverbal problem skills (Kovar et al., 2001). Tests included the Bayley Scales of Infant Development (BSID-II) and the Stanford-Binet Intelligence Scale (SB-IV) for younger children, and the Wechsler Adult Intelligence Scale (WAIS-III) for older children. Because of significant expressive language and motor impairments seen in HPE, a novel assessment tool (the Carter Neurocognitive Assessment or CNA) was developed at Rutgers University to minimize the effect of motor deficits (Leevers et al., 2005). The CNA has been administered for the past 3 years in a group of children with HPE (n ¼ 42; mean age 4 years 10 months; 19 boys, 26 girls). The analysis of the cognitive profile from this study (Roesler et al., unpublished data submitted for publication) revealed that performance was significantly associated with the severity of the brain malformation. HPE patients showed relative strength in social awareness and nonverbal communication and greatest weakness in the area of vocal communication. This has been noted incidentally during the clinical evaluations, as most of our patients, including many with alobar HPE, are very socially engaging and respond positively to faces and voices. A widely held misconception is that that children with HPE fail to show any developmental progress. However, our long-term follow-up studies reveal that children with HPE do acquire new skills, albeit slowly. Re-evaluations indicated small gains in development suggesting that the CNA assesses incremental changes in children with severe motor and expressive speech deficits and that it is likely that the brain does reorganize to promote optimal neurocognitive function in children with HPE. Children with seizures performed more poorly on the CNA. Children with HPE have been described as having behavior that fluctuates between calmness and irritability (Barr and Cohen, 1999). Children also have an exaggerated reaction to sudden or loud noises, which causes them to cry or scream. Their cry is often high-pitched and sounds distressed. 2.7.3. Seizures and epilepsy Approximately half of the children with HPE in this cohort had at least one seizure (Lewis et al., 2002;

HOLOPROSENCEPHALY Plawner et al., 2002). In the ongoing study of our cohort, 50 of 121 (41%) patients developed epilepsy that required treatment with antiepileptic medication, and 15% had frequent seizures (more than one per day). In this group of 50 patients with epilepsy, 30 experienced partial seizures with or without generalization, 10 had generalized convulsive seizures (i.e., tonic, clinic, or tonic-clonic), 9 had infantile spasms and 7 had myoclonic seizures, while 11 had partial seizures that were mixed or not well characterized. Children with seizures performed more poorly on the neuropsychological testing than those without seizures. Cortical malformations were more commonly observed in neuroimaging studies in patients with refractory seizures (Plawner et al., 2002). The relatively low frequency of refractory epilepsy in HPE is somewhat surprising, especially when compared to other conditions such as lissencephaly in which epilepsy is found in the vast majority of patients. The presence of epileptic foci might depend in part on the extent of the mediolateral gradient in the horizontal axis. The most severely disorganized cortex is located in the midline and paramedian regions on pathological studies (Sarnat, 1992) and thus is less likely to be associated with epilepsy than if the cortical malformations had extended far laterally or had involved a greater proportion of the total cortical area (Sarnat and Flores-Sarnat, 2001). However, when midline cortical malformations are large as those shown in Figure 2.8, epilepsy does occur. An electroencephalogram (EEG) and a high-quality MRI are usually obtained in patients who are suspected of having seizures to assess recurrence risks and etiology. MRI should include imaging in three planes and should include thin-sliced three-dimensional acquisitions to assess for cortical malformations. Patients should also have routine electrolytes testing with special attention to sodium concentrations. Sodium imbalance is a common cause of acute reactive seizures in HPE patients. Electroencephalographic studies have revealed various abnormalities. Common background abnormalities include hypersynchronous and b activity (Clegg et al., 2002; Hahn et al., 2003). Other electroencephalographic studies in HPE patients with frequent seizures have reported abundant paroxysmal activity consisting of low-amplitude fast activity that evolve into generalized, rhythmic, high-amplitude d activity (DeMyer and White, 1964; Watanabe et al., 1976). In a series of 18 HPE patients who had an EEG before any seizures, sharp transients were noted in five (28%), all in patients with alobar or semilobar HPE (Hahn et al., 2003). The presence of sharp transi-

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ents was not predictive of epilepsy but the study was limited by the small sample size. 2.7.4. Endocrinopathies Children with HPE commonly have endocrine disorders because the midline malformation also affects the development of the hypothalamus and the pituitary gland. Neuropathological studies have revealed variable degree of abnormalities of the hypothalamus and pituitary gland (Muenke, 1995). Endocrinopathies contribute significantly to the morbidity and mortality in HPE (Cameron et al., 1999). Diabetes insipidus, due to posterior pituitary dysfunction, is a common problem in HPE (Hasegawa et al., 1990; Van Gool et al., 1990; Takahashi et al., 1995; Plawner et al., 2002; Traggiai and Stanhope, 2002). In a recent study of endocrine disorders in 117 children with HPE, 70% of the patients with classic HPE had diabetes insipidus. The severity of the diabetes insipidus correlated with the grade of HPE and hypothalamic nonseparation (Hahn et al., 2005). This correlation suggests the possibility that the diabetes insipidus is central in origin and due to abnormal development of the hypothalamus. The hypothalamus is located medially and rostrally in the early developmental fate maps and is therefore more likely to be affected by the failure of cleavage than structures located further from the midline or more caudally. However, in some cases of HPE, diabetes insipidus may be due to abnormal hypothalamic osmoreception. A neuropathological study of the hypothalamus in severe HPE revealed neurons that were immunoreactive to vasopressin, but their distribution and expression levels were altered and diverse compared to controls (Hayashi et al., 2004). However, the significance of these findings is not clear, since none of the cases in this study had diabetes insipidus. Diabetes insipidus usually evolves slowly in HPE; many children seem to remain asymptomatic and the disorder is discovered incidentally on routine screening that reveals sodium concentrations greater than 160 mEq/l. For mild diabetes insipidus, fluid management may be the only intervention required. If they develop clinically significant diabetes insipidus, desmopressin (DDAVP) is an effective treatment. Anterior pituitary dysfunctions were less common in our study (Hahn et al., 2005). Hypothyroidism was identified in 11% of patients, hypocorticism in 7% and growth hormone deficiency in 5%. Others have noted growth delay to be common in children with alobar HPE (Barr and Cohen, 1999). However, it is not clear whether this is due to growth hormone deficiency, as no systematic studies have been performed in

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patients with HPE. Assessment of anterior pituitary function usually includes cortisol, adrenocorticotropic hormone (ACTH), thyroid-stimulating hormone (TSH), free thyroxine and IGF1. In contrast to classic HPE, none of the patients with MIH had posterior pituitary dysfunction (Lewis et al., 2002). Only one of 19 patients (5%) with MIH had anterior pituitary dysfunction (growth hormone deficiency) (Hahn et al., 2005). This observation may reflect the relative sparing of the hypothalamic nuclei in MIH (Lewis et al., 2002). 2.7.5. Temperature dysregulation Children with HPE often have erratic temperature regulation (poikilothermia) (Barr and Cohen, 1999). Temperature dysregulation was found in 32% of patients with classic HPE (Plawner et al., 2002). The presumption is that this may be due to a hypothalamic dysfunction, as the severity of the dysregulation correlated with the degree of hypothalamic nonseparation (Plawner et al., 2002). 2.7.6. Microcephaly Microcephaly was present in three-quarters of patients with classic HPE and approximately half of the patients with MIH (Lewis et al., 2002; Plawner et al., 2002). Microcephaly was present in a greater proportion of patients with semilobar and lobar HPE (81% and 83% respectively), when compared with alobar HPE patients (38%) and MIH patients (47%). Alobar HPE patients more commonly had hydrocephalus, which produced macrocephaly, rather than microcephaly (Plawner et al., 2002). However, their brain sizes were small. Other studies have noted that the head in a child with classic HPE is usually small unless there is an excess of cerebrospinal fluid around the brain (Barr and Cohen, 1999). Hence, if a child with classic HPE does not have microcephaly, neuroimaging studies to assess for hydrocephalus are warranted and the child should be monitored for signs of elevated intracranial pressure. Volumetric MRI studies have confirmed the abnormally small brain volumes (adjusted for age) in HPE patients (Takahashi et al., 2003, 2004). In patients with more severe forms (e.g. alobar) or those with large dorsal cysts, the reduction was profound (approximately 39% of normal) (Takahashi et al., 2004), whereas in semilobar HPE the reduction was modest (approximately 55% of normal) (Takahashi et al., 2003). In a small series consisting of both lobar and semilobar patients, the reduction was even smaller (80% of normal) (Takahashi et al., 2004).

2.7.7. Dorsal cyst and hydrocephalus Dorsal cysts were associated most commonly with alobar HPE (92%). In other types, dorsal cyst was found in 28% of semilobar cases, 9% of lobar cases and 40% of MIH patients. The pathogenesis of dorsal cyst in HPE is discussed in the Neuroimaging section. Cerebrospinal fluid shunting due to hydrocephalus was required in 19% of HPE patients. A much higher proportion of CSF shunting procedures were performed in alobar HPE patients (61%) and those with a dorsal cyst (45%). Therefore, a patient with dorsal cyst is at risk for developing symptomatic hydrocephalus and requires close follow-up for possible CSF shunting. When significant hydrocephalus is present, CSF shunting should be considered even in severe HPE. Deferring the procedure will only lead to progressive head enlargement and make caring for the child more difficult (Barr and Cohen, 1999). If a vertex encephalocele is present at birth in association with a dorsal cyst, it should be surgically excised. This type of encephalocele, which is unique to HPE, usually contains rests of ependymal cells of the dilated and herniated monoventricle and rarely contains any neural tissue. 2.7.8. Motor dysfunction Abnormalities of tone and movement affect patients with HPE of all types (Barr and Cohen, 1999; Lewis et al., 2002; Plawner et al., 2002). In our cohort, the proportion of patients having significant motor dysfunction (hypotonia, dystonia, spasticity and involuntary movements) varied considerably by type of HPE. In alobar and semilobar HPE patients, spasticity and dystonia were noted in greater than 80% and hypotonia was present in greater than 70%. Patients with lobar HPE had less frequent spasticity (64%) and dystonia (36%). Involuntary movements, manifesting as choreoathetosis, were most commonly seen in the semilobar group (41%). Patients with MIH had high rates of spasticity (87%) and axial hypotonia (60%). However, in MIH axial hypotonia was present in only 47% and involuntary movements were not observed in any patients (Lewis et al., 2002). Many of the children with classic HPE had a typical distribution of upper limb dystonia and lower limb spasticity. In alobar HPE, hypertonia and spasticity may increase with stimulation or excitement (Barr and Cohen, 1999). This phenomenon may represent a form of dystonia, since the hypertonicity varies with time. Holoprosencephaly patients with motor dysfunction usually receive physical and occupational therapy. For symptomatic dystonia, which often has a predilection

HOLOPROSENCEPHALY for the upper limbs, trihexyphenidyl has been used. This anticholinergic medication may improve the motor function of the hands and arms. 2.7.9. Oromotor dysfunction Feeding and swallowing difficulties are common in HPE patients with or without cleft lip/palate. These difficulties include choking spells and gagging during feedings, slowness in eating, and vomiting (Barr and Cohen, 1999). In classic HPE the severity of the feeding difficulties correlated with the grade of HPE (Plawner et al., 2002). All the alobar HPE patients had severe feeding problems, while only 9–13% of the patients with milder HPE types (lobar and MIH) had such problems. Approximately half of semilobar HPE patients were affected. Approximately two- thirds of patients with alobar and semilobar HPE required a gastrostomy tube, which may ensure sufficient caloric intake and also help achieve sufficient free-water intake when patients have diabetes insipidus. Trihexyphenidyl may improve oromotor function by decreasing secretions and improving swallowing. 2.7.10. Prognostication A wide spectrum of neurodevelopmental outcomes exists in HPE, which correlates with the severity of brain malformation. Therefore, caution is warranted when giving prognostic information to families with a child affected by HPE. The counseling on outcomes to parents of children with severe types such as alobar HPE would be quite different than that for milder types, such as lobar and MIH. This underscores the importance of accurate neuroradiological classification of HPE in guiding counseling. A common misperception is that children with HPE do not survive beyond infancy. While early mortality is common in severe types of HPE (especially when accompanied by severe craniofacial anomalies or chromosomal abnormalities), many patients with less severe types will survive into childhood and beyond. Of the 104 children with HPE evaluated at the Carter Centers, the mean age was 4 years and 15% were between 10 and 19 years of age (Stashinko et al., 2004). HPE patients who manifest brainstem dysfunction, severe disabilities and aspiration pneumonias are at greater risk of early deaths. 2.7.11. Prenatal diagnosis and genetic counseling The recurrence risk of sporadic, nonsyndromic, nonchromosomal HPE is estimated to be 6% (Roach et al., 1975). Since recurrence risks are higher in familial

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forms of HPE, a thorough family history is essential. Special attention should be paid to microforms of HPE, such as a single central incisor or anosmia. Families concerned about recurrence in future pregnancies should receive genetic counseling from a counselor experienced with HPE. Prenatal diagnosis and imaging are discussed in the Neuroimaging section.

2.8. Conclusion Holoprosencephaly is a complex developmental brain malformation. Advances in neuroimaging, genetics and neuropathology have dramatically improved our understanding of the etiology and pathogenesis of this condition. The details of the complex interplay of genetic and environmental factors involved in HPE are just emerging. Our growing understanding and recognition of the wide clinical spectrum of HPE should enable us to provide more accurate diagnoses and prognoses. This increased knowledge should lead to improved management of common medical complications and more optimal family counseling. Careful assessment of each affected individual and neuroimaging studies are essential when dealing with cases of brain malformations such as HPE. With advanced MRI we are no longer dependent on the evaluation of the face to predict the brain. As pointed out in an editorial by Patterson (2002), ‘the face predicts the brain; the image predicts its function.’

Acknowledgments This research was supported by the Carter Centers for Brain Research in Holoprosencephaly and Related Malformations, the Don and Linda Carter Foundation, and the Carter-Crowley Foundation. The author thanks Ms Jenny Jimison for assistance with the manuscript and Dr Nancy Clegg for assistance with the Carter Center database.

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Septo-optic-pituitary dysplasia PETER HUMPHREYS* Children’s Hospital of Eastern Ontario, Ottawa, Ontario, Canada

3.1. Introduction Septo-optic-pituitary dysplasia (SOPD) is a rare syndrome characterized by a combination of agenesis of the septum pellucidum, optic nerve hypoplasia and pituitary insufficiency. As is often the case with syndromic diagnoses, SOPD has enormous variability in clinical expression and many putative etiologies, some clearly identified, some hypothetical. That SOPD has continued to survive as a ‘discrete’ diagnostic entity is largely due to the fact that the early identification of optic nerve hypoplasia obliges the clinician to consider the simultaneous presence of hypopituitarism, a potentially lifethreatening disorder when symptomatic in infancy or early childhood. There are no published incidence or prevalence figures for SOPD but there are equivalent data for some of its component features. Ouvrier and Billson (1986) reported an incidence of 1.8–2.0:100 000 live births for congenital optic nerve hypoplasia. For congenital hypopituitarism the reported incidence figure is 1:29 000 or 3.4:100 000 live births (Hanna et al., 1986). August et al. (1990) found that 4% of 2331 children with growth hormone deficiency had ‘septooptic dysplasia’. Granted that congenital hypopituitarism is not equivalent to childhood growth hormone deficiency, if we extrapolate the results of the studies by Hanna et al. and August et al., it seems unlikely that the incidence of SOPD is much greater than 1:106 live births. Although an association between optic nerve hypoplasia and septum pellucidum agenesis was first recognized by Reeves (1941), it was De Morsier who reported the first case series, and who proposed the term ‘septo-optic dysplasia’ to characterize the syndrome (De Morsier, 1956). Although the syndrome has expanded consider-

ably since De Morsier’s report, many clinicians have continued to refer to SOPD as De Morsier syndrome, in honor of his contribution. An association between septo-optic dysplasia and congenital hypopituitarism was first described by Hoyt and collaborators (1970), leading to the expanded terminology now in use: septo-optic-pituitary dysplasia. Since 1970 it has become apparent that the three main features of SOPD may, in individual patients, occur in isolation or in any mathematically possible combination. Thus, in addition to De Morsier’s original combination of optic nerve hypoplasia and absent septum pellucidum without hypopituitarism, one may see optic nerve hypoplasia and hypopituitarism without septum pellucidum agenesis (Patel et al., 1975; Costin and Murphree, 1985; Brodsky and Glasier, 1993), and absent septum plus hypopituitarism with normal eyes (Morishima and Aranoff, 1986). At the same time, detailed pathological and imaging studies have further blurred the borders of the SOPD syndrome by describing a wide variety of associated brain anomalies and pathologies. Thus, for example, SOPD may be accompanied by lobar holoprosencephaly (Roessmann et al., 1987), schizencephaly (Aicardi and Goutie`res, 1981; Barkovich et al., 1989; Kuban et al., 1989), callosal dysgenesis or agenesis (Zeki et al., 1992; Stevens and Dobyns, 2004) and gray matter heterotopia (Brodsky and Glasier, 1993). Finally, to complete the blurring process, some of the major cerebral anomalies just described have been reported in association with congenital pituitary insufficiency (but without optic nerve hypoplasia). Examples include lobar and semilobar holoprosencephaly (Cameron et al., 1999), callosal agenesis (Cameron et al., 1999) and periventricular gray matter heterotopia (Mitchell et al., 2002).

*Correspondence to: Peter Humphreys, Professor of Pediatrics (Neurology), University of Ottawa, Children’s Hospital of Eastern Ontario, 401 Smyth Road, Ottawa, Ontario K1H8L1, Canada. E-mail: [email protected].

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Given this expanded spectrum of SOPD and the various outliers, it is likely that SOPD, as a construct, is simply a chimera resulting from ascertainment biases on the part of (as the case may be) ophthalmologists, endocrinologists and radiologists. Nevertheless, as noted at the outset, SOPD and its various satellites remain a useful construct: neurologists, pediatricians and ophthalmologists, when encountering patients having optic nerve hypoplasia and/or major brain anomalies, must consider the possibility of hypopituitarism, either already present covertly or developing later in the patients’ lives.

3.2. Clinical features 3.2.1. Optic nerve hypoplasia Hypoplasia of the optic nerves in SOPD may be bilateral or unilateral; i.e. pituitary insufficiency may still develop even when only one optic nerve is involved (Brodsky and Glasier, 1993). Impairment of vision is typically evident from an early age and may vary from bilateral and profound to unilateral and mild. When visual impairment is bilateral and severe it is often accompanied by an oscillatory or ‘searching’ nystagmus, evident from early infancy. While there is usually a correlation between the severity of nerve hypoplasia and the amount of visual impairment, it occasionally happens that an obviously hypoplastic nerve is nevertheless associated with normal visual acuity. In such cases, however, in-depth testing will usually uncover

a visual field defect and/or field constriction (Margalith et al., 1984; Hellstrom et al., 1999). On funduscopic exam, the cardinal feature of optic nerve hypoplasia is a small, pale optic nerve head. Typically the disc diameter is one-third to one-half normal (Fig. 3.1). In addition, the hypoplastic disc may be surrounded by a yellow halo, the color being distinct from that of the normal retina. As a result, a cursory inspection of the disc may suggest to the examiner that its size is normal when in reality it is not. On occasion, the halo is rimmed on its inner and outer edges by a band of pigment; this produces a striking phenomenon known as the ‘double ring sign’ (Margalith et al., 1984). In some cases, one optic nerve head is hypoplastic while the other is of normal size but abnormally pale. This combination of optic nerve hypoplasia and atrophy was reported in eight out of 51 in the case series reported by Margalith et al. (1984). Optic nerve hypoplasia can also be documented radiologically, both on computed tomography (CT) studies of the orbits, and on axial and coronal magnetic resonance imaging (MRI) sequences. The optic nerves, chiasm and tracts all appear abnormally small (Fig. 3.2). 3.2.2. Pituitary insufficiency In SOPD, symptoms of dysfunction of the hypothalamic–pituitary axis may appear at any time from the neonatal period to adolescence. In neonates, in whom

Fig. 3.1. Septo-optic dysplasia: a 12-year-old girl with unilateral optic nerve hypoplasia and agenesis of the septum pellucidum; no pituitary dysfunction. (A) Fundus photograph of hypoplastic optic nerve head. The limits of the optic disc are shown by arrowheads, those of surrounding corona by arrows. (Courtesy of Dr William Clarke.) (B) Axial CT image. The septum pellucidum is absent and the anterior horns have a squared-off appearance laterally.

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Fig. 3.2. Optic-pituitary dysplasia: a 2-year-old boy with severe bilateral optic nerve hypoplasia, blindness and panhypopituitarism. He also has brainstem dysplasia with congenital left facial paresis, bilateral sensorineural deafness and aqueductal stenosis. (A) Midsagittal T1-weighted MR image. There is mild compensated hydrocephalus with upward bowing of the corpus callosum and dilation of the proximal aqueduct (arrowhead). The anterior pituitary gland is present in the sella turcica but the posterior pituitary gland bright signal is absent (arrow). (B) Axial T1-weighted MR image. The optic nerves within the orbits are markedly hypoplastic (arrowheads).

the presence of optic nerve hypoplasia may not be immediately recognized, symptoms of pituitary insufficiency may be nonspecific but life-threatening (Patel et al., 1975; Margalith et al., 1985; Cameron et al., 1999). Deficiencies of growth hormone, corticotropin or antidiuretic hormone, either individually or in combination, may produce signs or symptoms as varied as recurrent, unexplained hypoglycemia, jaundice, seizures, apnea, respiratory distress and electrolyte disturbances. Often the neonatal manifestations of pituitary insufficiency are only recognized as such later in life. In a retrospective series of patients with optic nerve hypoplasia reported by Margalith et al. (1984), 25 out of 44 had a variety of neonatal symptoms, including six with hypoglycemia, 10 with jaundice and four with electrolyte disturbances. In a subsequent paper derived from the same patient series, Margalith et al. (1985) reported neonatal hypoglycemia as the presenting symptom in 13 of 16 patients with optic-pituitary dysplasia. In other SOPD cases, symptoms may appear for the first time at a later age, often around 4–5 years. One may see the insidious appearance of growth failure and/or hypothyroidism (Margalith et al., 1985; Costin and Murphree, 1985; Siatkowski et al., 1997). On the other hand, corticotrophin deficiency may appear with a catastrophic presentation in childhood, leading to sudden death. Brodsky et al. (1997) reported sudden death in five children (aged 9 months to 7 years) with

SOPD and corticotrophin deficiency; four of the five also had diabetes insipidus (Brodsky et al., 1997). Dwarfism, hypothyroidism, corticotrophin deficiency, diabetes insipidus, hyperprolactinemia and sexual infantilism may appear in any combination, with some children having panhypopituitarism (Costin and Murphree, 1985; Margalith et al., 1985; Cameron et al., 1999). On the other hand, in one specific instance, SOPD may present with a pituitary hormone ‘excess’ in the form of precocious puberty (Margalith et al., 1985). A proposed explanation for this apparent contradiction will be considered below. Overall, symptoms of pituitary insufficiency have been reported in 27–60% of children with optic nerve hypoplasia (Costin and Murphree, 1985; Margalith et al., 1985; Masera et al., 1994; Siatkowski et al., 1997; Miller et al., 2000). Corresponding figures for pituitary insufficiency in cases of agenesis of the septum pellucidum with normal eyes are lacking. 3.2.3. Agenesis of the septum pellucidum Absence of the septum pellucidum is usually apparent on CT images and axial MRI sequences but is best appreciated on MR coronal sequences. The frontal horns are often slightly enlarged and squared off laterally (Kuban et al., 1989) (see also Fig. 3.1). In some cases of SOPD, the septum is present but incomplete (Barkovich et al., 1989).

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Other septal anomalies may be associated with features of optic-pituitary dysplasia. Bodensteiner and Schaefer have reported cases of an abnormally wide (> 1 cm) cavum septum pellucidum having growth failure and/or optic nerve hypoplasia (Bodensteiner and Schaefer, 1990). While absence of the septum pellucidum, as a membranous structure, would not be expected to produce any neurological deficits, one could envisage symptoms related to dysgenesis of the adjacent septal area of the midline basal forebrain, dysgenesis that might not be apparent on imaging studies. Septal area lesions created in rats have produced abnormalities in navigational ability (Miller et al., 1977). Similar deficits have not been documented in individuals with septo-optic dysplasia. Groenveld et al. (1994) compared spatial abilities in six children with septo-optic dysplasia with 13 children having optic nerve hypoplasia and an intact septum; no significant differences were reported (Groenveld et al., 1994). Likewise, Williams et al. (1993) reported no significant mental, behavioral or neurological deficits (other than poor vision) in seven patients with absent septum pellucidum and optic nerve hypoplasia. In contrast, there is a single anecdotal report of major psychomotor deficits in patients with isolated septum pellucidum agenesis. Belhocine et al. (2005) reported six cases of apparently isolated septal agenesis, three of whom had mixed motor and mental deficits while three were asymptomatic; one of the six had endocrine dysfunction. 3.2.4. Other neurological pathology As was noted in the Introduction, radiological and pathological studies of SOPD have revealed a large number of developmental brain anomalies, as well as some pathological abnormalities acquired in utero after the putative time window during which SOPD is believed to develop (see Pathogenesis, below). A list of structural pathologies associated with SOPD is provided in Table 3.1, along with the relevant references; some are also illustrated in Figs. 3.3 and 3.4. Of the abnormalities listed, schizencephaly is particularly common (Fig. 3.4A). In two small SOPD series (Barkovich et al., 1989; Kuban et al., 1989), schizencephaly was reported respectively in four of nine and five of 11 cases. Barkovich et al. (1989) suggested that septo-optic dysplasia plus schizencephaly might be a distinct disorder in that none of their five cases had pituitary dysfunction. In the same vein, some authors have argued that SOPD with other major cerebral pathologies is qualitatively different from ‘pure’ SOPD and should be referred to as ‘SOPD-plus’

(Miller et al., 2000). It remains to be seen whether such distinctions have any clinical utility. Depending upon the type and location of the accessory neurological pathologies, a wide variety of other symptoms and signs may be present in addition to those already described for SOPD. Common examples include mental subnormality, specific learning difficulties, epilepsy, deafness, hypotonia, hemiparesis (e.g. in schizencephaly or porencephaly) and spastic diparesis (with periventricular leukomalacia).

3.3. Etiology and pathogenesis 3.3.1. Etiology With occasional exceptions, SOPD appears to be a sporadic disorder acquired in utero during the first trimester. Many putative etiological factors have been suggested but definitive proof for the role of these factors is lacking. Congenital optic nerve hypoplasia, with or without septum pellucidum agenesis and/or hypopituitarism, has been reported in infants of diabetic mothers (Patel et al., 1975) and in infants infected in utero with cytomegalovirus (Ouvrier and Billson, 1986). Maternal drug ingestion, whether for therapeutic or recreational reasons, is also an important potential cause: drugs implicated include valproate (McMahon and Braddock, 2001); phencyclidine (Michaud et al., 1982); ethanol, with or without marijuana (Margalith

Table 3.1 Central nervous system anomalies associated with septooptic-pituitary dysplasia Type of anomaly

References

Lobar holoprosencephaly Olfactory tract and bulb hypoplasia Schizencephaly

Roessmann et al., 1987 Levine et al., 2001

Corpus callosum dysgenesis Gray matter heterotopia Focal cortical dysplasia

Porencephaly Periventricular leukomalacia Rhombencephalosynapsis

Aicardi and Goutie`res, 1981; Barkovich et al., 1989; Kuban et al., 1989 Zeki et al., 1992; Stevens and Dobyns, 2004 Brodsky and Glasier, 1993 Nuri Sener, 1996; Miller et al., 2000; Stevens and Dobyns, 2004 Aicardi and Goutie`res, 1981; Kuban et al., 1989 Brodsky and Glasier, 1993 Michaud et al., 1982

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Fig. 3.3. Septo-optic-pituitary dysplasia: an 11-year-old girl with severe bilateral optic nerve hypoplasia, blindness, panhypopituitarism, mental subnormality and epilepsy. She also has agenesis of the corpus callosum, colpocephaly and a Dandy– Walker malformation. (A) Axial CT image. The lateral ventricles are parallel, typical of callosal agenesis. There is also colpocephaly with hypoplasia of the occipital cerebral mantle, and a simplified right sylvian fissure with adjacent cortical dysplasia (arrow). (B) Axial CT image. The cerebellar vermis is absent (star).

Fig. 3.4. (A) Coronal fast T1-weighted gradient echo MR image of a 3-week-old boy with bilateral open-lip schizencephaly, absence of the septum pellucidum and bilateral optic nerve hypoplasia. Pituitary function was normal to age 1. (B) Midsagittal T1-weighted MR image of an 11-month-old boy, ex-premature, with acute adrenal insufficiency. Optic nerves and septum pellucidum were intact. The posterior pituitary gland bright signal is located ectopically posterior to the optic chiasm (arrow). The corpus callosum is abnormally thin.

et al., 1984; Coulter et al., 1993); flunitrazepam (Orrico et al., 2002); cocaine and heroine (Dominguez et al., 1991; Orrico et al., 2002); amphetamine and phenylpropanolamine (Dominguez et al., 1991). Schizencephaly and porencephaly, both part of the expanded SOPD

spectrum, are also known to be associated with maternal ingestion of cocaine, amphetamine and phenylpropanolamine (Dominguez et al., 1991). Very young maternal age is an apparent etiological factor (Patel et al., 1975; Margalith et al., 1984);

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SOPD occurs more often in the first-born children of 13–19-year-old mothers. It is unclear, however, whether this association is related to maternal age itself, as is the case with many other developmental anomalies, or to the higher frequency of recreational drug use in very young mothers. Familial cases of SOPD have occasionally been reported, raising the possibility of genetic factors. Blethen and Weldon (1985) reported SOPD in first cousins; Benner et al. (1990) described a combination of bilateral optic nerve hypoplasia, septum pellucidum agenesis, pituitary insufficiency and midline callosal and cerebellar anomalies in dizygotic twins. Two siblings from a consanguineous parentage were reported by Wales and Quarrel (1996) to have absence of the septum pellucidum, callosal agenesis, neonatal hypoglycemia and panhypopituitarism. These latter two cases were subsequently found to have a homozygous Arg53Cys missense mutation in the homeodomain of the homeobox gene HESX1 (homeobox gene expressed in embryonic stem cells) (Dattani et al., 1998). After this ground-breaking report appeared, other mutations in the HESX1 gene have been described, sometimes in the heterozygous state, for the most part associated with milder phenotypes (Thomas et al., 2001; Cohen et al., 2003; Carvalho et al., 2003; Tajima et al., 2003). The relationship between HESX1 mutation type and phenotype is summarized in Table 3.2. In most sporadic cases of SOPD, no etiological factor(s) can be identified; it is possible that some of Table 3.2 HESX1 mutations Mutation (zygosity)

Phenotype

R160C missense (homozygous) 1684delG (homozygous) S170L substitution (heterozygous) T181A, Q6H substitutions (heterozygous) Frameshift 306/ 307insAG (heterozygous) I26T (homozygous)

SOPD, callosal dysgenesis*

*

Dattani et al., 1998. Cohen et al., 2003. { Thomas et al., 2001. } Tajima et al., 2003. } Carvalho et al., 2003. {

SOPD, callosal dysgenesis{ Growth hormone deficiency optic nerve hypoplasia{ isolated pituitary deficiency, ectopic/absent posterior pituitary{ pituitary insufficiency, unilateral optic nerve hypoplasia} isolated pituitary deficiency}

these cases are due to mutations in HESX1 or a number of other related genes (see below). 3.3.2. Pathogenesis 3.3.2.1. Embryological considerations In SOPD, the coexistence of developmental defects in the optic nerves, midline basal forebrain and hypothalamic pituitary axis suggests a regional disturbance in embryogenesis occurring at around 6 weeks gestational age. Optic nerve hypoplasia results from a failure of axonal growth cones projecting from retinal ganglion cells to penetrate the optic nerve head and grow centripetally along the optic nerves, a process that evolves between 4 and 8 weeks gestational age (Margalith et al., 1984; Ouvrier and Billson, 1986). In animal models of optic nerve hypoplasia, ganglion cell layer axons successfully grow in the direction of the optic nerve head but, having failed to detect an appropriate signal directing them to pierce the optic nerve head, then appear to wander off aimlessly to other areas of the retina (Oster et al., 2004). While defective axonal path finding may be the most common cause of optic nerve hypoplasia, other mechanisms may exist. In cases of SOPD accompanied by major cerebral encephaloclastic processes such as schizencephaly and porencephaly, optic nerve hypoplasia could result from transsynaptic degeneration in the optic pathway. In such cases the optic nerve hypoplasia would presumably develop later than 4–8 weeks gestational age. The fact that SOPD has occasionally been reported in association with optic atrophy lends support to this hypothesis (Margalith et al., 1984). The septum pellucidum normally forms during ventral induction of basal forebrain structures, again at around 6 weeks gestational age (Rakic and Yakovlev, 1968). Separation of the prosencephalon into discrete putative cerebral hemispheres, the development of the olfactory tracts and the differentiation of the lamina terminalis region into the septal area and corpus callosum anlage are all proceeding at around the same time. Disruption of these latter processes presumably explains the reported associations between SOPD and olfactory tract hypoplasia, arrhinencephaly and lobar holoprosencephaly (Roessmann et al., 1987; Levine et al., 2001), and between SOPD and agenesis of the corpus callosum (Zeki et al., 1992; Stevens and Dobyns, 2004). Pituitary gland development also occurs at around 5–6 weeks gestation, the anterior pituitary gland derived from Rathke’s pouch, a dorsal out-pocket originating in the primitive stomodeum, and the posterior pituitary gland derived from the infundibulum, a ventral projection of the basal forebrain (Mu¨ller and O’Rahilly, 1989). In most cases of septo-optic dysplasia with pituitary insufficiency, it is the latter process

SEPTO-OPTIC-PITUITARY DYSPLASIA that appears to be defective. MR studies of the sella turcica region usually reveal the anterior pituitary gland to be present but the normally bright signal of the posterior pituitary to be absent (Fig. 3.2A). At the same time, the pituitary stalk may be absent or hypoplastic, and the posterior pituitary tissue located ectopically near the mammillary bodies (Fig. 3.4B) (Kaufman et al., 1989; Brodsky and Glasier, 1993). Not evident on MR studies but revealed pathologically are concomitant structural deficiencies in hypothalamic cell clusters, e.g. absence of the supraoptic and paraventricular nuclei (Roessmann et al., 1987). The development of cerebral hemispheric structures implicated in some of the other SOPD-associated malformations occurs later than 4–8 weeks gestational age. In schizencephaly there is a segmental defect in the development of the cerebral mantle, a process involving both cell proliferation and cell migration that begins at around 8–10 weeks gestational age (Barkovich and Norman, 1988; Barkovich and Kjos, 1992). Gray matter heterotopia are the result of focal disruptions in neuronal migration, a phenomenon that primarily occurs between 2–4 months gestational age in humans (Rakic, 1972; Barkovich et al., 1992). Other associated pathologies develop even later in gestation: porencephaly is an encephaloclastic process that typically dates from the second or third trimesters (Volpe, 2001), while periventricular leukomalacia dates from the early third trimester (Banker and Larroche, 1962). These widely disparate timings during gestation suggest that some of the causes of SOPD begin to create mischief at around 6 weeks gestational age and continue to disrupt brain development at intervals throughout the remaining pregnancy. 3.2.2.2. Acquired pathogenetic mechanisms Considering the variety of potentially teratogenetic factors thus far linked to SOPD, factors as disparate as cytomegalovirus, maternal diabetes, ethanol and cocaine, it seems likely that sporadic SOPD may result from a number of distinct mechanisms. Thus far, however, the precise nature of these teratogenic mechanisms remains the subject of conjecture. Although reported studies of the hypothalamus in SOPD are rare, there is some evidence to support acquired injury as an important pathological phenomenon. Swaab described the post-mortem findings in a 4year-old girl with SOPD, the most important observations being the presence of scattered regions of astrocytosis (sometimes with calcifications) throughout the hypothalamus and virtual absence of the supraoptic and paraventricular nuclei (Swaab, 2005) In this instance the cause of the pathological abnormalities was not determined.

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There is compelling evidence to suggest that, of all the potential pathogenetic mechanisms for SOPD, the most common is likely to be vascular disruption. First, cocaine, amphetamine and phenylpropanolamine all produce vasospasm; maternal ingestion of these drugs can be followed by SOPD in the offspring (Dominguez et al., 1991). Second, focal or generalized vascular insults have been implicated in the pathogenesis of several of the cerebral anomalies associated with SOPD: schizencephaly (Barkovich and Kjos, 1992), polymicrogyria (Humphreys et al., 1991), porencephaly (Volpe, 2001), periventricular leukomalacia (Volpe, 2001). A strong proponent for a vascular pathogenesis of SOPD has been Lubinsky (1997a). Using the same logic outlined in the previous paragraph, Lubinsky proposed that SOPD is a vascular disruption sequence involving blood vessels that supply basal forebrain structures during their critical periods of ontogenesis. A candidate vessel is the anterior cerebral artery, which, prior to the origin of the anterior communicating artery, supplies the developing optic nerves and chiasm and, through perforating branches, the anterior hypothalamus, septal area and septum pellucidum. Thus, an appropriately-timed disruption of one or both anterior cerebral arteries could compromise the development of all the main structures defective in SOPD (Lubinsky, 1997a). Lubinsky also made the interesting observation that other prenatal vascular disruption sequences such as hydranencephaly and gastroschisis are likewise linked to young maternal age (Lubinsky, 1997b). Stevens and Dobyns (2004) have reported an illustrative case that lends support to the vascular pathogenetic hypothesis. Their patient, the infant of a 14-yearold mother, had SOPD, bilateral perisylvian cortical dysplasia, periventricular nodular heterotopia, callosal agenesis, multiple digit amputations and constriction rings. Of these, the last two were ascribed to vascular disruption with bleeding into the amniotic sac. Thus a vascular disruption occurring in embryonic or early fetal life could account for all the anomalies seen in this patient (Stevens and Dobyns, 2004). (c) Genetic mechanisms For familial, as well as some sporadic forms of SOPD, there is a wealth of recent evidence implicating a variety of gene-related mechanisms. Many of the genes identified are also involved in the pathogenesis of familial and sporadic cases of isolated optic nerve hypoplasia or pituitary insufficiency. Mention has already been made of the homeobox gene HESX1, the first gene shown to be associated with human SOPD (Dattani et al., 1998; Parks et al., 1999; Dattani and Robinson, 2002); HESX1 has been

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mapped to 3p21.1–21.2 (Dattani et al., 2000). In the mouse, hesx-1 is primarily expressed in developing forebrain tissue. Dattani and colleagues have developed an hesx-1-null mouse model that revealed a phenotype containing many components of human SOPD but with additional severe anomalies: reduced prosencephalon, anophthalmia or microphthalmia, defective olfactory maturation, Rathke’s pouch bifurcations and anomalies in all the cerebral commissures plus the septum pellucidum (Dattani et al., 1998). While HESX1 heterozygous mutations may produce symptoms (Table 3.2), hesx-1/ heterozygotes are usually asymptomatic, with only 1% of mice showing symptoms (Thomas et al., 2001). HESX1 contains a transcription repressor domain that appears to function in collaboration with a corepressor Groucho homolog/transducin-like repressor of split-1 (Gro/TLE1) (Cohen et al., 2003). Mutations in the HESX1 gene may lead to basal forebrain and optic nerve abnormalities through loss of function, gain of function or (potentially) dominant-negative mechanisms, as illustrated in the following examples. In the R160C HESX1 mutation, the mutation appears to disrupt the HESX1 DNA-binding domain, preventing the gene product from binding to DNA and thus interfering with transcription repression (Brickman et al., 2001). In contrast, the 1684delG mutation results in increased DNA binding and excessive repression of PROP-1-dependent gene activity (Cohen et al., 2003). The relationship between HESX1 and PROP-1 (prophet of PIT1) is examined below. The I26T mutation does not affect the DNA binding domain but, involving the Engrailed homology repressor domain (eh1), instead interferes with the ability of HESX1 to recruit its co-repressor Gro/TLE1, leading to an overall partial loss of repression (Carvalho et al., 2003). Finally, a potential dominant-negative mechanism may be seen through a second effect of the R160C mutation in that the gene product inhibits repressor activity of wild type HESX1 protein; the eh1-containing repressor domain is necessary for this effect (Brickman et al., 2001). Notwithstanding the fact that mutations in HESX1 are clearly related to familial and sporadic cases of SOPD (Dattani and Robinson, 2002), it has become apparent that most cases of SOPD that lack a history of maternal exposure to putative teratogenetic factors do not have HESX1 mutations. Two recent studies reporting genetic screening of sequential cases of SOPD (18 and 23 cases respectively) failed to reveal HESX1 mutations in any case (Antonini et al., 2001; Rainbow et al., 2005). These results suggest that, where genetic mechanisms are at play, many other genes are likely to be implicated.

Current evidence suggests that a large number of genes contribute to basal forebrain assembly, with some earlier-expressed genes helping to regulate processes as basic as cerebral hemisphere separation and others seemingly focused on specific portions of the forebrain such as the hypothalamic–pituitary axis. Defects in homeobox genes such as SHH, SIX3, TGIF and ZIC2 are known to be associated with holoprosencephaly, a major malformation that, in turn, may be accompanied by hypothalamic dysplasia and diabetes insipidus (Sarnat and Flores-Sarnat, 2001). Other homeobox genes (e.g. PROP1, POU1F1/PIT1, LHX4) have been linked to familial hypopituitarism without accompanying septo-optic dysplasia (Parks et al., 1999; Machinis et al., 2001; Dattani and Robinson, 2002). At least some of the genes just mentioned appear to work together, either in sequence, collaboration or competition, in early forebrain development. Cohen et al. (2003) have presented evidence, for example, that HESX1 and PROP1 function in a balanced competition, the former as an inhibitor of transcription, the latter as a promoter. While a number of genes have been linked to the pathogenesis of isolated hypopituitarism, the same cannot be said, thus far, for isolated optic nerve hypoplasia. In mouse models, however, there are at least two molecules implicated in the guidance of retinal ganglion cell axons into the optic nerve head: netrin-1 and DCC (deleted in colorectal cancer). Netrin-1 is an axon guidance molecule originally identified as a chemotropic factor attracting growth cones of commissural axons in the spinal cord; DCC belongs to the immunoglobulin superfamily and functions as a receptor for netrin-1. Knockout mouse models for both netrin-1 and DCC demonstrate optic nerve hypoplasia, as well as callosal dysgenesis and hypothalamic neuronal ectopia (Oster et al., 2004). It is thus conceivable that as-yet unidentified human homologues of netrin-1 and DCC could also play a role in the pathogenesis of SOPD. Finally, it appears that deficiencies in genes involved in basic cellular metabolic processes throughout the body may occasionally produce elements of SOPD along with a host of defects in other organs. Optic nerve hypoplasia has been reported in Down syndrome, Zellweger syndrome and hyperpipecolic acidemia (Ouvrier and Billson, 1986). There is also a case report of a patient with a mutation in the mitochondrially encoded cytochrome b gene (T14849C), associated with complex III deficiency, septo-optic dysplasia, cerebellar hypoplasia, retinitis pigmentosa, hypertrophic cardiomyopathy and acute rhabdomyolysis (Schuelke et al., 2002).

SEPTO-OPTIC-PITUITARY DYSPLASIA Thus, the enormous range of putative genetic mechanisms underlying SOPD and its individual components underscores the clinical heterogeneity of the syndrome.

3.4. Investigation and management The raison-d’eˆtre for SOPD as a diagnostic entity meriting its own chapter is the simple fact that the presence of any or all of optic nerve hypoplasia, septum pellucidum agenesis, lobar holoprosencephaly, callosal agenesis and schizencephaly (as well as other brain anomalies) are a clue to the possible existence of an initially silent and potentially deadly endocrinological disorder. It is thus imperative that the optic nerve and/or cerebral anomalies be correctly identified in a timely fashion. At the same time, while it is incumbent upon the clinician to consider the possibility of congenital hypopituitarism when provided with the appropriate clues, it must be remembered that most children with optic nerve hypoplasia or midline cerebral anomalies (or, for that matter, isolated hypopituitarism) do not have SOPD (Ouvrier and Billson, 1986). 3.4.1. The diagnosis of optic nerve hypoplasia The detection of bilateral optic nerve hypoplasia in a child with severe congenital visual impairment and searching nystagmus is usually not difficult. Problems arise, however, when only one nerve is hypoplastic or when the hypoplasia is mild and vision appears to be intact. The initial clue to unilateral optic nerve hypoplasia may be early-onset, persistent monocular strabismus (Margalith et al., 1984), as was the case in the patient illustrated in Figure 3.1. Mild unilateral optic nerve hypoplasia may be detected by disc diameter comparison using fundus photography. Additional clinical confirmation may be obtained by examination using red-free light (Manor and Korczyn, 1976) and, if the patient is old enough, by detailed visual field testing for segmental deficits. If optic nerve hypoplasia is suspected on clinical grounds, the diagnosis can be confirmed radiologically by CT or MR imaging of the orbits and the chiasmatic region (Margalith et al., 1984; Brodsky and Glasier, 1993) (see also Fig. 3.2). Electroretinographic studies may not detect any abnormality in optic nerve hypoplasia, probably because the retinal ganglion cell layer remains intact, with only its axons misguided. On the other hand, visual evoked responses are usually absent or delayed (Coupland and Sarnat, 1990). On occasion, the identification of optic nerve hypoplasia on funduscopic examination may be hampered by coexisting anterior optic abnormalities. Gunduz

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et al. (1996) have reported a child having a cataract in one eye and anterior segment dysgenesis in the other. The unsuspected presence of bilateral optic nerve hypoplasia, as well as absence of the septum pellucidum and callosal hypoplasia, was subsequently noted on MRI (Gunduz et al., 1996). 3.4.2. The identification of relevant cerebral anomalies In the absence of optic nerve hypoplasia, the diagnosis of septal-pituitary dysplasia or the identification of SOPD-associated major brain malformations may be difficult. Schizencephaly and porencephaly, depending upon their size and location, may present at a relatively early age with hemiparesis and epileptic seizures. Lobar holoprosencephaly and callosal agenesis may be associated with dysmorphic features and developmental delay. In contrast, closed-lip schizencephaly, gray matter heterotopia and focal cortical dysplasia, if they do not produce early-onset seizures, may only be detected in the later investigation of learning disabilities. Isolated agenesis of the septum pellucidum, as has been noted, may produce no neurological symptoms whatever (Williams et al., 1993). Clearly what is required is a high index of suspicion and the ability to think in a lateral fashion once a relevant malformation has been identified. As a rule, MRI, if available, is the preferred technique for the visualization of those cerebral malformations most often associated with SOPD; this is particularly true for smaller, relatively subtle anomalies such as focal cortical dysplasia and gray matter heterotopia. In addition, the degree of preservation of the corpus callosum (in cases of callosal dysgenesis) is best assessed by a midline sagittal MR image. The principal radiological criteria for the main cerebral anomalies associated with SOPD are summarized in Table 3.3 (see also Barkovich et al., 1989; Kuban et al., 1989; Zeki et al., 1992; Fitz, 1994; Siatkowski et al., 1997). In septo-optic dysplasia, agenesis of the septum pellucidum is typically detected on either a CT or MR image performed as a screening test in a child presenting with visual impairment and optic nerve hypoplasia (as in Fig. 3.1). With the advent of fairly routine highquality abdominal ultrasound examination during pregnancy, however, it is now feasible to detect isolated septum pellucidum agenesis prior to birth. Lepinard et al. (2005) reported two fetuses in whom agenesis of the septum pellucidum was identified on ultrasound at 29 and 30 weeks gestational age respectively. Both fetuses went on to have in utero MRI studies that confirmed the ultrasound diagnosis. One of the

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Table 3.3 Diagnostic radiological criteria for SOPD-associated cerebral anomalies Anomaly

Criteria

Absence of septum pellucidum

Open communication between anterior horns of lateral ventricles ‘Squared-off’ anterior horns Fornices displaced inferiorly Fusion of inferior frontal lobes (coronal MRI) Absence of anterior third ventricle absence of anterior corpus callosum partial fusion of basal ganglia Wide separation of lateral ventricles High-riding third ventricle Parallel anterior horns Absence of cingulate gyrus colpocephaly Cleft between lateral ventricle(s) and pial surface of hemisphere Close-lipped (sides apposed) or open-lipped Cleft walls lined with gray matter Irregular collections of gray matter lining lateral ventricle(s) or in central hemispheric white matter Irregular thickness of cortical gray matter Abnormally large or small gyri with irregular contour(s) open sylvian fissure(s) cortical infolding Cystic cavity in central hemispheric white matter communication with adjacent lateral ventricle

Lobar holoprosencephaly

Agenesis of corpus callosum

Schizencephaly

Gray matter heterotopia Focal cortical dysplasia Porencephaly

two was also found on MRI to have optic chiasm hypoplasia; fully expressed SOPD was confirmed post-natally (Lepinard et al., 2005). Posterior pituitary agenesis or ectopia, whether or not associated with other cerebral anomalies, is also best seen on mid-sagittal MRI (Kaufman et al., 1989; Brickman et al., 2001; Mitchell et al., 2002) (see also Figs. 3.2A and 3.4B). The same radiological findings may also be seen in some cases of isolated congenital hypopituitarism (Triulzi et al., 1994; Machinis et al., 2001). When present, posterior pituitary ectopia is predictive of pituitary dysfunction; many cases of the SOPD complex, however, have hypopituitarism without any radiological abnormality in the pituitary region (Abernethy et al., 1997).

3.4.3. The diagnosis and management of hypopituitarism in SOPD In the neonatal period, congenital hypopituitarism should be suspected in any infant presenting with unexplained recurrent hypoglycemia, prolonged jaundice or seizures (Patel et al., 1975; Margalith et al., 1984; Cameron et al., 1999). In males, micropenis may also be a clue to diagnosis. In jurisdictions where

such programs are available, presymptomatic neonatal hypopituitarism may be detected by neonatal screening of serum thyroxine and thyroid-stimulating hormone (TSH) levels (van Tijn et al., 2005). Later in childhood, symptoms of pituitary hormone insufficiency tend to present insidiously and in any conceivable combination from isolated hypothyroidism or growth failure to fully-expressed hypopituitarism (Margalith et al., 1985; Cameron et al., 1999; Geffner, 2002). The endocrinological testing procedures necessary to detect deficiencies of individual pituitary hormones or hypothalamic releasing factors are summarized in Table 3.4 (see also Geffner, 2002; Forest, 2003; Ranke, 2003; van Tijn et al., 2005). In cases of optic nerve hypoplasia and associated hypopituitarism, the latter problem is typically due to hypothalamic dysfunction rather than failure of the anterior pituitary gland itself; exogenous administration of hypothalamic releasing factors often produces normal elevations in levels of the respective pituitary hormones (Margalith et al., 1985). As a rule, impaired function of the hypothalamic–pituitary axis is more likely with bilateral, rather than unilateral, optic nerve hypoplasia (Skarf and Hoyt, 1984). Mention has already been made of the fact that, while sexual infantilism may occur in SOPD, precocious

SEPTO-OPTIC-PITUITARY DYSPLASIA Table 3.4 Diagnostic work-up of pituitary insufficiency Hormone category

Investigations

Growth hormone (GH)

# levels of serum IGF1, IBP3 # serum GH response to insulininduced hypoglycemia, glucagon, arginine or clonidine # free or total thyroxine Thyrotropin level may be low, normal or high Usually # response of thyrotrophin levels to TRH stimulation; occasionally normal or delayed " response # early-morning serum cortisol and serum corticotropin # cortisol response to low-dose ACTH, metapyrone, glucagon, CRH or insulin-induced hypoglycemia #levels of serum LH, FSH, estradiol, testosterone # response of LH, FSH to GnRH stimulation, but may be normal " serum prolactin levels " response to TRH stimulation Inability to concentrate urine with water deprivation Clinical response to argininevasopressin

Thyroid hormone

Corticotrophin

Gonadal hormones

Prolactin Antidiuretic hormone

ACTH, adrenocorticotropic hormone; CRH, corticotropin-releasing hormone; FSH, follicle-stimulating hormone; GnRH, gonadotropinreleasing hormone; IBP, insulin-like binding protein; IGF, insulin-like growth factor; LH, luteinizing hormone; TRH, thyrotropin-releasing hormone.

sexual puberty is quite common (Margalith et al., 1985). Indeed, in this respect, SOPD behaves differently from isolated congenital panhypopituitarism (Nanduri and Stanhope, 1999). Nanduri and Stanhope have postulated that gonadotrophin secretion in SOPD is usually preserved because gonadotrophin releasing hormone (GnRH) neurons, unlike other hypothalamic releasing hormone neurons, originate in the olfactory endothelium and migrate along the olfactory tract to the hypothalamic region. Their arrival in the hypothalamus does not occur until the end of the first trimester, weeks after the 4–8 weeks gestation time window during which SOPD is believed to develop (Nanduri and Stanhope, 1999). Treatment of specific pituitary hormone deficiencies, once identified, is usually the province of endocrinologists rather than neurologists. Indeed, management of endocrine dysfunction in SOPD is sufficiently

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complex and potentially hazardous that referral to an endocrinologist is strongly recommended. Only a brief outline of management strategies will be given here. Interested readers are referred to a recent consensus article by Stanhope et al., and a review article by Geffner (Stanhope et al., 2001; Geffner, 2002). Although, in principle, treatment of pituitary dysfunction in SOPD could consist either of pituitary hormones or, where available, hypothalamic releasing factors, the possible occurrence of pituitary gland dysplasia in SOPD makes the latter option, in general, less practical (Geffner, 2002). Recombinant human growth hormone is usually given by daily injection, the cumulative weekly dose being 0.18–0.3 mg/kg. Since growth hormone is, in normal individuals, primarily secreted during sleep, the doses are normally given during the evening (Geffner, 2002). As a rule, the dose is not increased at the time of puberty, whether the latter is spontaneous or induced (Stanhope et al., 2001). Thyroid replacement is in the form of oral thyroxine, at approximately 50 mg/m2/day, the dose being adjusted as required by the ongoing results of monitored serum thyroxine levels. For corticotrophin deficiency there are several potential options. The usual practice is to administer hydrocortisone by mouth, the average daily dose being 7–10 mg/m2/day, given in two or three divided doses (Geffner, 2002). In the Stanhope et al., 2001 consensus paper, it was suggested that the morning dosage should be larger than the others. During intercurrent illnesses, or other stresses such as surgery or injury, the daily dose of hydrocortisone should be doubled or tripled for the duration of the stress (Geffner, 2002). Prednisone, given as 25% of the milligram dose of hydrocortisone, is a practical alternative to hydrocortisone. Dexamethasone, in a dose of 1–4% of the hydrocortisone milligram dosage, is a third alternative, but there is a greater risk of inadvertent toxicity (Geffner, 2002). As mentioned above, sexual infantilism in SOPD is rare, with precocious puberty a more likely problem. The ongoing emergence of precocious puberty may be delayed, if necessary, using analogs of GnRH (e.g. leuprorelin) (Kletter and Kelch, 1994). Where sexual infantilism is present, puberty in girls may be induced by synthetic estrogen, administered orally or by skin patch, starting at age 11–12 years. Oral progesterone is added later, after 18–24 months of estrogen therapy (Stanhope et al., 2001). For boys, puberty is induced at around age 13–14 years with testosterone, usually in the form of a monthly injection (50– 100 mg/4 weeks). In adults with established puberty, alternate routes of testosterone administration include daily skin patches and a daily skin gel (Geffner, 2002).

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Diabetes insipidus is routinely managed with arginine-vasopressin given as a nasal spray (5–20 mg/ day), or with oral tablets of desmopressin acetate (100–400 mg/day). Larger doses of the oral format are required because much of the drug is inactivated by stomach acid. During surgery or severe illness, antidiuretic hormone is usually given by continuous intravenous infusion (Geffner, 2002). 3.4.4. Genetic analysis In SOPD cases where there is a family history of individuals with some or all of the characteristics of SOPD, HESX1 gene analysis is now commercially available; all other potentially relevant gene analyses are only available on a research basis.

Acknowledgment The author wishes to thank Drs Sarah Lawrence and Pranesh Chakraborty for helpful suggestions about the manuscript.

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Masera N, Grant DB, Stanhope R, et al. (1994). Diabetes insipidus with impaired osmotic regulation in septo-optic dysplasia and agenesis of the corpus callosum. Arch Dis Child 70: 51–53. Michaud J, Mizrahi EM, Urich H (1982). Agenesis of the vermis with fusion of the cerebellar hemispheres, septooptic dysplasia and associated anomalies. Acta Neuropathol (Berl) 56: 161–166. Miller MA, Innes WC, Enloe LJ (1977). Performance on a four-choice search task following septal lesions in rats. Physiol Psychol 5: 433–439. Miller SP, Shevell MI, Patenaude Y, et al. (2000). Septooptic dysplasia plus: a spectrum of malformations of cortical development. Neurology 54: 1701–1703. Mitchell LA, Thomas PQ, Zacharin MR, et al. (2002). Ectopic posterior pituitary lobe and periventricular heterotopia: cerebral malformations with the same underlying mechanism? Am J Neuroradiol 23: 1475–1481. Morishima A, Aranoff GS (1986). Syndrome of septo-opticpituitary dysplasia: the clinical spectrum. Brain Dev 8: 233–239. Mu¨ller F, O’Rahilly R (1989). The human brain at stage 16, including the initial evagination of the neurohypophysis. Anat Embryol 179: 551–569. Nanduri VR, Stanhope R (1999). Why is the retention of gonadotrophin secretion common in children with panhypopituitarism due to septo-optic dysplasia? Eur J Endocrinol 140: 48–50. Nuri Sener R (1996). Septo-optic dysplasia associated with cerebral cortical dysplasia (cortico-septo-optic dysplasia). J Neuroradiol 23: 245–247. Orrico A, Galli L, Zappella M, et al. (2002). Septo-optic dysplasia with digital anomalies associated with maternal multidrug use during pregnancy. Eur J Neurol 9: 679–682. Oster SF, Deiner M, Birgbauer E, et al. (2004). Ganglion cell axon pathfinding in the retina and optic nerve. Semin Cell Dev Biol 15: 125–136. Ouvrier R, Billson F (1986). Optic nerve hypoplasia: a review. J Child Neurol 1: 181–188. Parks JS, Brown MR, Hurley DL, et al. (1999). Heritable disorders of pituitary development. J Clin Endocrinol Metab 84: 4362–4370. Patel H, Tze WJ, Crichton JU, et al. (1975). Optic nerve hypoplasia with hypopituitarism: septo-optic dysplasia with hypopituitarism. Am J Dis Child 129: 175–180. Rainbow LA, Reest SA, Shaikh MG, et al. (2005). Mutation analysis of POUF-1, PROP-1 and HESX-1 show low frequency of mutations in children with sporadic forms of combined pituitary hormone deficiency and septo-optic dysplasia. Clin Endocrinol 62: 163–168. Rakic P (1972). Mode of cell migration to the superficial layers of fetal monkey neocortex. J Comp Neurol 145: 61–84. Rakic P, Yakovlev PI (1968). Development of the corpus callosum and cavum septi in man. J Comp Neurol 132: 45–72. Ranke MB (2003). Diagnosis of growth hormone deficiency and growth hormone stimulation tests. In: MB Ranke (Ed.),

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Rhombencephalosynapsis PETER G. BARTH* Emma Children’s Hospital/Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands

4.1. Introduction and history The first detailed description of rhombencephalosynapsis (RS) is generally attributed to the Viennese neuropathologist Obersteiner (1916) who presented his findings under the title ‘Ein Kleinhirn ohne Wurm’ (‘A cerebellum without vermis’). The brain belonged to a 28-year old male clerical employee who had committed suicide. No further details on the man’s life or medical history were available. Obersteiner’s illustrated study presents all the features of this malformation. There were no supratentorial abnormalities. On the dorsal surface of the cerebellum he noted the apparent absence of the vermis with the folia of the hemispheres following a smooth uninterrupted course across the midline. The incisura cerebelli posterior, normally marking the inferior limit of the vermis, was missing. The ventral view of the cerebellum showed that the tuber and pyramis of the vermis, normally visible between the semilunar and biventer lobules, were missing. The tonsils, however, were well separated and the nodule and the uvula between them, both underdeveloped, were present, forming the only macroscopic evidence of the vermis. On sectioning and microscopy, near-fusion of the dentate nuclei was found while the fastigial nuclei were absent. Midline abnormalities were not restricted to the cerebellum but could be traced upwards to the lower mesencephalon. The superior cerebellar peduncles were apposed occupying the place of the anterior medullary velum. The abnormal course of the superior cerebellar peduncles caused the trochlear nerves to pass through the superior cerebellar peduncles on the way to their decussation and exit. An abnormal position of the mesencephalic trigeminal tract lateral to the superior cerebellar peduncles was also noted. Decussation of the superior cerebellar peduncles was shown, albeit at a

slightly higher level than normal, and no abnormalities could be found upward from the level of the normalappearing red nuclei. The inferior colliculi were not well separated (Figure 5 in Obersteiner’s report). The name ‘rhombosynapsis’ was proposed by De Morsier (1955) to contrast this malformation with another entity, introduced by this author under the name ‘rhomboschizis’. In the latter entity the vermis is absent but the cerebellar hemispheres are separated by a cleft taking the place of the vermis. From 1916 until 1987 seven further cases of RS were contributed to the literature, all diagnosed by autopsy (Gross, 1959; Kepes et al., 1969; Michaud et al., 1982; Schachenmayr and Friede, 1982; Isaac and Best, 1987). Although computed tomography (CT) scanning became available around 1975 as a tool for neuroimaging, no case was diagnosed by this method. Inherent limitations of CT in the analysis of posterior fossa anomalies may have caused the delay in clinical awareness. The first reported patients diagnosed during life were published in 1991, with the help of magnetic resonance imaging (MRI) (Savolaine et al., 1991; Truwit et al., 1991). When we add the cases reported after 1991 until the middle of 2005, 58 cases have been reported in less or more detail, the majority by MRI (for references see Table 4.1). The incidence of the anomaly is unknown. Sener (2000) found four cases in his MR database of 3000, a frequency of 0.13%). Studies show an excess of cerebral- and extracerebral associated malformations. More clinical details of the affected persons were provided as MRI reporting took the lead over neuropathological reporting. In this chapter a description of the neuropathological and imaging features of RS will be presented, together with a summary of the recorded cerebral- and extracerebral associated anomalies and neurological findings.

*Correspondence to: Peter G. Barth MD, Emeritus Professor in Pediatric Neurology, Emma Children’s Hospital/Academic Medical Center, Room G8-211, PO Box 22700, 1100 DE Amsterdam, Netherlands., E-mail: [email protected], Tel: þ31-20-566-7508, Fax: þ31-20-691-7735.

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Table 4.1 Rhombencephalosynapsis and associated conditions Reference

Sex, age

Intracranial associations

Aydingoz et al., 1997 Bell et al., 2005 Brocks et al., 2000

f, 0.8 years f, 55 years m, 14 years

Hy* Hy*, SA, atrophy hippocampi

Danon et al., 2000 De Jong and Kirby, 2000

f, 34 weeks g.a. m, 35 weeks g.a. autopsy

Hy**, SA, angulation corpus callosum Hy*** (by description)

Demaerel et al., 1995 (1)

m, 6 years

Hypoplasia inferior olivary nuclei

Demaerel et al., 1995 (2)

m, 2 years

Demaerel et al., 2004

m, newborn

Hy**, SA, partial dysgenesis corpus callosum Hy**, SA, FF

Garfinkle, 1996

f, 11 years

Gross, 1959 (1)

f, 0.15 years autopsy

Gross, 1959 (2)

m, 2 years autopsy

Guyot et al., 2000 Isaac and Best, 1987 (1) Isaac and Best, 1987 (2) Kepes et al., 1969 Lespinasse et al., 2004

f, 39 years m, 11 years autopsy f, 35years autopsy m, 0.5years autopsy f, 2.5years

Litherland et al., 1993

m, 22 weeks g.a. autopsy

Extracranial associations

Prenatal factors

Asymmetric hand anomalies

Prenatal ultrasound detection of small cerebellum

Go´mez–Lo´pez-Hernańdez syndrome

FD, preaxial polydactyly right foot, rib anomaly, conotruncal defect, ambiguous genitalia

Previous pregnancy conjoined twins Ethosuximide first 4 months of pregnancy

Bilateral cryptorchidism, large VSD, imperforatio ani, Hirschsprung’s disease. Maternal juvenile onset diabetes mellitus

Craniosynostosis?, biparietal alopecia, keratitis

Features suggestive of Go´mez–Lo´pez-Hernańdez syndrome

Peripheral nerve palsies, turricephaly, undescended testicles, absent hair over temples

Agenesis right lung FD, short limbs, benign pulmonary stenosisb

Unbalanced subtelomeric translocation t(2p;10q)

P. G. BARTH

Absent ventricles, absent sylvian fissures, absent precentral and central sulci, cortex suggestive of polymicrogyria, synthalamus Hy***, SA, partially undifferentiated oliva inferior, abnormal accessory olivary nuclei, absent spinal tract and nucleus V Microcephaly, dilated lateral ventricles, hypoplastic temporal poles, midline thalamic fusion, cerebral white matter gliotic, partially undifferentiated oliva inferior, abnormal accessory olivary nuclei Hy* Hy*** Hy***, SA Hy***, midline thalamic fusion

Craniosynostosis, bitemporal alopecia, corneal clouding

f, 20 years

Hy*, SA, dysplastic hippocampi, supracollicular lipoma Focal dysplasia left temporal cortex Hy**, SA, septo-optic dysplasia

Mendonça et al., 2004 (2) Michaud et al., 1982

f, 14 years f, term newborn autopsy

Montull et al., 2000

f, 39 years

Munõz et al., 1997 (1)

f, 4years

Munõz et al., 1997 (2)

f, 14years

Munõz et al., 1997 (3)

m, 9

Napolitano et al., 2004 (1)

f, 32 weeks g.a.

Napolitano et al., 2004 (2) Napolitano et al., 2004 (3) Obersteiner, 1916

m, 25 weeks g.a. f, 28 weeks g.a. m, 28 years autopsy

Odemis et al., 2003

m, 0.5 years

Oei et al., 2001

f, 2 years

Hy***, SA, FF prenatal ultrasound

Romanengo et al., 1997 Savolaine et al., 1991 Schachenmayr and Friede, 1982

m, 16 years f, 21 years m, 0.3 years autopsy

Scroop et al., 2000 Sener and Ozelzite, 2003

f, 34 years m, 22 years

FD Hy** Hy**, right paramedian hemisphere defect, absent olfactory tracts and bulbs, mammillary bodies and pineal gland not identified Hy* Hy**, tectal beaking, Chiari II

Sergi et al., 1997

m, 23 weeks g.a. autopsy

Undivided frontal brain, absent forebrain ventricles, partial craniosynostosis,

Hy**, SA, FF, temporal lobe hypoplasia (hippocampal dysplasia?), underdeveloped anterior commissure

Hy**

Bilateral hand anomalies, left radial hypoplasia, anomalous venous return, large ASD, single-lobed right lung, partial duplication of left ureter, ectopic kidneys, multiple vertebral segmentation defects, rib defects Bilateral hand anomaly

Maternal phencyclidine during first 6 weeks of pregnancy

Lambdoid sutures prematurely closed, biparietal alopecia, corneal clouding Lambdoid sutures prematurely closed, biparietal alopecia Lambdoid sutures prematurely closed, biparietal alopecia, corneal clouding

Go´mez–Lo´pez-Hernańdez syndrome Go´mez–Lo´pez-Hernańdez syndrome Go´mez–Lo´pez-Hernańdez syndrome

Hy***, SA, increased thickness anterior commissure prenatal ultrasound Hy** prenatal ultrasound Hy*** prenatal ultrasound Partially undifferentiated inferior olivary nucleus, abnormal accessory olivary nuclei according to later review by Gross (1959) Capillary hemangiomas left pretibial skin Preauricular appendage left, short fifth fingers Consanguinity parents Partial fusion right eyelids Bifid spine C1-C4,

Maternal insulin dependent diabetes mellitus

55

Cervicothoracic meningomyelocele, tethered spinal cord, dermal sinuses FD, atresia right external ear, asymmetric bilateral upper extremity anomalies

RHOMBENCEPHALOSYNAPSIS

Mendonça et al., 2004 (1)

(continued)

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Table 4.1 (Continued) Sex, age

Intracranial associations

Extracranial associations

Siebert et al., 2005

f, 29 weeks

Hy** lobar holoprosencephaly

Left arm: absent radius, thumb and first metacarpal, Fallot’s tetralogy

Silit et al., 2002 Simmons et al., 1993 (1)

m, 1.5 years f, 4.5 years

Simmons et al., 1993 (2)

m, 28 years

Simmons et al., 1993 (3) Toelle et al., 2002 (1) Toelle et al., 2002 (2) Toelle et al., 2002 (3) Toelle et al., 2002 (4)

m, 10 years m, 3.5 years m, 1.5 years f, 3 years m, 1.5 years

Hy** Hy***, right sylvian cortical dysplasia, fused thalami, small frontal encephalocele Hy***, left parietal ‘cleft’, diverticulum atrium right ventricle Hy***, SA

Toelle Toelle Toelle Toelle

f, 5 years m, 5 years f, 5 years m, 6 years

et et et et

al., al., al., al.,

2002 2002 2002 2002

(5) (6) (7) (8)

Toelle et al., 2002 (9) Truwit et al., 1991 (1) Truwit et al., 1991 (2)

m, 5.5 years f, 4.5 years m, 12 years

Truwit et al., 1991 (3)

m, 4.2 years

Utsonomiya et al., 1998(1)

m, 4 years

Utsonomiya et al., 1998 (2) Verri et al., 2000 Von Boltenstern et al., 1995

m, 3.2 years m, 22 years m, 2 years

Yachnis, 2002

f, 29 years autopsy

Hy*, SA Hy*

FD, asymmetric hand anomalies

Hy, SA Hy*** Hy***, SA Encephalocele occiput, subcortical and periventricular heterotopias Hy***, retrocerebellar cyst Hy**, SA, FF Hy**, SA, FF, hypoplastic temporal lobes Hy***, SA, FF, small chiasma opticum, hypoplasia temporal lobes Hy**, SA, hypoplasia ant commissure, FF Hy** – (by description only) Hy**, SA, FF, hypoplasia parahippocampal gyri Hy**, SA

Both neuropathological and MRI studies included. FD: facial dysmorphia; FF: fused fornices; g.a. gestational age; Hy: hydrocephalus:*mild,

**

moderate,

***

Prenatal factors

Maternal use of clonazepam and valproate

Radial aplasia, 13 ribs

FD, corneal anesthesia High palate

Interstitial deletion chromosome 2q

Scoliosis

advanced, the latter including treated patients; SA: absent septum pellucidum.

P. G. BARTH

Reference


4.2. The confines of rhombencephalosynapsis defined by neuropathology RS implies the total or partial absence of the cerebellar vermis, including its lobules, the fastigial nuclei, and efferent and afferent connections. The cerebellar hemispheres are fused across the midsagittal plane without any intervening cleft or visible margin. Macroscopically the transverse diameter of the cerebellum is reduced (Gross, 1959), Schachenmayr and Friede (1982) described a pear-shaped narrow structure, with the broad part posteriorly situated (Fig. 4.1). The dentate nuclei are seen as an almost single structure consisting of contributions by left and right hemispheres (Fig. 4.2). The study by Obersteiner (1916) reported an abnormal, medial course of the superior cerebellar peduncles with the closely apposed left and right peduncles overlying the aqueduct. This aberrant course also altered the course of the nearby trochlear nerves and mesencephalic trigeminal tracts, although these structures are spared. He also noticed that the nodulus and the uvula were partly spared. Gross (1959) studied two further autopsy cases, a baby girl of 8 weeks and a boy of 2 years, and restudied the material previously reported by Obersteiner (1916). Cerebellar findings were essentially the same in all three. The transverse diameter of the cerebellum was decreased. RS was subtotal with

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the nodulus in all cases preserved, while in the case of Obersteiner the uvula was preserved as well. The dorsal portions of the dentate nuclei appeared to be fused, or at least in close apposition (Fig. 4.1). Nuclear masses, probably representing the globose and emboliform nuclei, were present in the hili while the fastigial nuclei were absent. Small heterotopic collections containing Purkinje cells were found in the central cerebellar white matter in all three cases. The convexity view of the cerebellar hemispheres was essentially normal, with all the lobes discernible. (Interestingly the flocculi were present as well, implying that RS cannot be simply defined as an ‘archicerebellar’ defect.) All major sulci could be recognized, both on the dorsal and the ventral sides. Normally oriented folia from the hemispheres fused across the midline in the absence of vermal markings. The incisura cerebelli posterior, which normally marks the posterior end of the vermis, was absent. The anterior medullary velum was also absent. The superior cerebellar peduncles were apposed or fused over the aqueduct of Sylvius. The nuclei of the trochlear nerves were positioned lateral to the superior peduncles instead of medial and the trochlear nerves were seen traversing the aberrant superior peduncular bundles. The trigeminal spinal nuclei and tracts were missing in case 1 of Gross (1959), and present in case 2. Gross remarked

Fig. 4.1. Transverse (perpendicular to the fourth ventricle) sections of the cerebellum of a 1.5-year-old boy with rhombencephalosynapsis who died from the complications of hydrocephalus. Sections stained with Luxol fast blue. (A) Higher pontine level. The superior cerebellar peduncles are too close to the midline (arrow). Absence of superior medullary velum. (B) Level of the middle cerebellar peduncles. Close approximation of dentate nuclei. A few possible remnants of the vermian cortex are visible in the midline. Notice the abnormal shape of the fourth ventricle. (C) Level of the lateral foramina (slightly asymmetrical).

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Fig 4.2. Coronal T1-weighted MRI from 1-year-old patient with rhombencephalosynapsis (same patient as Fig. 4.1). Collapse of ventricular system because of drainage of hydrocephalus. Abnormal ‘pear-shaped’ cerebellum. Absence of posterior incisure marks absence of the vermis. (Left) Left–right continuity of cortical structure. (Right) Left–right continuity of white matter.

on an abnormality of the olivary complexes. The dorsal and medial accessory olivary nuclei were absent or undeveloped, while the lower limb of the inferior olivary nucleus was unfolded. A further case was described by Gross and Hoff (1959). From the description and photographs this case must be identical to the first case of Gross (1959). Schachenmayr and Friede (1982) observed absence of the dorsal accessory olivary nuclei, reduction of the medial accessory olivary nuclei and normal aspect of the inferior olivary nuclei. Kepes et al. (1969) described forking of the aqueduct and an undivided collicular plate. Schachenmayr and Friede (1982) also observed fusion of the left and right halves of the inferior tectum. Narrowing of the aqueduct and periaqueductal gliosis was described by Isaac and Best (1987).

4.3. The confines of rhombencephalosynapsis defined by neuroimaging Magnetic resonance imaging is vastly superior to any other means of imaging in its ability to show the diverse features of RS and to differentiate RS from other cerebellar anomalies (Patel and Barkovich, 2002; Adamsbaum et al., 2005). Standard images useful for discriminating sufficiently between gray and white matter in two or three planes are sufficient for making the diagnosis. For a precise diagnosis knowledge of the lobular organization of the cerebellar vermis, especially in midsagittal projection, will be helpful (Courchesne et al., 1989; Adamsbaum et al., 2005). The following observations are important for diagnosis by MR.

In midsagittal section: a. Cerebellar parenchyma takes the place of the velum medullare anterius (Fig. 4.3A) b. Absence of part or all vermal lobules outlined by their limiting sulci c. An abnormal shape of the fourth ventricle, often with a rounded rather than tent-shaped fourth ventricle; the normal contour of the nodule may be present or absent (Fig. 4.3A, B). The shape of the fourth ventricle in midsagittal section may, however, be normal, indicating presence of the nodule (Utsonomiya et al., 1998; Demaerel et al., 2004) d. An abnormal shape of the collicular plate e. Absent flow void in the aqueduct as sign of aqueductal stenosis in case of hydrocephalus. In transverse sections: a. Continuity of hemispheric folia across the midline (Fig. 4.2A) b. Continuity or apposition of the middle cerebellar peduncles in the midline c. Fusion or apposition of the dentate nuclei d. Replacement of the usual horseshoe shape of the fourth ventricle with the open side of the horseshoe pointing in the posterior direction by a slitlike space. The resulting shape is often referred to as ‘keyhole’ or ‘teardrop’ appearance. The appearance results from absence of the vermis. (This abnormality can be seen on CT as well) e. Absence of the posterior cerebellar incisure, which normally marks the inferior end of the vermis (Fig. 4.2A, B)


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Fig 4.3. Midsagittal sections from two different patients with rhombencephalosynapsis show variability. (A) T1-weighted. Same patient as Figure 4.2. Angulation and discontinuity of corpus callosum possibly due to collapse after ventricular drainage for hydrocephalus. Black asterisk marks absence of anterior medullary velum. Notice abnormal shape of the fourth ventricle and absence of nodule. (B) Inversion recovery. 1-month-old patient with congenital hydrocephalus and rhombencephalosynapsis. Less severe involvement than (A). Notice presence of nodule (white asterisk) but absence of normal vermal sulci. (B, courtesy of Prof Dr LS de Vries, Utrecht.)

f. Cerebellar hypoplasia, with diminution of volume especially pronounced in the lateral directions. Sometimes a pear shape is specifically mentioned (Scroop et al., 2000). This shape, however, is present in many published axial or coronal projections (Fig. 4.2A, B). In coronal sections: a. Fusion of the inferior colliculi b. Close approximation or fusion of the superior cerebellar peduncles. Cerebellar cortical abnormalities resulting in abnormal orientation of folia, without other findings suggestive of RS, may pose differential diagnostic problems (Soto et al., 2004). A case with vertically oriented folia-like structures, partial fusion and multifocal cystic lesions was reported by Takanashi et al. (1999).

4.4. Associated intracranial findings 4.4.1. Hydrocephalus Hydrocephalus is the most common abnormality found in association with RS. In 58 reported patients the size of the lateral ventricles was increased in 39 (excluding a case of holoprosencephaly). Table 4.1 provides an overview. In 14 patients surgery, mostly ventricular shunting, was performed to treat hydrocephalus (Schachenmayr and Friede, 1982; Truwit et al., 1991 (one of

three cases); Simmons et al., 1993 (three cases); Von Boltenstern et al., 1995; Utsonomiya et al., 1998; Danon et al., 2000; Guyot et al., 2000; Oei et al., 2001; Toelle et al., 2002 (three cases); Yachnis, 2002). No followup data are available on the results of shunting. On the basis of available reports it is not possible to draw a clear line between mild dilatation, ventriculomegaly and ventricular dilatation due to obstructive hydrocephalus. Taking account of the number of surgical procedures, the minimum rate of obstructive hydrocephalus is about 25%. Hydrocephalus appears to be caused primarily by aqueductal stenosis. Specific mention of aqueductal forking and/or periaqueductal gliosis is made in some reports (Isaac and Best, 1987 (case 2); Kepes et al., 1969; Yachnis, 2002). A patent aqueduct was found by Gross (1959) (case 2). Absence of the aqueductal flow void on MRI indicating obstruction was reported by Truwit et al. (1991) (case 3) and Utsunomiya et al. (1998) (case 2). Other possible sites of obstruction included midline fusion of the thalami (Simmons et al, 1993; Kepes et al., 1969) and obstruction of the fourth ventricle by cerebellar tissue (Yachnis (2002). A trapped fourth ventricle was reported twice by Sener (2000), suggesting obstruction of the exits of the fourth ventricle as a possible mechanism of CSF flow obstruction. Congenital hydrocephalus is indeed the presenting problem in several reports (Schachenmayr and Friede, 1982; Simmons et al., 1993; De Jong and Kirby, 2000; Toelle et al., 2002 (cases 6,7)). Intrauterine diagnosis of hydrocephalus associated with RS by ultrasound

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could be made as early as the 23rd week of pregnancy (Napolitano et al., 2004). From these data it appears that ventricular dilatation is common in patients with RS and that, in a significant proportion, it is due to obstructed CSF flow and is not uncommonly present in the young fetus. 4.4.2. Infratentorial malformations associated with RS Gross (1959) observed decreased undulation of the ventral limb of the inferior olivary nucleus, suggesting lack of fetal differentiation, in two cases and, on restudying of the earlier material reported by Obersteiner (1916), in his case also. This author further observed hypoplastic development of the medioventral and dorsal accessory olivary nuclei in all three cases. Hypoplasia of the inferior olives detected by MR was reported by Demaerel et al. (1995). A patient described by Sener and Dzelzite (2003) had an operated cervicothoracic meningomyelocele and Chiari II malformation besides RS. A photographic series by Sener (2000) illustrates two cases of trapped fourth ventricle. 4.4.3. Aplasia of the septum pellucidum The most frequent association after hydrocephalus is absence/ aplasia of the septum pellucidum (SA). The cause of septal deficiency is equivocal in the case of obstructive hydrocephalus because severe hydrocephalus may cause septal disruption. However, SA in RS is also observed three times, together with mild ventricular dilatation (Table 4.1). Fusion of the fornices is specifically mentioned eight times (Table 4.1). Fusion of the fornices cannot be explained as a secondary phenomenon and may be regarded as evidence of a true midline defect. Septo-optic dysplasia was described for the first time by De Morsier (1956) as an entity in which septal aplasia combines with an embryonic disturbance affecting the chiasm, optic nerves and tracts. Septo-optic dysplasia was also found in the case of RS reported by Michaud et al. (1982), combined with hypoplasia of the optic nerves, chiasm and optic tracts, moderate hydrocephalus and agenesis of the posterior lobe of the pituitary. The increased incidence of septal aplasia, including fused fornices and a case of septo-optic dysplasia, suggests a link between RS and supratentorial midline malformations. 4.4.4. Hippocampal abnormalities Parahippocampal hypoplasia, in combination with septal aplasia, was reported by Von Boltenstern et al.

(1995). A similar MRI, reported as temporal hypoplasia, was reported by Montull et al. (2000) and Truwit et al. (2001) (case 1). 4.4.5. Cerebral commissures Dysgenesis of the corpus callosum was reported by Demaerel et al. (1995), together with septal aplasia. Abnormalities of the anterior commissure were described occasionally, including both hypoplasia (Truwit et al., 1991) and hyperplasia (Montull et al., 2000). Several patients had a dorsal ‘kink’ in the corpus callosum (Simmons et al., 1993), regarded as the result of drainage of hydrocephalus (Fig. 4.3A). However these authors also reported hypogenesis of the posterior parts of the corpus callosum. 4.4.6. Holoprosencephaly Garfinkle (1996) reported an 11-year-old girl combining RS with complete absence of ventricles, sylvian fissures and precentral and central sulci. There was cortical dysplasia suggestive of polymicrogyria and fusion of the thalami. ‘Telencephalosynapsis’ was described in the case of a male fetus with frontal hypoplasia, fusion of the hemispheres, fusion of the basal ganglia and absence of the lateral ventricles together with RS and a Dandy–Walker-like malformation (Sergi et al., 1997). Lobar holoprosencephaly, RS and left upper limb reduction defects were reported in a female fetus as part of a series with limb reduction defects and holoprosencephaly (Siebert et al., 2005). Manifest maternal diabetes mellitus was involved in the cases of Garfinkle (1996) and Sergi et al. (1997). 4.4.7. Neural tube defects A patient described by Sener and Dzelzite (2003) had an operated cervicothoracic meningomyelocele and Chiari II malformation besides RS. One of the patients of Toelle et al. (2002) had a small occipital encephalocele. Schachenmayr’s second patient possibly had a parietal encephalocele (Schachenmayr and Friede, 1982). 4.4.8. Neocortical dysplasia Only one report mentions periventricular neuronal heterotopia together with a small ‘occipital’ encephalocele (Toelle et al., 2002, patient 8). Patient 1 of Simmons et al. (1993) had a one-sided parietal cleft in the cortex, not in contact with the ventricle, covered by cortical tissue and reaching over the midline.


4.5. Neurological and psychological findings in rhombencephalosynapsis In most cases of RS the impact of the vermal abnormalities is probably or definitely obscured by such compounding factors as gross hydrocephalus and other variable associated malformations. Two cases had chromosomal anomalies (Truwit et al., 1991, case 2; Lespinasse et al., 2004) and their mental deficiency may have been due to contiguous gene defects. Therefore only a limited sample of all reported cases has uncomplicated RS with neurological findings described in sufficient detail. In the series of nine by Toelle et al. (2002), mild truncal ataxia was the most frequent neurological symptom (four out of nine), nystagmus was reported in two patients and strabismus in three. More severe impairments including pyramidal and extrapyramidal symptoms were present in three. Cognitive development was normal in four patients. In the series of Truwit et al. (1991), a 4.5-year-old girl had normal memory and vocabulary and balance problems, but these may have been compounded by sensory problems, including corneal insensitivity and clouding. The 6-year-old patient of Demaerel et al. (1995) was floppy at 8 weeks, could sit with support at 13 months, and walked independently at 22 months. He was referred for head rolling and on neurological examination had poor balance, poor hand coordination, no dysmetria, abnormal vertical eye movements and gaze paretic nystagmus with saccadic pursuit. Speech was normal, although reading was delayed. A 39-year old woman (a secretary) reported by Guyot et al. (2000) manifested a subtle abnormality of tandem gait. One of two patients reported by Mendonça et al. (2004), a woman of 20 years, was referred for MRI having psychiatric complaints. She was said to have normal intelligence (there was no formal testing) and was normal on physical neurological examination. A 39-year-old woman reported by Montull et al. (2000), had an IQ of 68 (WAIS), a squint, a broadbased gait with astasia–abasia, negative Romberg, dysmetria and dysdiadochokinesis. A man with normal intelligence was reported by Bell et al. (2005). This man, who was tested because of mood disturbance, had a broad range of neuropsychological tests. His overall performance IQ was 86, and verbal IQ 94. Only mild gait disturbance was found on neurological testing. Available evidence indicates ataxia as the major consequence in a limited number of studies. Nystagmus and strabismus are reported occasionally. In view of the affection of the vermis, further details on the impact on eye movements and vestibular studies would be interesting, but are lacking to date.

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4.6. Etiology 4.6.1. Prenatal exposure Prescription drugs used in the first trimester of pregnancy are mentioned in several reports. Phencyclidine was reported by Michaud et al. (1982). Antiepileptic drugs were reported twice: ethosuximide by Demaerel et al. (1995) and combined clonazepam and valproate by Toelle et al. (2002). Juvenile-onset diabetes mellitus (Garfinkle, 1996) and insulin-dependent diabetes mellitus (Sergi et al., 1997) during pregnancy were reported twice. The associated pathology in both cases was an holoprosencephaly variant with absent lateral ventricles, besides RS. 4.6.2. Chromosomal anomalies An unbalanced subtelomeric translocation t (2p;10q) was found by Lespinasse et al. (2004). Antenatally, the female fetus was observed to have increased nuchal translucency and a slightly hypoplastic cerebellum. A standard karyotype was normal. Rhombencephalosynapsis and a submicroscopic unbalanced subtelomeric translocation t(2p; 10q) were demonstrated after birth. In patient 2 described by Truwit et al. (1991) an interstitial deletion of chromosome 2q was found. The karyogram was not described in detail.

4.7. Inherited disorders No recurrence has been described in any of the reported cases. Parental consanguinity was reported only once (Romanengo et al., 1997). A well delineated syndromal association has become known as cerebello-trigeminal-dermal dysplasia or Go´mez–Lo´pezHernańdez syndrome. 4.7.1. A syndromal association: Go´mez–Lo´pez-Hernańdez syndrome (MIM 601853) A girl with brachycephaly, anesthesia in the distribution of the sensory trigeminal nerve causing keratitis, weakness of masseter muscles, bandlike alopecia on the parietal and occipital skin was reported in 1979 by Go´mez. Beside this, the patient was mentally retarded, had positive Babinski reflexes and ataxia. The author pointed out that the association with bandlike alopecia and facial anesthesia could be explained by abnormal development of the placode that gives rise to the semilunar (gasserian) ganglion, as this placode has a dual origin from the embryonic epidermis and neural crest tissue. Similarly, Go´mez sought to explain the

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ataxia from developmental failure of the cerebellum, which arises at the same level, although no radiological evidence was available for his patient. Two similar unrelated female patients were described by Lo´pezHernańdez (1982), who was apparently unaware of the previous paper. The description has the following features:

Towerlike skull with absent sutures on skull X-rays, midface hypoplasia Bilateral parietal areas of alopecia Low-set posterior rotated ears Hoarse voice Hypoplasia of the labia majora Fifth fingers with clinodactyly (toe koilonychia in one) Short stature Mental and motor delay Trigeminal facial anesthesia with absent corneal reflexes and keratitis associated with automutilation leading to blindness in one.

Computed tomography of the brain showed enlargement of the lateral and third ventricles in one and normal ventricular width in the other. The cerebellar hemispheres appeared underdeveloped, and pons and cerebellum could not be separated, both appearing to form part of a single mass. Hence the anomaly was characterized as pons–vermis fusion anomaly (atresia of the fourth ventricle). A further case described as ‘displasia cerebelotrigeminal’ was contributed by I. Pascual-Castroviejo in 1983, cited by Verońica Munõz et al. (1997). Munõz et al. (1997) described another three children, two girls and a boy, with external and neurological features strikingly similar to those previously described. All three had a characteristic turricephaly with lambdoid sutural stenosis and abnormally thickened occipital bones. Short stature was present in each. All had trigeminal anesthesia and two had corneal clouding. Latency of the blink reflex was tested in two and found to be increased in each. All three had delayed motor development and ataxia. Two of the three also had mental retardation; one was described as bright. RS was diagnosed in each by MRI. There was overt ventricular dilatation in one and a normal sized supratentorial ventricular system was reported in the other two (Fig. 4.4). More MRI findings on the same patients were detailed in a further paper by Munõz et al. (2004), including the finding of supracollicular lipoma in one. Brocks et al. (2000) contributed the oldest patient in whom the diagnosis has been made: 19 years, including MRI confirmation of RS. This man had bilateral lambdoid sutural stenosis, for which surgery was undertaken.

Fig 4.4. Go´mez–Lo´pez-Hernańdez syndrome. 8-year-old girl with turricephaly, bilateral alopecia (not shown here) and anesthesia in the distribution of the sensory trigeminal nerve. The scars on her forehead and corneal clouding on the right are due to self-inflicted injury. The patient had rhombencephalosynapsis confirmed by MRI. Details previously described (Munõz et al., 1997 (patient 1); Munõz et al., 2004).

Summarizing the data on patients reported in the English literature, the following findings appear typical for the syndrome: a. Craniosynostosis affecting the lambdoid and possibly other sutures, causing a brachycephalic external aspect b. Trigeminal anesthesia, provoking automutilating behavior, including corneal lesions c. Rhombencephalosynapsis. Supratentorial involvement may entail widened ventricles, and abnormal hippocampal development, as illustrated in Munõz et al., 1997 d. Circumscribed bilateral parietal or temporal alopecia. Skin biopsies in these regions, first reported by Lo´pez-Hernańdez (1982), showed underdeveloped hair follicles and sebaceous glands with either normal or underdeveloped hair shafts (Munõz et al., 1997, 2004) e. Clinical neurological findings include mental retardation with delayed speech, behavioral abnormalities and ataxia. In retrospect at least one neuropathological case described by Gross (1959) is of particular interest versus Go´mez–Lo´pez-Hernańdez syndrome. His patient 1, a girl who died 5 weeks after term birth, had brachycephaly and thickened occipital bone both on palpation and

RHOMBENCEPHALOSYNAPSIS skull X-ray, purulent keratitis and a bilateral streak of absent hair on both sides of the skull. Among the other RS reports, case 2 of Gross (1959) had turricephaly, absent hair on the temples and asymmetric peripheral palsies. No mention is made of corneal insensitivity. Case 1 of Truwit et al. (1991) had corneal anesthesia but no reference is made to craniostenosis or localized alopecia. While the etiology of case 1 of Truwit and case 2 of Gross remain enigmatic, the combined features of the first case of Gross (1959) indicate that she must have been the first documented example of Go´mez–Lo´pezHernańdez syndrome. It constitutes the first report of this syndrome with a detailed neuropathological examination. The neuropathological findings do not differ in essence from other reports with respect to the cerebellum. Gross observed, however, that the spinal trigeminal nucleus and its tract were absent. The mesencephalic trigeminal root was present; the mesencephalic trigeminal nucleus is omitted from description. This observation was not made in his second case, nor in his restudy of the first case of Obersteiner (1916). No description is available on the trigeminal ganglion. It may be recalled that the sensory trigeminal fibers in the brainstem have a dual embryonic origin. The afferent spinal tract fibers have their first synapse in the semilunar ganglion and are postganglionic while the mesencephalic fibers only pass through the trigeminal ganglion and have their first synapse in the mesencephalic trigeminal nucleus. It may also be remembered that the semilunar ganglion of the trigeminal nerve has a dual origin, which is in part the Gross (1959) may be significant and supports the ‘placodal’ theory formulated by Go´mez (1979). No familial cases and no parental consanguinity are on record, and none of the parents has been described as harboring mild features of the disease. This leaves the mode of inheritance unsettled for the moment.

4.8. Consideration of genetic factors There is no animal model expressing an RS-like phenotype. In humans the phenotype is remarkably different from other cerebellar midline anomalies such as molar tooth disorders (including Joubert syndrome) and Dandy–Walker malformation (Patel and Barkovich, 2002; Adamsbaum et al., 2005). In all these disorders the vermis is reduced in mass or completely absent, causing a cleft between the cerebellar hemispheres. In RS the cerebellar hemispheres seem to ‘ignore’ the missing vermis. They fuse seamlessly, restoring transverse continuity. When considering a mechanism that might explain this, it is useful to recall that in RS the transverse diameter of the cerebellum is narrower than one would expect from absence of the

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vermis only (e.g. the well illustrated case of Schachenmayr and Friede (1982)). Hypoplasia of the cerebellar hemispheres was also reported by Sener (2000). The lack of transverse differentiation/growth may affect the cerebellum over its complete length or with an anteroposterior gradient, in the latter situation giving the cerebellum a pear shape, with the smallest diameter at the anterior end (see also Fig. 4.2A, B). This suggests some involvement of the cerebellar hemispheres in the pathogenesis of RS. An organization of the cerebellum in sagittal strips or modules is present from an early stage, linking, for example, the paramedian cerebellar cortex, fastigial nucleus and parts of the ipsilateral olivary nuclei in a single functional sagittal unit. Parallel longitudinal strips of slices that comprise cerebellar cortex, central nuclei and olivary nuclei alternately express positive and negative responses to various immunostains (reviewed by Voogd and Glickstein, 1998). This medio-lateral organization in longitudinal strips of various lengths similarly reflects the embryonic spatial organization of the murine analogs of Drosophila segment polarity genes (Millen et al., 1995; Herrup and Kuemerle, 1997). These sagittal compartments form modules that alternately express or skip expression of developmental genes, including en1, en2, wnt-7B, and PAX-2. By observing alternate expression patterns, six distinct bilateral domains across the cerebellum (extending also to the midbrain) and one shared midline band were found. However, no knock-out study of any of these genes has resulted in a phenotype exhibiting restricted growth/ development in a pure parasagittal pattern resembling RS. It should be mentioned here that the same developmental genes also have spatially restricted expression imparted by the organization in rhombomeres (Lumsden, 1990; Lumsden and Krumlauf, 1996). It may be opportune to briefly discuss here what can be learned from Go´mez–Lo´pez-Hernańdez syndrome. In this syndrome there is an association between RS and developmental failure of the trigeminal placodes. The cerebellum develops within rhombomere 1 (Wingate and Hatten, 1999). The rhombic lips that line the anterior half of the roof plate provide the neurons destined for cerebellum, inferior olivary nucleus and lateral pontine nuclei. The trigeminal nerve and nucleus originate in part from a neurogenic placode developing in the ectoderm close to rhombomere 2. The other part develops from the cranial neural crest, which derives from the anterior part of the neural tube at the time of its closure (Lumsden et al., 1991). Spatial proximity between the trigeminal placodes, cranial neural crest and rhombic lips may not be the answer to the coincidence of placodal and cerebellar anomalies. Shared expression of developmental

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genes may be more relevant. A number of transcription factors have been associated with development of the trigeminal placode in different vertebrate species, including genes with shared expression in placodes, neural crest and neural tube (see Baker and Bronner-Fraser, 2001 for review). The homeobox genes tlx and HOX11 provide an example that may be relevant (Logan et al., 1998). tlx-1 and tlx-3 are both expressed in placode-derived cranial neurons, including the trigeminal placodes, and the hindbrain in chicken. tlx-3 is also expressed early in a streak-like longitudinal domain within the hindbrain that includes rhombomere 1, the territory of the developing cerebellum. This, and other genes that are expressed early in both the trigeminal placodes and the cerebellar primordium, may help to focus on potential candidate genes for Go´mez–Lo´pez-Hernańdez syndrome – and may be relevant for nonsyndromic RS as well. The presence of septal aplasia in a large proportion of patients with RS, including a unique case with septo-optic dysplasia (Michaud et al., 1982) raises the possibility of a common genetic background. The only gene associated with septo-optic dysplasia in man and mouse until now is HESX1. The expression of the gene is restricted to the forebrain and mouse mutants lacking the normal gene have no malformation caudal to the forebrain (Dattani et al., 1998).

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De Jong G, Kirby PA (2000). Defects of blastogenesis: counseling dilemmas in two families. Am J Med Genet 91: 175–179. De Morsier G (1955). Etudes sur les dysraphies cranioence´phaliques: II: Ageńe´sie du vermis ce´re´belleux. Dysraphies rhomboence´phalique me´diane (rhomboschizis). Monatsschr Psychiatr Neurol 129: 321–334. De Morsier G (1956). Etudes sur les dysraphies cranioence´phaliques: III. Ageńe´sie du septum lucidum avec malformation du tracturs optique. La dysplasie septo-optique. Schweiz Arch Neurol Psychiatr 77: 269–292. Demaerel P, Kendall BE, Wilms G, et al. (1995). Uncommon posterior cranial fossa anomalies: MRI with clinical correlation. Neuroradiology 37: 72–76. Demaerel P, Morel C, Lagae L, et al. (2004). Partial rhombencephalosynapsis. Am J Neuroradiol 25: 29–31. Garfinkle WB (1996). Aventriculy: a new entity? Am J Neuroradiol 17: 1649–1650. Go´mez MR (1979). Cerebellotrigeminal and focal dermal dysplasia: a newly recognized neurocutaneous syndrome. Brain Dev 1: 253–256. Gross H (1959). Die Rhombencephalosynapsis, eine systemisierte Kleinhimfehlbildung. Arch Psychiatr Nervenkr 199: 537–552. Gross H, Hoff H (1959). Sur les dysraphies cranio-ence´phaliques. In: G Heuyer, M Feld, J Gruner (Eds.), Malformations Congeńitales du Cerveau. Masson, Paris, pp. 287–296. Guyot LL, Kazmierczak CD, Michael DB (2000). Adult rhombencephalosynapsis. Case report. J Neurosurg 93: 323–325. Herrup K, Kuemerle B (1997). The compartmentalization of the cerebellum. Annu Rev Neurosci 20: 61–90. Isaac M, Best P (1987). Two cases of agenesis of the vermis of cerebellum, with fusion of the dentate nuclei and cerebellar hemispheres. Acta Neuropathol (Berl) 74: 278–280. Kepes JJ, Clough C, Villanueva A (1969). Congenital fusion of the thalami (atresia of the third ventricle) and associated abnormalities in a 6-month-old infant. Acta Neuropathol (Berl) 13: 97–104. Lespinasse J, Testard H, Nugues F, et al. (2004). A submicroscopic unbalanced subtelomeric translocation t(2p;10q) identified by fluorescence in situ hybridization: fetus with increased nuchal translucency and normal standard karyotype with later growth and developmental delay, rhombencephalosynapsis (RES). Ann Genet 47: 405–417. Litherland J, Ludlam A, Thomas N (1993). Antenatal ultrasound diagnosis of cerebellar vermian agenesis in a case of rhombencephalosynapsis. J Clin Ultrasound 21: 636–638. Logan C, Wingate RJ, McKay IJ, et al. (1998). Tlx-1 and Tlx-3 homeobox gene expression in cranial sensory ganglia and hindbrain of the chick embryo: markers of patterned connectivity. J Neurosci 18: 5389–5402. Lo´pez-Hernańdez A (1982). Craniosynostosis, ataxia, trigeminal anaesthesia and parietal alopecia with pons– vermis fusion anomaly (atresia of the fourth ventricle). Report of two cases. Neuropediatrics 13: 99–102. Lumsden A (1990). The cellular basis of segmentation in the developing hindbrain. Trends Neurosci 13: 329–335.

RHOMBENCEPHALOSYNAPSIS Lumsden A, Krumlauf R (1996). Patterning the vertebrate neuraxis. Science 274: 1109–1115. Lumsden A, Sprawson N, Graham A (1991). Segmental origin and migration of neural crest cells in the hindbrain region of the chick embryo. Development 113: 1281–1291. Mendonça JL, Natal MR, Viana SL, et al. (2004). Rhombencephalosynapsis: CT and MRI findings. Neurol India 52: 118–120. Michaud J, Mizrahi EM, Urich H (1982). Agenesis of the vermis with fusion of the cerebellar hemispheres, septooptic dysplasia and associated anomalies. Report of a case. Acta Neuropathol (Berl) 56: 161–166. Millen KJ, Hui CC, Joyner AL (1995). A role for En-2 and other murine homologues of Drosophila segment polarity genes in regulating positional information in the developing cerebellum. Development 121: 3935–3945. Montull C, Mercader JM, Peri J, et al. (2000). Neuroradiological and clinical findings in rhombencephalosynapsis. Neuroradiology 42: 272–274. Munõz RMV, Dos Santos AC, De Pina Neto JM (2004). Cerebello-trigemino-dermal dysplasia. In: ES Roach, VS Miller (Eds.), Neurocutaneous Disorders. Cambridge University Press, Cambridge, pp. 306–312. Munõz RMV, Santos AC, Graziadio C, Pina-Neto M (1997). Cerebello-trigeminal-dermal dysplasia (Go´mez–Lo´pezHernańdez syndrome): description of three new cases and review. Am J Med Genet 72: 34–39. Napolitano M, Righini A, Zirpoli S, et al. (2004). Prenatal magnetic resonance imaging of rhombencephalosynapsis and associated brain anomalies: report of 3 cases. J Comput Assist Tomogr 28: 762–765. Obersteiner H (1916). Ein Kleinhirn ohne Wurm. Arbeit Neurol Inst Univ Wien 21: 124–136. Odemis E, Çakir M, Aynaci FM (2003). Rhombencephalosynapsis associated with cutaneous pretibial hemangioma in an infant. J Child Neurol 18: 225–228. Oei AS, Vanzieleghem BD, Kunnen MF (2001). Diagnostic imaging and clinical findings in rhombencephalosynapsis: case report and literature review. JBR-BTR 84: 197–200. Patel S, Barkovich AJ (2002). Analysis and classification of cerebellar malformations. Am J Neuroradiol 23: 1074– 1087. Romanengo M, Tortori-Donati P, Di Rocco M (1997). Rhombencephalosynapsis with facial anomalies and probable autosomal recessive inheritance: a case report. Clin Genet 52: 184–186. Savolaine ER, Fadell RJ, Patel YP (1991). Isolated rhombencephalosynapsis diagnosed by magnetic resonance imaging. Clin Imaging 15: 125–129. Schachenmayr W, Friede RL (1982). Rhombencephalosynapsis: a Viennese malformation? Dev Med Child Neurol 24: 178– 182.

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Scroop R, Sage M, Voyvodic F (2000). Rhombencephalosynapsis. Australas Radiol 44 (2): 225–227. Sener RN (2000). Unusual MRI findings in rhombencephalosynapsis. Comput Med Imaging Graph 24: 277–282. Sener RN, Dzelzite S (2003). Rhombencephalosynapsis and a Chiari II malformation. J Comput Assist Tomogr 27: 257–259. Sergi C, Hentze S, Sohn C, et al. (1997). Telencephalosynapsis (synencephaly) and rhombencephalosynapsis with posterior fossa ventriculocele (‘Dandy–Walker cyst’): an unusual aberrant syngenetic complex. Brain Dev 19: 426–432. Siebert JR, Schoenecker KA, Resta RG, et al. (2005). Holoprosencephaly and limb reduction defects: a consideration of Steinfeld syndrome and related conditions. Am J Med Genet A 134: 381–392. Silit E, Mutlu H, Ozturk T (2002). A rare cerebellar malformation: rhombencephalosynapsis. J Neuroradiol 29: 208–210. Simmons G, Damiano TR, Truwit CL (1993). MRI and clinical findings in rhombencephalosynapsis. J Comput Assist Tomogr 17: 211–214. Soto AG, Deries B, Delmaire C, et al. (2004). Cerebellar cortical dysplasia: MRI aspects and significance. J Radiol 85: 729–740. Takanashi J, Sugita K, Barkovich AJ, et al. (1999). Partial midline fusion of the cerebellar hemispheres with vertical folia: a new cerebellar malformation? Am J Neuroradiol 20: 1151–1153. Toelle SP, Yalcinkaya C, Kocer N, et al. (2002). Rhombencephalosynapsis: clinical findings and neuroimaging in 9 children. Neuropediatrics 33: 209–214. Truwit CL, Barkovich AJ, Shanahan R, et al. (1991). MR imaging of rhombencephalosynapsis: report of three cases and review of the literature. Am J Neuroradiol 12: 957–965. Utsunomiya H, Takano K, Ogasawara T, et al. (1998). Rhombencephalosynapsis: cerebellar embryogenesis. Am J Neuroradiol 19: 547–549. Verri A, Uggetti C, Vallero E, et al. (2000). Oral self-mutilation in a patient with rhombencephalosynapsis. J Intellect Disabil Res 44: 86–90. Von Boltenstern M, Konrad A, Jest W, et al. (1995). Rhombenzephalosynnpsis. Rofo Fortschr Geb Ro¨ntgenstr Neuen Bildgeb Verfahr 163: 91–93. Voogd J, Glickstein M (1998). The anatomy of the cerebellum. Trends Neurosci 21: 370–375. Wingate RJ, Hatten ME (1999). The role of the rhombic lip in avian cerebellum development. Development 126: 4395–4404. Yachnis AT (2002). Rhombencephalosynapsis with massive hydrocephalus: case report and pathogenetic considerations. Acta Neuropathol (Berl) 103: 301–304.



Embryology and malformations of the forebrain commissures HARVEY B. SARNAT* University of Calgary Faculty of Medicine and Alberta Children’s Hospital, Calgary, Alberta, Canada

5.1. Introduction and definition Agenesis of the corpus callosum is the most common major cerebral malformation that is neither lethal nor produces major neurological disabilities in all cases. It has been recognized for more than a century (Bruce, 1889). Callosal agenesis is traditionally classified as a ‘midline defect’ of the brain, but this interpretation is only partially true. A more correct embryological classification based upon pathogenesis is that it is a disturbance of axonal pathfinding in the developing brain. The midline defect is the failure to develop a bridge over which callosal axons may cross to project to the other hemisphere. A similar defect in a genetic murine model is due to failure of apoptosis of a midline glial septal wall just before the arrival of the first commissural axons (Zaki, 1985). Whether this same mechanism occurs in the human condition remains speculative, but it is a plausible explanation well demonstrated in another mammalian species. The corpus callosum is a commissure. Commissures differ from decussations because they interconnect corresponding structures in the two halves of the brain and their fibers neither ascend nor descend in the longitudinal axis of the neural tube. Decussations, by contrast, are a midline crossing of longitudinal projections that do not reciprocally connect similar structures but rather ascend or descend to project to different structures of the brain or spinal cord. The principal commissures of the human brain are: a) the corpus callosum, which projects axons from a widespread area of cerebral neocortex to the corresponding regions of the other hemisphere; b) the anterior commissure, which interconnects the rostral temporal lobes; c) the hippocampal commissure, or psalterium; d) the tectal commissures,

which interconnect the left and right superior colliculi of the rostral midbrain and inferior colliculi of the caudal midbrain; e) the posterior commissure, which is really the rostral end of the medial longitudinal fasciculus with contributions of a few additional fibers from the diencephalon, which cross the midline; the posterior commissure is only partly commissural, because some projections are to different brainstem nuclei on the side opposite their origin. Some brainstem and spinal cord axonal bundles that cross the midline resemble commissures but are really decussations: the trapezoid body of the auditory system; the decussation of the corticospinal tract at the caudal end of the medulla oblongata; the axons that cross between the ventral funiculi of the spinal cord, ventral to the central canal. Two commissures interconnect the cerebral neocortex, a large dorsal commissure, the corpus callosum, and a smaller ventral commissure, the anterior commissure (Fig. 5.1). No other direct connections exist between corresponding regions of neocortex on the two sides, though some multisynaptic circuits may find their way across the midline by involving descending axons to the thalamus and upper brainstem and, after synaptic relay, ascend to the cortex on the opposite side, particularly as part of the reticular formation (Sarnat, 2004a).

5.2. Phylogeny of commissures Bilateral symmetry as a body plan for most animals is an ancient development that began in evolution in the pre-Cambrian period; the oldest known fossils to show this body architecture is Vernanimalcula, a small invertebrate that lived in the sea some 580–600 million years ago (Bottjer, 2005). Commissural fibers are prominent in the brains of both invertebrates and vertebrates, and even occur in the brain of the planarian

*Correspondence to: H. B. Sarnat MD, FRCPC, Professor of Paediatrics, Pathology (Neuropathology) and Clinical Neurosciences, Alberta Children’s Hospital, 2888 Shaganappi Trail NW, Calgary, Alberta T3B 6A8, Canada., E-mail: [email protected], Tel: þ1-403-955-7131, Fax: þ1-403-955-2922.

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Fig. 5.1. (A) Neuropathological section of normal mature human brain in coronal plane at level of head of caudate nucleus. A large corpus callosum (cc) is seen as a horizontal axonal bundle dorsal to the lateral ventricles. The smaller anterior commissure (ac) is seen ventral to the anterior limb of the internal capsule as another horizontal commissure. On the left, the anterior commissure is divided into two bundles, a minor normal variation, and the actual crossing of fibers at the midline is rostral to the plane of this section. A thin layer of gray matter, the indusium griseum, lies at the dorsal surface of the corpus callosum, and the large cingulate gyrus is above it, in the medial wall of the cerebral hemispheres. (B) Mature human brain with agenesis of the corpus callosum as an isolated anomaly, sectioned at a level similar to that of (A). The corpus callosum is absent but the anterior commissure is large and well formed in its normal ventral position. The lateral ventricles are displaced laterally by the large bundle of Probst (bP), an abnormal fasciculus that courses rostrocaudally in the dorsomedial wall of the hemisphere and contains aberrant callosal axons that were unable to cross the midline at 74 days gestation. The hippocampus is not included in the anterior temporal lobes of these sections because it is more caudally situated, although its most rostral end is seen on the left in (B).

(flatworm), a simple invertebrate that evolved before the divergence of advanced invertebrates and vertebrates and is a candidate ancestor of the vertebrate central nervous system (Sarnat and Netsky, 1985, 2002). Among vertebrates, the corpus callosum is a structure only of the brains of placental mammals; marsupials, monotremes, birds, reptiles, amphibians, fishes, cyclostomes and protochordates all lack a corpus callosum (Sarnat and Netsky, 1981) (Fig. 5.2).

5.3. Embryology of the corpus callosum Ontogenesis is the basis for understanding all developmental malformations, and agenesis of the corpus callosum is no exception. For this reason, it is useful to briefly review the embryological formation of the human corpus callosum. The neural placode of embryonic vertebrates, beginning at the time of the primitive node of Hensen and primitive streak, also exhibits bilateral symmetry as it forms the neural tube. With the further development of the central nervous system, some axonal projections connect the two mirror-image halves of the brain to form commissures. In the human brain, the corpus callosum is the largest of these commissures.

A commissural plate differentiates within the dorsal third of the lamina terminalis at about 39 days gestation. This plate consists of glioblasts and serves as a passive bed for axonal passage, growing rapidly after axons begin to traverse it. The interhemispheric projection of the growth cones of these first axons is preceded by a precisely timed microcystic degeneration in the cortical plate deep in the interhemispheric fissure and death of astrocytes within this plate, probably a physiological process of apoptosis (Loeser and Alvord, 1968; Zaki, 1985). This event is well demonstrated in the mouse (Zaki, 1985) and is very likely in humans as well, though not as well documented. Subependymal primitive glial cells at the medial angles of the lateral ventricles proliferate and migrate to assist the passage of pioneering callosal axons (Zaki, 1985). At the midline and just rostral to the lamina terminalis, these cells unite to form a bridgelike ‘sling’ suspended below the interhemispheric fissure. The pioneering callosal axons grow along the surface of this cellular bridge as the cross to the other hemisphere, and the sling disappears after the corpus callosum is formed (Silver et al., 1982; Sarnat, 1992). Other mechanisms of axonal pathfinding, including attractive and repulsive

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Fig. 5.2. Coronal section of the normal brain of an opossum, a marsupial mammal. The hippocampi are dorsal structures, as in the rodent brain. Unlike placental mammals, marsupials lack a corpus callosum but possess a large anterior commissure (not seen in this plane of section) that presumably functions as a corpus callosum as well.

molecules in the extracellular matrix, also play a role in guiding axonal growth cones of developing callosal fibers and may be defective in callosal agenesis (Shen et al., 2002; Shu et al., 2003; Richards et al., 2004). In a similar regard, the homeodomain protein Vax1 may play a major role in the development of the corpus callosum by regulating the glial guidance of axonal growth cones across the callosal plate (Bertuzzi et al., 1999; Richards et al., 2004). Axons for the corpus callosum and anterior commissure are generated from the small pyramidal neurons of layer 3 of the mature cortex, by contrast with the corticobulbar and corticospinal tracts, which emanate from the large pyramidal neurons of layers 5 and 6. The corpus callosum involves axons from a widespread area of cerebral neocortex that includes frontal, parietal, temporal and occipital lobes, whereas the anterior commissure has fibers mainly from the anterior temporal lobes only. The first axons cross that callosal plate at 74 days (10.5 weeks) gestation, passing anterior and inferior to the foramen of Monro. These pioneer axons cross about 3 weeks later than the first axons of the anterior commissure, which cross at 54 days (8 weeks) gestation. In both cases, these axons are projected from the unlaminated cortical plate, which is not yet even complete with its full complement of radial migratory neuroblasts from the subventricular zone, and before there is synaptic activity in the cortical plate, except for connections between the Cajal–Retzius neurons of the molecular zone and neurons of future layer 6, the latter representing the earliest wave of radial neuroblast migration. Axons, but not dendrites, start projecting from migratory neuroblasts even before they reach their final

destination within the cortical plate. The earliest callosal fibers in the human emanate in the region of the primordial hippocampus (Rakic and Yakovlev, 1968); if this is true also in the human fetus, in whom the hippocampus is still the dorsal structure that it remains in the mouse, the origin of these first callosal axons may be from what will later become the neocortical parahippocampal gyrus. In the murine brain, these early axons pass through the septum (Zaki, 1985), which in the mouse is a relatively larger and more cellular structure than in the human, even during embryonic and fetal life. In a genetic strain of mouse with callosal agenesis, it is the failure of apoptosis of the glial septum to form a callosal plate or bridge over which callosal axons may pass to the other hemisphere that is the embryological and anatomical defect (Zaki, 1985). In mice in which callosal agenesis is induced by radiation at the critical time in development, the defect also arises from failure of the cortical plate to form and provide a pathway for axonal grown, but extensive death of pyramidal neurons in layer 3 of the neocortex and postnatal axonal elimination are also contributory factors (Abreu-Villaca and Schmidt, 1999). The sequence of callosal axons traversing the midline follows a rostrocaudal gradient, with fibers of the rostrum preceding those of the splenium. The rostrum thus appears first but both the genu and splenium may be recognized in the human fetus at 84 days gestation and the mature morphology is achieved by 115 days (Loeser and Alvord, 1968). Initially, there are many more axons than later because of many axonal collaterals that sprout from the principal axon to form diffuse connections in the contralateral cortical plate, but many of these collateral axons are later

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retracted to leave fewer, but more specific, connections as the brain matures (Innocenti, 1981; O’Leary et al., 1981; Gravel and Hawkes, 1990). This collateral retraction is a progressive process beginning in the second half of gestation and continuing well into the postnatal period. Because the growth cones of callosal axons follow the most direct path between the hemispheres, they separate and isolate scattered neurons of the subiculum (the transitional cortex between Ammon’s horn of the hippocampus and the neocortical parahippocampal gyrus); this narrow band of sparse gray matter dorsal to the corpus callosum becomes the indusium griseum (Abbie, 1939; Sarnat, 1992). In the mature human brain, the indusium griseum continues to lie between the corpus callosum below and the cingulate gyrus above, on either side of the midline. Myelination of the corpus callosum begins at 2 months postnatally by histological stains (Yakovlev and Lecours, 1967; Sarnat, 1992) and by electron microscopy, but is not detected by magnetic resonance imaging (MRI) until 4 months of age (Barkovich, 2000). The myelination cycle is long, over several years, and is not completed until mid-adolescence. As with the initial axonal projection across the midline, early myelination follows a rostrocaudal gradient, but this gradient is not evident after several months and the entire corpus callosum appears to myelinate uniformly. The mature corpus callosum contains 180 million axons, but only 40% are myelinated, even in the adult (Tomasch, 1954). Table 5.1 is a summary of the normal ontogenesis of the forebrain commissures. Convolutions of the brain do not begin in the first half of gestation, and at 20 weeks only the sylvian and calcarine fissures are formed and an incipient parieto-occipital sulcus may be evident. The cingulate gyrus and other gyri on the medial side of the cerebral

hemisphere are unform. In the absence of the corpus callosum, medial hemispheric gyri do not form with their long axis parallel to the dorsal surface of the brain but rather are perpendicular to it and to where the corpus callosum should have been. This gyral orientation is never found in normal brains. In brains with partial callosal agenesis it occurs only in the region of the deficient corpus callosum. The cingulate gyrus in normal brains and abnormal vertical gyri in callosal agenesis develop between 24 and 30 weeks gestation. Figs. 5.3–5.5 illustrate the normal corpus callosum and agenesis of the corpus callosum as seen in MRI in living patients.

5.4. Vascular supply of the corpus callosum 5.4.1. Arterial supply The rostrum and anterior part of the body of the corpus callosum are supplied by branches from the anterior cerebral artery. Its pericallosal branch lies in a sulcus dorsal to the corpus callosum and sends branches superiorly to the cingulate gyrus and inferiorly to the corpus callosum. One or more branches of the anterior choroidal artery, which itself arises from either the internal carotid or the middle cerebral arteries, provides blood to the splenium. Other branches to the splenium and posterior third of the body of the corpus callosum are from branches of the posterior cerebral arteries, so that the corpus callosum receives most of its arterial blood from the anterior circulation, but the posterior circulation also contributes (Crosby et al., 1962; Parent, 1996). The posterior body of the corpus callosum is thus a watershed zone between the anterior and posterior circulations, but watershed infarcts are not specifically identified in the corpus callosum without more extensive infarction in the cerebral hemispheres.

Table 5.1 Development of the corpus callosum Age

Event

39 days gestation 54 days gestation 71 days gestation 74 days gestation 84 days gestation 115 days gestation 24–40þ weeks gestation 38 weeks gestation 2 months 17 years

Commissural plate differentiates from lamina terminalis First pioneer axons cross midline in anterior commissure and hippocampal commissure (psalterium) Glial cells of callosal commissural plate begin programmed apoptosis First pioneer axons cross midline in rostrum of future corpus callosum Genu and splenium of corpus callosum identified Mature morphology of corpus callosum Retraction of callosal axonal collaterals to form fewer, but more specific, connections Initial myelination of anterior commissural axons Initial myelination of callosal axons Myelination of corpus callosum complete (40% of total axons)


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Fig. 5.3. Magnetic resonance images of the normal brain of an 11-year-old girl. (A) Sagittal section in T1 shows a well developed and complete corpus callosum. The cingulate gyrus is oriented parallel to the corpus callosum, just dorsal to it. (B) Coronal section in T1 shows both the corpus callosum dorsally and the anterior commissure more ventrally (compare with Figure 1). The lateral ventricles are seen on either side of the midline without a bundle of Probst (see Figs. 5.4 and 5.5).

5.4.2. Venous drainage The veins collecting blood from the corpus callosum are part of the deep venous drainage system of the cerebral hemispheres. Paired anterior callosal veins accompany the pericallosal artery on each side of the midline to drain blood from the anterior medial hemispheric surface and from the anterior corpus callosum. The paired posterior callosal veins are short vessels that pass caudally beneath the splenium of the corpus callosum to drain blood from the posterior medial surfaces of the cerebral hemisphere and from the posterior corpus callosum; they empty caudally into the unpaired vena magna or great vein of Galen and eventually into the straight sinus, mixing venous blood with contributions from the internal occipital vein and veins from the tentorium cerebelli and superior surface of the cerebellum (Crosby et al., 1962; Parent, 1996). There are no lymphatics in the central nervous system. 5.4.3. Ischemic atrophy and infarction Occlusions of the anterior cerebral artery are uncommon as isolated events, and rarer still in children. When they do occur, infarction of the anterior corpus callosum may occur, because the collateral circulation

is limited from the anterior cerebral or pericallosal artery on the other side. Vertebrobasilar insufficiency in the fetus and neonate may lead to tegmental watershed infarcts in the brainstem and to Mo¨bius syndrome, which also results in focal ischemic atrophy of the posterior third of the corpus callosum because of the transient bilateral poor perfusion through the posterior cerebral arteries (Sarnat, 2004b). Chronic or recurrent hypoperfusion of cerebral arteries in the fetus or neonate may sometimes result in infarction of the corpus callosum, but this is not an isolated infarct and is usually accompanied by extensive infarction resulting later in multicystic encephalomalacia or at least infarction of deep cerebral structures such as the thalami and deep telencephalic nuclei (i.e. basal ganglia). Chronic ischemia may lead to atrophy of the corpus callosum, but atrophy of this commissure usually results from loss of axons due to neuronal death in the cerebral cortex. The corpus callosum itself is only the midtrajectory of their axons. In fetuses and neonates, laminar necrosis of the neocortex often includes layer 3, the neurons from which the callosal axons emanate, and callosal atrophy is a chronic result that may be seen both by imaging during life and neuropathologically. Thinning of the corpus callosum that resembles dysgenesis is often an acquired ischemic change

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Fig. 5.4. Magnetic resonance images of the brain of a 6-week-old girl born at term with total agenesis of the corpus callosum. (A) Midline sagittal section in T1 shows the lack of a corpus callosum and, rather than a cingulate gyrus, the gyri of the medial side of the hemisphere are oriented perpendicular to the region normally occupied by the corpus callosum (compare with Fig. 5.3A). (B) Parasagittal section in T1 reveals the enlarged occipital horn of the lateral ventricle, known as colpocephaly. (C) Axial section in T1 demonstrates that the lateral ventricles are parallel, rather than curving convexly toward the midline and that a mass of white matter separates the ventricles from the interhemispheric fissure; this tissue is an abnormal large fasciculus, the bundle of Probst, and is composed of ipsilateral callosal axons that were unable to cross the midline. (D) Axial section in T1 more dorsal than that of (C) discloses the dilated occipital horn, as also seen in (B).


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Fig. 5.5. Magnetic resonance images of a 13-month-old girl with partial agenesis of the corpus callosum. (A) Midsagittal section in T1 reveals a tiny remnant of the corpus callosum near the expected position of the rostrum. Gyri of the medial side of the hemisphere are abnormally perpendicular in orientation. (B) Coronal section in T2 showing widely spaced lateral ventricles and bundle of Probst. (C, D) Axial sections in T1 show widely separated and parallel lateral ventricles, separated from the interhemispheric fissure by the bundle of Probst, coursing parallel to the ventricles. The occipital horns are dilated as colpocephaly.

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(Njiokiktjien et al., 1988). Ischemia of the splenium of the corpus callosum in living neonates is well demonstrated by diffusion-weighted MRI, which shows a markedly reduced apparent diffusion coefficient but does not distinguish moderate from severe injury (Solomon et al., 2005, 2007). 5.4.4. Hydrocephalus Acquired nonvascular lesions that may rupture or thin the corpus callosum to give an appearance of dysplasia include traumatic injuries and chronic hydrocephalus (Njiokiktjien et al., 1988). Congenital hydrocephalus in particular causes thinning of the corpus callosum, in part because of stretching and in part because of atrophy; this thinning occurs in both hydrocephalus-ex-vacuo with normal intraventricular pressure and obstructive hydrocephalus associated with increased pressure in the lateral ventricles. Neoplasms of the corpus callosum are rare.

5.5. Functions of the corpus callosum The functional importance of the corpus callosum in the human brain has been the topic of interest for many years and the theme of international symposia (Ettlinger, 1965). Interhemispheric transfer of information is the principal understood function of the corpus callosum but it also serves to synchronize the two cerebral hemispheres in terms of electrophysiological activity as recorded by electroencephalography (EEG). Sleep spindles, which are normally synchronous after 18 months of age, fail to synchronize (Lynn et al., 1980). If a transfer role is an important function, it would be anticipated that interhemispheric delays between bilaterally synchronous spike-wave discharges would be longer than the interhemispheric axonal conduction time of approximately 20 ms in the mature brain and a preceding cortical spike discharge would produce a callosal compound action potential sooner than a contralateral one, but this was not confirmed in a recent study (Ono et al., 2002).These authors suggested that the interhemispheric recruitment of the epileptogenic state involves a different role of the corpus callosum. More than 80% of callosal fibers are inhibitory. The precise role of the corpus callosum in generating secondary bilateral synchrony in the EEG and in epileptogenesis remains incompletely defined. Visual evoked potential generally remains normal in callosal agenesis, even if the entire posterior fornix and splenium are absent (Coupland and Sarnat, 1990). The importance of the corpus callosum in the interhemispheric transfer of information not of an epileptic nature is demonstrated by the clinical neurological

examination of patients with callosal agenesis who are old enough to cooperate. Stereognosis, the recognition of objects placed in the hand by their shape, and naming them, is a good example. In a patient with callosal agenesis who is left hemispheric dominant for language, an object, such as a coin, key or paper clip, placed in the right hand is easily identified and named because the epicritic somatosensory impulses are received in the left parietal lobe and transferred to the speech area of the same hemisphere. If the object is placed in the left hand, however, it is perceived and recognized in the right parietal lobe but the information must be transferred across the corpus callosum to the speech areas of the left hemisphere to be named. Patients with total callosal agenesis often have difficulty with this and a marked asymmetry of the two sides in this regard. Ability to easily name objects placed in either hand indicates that the aberrant pathways for callosal axonal projections are at least partially formed and functional. In addition, subtle deficits in visual-fine motor functions may be demonstrated by detailed testing (Gott and Saul, 1978; Bruyer et al., 1985; Joseph and Bannister, 2001). Visual processing, subtle linguistic functions such as comprehension of idiomatic expressions and deficits in social skills also may be deficient in patients with callosal agenesis (Bayard et al., 2004; Paul et al., 2005; Huber-Okrainec et al., 2005).

5.6. Aberrant projections of callosal axons 5.6.1. Anterior commissure In the majority of brains with callosal agenesis, neuropathological examination demonstrates that the anterior commissure is enlarged to as much as four times its normal size, representing aberrant callosal axons that found an alternative site to cross the midline, in addition to the expected anterior commissural fibers, which normally interconnect the anterior temporal lobes only. It is of interest that marsupial mammals, which lack a corpus callosum, have a very large anterior commissure, so the aberrant pathway in humans may be a reversion to an earlier phylogenetic condition. Reciprocal or combined hypoplasia of both commissures may be the result of defective expression of the PAX6 gene (see Genetics, below). 5.6.2. Bundle of probst An aberrant tract, known as the bundle of Probst, is composed of callosal axons that cannot cross the midline at their intended site and follow the path of least resistance to form a large ipsilateral fascicle in the dorsomedial part of each cerebral hemisphere, just

EMBRYOLOGY AND MALFORMATIONS OF THE FOREBRAIN COMMISSURES beneath the cingulate gyrus, that courses posteriorly (Probst, 1974; Lemire et al., 1975) (Figs. 5.4 and 5.5). Because of its position, interposed between the medial wall of the hemisphere and the lateral ventricle, it causes the characteristic straightening of the ventricular form and displacement away from the medial wall. In a genetically mutant acallosal mouse, fusion of the septal midline is delayed 72 hours and the sling does not form. The intended callosal axons approach the median barrier and whorl into a pair of large neuromas, homologous with the human bundle of Probst (Silver and Ogawa, 1983). Surgical transaction of the sling in normal murine embryos at precallosal stages of development results in the callosal axons also forming a pair of neuromas adjacent to the interhemispheric fissure, but these aberrant fibers retain a potential in the neonatal period of regrowing across the midline if a properly aligned, glia-covered artificial scaffold spanning the hemispheres is provided (Silver and Ogawa, 1983). 5.6.3. Hippocampal commissure (psalterium) In callosal agenesis, the hippocampal commissure is well formed but is displaced anteriorly (Lemire et al., 1975). It is of normal size but may contain a few aberrant callosal axons. Because the earliest pioneer axons in the corpus callosum arise in the region of the hippocampus in the mouse (Rakic and Yakovlev, 1968) and probably also in the human (Sarnat, 1992), some callosal axons may be added to those of the hippocampal commissure if they cannot cross between the hemispheres in their normal site, although the hippocampal commissure normally does not include axons from neocortex, only from paleocortex and subiculum. 5.6.4. Ventral corticospinal tract In some cases of callosal agenesis, in addition to or instead of a bundle of Probst, some callosal axons enter the internal capsule and form what appears to be an enlarged corticospinal tract. The entire trajectory of this pathway is enlarged to double or triple its normal size, including the middle third of the midbrain cerebral peduncles, the longitudinal axonal bundles in the basis pontis and enlarged medullary pyramids; in the spinal cord, these fibers join mainly the ventral corticospinal tract in the ventral funiculus (Fig. 5.6). Most of the normal corticospinal axons of the ventral funiculus do not decussate at the caudal end of the medulla oblongata, as do those that form the larger corticospinal tract in the lateral funiculus, but rather remain ipsilateral and decussate ventral to the central canal within the spinal cord, near to their sites of termination. Whether aberrant callosal axons that join

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the ventral corticospinal tract do the same is uncertain, but is likely. 5.6.5. Additional small aberrant sites of interhemispheric projection Some callosal axons undoubtedly cross the midline in a variety of sites as individual fibers or small groups not large enough to identify as commissures or fascicles. These sites have not been well studied and are thus poorly documented neuroanatomically; they probably are microscopic and below the resolution of imaging. Their course and destination in holoprosencephaly are unknown but they spread out within the subcortical white matter.

5.7. Genetics of callosal agenesis Because agenesis of the corpus callosum is associated with a large number of other malformations of the central nervous system, with various chromosomal and genetic diseases, and at times with malformations of other organ systems, it is really a syndrome rather than a single disease; hence the genetic basis is multiple and varied. However, isolated callosal agenesis without other abnormalities of the brain or other organs may have one or more genetic mutations as its etiology. Haplo-insufficiency of the PAX6 gene is associated with agenesis or hypoplasia of the corpus callosum and anterior commissure; affected patients have problems with interhemispheric transfer for auditory processing and also with olfactory function (Sisodiya et al., 2001; Bamiou, 2004). There may be a reciprocal relation between the corpus callosum and anterior commissure in PAX6 mutations, because some patients may have isolated anterior commissural agenesis with a normal corpus callosum or there may be a reduction in volume of both commissures (Bamiou et al., 2004). Defective PAX6 expression also can result in ocular dysgenesis or even anophthalmia, and also pituitary agenesis. Another genetic mutation, found in several female patients with isolated callosal agenesis whose male relatives had X-linked recessive lissencephaly with abnormal genitalia, is the ARX gene (Kato et al., 2004). The complex CRASH syndrome, which includes callosal agenesis as one of the constant features, is due to mutations in a single gene, L1-CAM (Fransen et al., 1995). Defective expression of the L1-CAM gene at the Xq28 locus also is associated with a neuroblast migratory disorder with pachygyria and congenital hydrocephalus usually due to aqueductal stenosis, transmitted as an X-linked recessive trait. Callosal agenesis is an additional feature in some human cases (Schrander-Stumpel et al., 1995) and in a murine model (Itoh et al., 2004).

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Fig. 5.6. A variant of an aberrant pathway of callosal axons in agenesis of the corpus callosum, much less frequent than the bundle of Probst, is callosal axons joining the internal capsule and projecting with the corticospinal tract into the cerebral peduncles, basis pontis (A), medullary pyramids, and continuing to descend in the ventral funiculus of the spinal cord (B). In this female fetus of 23-weeks gestation, the ventral corticospinal tract, seen in transverse section, is many times larger than normal because it contains axons originally destined for the corpus callosum. It also is mildly asymmetrical: larger on the right before decussation (A) and on the left caudal to the point of decussation at the medullospinal junction (B). The normal ventral part of the corticospinal tract in the spinal cord contains many uncrossed axons that do not decussate until they reach the level of their termination. Grey matter appears dark. This fetus had trisomy 13 and mild lobar holoprosencephaly as well. Synaptophysin, 40.

The zinc-finger homeobox gene ZFHX1B is defective in patients with the rare Mowat–Wilson syndrome and callosal agenesis is seen in these children (Sztriha et al., 2003). The genes well documented with associated specific disorders of neuroblast migration, including FLMA, DCX, LIS1, EMX2 and RLN, do not have an identified role in the development of the corpus callosum and callosal agenesis is not a constant feature of their mutations and deletions, although hypoplasia of the corpus callosum is frequent in these diseases. More probably these defective genes affect other, downstream genes in the various cascades in which downregulation of one gene results in the underexpression of others. In all anatomical forms of holoprosencephaly, agenesis of the corpus callosum (or hypoplasia in the mildest lobar form) is an integral part of the malformation, regardless of which of the six identified genes has the deletion or mutation. In the mouse, additional genes important in the formation of the corpus callosum include slit, robo, netrin1, Nfia, Emx-1 and Gap-43 (Ren et al., 2006), but correlative defects in these genes in the human are not proved. Isolated callosal agenesis is usually sporadic and is not transmitted as a mendelian trait. Cases of Shapiro syndrome (see below) are sometimes found in siblings of consanguineous parents, suggestive of autosomal recessive inheritance (Pineda et al., 1984). Many genetic syndromes include callosal agenesis as a cardinal feature, the Aicardi syndrome and Andermann syndrome being prototypical examples (see below).

5.8. Neuroanatomical appearance of the brain with total and partial agenesis of the corpus callosum 5.8.1. Pneumoencephalography and cerebral angiography The anatomical features of the normal, hypoplastic and agenetic corpus callosum are well demonstrated by all types of cerebral imaging. In the years preceding the advent of computed tomography (CT) in the early 1970s, pneumoencephalography was frequently employed and well demonstrated the characteristic lateral displacement of the lateral ventricles by the bundle of Probst, the straightening of the lateral ventricles so that they were parallel and the rising of the third ventricle between them to a more dorsal plane than the lateral ventricles because the roof of the third ventricle was not restricted by the corpus callosum (Davidoff and Dyke, 1934; van Epps, 1953; Sarnat, 1992). The massa intermedia of the thalamus, which is not a commissure but an uncleaved part of the medial thalamic nuclei with continuity of the two sides, is not formed in more than half the cases (Probst, 1973). Angiography of the brain also shows absence of the corpus callosum, interhemispheric lipomas (see below) and the aberrant course of the anterior cerebral arteries and the inconstant appearance of the pericallosal branch (Sabouraud et al., 1967). Arteriography is now performed only for another indication but, in

EMBRYOLOGY AND MALFORMATIONS OF THE FOREBRAIN COMMISSURES the years before imaging, was another standard approach for demonstrating cerebral malformations. Magnetic resonance angiography (MRA) may be done at the time of MRI of the brain and is able to demonstrate these vascular anomalies less invasively. Colpocephaly, a selective pathological dilatation of the occipital horns of the lateral ventricles, is a frequent but not universal feature accompanying agenesis of the corpus callosum, and may be demonstrated with any of the imaging techniques (Sarnat, 1992) (Figs 5.4 and 5.5). It is attributed to loss of white matter in the posterior cerebral hemispheres, due to absence of the splenium and of callosal axons in the white matter emanating from the occipital lobes. This explanation may be an oversimplification, because many of those axons are still projected from striate and visual associate cortex but are in aberrant positions with aberrant trajectories. Reorganization of the visual cortex and pathways in patients with callosal agenesis and colpocephaly has been demonstrated (Bittar et al., 2000). Other causes of colpocephaly, not involving agenesis of the corpus callosum, include primary dysplasias of the occipital lobes and atrophy of white matter secondary to periventricular leukomalacia, the latter mainly a complication of fetal cerebral ischemia in the late second and early third trimesters of pregnancy and of prematurity. 5.8.2. Sonography Advances in ultrasonography now make this technique reliable for diagnosing callosal agenesis and also colpocephaly in the fetus in utero during the second half of gestation and sometimes earlier; it also may be confirmatory in the postnatal period in early infancy (Gebarski et al., 1984; Lockwood et al., 1988; Volpe et al., 2006). Disadvantages are that lateral structures, such as the cerebral cortex, and posterior fossa structures, are poorly visualized or distorted. Images in the sagittal plane are an advantage of ultrasound over CT.

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from providing less precise information than MRI about cortical gyration and posterior fossa structures, is that a sagittal plane is not feasible. 5.8.4. Magnetic resonance imaging Magnetic resonance imaging provides the most detailed documentation of this cerebral malformation and any associated malformations of the brain, both supra- and infratentorial, in the living patient. Midsagittal images (Figs. 5.3–5.5) show the entire cross-section of the corpus callosum to determine whether any residual callosal fibers are evident and, in the case of partial agenesis or ischemic atrophy, to compare each part and determine whether there is selective hypogenesis or atrophy in the rostrum, middle portion or splenium (Curnts et al., 1986; Schaefer et al., 1991; Bodensteiner et al., 1994; Barkovich, 2000). The anterior commissure also may be visualized in the sagittal plane to determine if it is enlarged. Finally, the height of the third ventricle can be determined and anomalies associated with callosal agenesis, such as interhemispheric lipomas and arachnoidal cysts, are demonstrated. In axial and coronal planes, the various parts of the corpus callosum also may be seen and it is often useful to see the rostrum and splenium in these views because the lateral extent of the fibers, the bundle of Probst and the size and shape of the lateral and third ventricles can be defined. MRA may be performed at the time of MRI to demonstrate anomalous positions of the anterior cerebral and pericallosal arteries, as well as the anomalous venous drainage in the absence of the corpus callosum. Detailed descriptions of the neuroimaging features of total or partial callosal agenesis are provided in textbooks of pediatric neuroradiology (Barkovich, 2000) and in articles (Byrd et al., 1990; Schaeffer et al., 1991; Bodensteiner et al., 1994; Ren et al., 2006; Volpe et al., 2006). 5.8.5. Neuropathological studies

5.8.3. Computed tomography Computed tomography in axial images provides reliable diagnostic information with greater precision than does ultrasonography (Larsen and Osborn, 1982; Guibert-Tranier et al., 1982; Jeret et al., 1987) (Fig. 5.5). At times CT may be a satisfactory substitute for MRI, particularly in patients who would tolerate poorly the anesthetic required for MRI in young children. CT has an advantage over MRI of no anesthetic risk, but the disadvantage of exposing the child to a small amount of radiation. CT also is capable of demonstrating interhemispheric lipomas and arachnoid cysts (Fig. 5.4). One of its greatest limitations, apart

Direct pathological studies of the corpus callosum are limited to postmortem examination. The brain above the tentorium should be sectioned sagittally to visualize the medial sides of the two hemispheres and to measure the diameter of the anterior commissure and hippocampal commissure. Both hemispheres can then be sectioned in the traditional coronal plane, or one hemisphere cut coronally and the other in another plane, assuming that the brain is relatively symmetrical. In asymmetrical brains, such as those with porencephaly or with hemimegalencephaly, both hemispheres should be sectioned in the same way, preferentially coronally. Microscopic neuropathological examination of the

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cerebral cortex should pay special attention to the small pyramidal neurons in layer 3, using immunocytochemical markers to supplement histological stains; examples of such neuronal markers include neuronal nuclear antigen (NeuN), microtubule associated protein-2 (MAP2), chromogranin-A (CgrA), neurofilament proteins (NFP); neuron-specific enolase (NSE) and PGP-9.5, but not all such markers need be applied. Synaptophysin and other markers of synaptic vesicles provide information about the density of synapses on neurons in layer 3. Golgi impregnations of the cortex may provide dramatic identification of not only the neurons of origin but also their normal or abnormal dendritic arborizations, but are difficult studies and usually reserved for investigative protocols. DiI markers may show the projection of neuronal axons and demonstrate aberrant trajectories, but this also remains a research tool at this time and requires special preparation at the time of gross neuropathological examination.

5.9. Clinical presentation of syndromes of callosal agenesis, hypogenesis and dysgenesis 5.9.1. Isolated total agenesis of the corpus callosum This condition may occur as an isolated condition without other central nervous system malformations or anomalies in other organ systems, or may occur in the context of other cerebral dysgeneses and/or multiple developmental abnormalities of other organs and the extremities. The clinical expression depends upon the associated conditions, both neuroanatomical and systemic. In some cases, chromosomal disorders, recognizable genetic syndromes or metabolic diseases are present. In general, it is useful to segregate cases of isolated callosal agenesis, with or without associated interhemispheric lipomas or arachnoidal cysts (see below), from callosal agenesis as part of a more extensive forebrain dysgenesis such as holoprosencephaly (see below) or frontal encephaloceles (Sztriha, 2005). Hypertelorism is an important facial dysmorphism seen in many, but not all, children with total agenesis of the corpus callosum. It provides a useful clinical marker that may raise the suspicion of this malformation and justify further neuroimaging. The hypertelorism is usually associated with exophoria and sometimes with overt exotropia and inability to converge (Sarnat, 1992). From a developmental perspective, hypertelorism stands in contrast to the hypotelorism and even cyclopia in its most extreme form, which frequently denotes holoprosencephaly. Hypotelorism and midfacial hypoplasia results from defective mesencephalic neural crest induction of nonneural craniofacial structures because the rostrocaudal gradient of genetic

expression has reaches as far caudally as the midbrain and noncleavage of the superior colliculi often is noted neuropathologically (Sarnat and Flores-Sarnat, 2001) and demonstrated in some cases by MRI (see Ch. 2). Hypotelorism is rarely seen in callosal agenesis, except in holoprosencephaly, but hypertelorism is common. This facial dysmorphism also is a problem of neural crest formation, migration or terminal differentiation but involves prosencephalic, rather than mesencephalic, neural crest. The prosencephalic neural crest arises from the region of the lamina terminalis, the same embryological zone that gives origin to the commissural plate, which, when defective, impedes passage of callosal axons in callosal agenesis. The prosencephalic crest forms, among other structures, the intercanthal ligament, which holds the fetal orbits together during development so that the eyes face forward instead of being on the sides of the head, as they are in many animals and in early human fetuses (Sarnat et al., 2007). Neurological findings depend upon associated lesions (Parrish et al., 1979; Schaeffer et al., 1991). Global developmental delay may be mild or moderate. Mental retardation is common in children with callosal agenesis but is not universal (Serur et al., 1988). Even in patients with no obvious focal neurological or intellectual deficits, subtle findings of impaired transfer of information between the hemispheres may be demonstrated, for example a difference between the two hands in stereognosis and naming of objects (see Functions of the Corpus Callosum, above). This deficit resembles a receptive (Wernicke’s) or conduction aphasia in the posterior temporal lobe more than a motor aphasia arising in Broca’s area of the frontal lobe. Other subtle perceptual and neuropsychological deficits may be demonstrated by specific testing (Gazzaniga and Freedman, 1973; Bruyer et al., 1985). Even children within the normal IQ range may have specific learning disabilities and perceptual problems and should be assessed by neuropsychological examination. Many behavioral deviations are seen in children with callosal agenesis, even in the absence of mental retardation or hard neurological deficits, and these are attributed in part, by neuropsychologists, to disconnection and asymmetries between the hemispheres (Lassonde et al., 1995; Jancke et al., 1997). Microcephaly is seen in some, but this finding is exceptional. Callosal agenesis does not cause motor deficits, even if the entire rostrum and frontal lobe fibers are absent, except for motor apraxias in some cases. Neither hypotonia nor spastic diparesis can be attributed to agenesis of the corpus callosum alone. Impairment of coordination, ataxia, intention tremor and dyssynergia imply an associated cerebellar malformation or hypoplasia. Arthrogryposis multiplek congenital is a rare association with posterior callosal agenesis (Voorhies et al., 1984).

EMBRYOLOGY AND MALFORMATIONS OF THE FOREBRAIN COMMISSURES Epilepsy is found in a higher incidence than in the control population, particularly in infancy and early childhood (Lacey, 1985; Serur et al., 1988). Seizures may be generalized or partial, but benign partial epilepsy with rolandic spikes in the EEG is the most common pattern (Santanelli et al., 1989). The reason for epilepsy may in part be related to loss of the largely inhibitory influence of callosal axons (Gastaut et al., 1980) but focal cerebral cortical dysplasias also may be present and expressed as epileptogenic foci. Treatment with antiepileptic medication depends upon the type and frequency of seizures and EEG findings. Feeding difficulties, dysphagia and apraxia of swallowing may present in the neonatal period or early infancy but affect a minority of patients (Ng et al., 2004). Neuroendocrinopathies are an infrequent associated abnormality, particularly important not to overlook because they are a treatable complication. Isolated growth hormone deficiency is the most frequent aberration (Liapi et al., 1985), similar to septo-opticpituitary dysplasia. Panhypopituitarism is even rarer in callosal agenesis. Autonomic dysfunction is a serious complication in some older patients. Recurrent episodes of hypothermia and diaphoresis, when associated with agenesis of the corpus callosum, are sometimes called Shapiro syndrome (Shapiro et al., 1969; Guibert-Tranier et al., 1971; Fox et al., 1973; Noe¨l et al., 1973; LeWitt et al., 1983; Pineda et al., 1984). For uncertain reasons, these symptoms do not present in infancy but rather in late childhood or adolescent or even as late as 46 years of age, and relapses may be separated by months or years (Guibert-Tranier et al., 1982). Severe hypothermia may be fatal (Noe¨l et al., 1973). Shapiro syndrome in infancy is described in siblings of the opposite sex of consanguineous parents, with cyanotic and apneic episodes at 2 months and the death of one at age 4 months (Pineda et al., 1984). Though these paroxysmal episodes have been called diencephalic epilepsy (Fox et al., 1973), their pathogenesis as a seizure disorder is not substantiated by clinical or EEG evidence of epilepsy. Shapiro syndrome also has been found in some patients with interhemispheric lipoma and partial callosal agenesis (LeWitt et al., 1983), and rarely in patients with a normal-appearing corpus callosum (Fox et al., 1973). 5.9.2. Partial agenesis of the corpus callosum The rostrum or splenium of the corpus callosum may be preferentially affected, with preservation of the other part. Since each part develops at a slightly different time, in general following a rostrocaudal gradient, this selectivity suggests different timing of the insult. Hypoplasia of the middle section of the corpus callosum

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is a special case. This rare abnormality usually represents a rare anatomical form of holoprosencephaly originally known as the ‘middle interhemispheric fusion variant’ but later shortened to ‘middle interhemispheric variant’ because ‘fusion’ is not an embryologically correct term (Barkovich and Quint, 1993; Lewis et al., 2002; Simon et al., 2002; see also Ch. 2). Occasionally, selective partial agenesis of the middle of the corpus callosum with preservation of the rostrum and splenium is demonstrated in patients who do not have holoprosencephaly, including the middle interhemispheric variant, but who have other anomalies such as Chiari II malformation (Sarnat, 1992). It is important to recognize that slightly parasagittal views in MRI in which a column of the fornix is seen may give the appearance of focal hypoplasia in the posterior part of the body of the corpus callosum, but this is normal and is not seen in midsagittal views of the same brain. The clinical manifestations are similar to those of total callosal agenesis but often less severe because some connections across this commissure are preserved. Difficulties with interhemispheric transfer of stereognosis and naming information is more expected with focal agenesis of the splenium than of the rostrum, because the somatosensory reception is in the parietal lobe and Wernicke’s area for speech is in the posterior temporal lobe, though articulation must be transferred to Broca’s area of the frontal lobe. The outcome, in terms of developmental delay and cognitive and learning disabilities, is similar in partial as in total callosal agenesis (Schaeffer et al., 1991; Paul et al., 2004;Volpe et al., 2006). The availability of prenatal diagnosis has led to a high rate of elective terminations of pregnancy (Volpe et al., 2006). 5.9.3. Holoprosencephaly Holoprosencephaly is a malformation in which the corpus callosum usually is absent and there is no apparent bundle of Probst or other aberrant fibre tracts where callosal axons might have wandered to find a means of crossing the midline. Nevertheless, in those lateral parts of the cortex where recognizable lamination is organized following a mediolateral gradient, layer 3 with small pyramidal cells may be identified and its neurons are not degenerating or reduced in number (Sarnat and Flores-Sarnat, 2001). The trajectory and destination of the axons of these layer 3 neurons are unknown but it was noted more than half a century ago by Lichtenstein and Maloney that, in holoprosencephaly, ‘transverse callosal fibers develop as a diffuse structure in the white substance, rather than as a compact corpus callosum’ (Lichtenstein and Maloney, 1954). In mild cases of lobar holoprosencephaly,

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noncleavage of the frontal poles may displace the rostrum and anterior body of the corpus callosum such that it appears absent by imaging but is revealed to be present by histopathological examination (Schaefer et al., 1991). The middle interhemispheric fusion variant of holoprosencephaly is discussed above (see Partial agenesis of the corpus callosum). The absence of the corpus callosum in holoprosencephaly is well illustrated in Chapter 2. 5.9.4. Hemimegalencephaly In hemimegalencephaly, the corpus callosum may appear normal but is more often asymmetrically enlarged in the hemisphere with the dysplasia and is usually asymmetrical in position and orientation, as is shown well by MRI (Flores-Sarnat, 2002). Epidermal nevus, a neurocutaneous syndrome of neural crest origin, is associated with callosal dysgenesis if complicated by hemimegalencephaly (see Chapter 10) (Flores-Sarnat, 2002). Corpus callosotomy for intractable epilepsy in hemimegalencephaly has the worst seizure outcome and the most frequent surgical complications when compared with other underlying conditions (Shimizu, 2005). 5.9.5. Disorders of neuroblast migration Callosal agenesis, total or partial, or dysgenesis, may be an additional neuropathological finding in many disorders of neuroblast migration with different genetic bases. These include periventricular nodular heterotopia, subcortical laminar (‘band’) heterotopia (Vossler et al., 1999; Caksen et al., 2003), and pachygyria/lissencephaly syndromes (Sarnat, 1992; Da´vila-Gutie´rrez, 2002; Miyata et al., 2004; see also Ch. 13). Some of these conditions additionally involve posterior fossa neural defects, such as cerebellar hypoplasia, Dandy–Walker malformation and brainstem dysplasias. Extreme microcephaly with abnormal cerebral convolutions may also include callosal agenesis (Sztriha et al., 2005). 5.9.6. Other midline neuroanatomical defects In agenesis of the corpus callosum not associated with defined genetic malformations, such as holoprosencephaly and disorders of neuroblast migration, other brain structures are inconstantly dysplastic. These are usually midline or paramedian structures and include hypoplasia of the optic nerves and chiasm, heterotopia and posterior fossa neural defects. Absence of the septum pellucidum is universally reported in MRI and other imaging studies and the septum pellucidum may not be evident even at postmortem examination;

careful neuropathological examination may show, however, that the two leaves of the septum are actually present and lie laterally and on top of the thalamus and basal ganglia; they indeed would not be detected by imaging (Sarnat, 1992). Cerebellar malformations include hypoplasia of the vermis or its total absence, similar to Joubert syndrome, or partial absence with ballooning of the posterior fossa posteriorly to form a Dandy–Walker malformation. Some patients with either total or partial callosal agenesis also have Chiari malformations, despite the absence of a meningomyelocele (Sarnat, 1992). 5.9.7. Genetic syndromes of callosal agenesis Several well defined genetic diseases are characterized in part by agenesis of the corpus callosum. Numerous genetic and dysmorphic syndromes not due to chromosomopathies are reported with occasional cases lacking a corpus callosum (Da´vila-Gutie´rrez, 2002; Bodensteiner, 2005). Andermann syndrome, transmitted as an autosomal recessive trait mainly in French-Canadian families, is a peripheral motor–sensory neuropathy with mental retardation and absence of the corpus callosum (Andermann et al., 1972, 1975, 1977; Larbrisseau et al., 1984). The neuropathy is the most constantly expressed feature in other members of the same families, with or without callosal agenesis (Mathieu et al., 1990). Aicardi syndrome is agenesis of the corpus callosum, chorioretinal lacunae, vertebral anomalies, mental retardation and myoclonic epilepsy including infantile spasms, and an extremely abnormal EEG with asymmetrical or lateralized paroxysmal burst-suppression activity (Aicardi et al., 1969; Dennis and Bower, 1972; de Jong et al., 1976; Fariello et al., 1977). Myoclonic seizures are nearly always preceded by other types of seizures (Bour et al., 1986). The electroretinogram is abnormal in about half the cases. Other, inconstant neuropathological lesions include agenesis of the anterior commissure, hypoplasia of the inferior cerebellar vermis, abnormal orientation of the hippocampi, periventricular heterotopia and nonlaminated polymicrogyria of the neocortex (Ferrer et al., 1986). Colobomas of the optic disk are common, in addition to retinal lacunae, which are focal dysplasias, not infarcts. The disorder is found almost exclusively in girls, an Xlinked dominant trait with a chromosomal deletion or autosomal translocation at the Xp22 locus but with molecular heterogeneity so that phenotype severity does not always correlate well with genotype (Neidich et al., 1990). Rarely, boys with a normal 46XY karyotype are reported (Curatolo et al., 1980) and Aicardi syndrome also was described in a male with 47XXY chromosomes (Hopkins et al., 1979). Aicardi syndrome has been

EMBRYOLOGY AND MALFORMATIONS OF THE FOREBRAIN COMMISSURES reported in association with a variety of tumors, both neural and extraneural, but this association occurs in a minority of cases and may be fortuitous (Sarnat, 1992). The Aicardi–Goutie`res syndrome is distinct from the Aicardi syndrome, and involves symmetrical calcifications of the basal ganglia, cerebral cortical atrophy and white matter demyelination, cerebellar hypoplasia and also agenesis or hypogenesis of the corpus callosum in some (Aicardi and Goutie`res, 1984; Lanzi et al., 2002; Abdel-Salam et al., 2004; stergaard and Christensen, 2004). Cerebrospinal fluid lymphocytosis with high levels of a-interferon, in the absence of infection, is a constant feature. It is an autosomal recessive trait affecting both sexes, with consanguinity frequently disclosed in the family history. Clinically, it presents as a progressive encephalopathy from early infancy, with feeding difficulties, spastic diplegia, irritability and unexplained episodic fevers. Other hereditary cases of agenesis of the corpus callosum, unrelated to Aicardi syndrome, are also described. Autosomal dominant (Lynn et al., 1980) and X-linked recessive (Menkes et al., 1964) transmission is documented. It is a frequent associated anomaly in autosomal dominant Apert syndrome and sometimes Crouzon syndrome (Gross and Hoff, 1959; de Leo`n et al., 1987). The corpus callosum is often hypoplastic in cases of ocular albinism with disturbances of neuroblast migration (Bodensteiner et al., 1990). It is found in the Juberg–Hayward syndrome, an autosomal recessive disease with growth retardation, microcephaly, cleft lip and palate and radial ray anomalies of the fingers and toes; neuropathological findings, diagnosed prenatally and postnatally, include cerebellar hypoplasia, agenesis of the corpus callosum and sometimes hydrocephalus (Couvreur-Lionnais et al., 2005). The acrocallosal syndrome is an autosomal recessive disorder consisting of agenesis of the corpus callosum, macrencephaly, severe mental retardation, facial dysmorphism and postaxial polydactyly of the fingers and toes (Schinzel, 1979, 1988; Schinzel and Kaufmann, 1986; Gelman-Kohan, 2005). Consanguinity has been found in a few families. Variable anomalies occur in other systems, including congenital cardiac defects such as tetralogy of Fallot, renal cysts, urogenital malformations including micropenis, imperforate anus and malignant hyperpyrexia (Ikbal et al., 2004; GelmanKohan, 2005). Though clinical features overlap with the autosomal dominant cephalopolysyndactyly syndrome of Greig, allelism of these two syndromes was shown by linkage analysis to be unlikely (Brueton et al., 1992). Among chromosomal disorders, callosal agenesis is common in trisomy 8 and its mosaic variants, trisomy 11, trisomy 13 and, less frequently, trisomy 18 (Serur

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et al., 1988; Jeret et al., 1987). Agenesis of the anterior commissure, by contrast, is frequent in trisomy 18 (Sumi, 1970) but rare in trisomy 13. Other trisomies that occasionally have callosal agenesis include trisomy 4p, trisomy 6, XO Turner syndrome and XXY Klinefelter syndrome (Serur et al., 1988; Bodensteiner, 2005). In total, more than 65 chromosomal deletions, duplications and translocations involve total or partial agenesis of the corpus callosum in some cases, but rarely is it a constant feature (Dobyns, 1996). 5.9.8. Association of callosal agenesis with metabolic diseases It has been recognized for more than a quarter-century that agenesis of the corpus callosum is a feature of many inborn metabolic diseases and it was proposed that this anatomical defect be used as a clinical marker of some biochemical diseases and to help focus the laboratory investigation of children with callosal agenesis (Bamforth et al., 1988; Kolodny, 1989; Bodensteiner, 2005). Some of the diseases in which agenesis of the corpus callosum occurs with a high frequency are nonketotic hyperglycinemia (Dobyns, 1989), some aminoacidurias, loss of cytochrome-c-synthetase, which leads to a secondary mitochondrial cytopathy (Prakash et al., 2002); it has also been reported in Leigh’s encephalopathy, another mitochondrial disease. Induced metabolic factors, including fetal toxins and drug exposures, also may result in callosal agenesis or dysgenesis at the critical time of about 4 weeks gestation (Da´vila-Gutie´rrez, 2002), shortly after neural tube closure. These include the fetal alcohol syndrome (Church and Gerkin, 1988). Isolated callosal agenesis is not reported as a result of maternal anticonvulsant use in the first trimester of pregnancy but may accompany neural tube defects, which are a common teratogenic effect of antiepileptic drugs such as valproic acid and phenytoin (Ng et al., 2005).

5.10. Lipomas and arachnoidal cysts of the interhemispheric fissure It is not infrequent to find on CT or MRI that total or, more commonly partial, agenesis of the corpus callosum is associated with either a lipoma (Fig. 5.7) or an arachnoidal cyst. Focal hypoplasia or agenesis of the corpus callosum may be limited to the site of these lesions. Lipomas may be found in half of cases of partial callosal agenesis; they usually are nonprogressive but some exhibit rapid and aggressive growth despite the fact that all are congenital lesions (Sabouraud et al., 1967). While these lesions often are referred to as ‘lipomas or cysts of the corpus callosum’, they

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Fig. 5.7. Magnetic resonance imaging of a 17-year-old girl with an interhemispheric lipoma and partial absence of the corpus callosum in the region of the lipoma. Sagittal section in T1 (A), Axial section in T1 (B) and coronal section in T2 (C) demonstrate the great extent of the lipoma. A previous study at 5 years of age showed a much smaller lipoma surrounding and partially replacing only the splenium; the rostrum and body of the corpus callosum were otherwise well formed. Such lipomas often grow slowly over many years. This patient has never had surgery of the brain and she is neurologically and intellectually normal, a good student in grade 12.

EMBRYOLOGY AND MALFORMATIONS OF THE FOREBRAIN COMMISSURES actually do not form in the corpus callosum or arise from callosal tissue. They originate in the primitive meninx that invades the interhemispheric fissure during cleavage of the prosencephalon at about 4 weeks gestation, hence are lipomas or loculated cysts of the arachnoid, which is a mesencephalic neural crest derivative (Zettner and Netsky, 1960; Sarnat, 1992). The presence of frontonasal dysplasia or extraosseous lipoma of the forehead midline, or even an intracranial abnormal bony vertical bar, in the midline of some children with interhemispheric lipoma (Pascual-Castroviejo et al., 1985) is further evidence of a neural crest origin of this ectopic tissue. Nevertheless, the frequency of hypertelorism in focal dysgenesis of the corpus callosum associated with cysts and lipomas is much less than in isolated total callosal agenesis. The combination of agenesis of both the corpus callosum and the anterior commissure with interhemispheric lipoma suggests a disturbance of genetic programming. Ultrasonography in the preterm infant may mistakenly lead to the identification of such lipomas as focal subependymal hemorrhages (Imaizumi et al., 1988).

5.11. Role of the corpus callosum and callosotomy in epilepsy The corpus callosum has an important function in synchrony between the two hemispheres. It synchronizes the sleep spindles after 18 months and patients with agenesis of the corpus callosum continue to have independent sleep spindles in the two hemispheres, although there is no overall asymmetry in their frequency or total number (Sarnat, 1992). Corpus callosotomy is a surgical means of stopping the spread of an epileptic focus across the midline to produce secondary bilateral synchrony, and this procedure is used in selected children with intractable epilepsy (Sakas and Phillips, 1996; Sorenson et al., 1997; Snead, 2001; Guenot, 2004; Shimizu, 2005). Generalized tonic-clonic seizures and drop attacks are the types of epilepsy most benefited by corpus callosotomy: about 60% become seizure-free and another 30% have a reduction in the frequency, duration and severity of the seizures, although the procedure may not abolish seizures completely. In various series, only 10–25% of patients have no change in their epilepsy after corpus callosotomy. Some patients with drop attacks also may benefit from this procedure. Quantitative histopathological studies of two patients who died decades after corpus callosotomy revealed numerous atypical pyramidal neurons (of layer 3) with abnormal basal dendrites; these changes might reflect deafferentation after callosotomy but might also be related to the underlying epilepsy (Jacobs et al., 2003). Changes

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are demonstrated by MRI in the subcortical white matter, distant from the corpus callosum itself, following callosotomy and unrelated to surgical hemorrhage or trauma; wallerian degeneration of axons in the centrum semiovale could account for these signal changes (Khurana et al., 1999). Corpus callosotomy may also be significantly beneficial in seizure control in some patients with other types of neuroblast migratory disorders of the cerebrum, such as subcortical laminar heterotopia (Vossler et al., 1999). The corpus callosum also is needed to produce the synchronous 3 Hz spike-wave complexes of absence epilepsy. That this seizure disorder, sometimes genetically determined, usually resolves spontaneously in mid- to late adolescence and may be related to the chiefly inhibitory function of callosal fibers and the late myelination of some projections (Sarnat, 1992). Interhemispheric lipomas associated with partial callosal agenesis are usually associated with seizures (Gastaut et al., 1980). Agenesis of the corpus callosum is not a malformation amenable to surgical treatment but occasionally a rapidly growing interhemispheric lipoma or arachnoidal cyst may require resection.

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Disorders of segmentation of the neural tube Chapter 6

Disorders of segmentation of the neural tube: Chiari malformations HARVEY B. SARNAT* University of Calgary Faculty of Medicine and Alberta Children’s Hospital, Calgary, Alberta, Canada

6.1. Introduction Chiari malformations are congenital downward displacements of the posterior part of the cerebellum, either alone, as in some Chiari I cases or together with the lower (Fig. 6.1) medulla oblongata (some Chiari I and all Chiari II and III cases), through the foramen magnum into the spinal canal. They may be associated with neural tube defects: lumbosacral or thoracic meningomyelocele (Chiari II, III) and posterior encephalocele (Chiari III). Segmental hydromyelia (all types) and intrinsic dysgeneses of the cerebellum and brainstem (all types), including congenital aqueductal atresia, are other frequently associated features. A low torcula with ventral displacement of the tentorium cerebelli and a small posterior fossa is a constant feature of the Chiari II malformation. Fetal and/or postnatal hydrocephalus is a frequent clinical complication. Although the rostrocaudal displacement of the cerebellum, with or without additional protrusion of the medulla oblongata, is frequently called ‘herniation’, this term should be avoided because it implies a mechanism of neural tissue being forced through the foramen magnum, a popular theory of the 20th century that is now being challenged. True herniation of the tonsils of the cerebellum due to intracranial hypertension is not a Chiari malformation, although some authors persist in regarding it as such, and other authors use the term ‘acquired Chiari deformity’ with a variety of mass lesions, including meningiomas (Blaivas and Bebarski, 2005). Anatomically, the ‘tonsils’ of the cerebellum are the posterior medial part of the cerebellar hemispheres, continuous with the

uvula of the vermis and rostral to the flocculus. This is the part that herniates in acquired conditions, but in Chiari malformation it is largely the posterior vermis in addition that protrudes. The pathogenesis of Chiari malformations has been passionately debated since the initial meticulous neuropathological descriptions by Hans Chiari of Vienna in 1891 and 1896, observations that remain as valid today as they were more than a century ago (Chiari, 1891, 1896). The principal hypotheses that have been proposed until recently have all been based upon mechanical events. None satisfactorily addresses all features of the Chiari malformations. A molecular genetic hypothesis was proposed recently that could explain such aspects as the shallow posterior fossa, intrinsic focal malformations of the brainstem and cerebellum, and myelodysplasia including meningomyelocele and segmental hydromyelia. This chapter focuses on pathogenesis more than on the clinical features of Chiari malformations, and each of the above theories is considered in the context of both traditional and new neuropathological, clinical and neuroimaging findings.

6.2. Mechanical and hydrodynamic theories of pathogenesis 6.2.1. Traction theory The spinal cord is tethered by the lumbosacral meningomyelocele and pulls the lower brainstem and cerebellum through the foramen magnum because of the differential growth of the vertebral column and the spinal cord, with more rapid growth of the bony than the neural elements

*Correspondence: H. B. Sarnat MD, FRCPC, Alberta Children’s Hospital, 2888 Shaganappi Trail NW, Calgary, Alberta T3B 6A8, Canada. E-mail: [email protected], Tel: þ1-403-955-7131, Fax: þ1-403-955-2922.

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(Penfield and Coburn, 1938; Lichtenstein, 1940, 1942). This theory was definitively discredited by Goldstein and Kepes, who provided experimental proof that traction at the caudal end of the spinal cord dissipated within four segments (Goldstein and Kepes, 1966). It also cannot explain the presence of Chiari malformations not associated with neural tube defects or tethering. 6.2.2. Pulsion theory Downward pressure is exerted upon the brainstem and cerebellum by early and progressive fetal hydrocephalus (Gardner, 1973, 1977; Gardner et al., 1975; Caviness, 1976; Masters, 1978). The pulsion theory was the mechanism originally proposed by Chiari (1891, 1896). A variant of the hydrodynamic pulsion theory was based upon the assumption that the developing ventricular system communicated with the amniotic sac through a hydromyelic spinal cord and resulted in CSF leakage into the amnion with secondary hindbrain herniation (Cameron, 1957), but Peach dispelled this hypothesis by pointing out that hydrodynamic pressure gradients are distributed equally and that amniotic fluid movement in the opposite direction frequently is found in fetuses with meningomyelocele (Peach, 1964, 1965a, 1965b). A minority of cases of Chiari malformation do not show associated spina bifida or caudal neural tube defects (Peach, 1965a; Case et al., 1977) and, conversely, rare cases of myelodysplasia do not exhibit a Chiari II malformation (Osaka et al., 1978). 6.2.3. Hydrodynamic oligo-CSF theory The oligo-CSF or unified theory attributes a small posterior fossa and cerebral dysgenesis to a paucity of sufficient fluid to distend the cerebral vesicles during early fetal development because the open neural tube allows leakage and prevents the accumulation of fluid within the ventricular system (Padget, 1972; Padget and Lindenberg, 1989; McClone and Knepper, 1989). Some cases of Chiari malformation detected prenatally have had successful intrauterine surgical repair of the meningomyelocele, with subsequent reversal of either hydrocephalus or of oligo-CSF and also of cerebellar tonsillar protrusion through the foramen magnum (Bruner et al., 1999; Olutoye and Adzick, 1999); these results are sometimes cited as evidence in support of this hypothesis but the successful correction of anatomical defects does not necessarily confirm the original mechanism of formation of these defects. Hydrodynamics may well play a role, particularly in late gestation and postnatally, in potentiating and further exaggerating the anatomical Chiari malformation, but are not likely to be the primary factor in pathogenesis.

6.2.4. Crowding theory The crowding theory of Marıń-Padilla is based upon the premise that the posterior fossa is too small; rapid growth of neural tissues within it causes them to be squeezed through the foramen magnum as a ‘toothpaste tube’ phenomenon. Although the differential growth of osseous and neural structures usually is thought of as the vertebral column growing in the longitudinal axis faster than the spinal cord, in this case the neural tissue (cerebellum and brainstem) grows faster than the surrounding cranial bones (MarıńPadilla and Marıń-Padilla, 1981; Naidich et al., 1982; Friede, 1989; Marıń-Padilla, 1991; Nishikawa et al., 1997). The Marıń-Padilla theory regarded the primary defect as mesodermal, involving the cranial base rather than neuroectodermal tissue. The crowding theory has some merit because the posterior fossa is indeed too small and the torcula is displaced downward in Chiari malformations. Many cases of well formed Chiari II malformations are described at midgestation and earlier (Sarnat, 1992a), and the cisterna magna is absent in first trimester ultrasound studies of Chiari malformations, attributed to early malpositioning of the cerebellar vermis and medulla oblongata within the cervical spinal canal (Goldstein et al., 1989). The occipital bone and basal chondrocranium are underdeveloped, probably due to hypoplasia of the basioccipital bone originating from the paraxial mesoderm (see Molecular genetic theory, below), also causing basilar impression (Marıń-Padilla, 1991; Nishikawa et al., 1997). Crowding is indeed very probably a contributory factor in the late stages of gestation and postnatally, with rapid growth of posterior fossa neural structures, but it cannot explain the Chiari malformation as the primary mechanism in the young fetus before midgestation. 6.2.5. Birth trauma theory Molding of the head during delivery distorts the posterior fossa (Williams, 1991). Many cases of meningomyelocele are detected prenatally by ultrasound and are delivered by cesarian section; others are well documented earlier than midgestation, which is inconsistent with a traumatic hypothesis (Sarnat,1992). The anatomical placement of the torcula is not an acute defect occurring at the time of birth but a developmental anomaly dating from about 8–10 weeks gestation. This theory is completely untenable.

6.3. Molecular genetic theory of pathogenesis The novel and recent data of the last decade about molecular genetic programming of the developing

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Fig. 6.1. MRI-T1 in (A) midsagittal and (B) parasagittal views of Chiari I malformation, showing a 2.2 cm protrusion (not herniation) of cerebellar tissue through the foramen magnum to cover the dorsal surface of the upper cervical spinal cord in a 4year-old boy with syncope and headaches but no vomiting. He has no evidence of a lumbosacral meningomyelocele. The MRI of the brain was otherwise normal.

brain and other tissues provide a new opportunity to reinterpret the Chiari malformations and propose a mechanism of pathogenesis that does not rely on the inadequate mechanical theories of the past. Beginning with the original pathological descriptions of Cleland (1883) and of Chiari (1891, 1896), many subsequent authors have also noted a disruption of normal histological architecture in the medulla oblongata, cervical spinal cord, cerebral aqueduct and cerebellum, and speculated that the Chiari malformations may be a primary dysgenesis of neural tissues, but they could not correlate these observations with any of the mechanistic theories. The explanation of primary neural dysgenesis also was considered but rejected by many authors because the Chiari malformation does not correspond to an arrest in any normal stage of ontogenesis. The concept of genetic expression as focal dysgenesis limited to portions of the neural tube was poorly understood before the identification of homeobox genes for segmentation of the hindbrain and a potential for ectopic expression under some conditions. On the basis of an extrapolation of these data to the Chiari malformation, and the additional demonstration of abnormalities in intermediate filament protein expression in ependymal cells focally in the regions of dysgenesis in Chiari malformation that probably represent a secondary upregulation, an hypothesis emerged that the Chiari malformations are primary defects in the genetic programming of hindbrain

segmentation and of growth of associated structures of the chondrocranium (Sarnat, 2004a). The theoretical substrate for this hypothesis is reviewed here. 6.3.1. Segmentation of the embryonic neural tube and intrinsic neural dysgeneses in Chiari malformations Segmentation of the axial mesoderm, such as somite and sclerotome formation, was recognized in the earliest studies of vertebrate embryology in the 19th century, but the lack of apparent segmentation of the neural tube, and of the spinal cord in particular, intrigued and confused embryologists for more than a century. In 1890, Professor CFW McClure, a leading anatomist and embryologist of his time, wrote: ‘The primitive segmentation of the vertebrate brain is a problem which has probably attracted as much of the attention of morphologists as any one of the great, unsettled questions of the day, and many views have been advanced’ (McClure, 1890). More contemporary students of development were no less fascinated and perplexed by the lack of visible anatomical landmarks demarcating compartmentalization of the early neural tube, until the late1980s and 1990s, when new molecular genetic techniques disclosed just the hindbrain compartments that had been predicted. These segments, comprising eight rhombomeres, a mesencephalic neuromere and six diencephalic and telecephalic

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Fig. 6.2. For full color version, see color plate section. Atresia of the cerebral aqueduct of the midbrain in a 9-week-old infant born at term with meningomyelocele and Chiari II malformation. The aqueduct is replaced by residual ependymal rosettes and clusters but does not allow a flow of CSF from the third to the fourth ventricle. These ependymal cells, and also those lining the fourth ventricle, are strongly immunoreactive for vimentin in the regions of dysgenesis but not in the third or lateral ventricles or the normal central canal sectors of the spinal cord; the same ependyma is nonreactive for glial fibrillary acidic protein (GFAP) and for S-100b protein; hence this overexpression of vimentin is not a simple maturational delay and is probably upregulation secondary to another gene of hindbrain segmentation (e.g. HOX) being downregulated as the molecular defect. Vimentin, 250.

prosomeres, appear to form to define zones of cellular proliferation and to restrict cellular migration across boundaries separating the compartments (Keynes and Stern, 1984, 1986; Keynes and Lumsden, 1990; Wilkinson and Krumlauf, 1990; McGinnis and Krumlauf, 1992; Guthrie and Lumsden, 1991; Puelles and Rubinstein, 2003). Without such boundary restrictions, similar neurons could not be concentrated to form cranial nerve nuclei and other intrinsic structures with common functions. Many genes and gene families have now been identified that interact with each other to program the neuromeres and intervening boundaries. The best studied examples are the HOX family, Krox-20 (a mouse gene homologous with EGR-2 in the human), the Wnt (wingless) family, the En (Engrailed) family, and also Shh (Sonic hedgehog), which influences CNS polarity gradients, among its multiple other functions (McMahon, 1992; Augustine, 1993; Echelard et al., 1993; Riddle et al., 1993; Rijii et al., 1993; Barrow et al., 2000; Sarnat and Menkes, 2000). The experimental inactivation or deletion of specific homeobox genes in the chick and mouse embryos results in the loss of hindbrain segmentation, deletion of entire neuromeres, disruption of the normal anatomical architecture of the brainstem and ectopic expression in domains rostral or

caudal to their normal rhombomeric sites of expression (Chisaka and Capecchi, 1991; Lufkin et al., 1991; McMahon et al., 1992; Schneider-Maunouory et al., 1993). In the amphibian embryo, interference with expression of a particular homeobox protein causes a phenotype in which the anterior spinal cord is transformed into a hindbrain-like structure and the fourth ventricle extends caudally into the cervical canal (Wright et al., 1989), strongly reminiscent of the human Chiari II malformation. Some exogenous toxins act as teratogens in the developing embryonic hindbrain by interfering with the genetic regulation of rhombomere formation. One of the best studied such substances is vitamin A or retinol, the alcohol of retinoic acid. Retinoic acid is normally synthesized by the notochord and by the floor plate ependyma (Wagner et al., 1990); other ependymal cells do not secrete retinoic acid but do have retinoic acid receptors (Maden et al., 1989, 1991; Ruberte et al., 1991). A small amount of retinoic acid continues to be synthesized even in the adult brain (Yasuda et al., 1989; Dev et al., 1993). During the stage of rhombomere formation, the chick and mouse embryo exposed to excessive retinoic acid undergoes disruption of hindbrain segmentation, transformation of rhombomeres 2/3 to a more caudal 4/5 identity, and neural tube defects including multiple or defective ventricular diverticula and canals; these effects appear to be mediated by alteration of HOX codes (Durston et al., 1989; Yasuda et al., 1989; Alles and Sulik, 1990; Morris-Kay et al., 1991; Marshall et al., 1992). HOX2.9, for example, is normally expressed only in the domain of r4 but, in the presence of excessive retinoic acid, its domain of expression is extended throughout the preotic hindbrain, whereas Krox-20 becomes totally inhibited from its normal expression in r3 (Morris-Kay et al., 1991). Retinoic acid anatagonizes the expression of HOX genes and thus may be regarded as yet another regulator of hindbrain segmentation (Kessel, 1993). Another family of candidate genes capable of inducing anterior to posterior transformation of the rostrocaudal gradient in the neural tube of the mouse is the orthodenticle (Otx) family; reduction of Otx-2 expression correlates with hindbrain expansion with rostral extension of the HOXb1 domain, also induced by retinoic acid (Simeone et al., 1995). Regionalization within a rhombomere of gene expression also occurs, for example the localization of HOXd10 in the developing lumbosacral spinal cord of r8, precisely the most frequent site of meningomyeloceles (Lance-Jones et al., 2001). Excessive vitamin A is a well documented teratogen in rodent embryos and even a single dose at the critical time produces neural tube defects very similar to human meningomyeloceles and Chiari II

DISORDERS OF SEGMENTATION OF THE NEURAL TUBE: CHIARI MALFORMATIONS 93 cell but only in a few cells in the adult, such as endomalformations (Shenefelt, 1972; Marıń-Padilla and thelial cells and fibroblasts. In the human, vimentin is Marıń-Padilla, 1981; Kohga and Obata, 1992; Chen weakly expressed in undifferentiated neuroepithelial et al., 1995). Exogenous maternal stress further cells but becomes strongly expressed in fetal ependyenhances the teratogenicity of retinoic acid in mice mal cells, appearing and regressing in a precise tempo(Rasco and Hood, 1995) and also may play a role in ral and spatial sequence. In the floor and roof of the humans. As will be discussed below, Chiari malformafourth ventricle, ependymal vimentin immunoreactivition and neural tube defects are not usually inherited as ty is strong until 34 weeks gestation and thereafter a mendelian trait. Another well recognized exogenous diminishes to only weak expression in less than half factor leading to neural tube defects and Chiari malforthe cells by term, and soon disappears from all fourth mation is deficiency of folic acid early in gestation ventricle ependymal cells (Sarnat, 1992b). In the cere(Smithells, 1983; Oakley et al., 1983). bral aqueduct vimentin normally shows no further The molecular genetic hypothesis of Chiari malforimmunoreactivity by 20 weeks gestation (Naidich mations is that the dysgenesis of the hindbrain and spiet al., 1991; Sarnat, 1992b). In the spinal central canal, nal cord are due to abnormal segmentation resulting ependymal vimentin becomes weak after 22 weeks from defective or ectopic expression of one or more gestation, disappearing in the ependyma in the zone of the homeotic genes that regulate rhombomere and of the basal plate sooner than in the region of the alar mesencephalic neuromere formation. The cause may plate. The basal processes of the roof plates and floor be a spontaneous mutation or deletion in a homeobox plates, which radiate to form the dorsal and ventral gene, probably one of the HOX family, or a genetic median septa, consistently show the strongest immumutation induced by an exogenous teratogen. noreactivity in young fetuses of 10–14 weeks gestation and continue to show strong expression until these pro6.3.2. Ependymal maturation and abnormalities cesses fully retract in the first few postnatal months in Chiari malformations (Stangaard and Mllga˚rd, 1989; Takano and Becker, 1997). The presence of vimentin in ependymal cells The fetal ependyma is a much more dynamic structure is unrelated to the mitotic cycle and differentiated in fetal development than in adult life, participating in ependymal cells in the fetus no longer undergo the several processes of normal ontogenesis (Sarnat, mitosis at the ventricular wall that undifferentiated 1992c). Its differentiation from neuroepithelium and neuroepithelium exhibits (Sarnat, 1992c, 1995). its maturation from a pseudostratified columnar to a Glial fibrillary acidic protein (GFAP) is not simple cuboidal epithelium are accompanied by expressed in undifferentiated neuroepithelial cells. It changes in concentration and immunocytochemical appears transiently in the fetal ependyma, coexpressed reactivity for several intermediate filament proteins with vimentin, and persists longer, replacing it as the and secretory molecules of fetal life that are no longer principal intermediate filament protein of these cells. synthesized at maturity and follow a precise regional It disappears entirely from all ependymal cells late in and temporal sequence of maturation (Friede, 1961; gestation (37–40 weeks) and in the first few weeks of Dooling et al., 1977; Roessmann et al., 1980; Sasaki postnatal life (Roessmann et al., 1980; Sasaki et al., et al., 1988; Stangaard and Mllga˚rd, 1989; Gould 1988; Sarnat, 1992b; Yamada et al., 1992). A unique et al., 1990; Sarnat, 1992b, 1998a). feature of the ependymal distribution of GFAP is that, Abnormal concentrations of the intermediate filaunlike vimentin, it is never expressed at any gestational ment proteins, in particular the upregulation of vimenage in floor plate cells (Sarnat, 1992b). S-100b protein tin in ependymal cells, is demonstrated in fetuses and initially has a much more restricted distribution in the neonates with Chiari II malformation, but these alterafetal ependyma than either vimentin or GFAP but it tions are restricted to zones of dysgenesis such as the too is transient and not expressed in the mature epenfourth ventricle, atretic cerebral aqueduct and hydrodyma. It persists longer than vimentin and GFAP but myelic portions of the spinal central canal (Sarnat, disappears in the first few postnatal weeks. 2004a). It is more likely that this focal overexpression In Chiari II malformation, a striking and consistent of vimentin is a secondary upregulation of the vimentin aberration in the regional distribution of ependymal gene than a primary mechanism of pathogenesis in the vimentin is demonstrated in fetuses and young infants, Chiari malformation. The technique employed for the (Takano and Becker, 1997; Sarnat, 2004a). The most immunocytochemical demonstration of vimentin as a important feature in fetal life and early infancy can maturational marker may differ from that used for other be summarized as a focal persistence of strong epenpurposes (Sarnat, 1998b). dymal vimentin expression, restricted to regions of Vimentin is a cytoskeletal intermediate filament dysgenesis: roof and floor of the fourth ventricle, protein expressed transiently in many types of fetal

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stenotic or atretic cerebral aqueduct (Fig. 6.2) and hydromyelic levels of spinal cord but not in the third or lateral ventricles despite ventriculomegaly or in the normal central canals at nonhydromyelic levels of spinal cord. A more generalized upregulation of vimentin in the ependyma in other sites, such as the third and lateral ventricles and normal regions of spinal cord, occurs only later in childhood as a secondary phenomenon. Congenital hydrocephalus due to other causes does not exhibit this same local ependymal vimentin immunoreactivity in the perinatal period, although in later childhood it becomes more diffusely expressed and not so localized (Sarnat, 2004a). The overexpression of vimentin without cooverexpression of GFAP or S-100b protein indicates that increased vimentin in Chiari malformation is not merely a nonspecific maturational delay in the ependyma. It also provides a contrast with lissencephaly/ pachygyria. In primary genetic disorders of neuroblast migration, including lissencephaly type 1 (Miller– Dieker syndrome) and type 2 (Walker–Warburg syndrome), and in cerebrohepatorenal disease of Zellweger, also associated with heterotopia, abnormal cortical lamination and abnormal ependymal architecture, GFAP and S-100b protein, but not vimentin, are upregulated (Sarnat et al., 1993a,b). Another reason why ependymal vimentin in Chiari malformation is not a simple maturational delay is its precise localization to the zones of dysgenesis and not in other areas, even in the presence of secondary hydrocephalus and tearing of the ependyma lining the expanding lateral ventricles. The upregulated vimentin expression in the ependyma of the involved regions in Chiari malformations is not interpreted to be a primary defect but rather as a secondary phenomenon that serves as a convenient neuropathological marker of defective programming of the hindbrain in cases of Chiari II malformation. The rodent is a poor model for studying this phenomenon because, unlike the human, vimentin continues to be expressed in ependymal cells throughout life (Didier et al., 1986; Maresˇ et al., 1988; Oudega and Marani, 1991; Bodega et al., 1994).

just caudal to the posteriorly displaced cervicomedullary junction (MacKenzie and Emergy, 1971). Hydromyelia also complicates some cases of Chiari I malformation without hydrocephalus or spina bifida (Isu et al., 1990). A variety of ependymal abnormalities occur in the spinal central canal of mice with experimentally induced hydrocephalus (James et al., 1977; Torvik and Murthy, 1977; Griebel et al., 1989) and in human hydrocephalic infants (Murthy and Deshpande, 1980; Takano and Becker, 1997). The causes of stenosis or atresia of the cerebral aqueduct are multiple and include genetically programmed defects as well as acquired congenital or postnatal viral infections such as mumps and gliotic obstruction due to the widespread ependymitis of neonatal bacterial meningitis with ventriculitis or posthemorrhagic hydrocephalus in premature infants. The most frequent cause of congenital aqueductal atresia, however, is its association with Chiari II malformation (Marıń-Padilla, 1991; Sarnat, 2004a). The cerebral aqueduct may be extensively kinked, forked or atretic, represented by a narrow channel or only residual clusters and rosettes of ependymal cells (Globus and Bergman, 1946; Beckett et al., 1950; Marıń-Padilla, 1991). The aqueductal stenosis of Chiari II malformation is probably an intrinsic part of the malformation and the hypothesis that the primary malformation is due to defective genetic programming of the hindbrain could include the aqueduct. It should be remembered that the normal cerebral aqueduct of the fetus is a much larger canal than in the term neonate or the adult. The collicular plate also is poorly formed in some cases of Chiari II (Sarnat, 2004a). Chick embryos subjected to physical puncture or lacerating wounds of the anterior medullary velum at various stages of development show impaired midbrain neuroepithelial wound healing if the CSF pressure is increased (Lawson and England, 1996). This experimental condition is not likely to be related to the pathogenesis of the Chiari malformation.

6.3.3. Hydromyelia and aqueductal atresia in Chiari malformations

The origins of membranous bone and endochondral bone are different and involve different genetic programs. Membranous bone, which forms most of the craniofacial structures including the bones of most of the calvaria, is a neural crest derivative. Endochondral bone, which comprises the cranial base and also the basioccipital, exoccipital and supraoccipital bones, is derived from paraxial mesoderm. These bones of the chondrocranium require genes of the HOX family for their normal development, the same genes that are essential for neural tube segmentation; membranous

Hydromyelia of the spinal central canal was recognized in the cervical cord by Cleland (1883) and Chiari (1891, 1896). It is more commonly associated with Chiari II malformation than with any other developmental anomaly, and sometimes extends as far caudally as the thoracic spinal cord (MacKenzie and Emergy, 1971; Wisoff, 1988; Sarnat, 2004a). Syringomyelia also is present in about 20% of cases and the cavity is usually

6.3.4. Development of the cranial base and the reason for a low torcula in Chiari malformations

DISORDERS OF SEGMENTATION OF THE NEURAL TUBE: CHIARI MALFORMATIONS bones from neural crest do not require HOX genes. The same neural segmentation genes that cause dysgenesis of the developing neural tube can, therefore, result in hypoplasia of the basioccipital and supraoccipital bones, causing the torcula to form in too low a position because the posterior wall of the posterior fossa is too low. Hypoplasia of these bones is exactly the finding in Chiari malformations. Occipital hyperplasia, the opposite condition, may cause cervical myelopathy (Ohaegbulam et al., 2005) and very probably it is due to upregulation of the same HOX family gene that is involved in Chiari malformation. 6.3.5. Inconstant involvement of supratentorial structures in Chiari malformations Supratentorial structures of the brain appear uninvolved except for the secondary effects of obstructive hydrocephalus. Occasional cases of Chiari malformation are reported with agenesis of the corpus callosum, absence of the septum pellucidum, subventricular nodular heterotopia and focal zones of cerebral cortical microdysgenesis with abnormal lamination and polymicrogyria (Peach, 1965b; Marıń-Padilla and Marıń-Padilla, 1981; Gilbert et al., 1986; Martıńez and Alvarado-Mallart, 1989; Sarnat, 1992a; Norman and Cochran, 1995), but represent a small minority. 6.3.6. Intrinsic dysgeneses of the brainstem and cerebellum in Chiari malformations Many authors have described microdysgeneses of the brainstem and cerebellum in Chiari malformations (Chiari, 1891, 1896; Cameron, 1957; Peach, 1965a,b; Friede, 1989; Sarnat, 1992a; Caldarelli et al., 2002; Pueyrredon et al., 2004). The first report of Chiari II malformation, by Cleland in 1883, was the first reference to brainstem dysgenesis, which he described as ‘the pontomedullary junction is indistinct, with an illdefined, rod shaped pons . . . a beak-like deformity of the quadrigeminal plate, directed backward and downward to a point found by fusion of the inferior colliculi’. In 1958 Daniel and Strich observed that the cerebellar hemispheres were frequently asymmetrical and that they sometimes extended around the brainstem ventrally to meet in the ventral midline; the vermis was buried between them (Daniel and Strich, 1958). Cases of Chiari II malformation associated with rhombencephalosynapsis (agenesis of the vermis with fusion of the medial walls of the cerebellar hemispheres) have now been documented (Sener and Dzelzite, 2003). Gilbert et al. (1986) described ‘hypoplasia of cranial nerve nuclei, olivary nuclei and pontine nuclei’ in young infants. The medulla oblongata may

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appear juxtaposed with the pons. Naidich et al. attributed the supratentorial gray matter heterotopia, disorganization of the cerebral gyri and dysgenesis of the corpus callosum to collapse of the cerebral ventricles (Naidich et al., 1991). The cerebellar tissue protruding through the foramen magnum in Chiari II malformations varies in length from short to long extensions and involves the nodulus, pyramis and uvula in that order (Gilbert et al., 1986). The cerebellar tonsils may protrude with the posterior vermis. The choroid plexus of the fourth ventricle also may protrude, as well as the most caudal part of the fourth ventricle itself. The cerebellar vermis in Chiari II is surprisingly associated with an expansion, rather than the anticipated reduction, in vermal volume, as determined in living patients with midsagittal MRI (Salman et al., 2004); these authors attributed this paradox to compressive displacement of midline structures within the confines of a small posterior fossa. Because no tissue was available for histopathological examination, primary dysgenesis as an alternative hypothesis could not be addressed. The most frequent microscopic findings are as follows: a) Focal dysgenesis of the cerebellar cortex, not only in the portions of cerebellum protruding below the foramen magnum but also deep heterotopia in the white matter of the vermis consisting of architecturally disorganized mature neurons, immature neuroblasts of the external granular layer and glial cells including Bergmann glia, with only rudimentary or no lamination; b) Elongation of the posterior part of the medulla oblongata with extension through the foramen magnum into the cervical spinal canal; this protrusion of the medulla may displace the cervical spinal cord and stretch its nerve roots, or it may be folded over the upper cervical spinal cord as an S-shaped curve in half the cases of Chiari II, sometimes associated with a sac-like cavity that does not communicate with the dilated central canal or have an ependymal lining (Sarnat, 1992a); c) Tectal beaking is commonly seen in imaging studies and confirmed neuropathologically; the caudal surface of the inferior collicular plate is curled dorsally; d) Aqueductal stenosis and replacement of the cerebral aqueduct by a series of ependymal rosettes and clusters that do not communicate fluid between the third and fourth ventricles; e) Ependymal abnormalities in the regions of dysgenesis, particularly the fourth ventricle (Sarnat, 2004a); f) Intrinsic dysgeneses of the architecture of the brainstem, including heterotopia and incompletely migrated inferior olivary nuclei from the rhombic lip of His and aberrant positions of some cranial nerve nuclei; g) Altered positions of cervical nerve

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roots, which course rostrally rather than caudally; h) Generally normal supratentorial structures in Chiari I and II malformations, except for the secondary effects of obstructive hydrocephalus; rare patients have hypoplasia of the corpus callosum; in Chiari III malformations there is protrusion of occipital lobe tissue as well as of infratentorial structures.

6.4. Clinical genetic aspects of Chiari malformations Chiari malformations are not transmitted as mendelian traits, either autosomal or linked to the X-chromosome, and their association with common chromosomal diseases is rare, although they occasionally complicate trisomy 18 without spina bifida (Case et al., 1977). Several authors have reported an association of Chiari I malformation and the Pierre–Robin sequence (Lee et al., 2003; Tubbs et al., 2004b; Holder-Espinasse et al., 2001). Some cases of this syndrome may be of genetic origin, as suggested by autosomal dominant or recessive inheritance or association with chromosomal aberrations (Stalker et al., 2001; Szewka et al., 2006). Syringomyelia is reported in twins discordant for Chiari I malformation (Tubbs et al., 2004b). Defective collagen XI is demonstrated in a few cases (Melkoniemi et al., 2003). A majority of cases, however, are probably acquired as columnar watershed infarcts in the fetal brainstem, in which symmetrical tegmental infarcts occur in the pons and medulla oblongata (Sarnat, 2004b). Involvement of the trigeminal motor nuclei in the zone of infarction leads to denervation of masticatory muscles with deficient subsequent growth of the mandible and, by the time of birth, micrognathia, cleft palate and partial or complete ankylosis of the temporomandibular joint, the essence of the Pierre Robin sequence. Such tegmental infarcts also alter the development of the lower brainstem, as the hypoglossal nuclei often are involved and might predispose to a Chiari-like malformation. There is an 8.6% incidence of Chiari 1 malformation associated with neurofibromatosis 1, shown by several authors and not likely to be spurious and coincidental (Tubbs et al., 2004a). Chiari malformation with syringomyelia (more probably hydromyelia) may also occur in Noonan syndrome (Peiris and Ball, 1982; Colli et al., 2001; Holder-Espinasse and Winter, 2003). The reason for these associations of unrelated disorders is unknown. With the exception of the Pierre–Robin sequence, Noonan syndrome and neurofibromatosis 1, no other recognized dysmorphic syndrome is associated with Chiari malformations. Among known exogenous teratogens, with the exception of retinoic acid (vitamin A) and

deficiency of folic acid as discussed above, no chemicals, hormones, alcohol, opiates, amino acids in high concentration or drugs are known to produce Chiari malformations in animals or humans (Swaab and Boer, 2001).

6.5. Clinical correlates in Chiari malformations Though this present chapter is mainly focused on the pathogenesis of Chiari malformations, it is instructive to briefly review the clinical manifestations to put the pathological and imaging findings in the context of patient care. The three forms of Chiari malformation present differently, and the relationship between these forms and whether they are really a continuum or different disorders remains unresolved. 6.5.1. Diagnosis Imaging is the definitive means of diagnosis during life, and MRI is the best available method (Gabrielli et al., 1990), in part because it provides sagittal and parasagittal views of the brainstem and spinal cord. T1- and T2weighted images are satisfactory and special techniques such as diffusion-weighted imaging and functional imaging are not required, although they may provide information about altered CSF flow patterns. MRI also is an effective means of demonstrating hydromyelia in children (Lima et al., 2005). At times, the diagnosis of Chiari malformation may be made in utero by fetal ultrasonography, which can assess not only ventriculomegaly but also the clivus–supraoccipital angle to determine the shape and size of the fetal posterior fossa (D’Addario, 2004; D’Addario et al., 2001). Plain X-rays may sometimes be helpful in showing hypoplasia of the basioccipital bone and cranial base. They also demonstrate Lu¨ckenscha¨del or lacunar skull of the membranous bones of the cranial vault. The growth of the cranial base may be altered in children with even mild Chiari I malformations (Kountouri et al., 2004). Bony vertebral anomalies are an inconstant and infrequent feature, but basilar impression, atlas assimilation, atlantoaxial dislocation and Klippel–Feil syndrome are occasionally found in any of the Chiari types. Apart from imaging studies, abnormal eye movements characteristic of Chiari II malformations, particularly saccadic adaptation, also provide useful clinical data (Salman et al., 2005). Somatosensory evoked potentials also may be altered (Boor et al., 2004). EEG is of limited value, as Chiari malformations do not affect the cerebral cortex except secondarily due to hydrocephalus. No molecular markers in blood,

DISORDERS OF SEGMENTATION OF THE NEURAL TUBE: CHIARI MALFORMATIONS cultured fibroblasts or other tissues are available to confirm the diagnosis. 6.5.2. Symptoms and signs The principal symptoms and signs of Chiari malformations may be summarized as: a) neurological deficits related to the lumbosacral meningomyelocele, including motor and sensory impairment in the lower extremities from involvement of the cauda equina as well as spinal cord dysplasia, and flaccid neurogenic bladder; b) obstructive hydrocephalus, at the level of the fourth ventricle and foramen magnus or because of aqueductal stenosis or atresia, which may provoke vomiting, Parinaud syndrome, or setting sun sign in infants, and lethargy; c) nystagmus, abnormal saccades and other abnormal eye movements, including downbeat nystagmus and seesaw nystagmus; Chiari malformation is the cause of 3% of cases of internuclear ophthalmoplegia (Bolanõs et al., 2004) but palsies of the oculomotor, trochlear and abducens nerves are not typically found; d) cerebellar deficits, although these are sometimes minor because most of the vermis and lateral hemispheres are preserved; most patients have some degree of ataxia after achieving the age of walking; e) epilepsy is not a characteristic of Chiari malformations, unless the cerebral cortex is frankly dysplastic as in Chiari III malformation; increased intraventricular pressure alone is not epileptogenic. 6.5.3. Chiari I malformation This is the mildest form and is not associated with neural tube defects such as meningomyelocele. Clinical presentation is often delayed until later childhood or even adult life, being asymptomatic in infancy and prepubertal childhood. Symptoms result from either direct pressure on the medulla oblongata, which may protrude through the foramen magnum, although less than in Chiari II malformation, or from compression of the vertebral arteries or smaller vessels, causing ischemia of the lower medulla and upper cervical spinal cord. Hydrocephalus from aqueductal stenosis also may occur. Hydromyelia may remain relatively asymptomatic despite its dramatic evidence in spinal cord MRI. With obstruction of the foramen magnum, torticollis, opisthotonus and cervical transverse myelopathy may appear, with headache, vertigo, laryngeal paralysis, progressive cerebellar deficit and sometimes lower cranial nerve deficits that often are asymmetrical (Nohria and Oakes, 1991; Pascual et al., 1992). Pierre–Robin sequence may be associated (Tubbs and Oakes, 2004) probably from involvement of the motor trigeminal nucleus and deficient innervation of mus-

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cles of masfication that are needed for mandibular bony growth in late fetal life (Sarnat, 2004b).The mean age at presentation is 17.5 years; hydromyelia is seen in 65%, scoliosis in 30% and hydrocephalus in only 12% (Nohria and Oakes, 1991). 6.5.4. Chiari II malformation The most frequent of the Chiari malformations, Chiari II, is nearly always associated with lumbosacral meningomyelocele, hence is diagnosed at birth if not prenatally. The close association of Chiari II malformation with lumbosacral meningomyelocele was noted by several early investigators including Chiari (1891, 1896), but it was first well documented by Adams et al. (1941), who demonstrated the lesion by intraspinal injection of lipiodol. Surgical attention is required in the neonatal period to closure of open neural tube defects and the prevention of infection, and acute or subacute treatment of the associated obstructive hydrocephalus. Dysphagia is seen in 71% of infants, with aspiration in 12%, stridor in 59%, apneic spells in 29% and arm weakness in 53% (Vandertop et al., 1992). Hydrocephalus requiring shunt maintenance is a major problem in later infancy and childhood. Scoliosis management may be problematic in some children. 6.5.5. Chiari III malformation The most severe and also the rarest form, this consists of a neural tube defect not only at the caudal end of the spinal cord but also a posterior encephalocele that contains both posterior fossa tissues and supratentorial tissues, including most of the cerebellum and often also occipital cortex and sometimes part of the occipital horn of the lateral ventricle (Caldarelli et al., 2002). Rhombencephalosynapsis may complicate some Chiari III malformations (Schachenmayr and Friede, 1982). These children have all the problems faced by those with Chiari II malformations, and an additional problem of posterior encephalocele with supratentorial compartment involvement, hence they have the poorest prognosis for survival beyond the neonatal period and for severe neurological sequelae, including cortical visual, cognitive and intellectual impairment. 6.5.6. Treatment The management of children with Chiari malformations, with or without associated meningomyelocele, is complex and difficult but includes closure of open neural tube defects soon after birth, compensation for hydrocephalus, usually by shunting if the child is old enough to support a shunt, decompression of tight

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bony structures in the region of the foramen magnum, management of neurogenic bladder and treatment of neonatal feeding difficulties, respiratory insufficiency and apnea. In some cases, fetal surgery is now performed to begin the treatment of neural tube defects and relieve intracranial hypertension. The multifaceted and difficult treatment of this condition is beyond the scope of this chapter. 6.5.7. Non-Chiari aqueductal atresia and hydromyelia Both congenital stenosis or atresia of the cerebral aqueduct of Sylvius and hydromyelia also occur in the absence of hindbrain malformations characteristic of the Chiari malformations and in the absence of neural tube defects. A relation to the Chiari malformations is suggested by the presence of selectively upregulated vimentin in the ependyma of these dysplastic zones in such cases, similar to that seen in Chiari malformations; rosettes of blind tubules in the atretic aqueduct also show this change (Sarnat, 2004a). Ectopic expression of a homeobox segmentation gene could account for aqueductal stenosis and distortion of the central canal with or without Chiari malformation, at least in some cases.

6.6. Reclassification of the Chiari malformations In the recently proposed reclassification of CNS malformations based upon patterns of genetic expression, rather than on traditional descriptive morphogenesis or on specific genetic defects without regard to anatomy, the Chiari malformation is considered to be an intrinsic disorder of genetic programming of the brainstem, particularly one of segmentation with probable upregulation and ectopic expression of a still unidentified homeobox gene (Sarnat, 2000; Sarnat and FloresSarnat, 2001, 2004). The original anatomical classification of Chiari (1891, 1896) is still used, except that his Chiari IV malformation is now recognized as cerebellar hypoplasia unrelated to the other Chiari malformations; hence this designation is presently of historical interest only. Chiari I is the least severe form, with mainly downward displacement of the cerebellar tonsils, little or no macroscopic distortion of the brainstem and not associated with meningomyelocele. Chiari II is the most frequent and is nearly always a complication of meningomyelocele in the lumbosacral region or occasionally at a higher level of spinal cord; it involves macroscopic displacement of the lower medulla oblongata through the foramen magnum as well as of cerebellar tissue. Chiari

III malformation involves a posterior cephalocele containing posterior fossa contents but usually without a lumbosacral spina bifida. All three forms involve microscopic dysgenesis of the neural tissue (see above).

6.7. Historical note The malformation was briefly described by Cleland in 1883, who first documented and also illustrated a downward displacement of the cerebellar vermis, deformity of the medulla oblongata and the tectal plate (Cleland, 1883). Chiari’s descriptions in two papers of 1891 and 1896 were meticulously detailed neuropathological studies that remain as accurate today as when first published, and he acknowledged Cleland’s earlier recognition of this malformation. Arnold added a minor note in 1894 about a single infant with a sacral teratoma who also had a ‘ribbon-like’ cerebellar herniation extending to the midcervical level (Arnold, 1894); the site of origin of the cerebellar tissue and a description of the brainstem were not provided. Two of Arnold’s students, Schwalbe and Gredig, published a paper in 1907 in the same journal that Arnold had published his, attempting to ‘honor’ their professor by maliciously adding his name ahead of that of Chiari and implying that Arnold’s meagre contribution was equal to or better than that of Chiari, while ignoring Cleland altogether (Schwalbe and Gredig, 1907). Arnold himself remained silent about the undeserved imposition of his name, only many years later admitting that it was vanity and national pride that prevented him from speaking out. The historical injustice is now recognized, and the eponym of Arnold should be dissociated from the Chiari malformations.

6.8. Conclusion Most theories of pathogenesis of the Chiari malformations throughout the late 19th and 20th centuries were mechanical: traction; pulsion; oligo-CSF; crowding in the posterior fossa; birth trauma. A molecular genetic hypothesis is here proposed that explains many features not addressed by the mechanical theories: a) small posterior fossa because of hypoplasia of the basioccipital bone, derived from paraxial mesoderm rather than from neural crest, and programmed by the HOX family and other homeobox genes; b) intrinsic dysgeneses in the brainstem and cerebellum because of ectopic expression and imperfect hindbrain segmentation and rhombomere formation. The spinal cord is a single rhombomere (r8) and segmental hydromyelia and defective neural tube closure caudally also are consistent with a molecular genetic cause; c) ependymal abnormalities in restricted regions of the ventricular system, principally those with

DISORDERS OF SEGMENTATION OF THE NEURAL TUBE: CHIARI MALFORMATIONS neural dysplasias; d) little or no involvement of supratentorial forebrain structures in most cases, except for secondary effects of hydrocephalus or in Chiari III, in which a posterior encephalocele includes both infratentorial and supratentorial neural tissues; rare patients have hypoplasia of the corpus callosum. If the molecular genetic hypothesis is confirmed in further studies in progress, Chiari malformations may be reclassified in the scheme of CNS malformations as disorders of genetically programmed segmentation of the neural tube.

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Disorders of segmentation of the neural tube: agenesis of selective neuromeres HARVEY B. SARNAT* University of Calgary Faculty of Medicine and Alberta Children’s Hospital, Calgary, Alberta, Canada

7.1. Introduction and historical context After gastrulation and formation of the primitive streak, which establish the three principal axes of both the body of the embryo and its neural plate, the neural tube is formed by folding the neural plate. The next major event in development is segmentation of the mesoderm, with the formation of paired somites with their dorsal myotomes and dermatomes and ventral sclerotomes, the latter forming the primordial segmental vertebral bodies of the spinal column. Shortly thereafter, the neural tube achieves its segmentation with the creation of compartments that limit the extent of cellular migration in the longitudinal axis (Sarnat, 1992). Not all multicellular animals are segmented, even if they exhibit one or more of the primitive axes in their bodies. Examples include sponges, coelenterates (jellyfish, medusae and hydras) and roundworms (e.g. Ascaris). Platyhelminths (flatworms) show either internal segmentation, as with the ladder-like periodic commissures between the longitudinal neural cords of the freeliving turbellarians (e.g. planarians), or overt external segmentation, as with the parasitic cestodes (tapeworms). Some invertebrates, such as mollusks (e.g. clams, snails, octopus) do not exhibit segmentation as adults but show it in their larval stages of development. The majority of invertebrates show distinct segmentation both as embryos and as adults: annelids (e.g. earthworms, leeches), arthropods (e.g. lobsters, insects, spiders). The segmentation of vertebrate embryos, as well as of invertebrates, fascinated and perplexed biologists from the 18th century or earlier. In 1890, Professor C.F.W. McClure, President of the Royal Biological Society of the UK, stated in his address to the Society in

its annual meeting of that year: ‘The primitive segmentation of the vertebrate brain is a problem which has probably attracted as much of the attention of morphologists as any one of the great, unsettled questions of the day, and many views have been advanced.’ Beginning in the mid-1980s, an understanding of the segmentation of the vertebrate embryo in general, and of the neural tube in particular, advanced from the long period of descriptive histological morphogenesis to a new phase of an appreciation of genetic programming. Families of genes were identified that were committed to segmentation of the neural tube during this phase of development: Homeobox (HOX) genes, Wingless (Wnt), Engrailed (En) and Paired homeobox (Pax) genes as well documented examples. The HOX family is particularly important for segmentation and also is involved in the intermediate trajectories of developing long axonal pathways. This family consists of 38 distinct genes, of which 13 are expressed in the neural tube but each associated with some segments (rhombomeres, see below) and not others, in a precise and predictable pattern (Keynes and Krumlauf, 1994; Stern and Foley, 1998). Knockout mice lacking hoxA/hoxD gene function not only exhibit abnormalities in segmentation of the neural tube but also the programming of limb development because of defective segmentation of the paraxial mesoderm and for shh signaling in developing extremities (Kmita et al., 2005).

7.2. Neuromere formation With closure of the neural tube, a row of three distinct bulges appears at the rostral end, to become the prosencephalon, mesencephalon and rhombencephalon.

*Correspondence: H. B. Sarnat MD, FRCPC, Alberta Children’s Hospital, 2888 Shaganappi Trail NW, Calgary, Alberta T3B 6A8, Canada. E-mail: [email protected], Tel: þ1-403-955-7131, Fax: þ1-403-955-2922.

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They are initially continuous but become divided into compartments or segments. The rhombencephalon or hindbrain is soon divided into a more rostral metencephalon, which later becomes the rostral pons, and the myelencephalon, which becomes the caudal pons and medulla oblongata. Compartmental boundaries in the neural tube are both physical and chemical barriers to longitudinal cellular migration. The first transverse boundary to form in the neural tube is between the mesencephalic vesicle and the hindbrain, later to become the meso-metencephalic junction, also known as the midbrain–hindbrain boundary or isthmus. Other transverse junctions then form, both caudal and rostral to this initial boundary, and each perpendicular to the longitudinal axis. The mesencephalic–metencephalic junction is regarded by some authors to be a master boundary, specifically the midbrain–hindbrain organizer, required for the formation of other junctions as segmentation of the neural tube proceeds (Krauss et al., 1992; Danielian and McMahon, 1996; Joyner, 1996; Araki and Nakamura, 1999; Acampora et al., 2001; Rhinn and Brand, 2001). This conclusion is largely based upon embryonic mouse and chick studies. The meso-metencephalic boundary is the zone associated with a larger number of genes and transcription factors than any other neuromeric junctions (Sarnat and Menkes, 2000). The first gene expressed at this site is Pax2; other genes involved with mesencephalic and metencephalic formation, and that are expressed at this first boundary to initiate segmentation of the neural tube, include Pax5, Pax8, Otx1, Otx2, Gbx2, Nkx2.2 and Lmx1b; fibroblast growth factor 8 (FGF8) and other trophic factors also interact. Animal models that manipulate the expression of these other genes and factors do not produce dysgeneses that resemble the human cases and are often associated with additional malformations of other organ systems and eyes that human subjects do not exhibit. The neuromeres are associated with specific pharyngeal pouches (branchial arches in vertebrates with gills) and this correspondence is constant within a species, but there is a mild interspecific difference, particularly in simple vertebrates. Agenesis of the mesencephalon and metencephalon in humans, however, does not seem to impair the formation of other neuromeres in the neural tube and the borders between the compartments are well defined (see below). The genes crucial to the formation of the initiating meso-metencephalic junction are expressed even in amphioxus, even though further hindbrain segmentation does not occur or is rudimentary in this simple protochordate, indicating early evolution and conservation of a primordial feature in the programming of vertebrate brain development (Wada and Satoh, 2001).

The segments of the embryonic vertebrate brain, including the human brain, are invariable, but the total number recognized has undergone revision. Segments of the embryonic neural tube are called neuromeres. The most widely accepted concept is that there are eight hindbrain neuromeres, also known as rhombomeres (r1–r8), one mesencephalic neuromere (r0) and four prosencephalic neuromeres or prosomeres, two diencephalic and two telencephalic (Keynes and Lumsden, 1990; Guthrie, 1996; Wolpert, 1998; Rao and Jacobson, 2005). The most caudal neuromere, r8, includes the most caudal part of the medulla oblongata and the entire spinal cord. The spinal cord is not intrinsically a segmented structure, although different regions show unique characteristics, such as preganglionic parasympathetic neurons limited to the cervical and sacral zones and the column of Clarke with its neurons for ascending spinocerebellar axons found only in the thoracic and lumbar regions. The apparent segmentation of the spinal cord from its external appearance is really an illusion due to clustering of nerve roots imposed by the true segmentation of adjacent mesodermal structures of the vertebrae and myotomes. More recently, some investigators interpret their studies as showing more neuromeres, with the spinal cord being divided into four neuromeres, the midbrain into two and the forebrain into six (Puelles and Rubinstein, 2003). The transverse junctions between neuromeres are physical barriers to cellular migration, with a thin wall of extensive cellular processes resembling radial glia; these cells also secrete a chemical barrier, with molecules that repel cells attempting to migrate in the longitudinal axis (Guthrie et al., 1991; Mai et al., 1998). Cell adhesion is increased in the boundary junctions. Mitotic activity of the neuroepithelium is less frequent at junctions than in other sites within the neuromere. Though cells still divide at the boundaries, their nuclei remain near to the ventricle during mitotic cycling and move centrifugally very little, within their radial strand of cytoplasm, during the interkinetic gap phases (Guthrie et al., 1991). The normal function of boundaries between rhombomeres also depends upon the expression of transcription factor Oct (homologous with Pou2 of invertebrates) (Burgess et al., 2002) and bidirectional signaling mediated by Eph receptors and their ligands (Klein, 1999).

7.3. Agenesis of isolated neuromeres 7.3.1. Agenesis of the mesencephalon and metencephalon with cerebellar hypoplasia Congenital absence of the midbrain and upper pons, with cerebellar aplasia or hypoplasia, is a rare human

DISORDERS OF SEGMENTATION OF THE NEURAL TUBE malformation with only a few case reports (Wang et al., 1983; Robinson et al., 1993; Mamourian and Miller, 1994; Van Coster et al., 1998; Velioglu et al., 1998; Sarnat et al., 2002; Bednarek et al., 2005). Some of these have been assumed to be due to destruction of a previously well formed brainstem in late fetal life (Robinson et al., 1993) but the evidence for this conclusion is weak. In one case, an organoid nevus was associated and a neurocutaneous syndrome was suspected (Wang et al., 1983). Rarer still are cases of isolated agenesis of the cerebellum (Sebahattin et al., 2005). Complete isolated cerebellar agenesis probably does not occur; in most cases there remains a small amount of residual cerebellar tissue (Glickstein, 1994; Leestma and Torres, 2000; Boltshauser, 2004). In reporting two unrelated infants with agenesis of the midbrain and pons with global cerebellar hypoplasia, we suggested that this unique malformation (Figs. 7.1, 7.2) may result from defective expression of the homeobox gene Engrailed-2 (EN2), extrapolating from evidence from experimental murine models of knockout organizer genes (see below) (Sarnat et al., 2002). This evidence is confirmed by other independent authors (ten Donkelaar et al., 2007). Several genes identified in transgenic mice are essential to the formation of a midbrain neuromere. PAX2 is one of the first genes to be expressed, as early as in Hensen’s node before the neuroepithelium even differentiates (Nornes et al., 1992; Puschel et al., 1992; Rowitch and McMahon, 1995; Rowitch et al., 1999a; Sarnat, 2000). Inactivation of pax-2 results in agenesis of the cerebellum and posterior midbrain in mice, and the same human PAX2 mutation results in renal anomalies, colobomata of the eyes and developmental defects of the brain (Favor et al., 1996). The next Pax gene expressed in the mesencephalon is PAX5. Deletions of pax-5 alone have little effect on cerebellar development, but when a pax-5 knockout mouse is combined with a hemizygous (i.e. incomplete) deletion of pax-2, neither the cerebellum nor the inferior colliculi forms (Urbańek et al., 1997). Another gene essential to the specification of the hindbrain organizer (i.e. meso-metencephalic boundary) is Unplugged (gbx-2) (Wassarman et al., 1997; Liu et al., 1999; Rhinn and Brand, 2001). This gene is activated by fibroblast growth factor-8 (FGF8), which, if upregulated, transforms regions of the rostral brain of the mouse embryo into a hindbrain fate (Liu et al., 1999) by ectopic expression or reversal of the rostrocaudal gradient of segmentation. Gbx-null mutant mice often have an enlarged, rather than an aplastic midbrain, however, and agenesis of the cranial nerve nuclei that form in rhombomeres 1–3 (Acampora et al., 2001). The Orthodenticle (otx-1 and

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otx-2) genes in mice are also essential for mesometencephalic boundary formation, and interact with gbx and the hox genes, which are primordial for the further segmentation of the hindbrain, but otx genes also help to program the rostral neural tube and cerebral cortical defects occur in murine homozygous deletions (Acampora et al., 2001; Rhinn and Brand, 2001). Deficiencies in these molecular genetic factors, Gbx2 and Otx-1, -2) in mutant mice do not correspond to the anatomopathological findings in human cases; the extent of the neural tube defect is not limited to the meso-metencephalic boundary that fails to develop. Three organizer genes are now well documented as indispensable for the programming of both the mesencephalic neuromere and the first rhombomere (r1). R1 forms the metencephalon and provides all progenitor cells of the entire cerebellar cortex; the deep cerebellar nuclei derive from r2, however (Sarnat and Menkes, 2000). Wingless-1 (Wnt1) is the first of these to be expressed, and activates Engrailed-1 (En1) and Engrailed-2 (En2). Hemizygous (incomplete) deletions in the transgenic mouse embryo leads to no morphological abnormalities in the developing brain; homozygous (complete) deletions, by contrast, result in agenesis of the murine mesencephalon and metencephalon (McMahon et al., 1992; Wurst et al., 1994; Joyner, 1996; Mastick et al., 1996; Saint-Jeannet et al., 1997; Wassef and Joyner, 1997). Engrailed that is upregulated and ectopically expressed causes a rostral shift of the dimesencephalic boundary and transformation of the dorsal diencephalon into midbrain tectum, simultaneously repressing Pax6, a diencephalic marker that precedes the expression of mesencephalic genes; Engrailed thus defines the position of the dorsal dimesencephalic boundary by directly repressing the diencephalic fate (Araki and Nakamura, 1999). Despite its crucial role, the Engrailed-2 gene itself has a very simple molecular structure, with only two exons and one intron. Two pax-2/-5/-8 binding sites in en-2 are required for initiation of mesencephalic and hindbrain expression in mice, further complicating mesencephalic–metencephalic ontogenesis (Song and Joyner, 2000). Mice with wnt-1 or en-1 deletions also have total cerebellar aplasia, whereas en-2 deletions are less severe and cause cerebellar hypoplasia with abnormal foliation (Millen et al., 1994, 1995; Kuemerle et al., 1997; Goldowitz and Hamre, 1998). This constellation of agenesis of the midbrain and upper pons with cerebellar hypoplasia corresponds closely to the ontogenetic defects found in our human cases and, for this reason, we speculated that our patients had a mutation, deletion or altered expression of the EN2 gene (Sarnat et al., 2002). The abnormal rostral displacement of the small

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Fig. 7.1. Full-term male neonate with agenesis of the midbrain and upper pons (mesencephalon and metencephalon) with rostral displacement of a hypoplastic cerebellum. He died at 5 days because of apnea. (A) Sagittal MRI, T1 to show anatomy during life; (B) Cranial base after removal of brain. The fossae are formed. (C) A ventral midline cord of neural tissue connected the diencephalon with the caudal pons and passed through aberrant foramina in the midline of the clivus and contained the hypoplastic and fused corticospinal tracts. (D) Base of the brain showing normal olfactory bulbs and tracts, optic nerves and chiasm, infundibulum and absence of the midbrain and upper pons (arrow). The ventral frontal and temporal gyri appear normal. (E) dorsal view of hypoplastic cerebellum and lower brainstem. The vermis is poorly developed and the folia of the lateral hemispheres are focally malformed. (F) Abnormal villi of ependyma, with small connections between them, line the tissue facing the empty space where the midbrain should have been, further confirming that this is a developmental malformation and not the result of an infarct. Haematoxylin-eosin, 400. (Reproduced with permission from Sarnat et al. 2002.)

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Fig. 7.1. (Continued)

cerebellum in both human and murine mesometencephalic agenesis is consistent with a rostral shift of the dimesencephalic boundary. The timing of such a defect would be during neural tube segmentation, at about 4 weeks gestation. Hypoplasia of the dentate and inferior olivary nuclei denotes additional, though incomplete, involvement of rhombomere 2; partial involvement of an adjacent neural tube segment does not detract from the premise of a primary EN2 mutation. The ectopic expression of En-2 in Purkinje cells of mice also may interfere with late-onset sagittal banding patterns, which might explain the imperfect lamination of the cerebellar cortex in humans (Baader et al., 1999). One difference between the murine model and the human cases is that, in the mouse embryo, the diencephalon is anatomically continuous with the myelencephalon without an intervening space, as occurs in human cases; this difference may be due to the embryonic and early fetal ages at which the mice were examined. The reported human cases were full-term neonates who lived a short time after birth. A thin neural cord containing hypoplastic, fused corticospinal tracts and nests of disorganized neural parenchyma

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provides a thin anatomical continuity between rostral and caudal structures in human cases. Another neonate was described with MRI evidence of a very narrow or absent pons, preserved midbrain and global cerebellar hypoplasia, but the images do not closely resemble our cases and no pathological confirmation was available (Mamourian and Miller, 1994). The DNA-binding transcription factor forkhead/ hepatocyte nuclear factor-3b (HNF3b), is not normally expressed in the midbrain or hindbrain but in transgenic mice may become expressed ectopically by using an En-2 promotor/enhancer, and the resulting embryos exhibit cerebellar hypoplasia and absence of the inferior colliculi; HNF3b also suppresses the dorsalizing gene Pax-3 (Sasaki and Hogan, 1994). These might be additional factors involved in the malformations in human cases, but again invoke the homeobox gene EN2 that we proposed as primary in pathogenesis. Engrailed genes also regulate the neuronal or glial cell fates in the midline of the neural tube (Condron et al., 1994), hence might explain an apparent gliosis without implicating a mobilization of ‘reactive’ astrocytes as occurs with infarction. Other late developmental events are particularly associated with ectopic expression of En-1 in transgenic mice. These include cystic malformations of the posterior cerebellar vermis reminiscent of the Dandy–Walker malformation in humans (Rowitch et al., 1999b) and the regulation of axonal pathfinding by interneurons that project to motor neurons, by the interaction of En-1 with netrin-1 (Saueressig et al., 1999). Neither of these anomalies was evident in the human cases. Infants with mesometencephalic agenesis also have ectopic and dysplastic thyroid tissue. An antagonistic relation of Engrailed homeodomains to thyroid transcription factor 1 is suggested by some studies (Damante et al., 1996; Bruno et al., 2000). EN2 messenger RNA has been demonstrated by fluorescent in situ hybridization in the human brain at midgestation (Zec et al., 1997) but this type of demonstration is not feasible in formalin-fixed human brain tissue and even the normal gene would no longer be strongly expressed at term or postnatally. The EN2 gene also is implicated in some cases of autism in children with a normal brainstem by imaging (Nabi et al., 2005) but if EN2 is indeed involved with such cognitive functions, it would be a much later re-expression and in different regions of the neuraxis, more likely involving the telencephalon and possibly diencephalon than the brainstem or cerebellum. Finally, absence of the midbrain and upper pons with cerebellar hypoplasia cannot be due to infarction from a vascular anomaly because the territory does not correspond to any single vessel and basilar artery

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occlusion would certainly have caused extensive infarction both in the brainstem and in the occipital cortex, supplied by the posterior cerebral arteries; neither the brainstem caudal to the lesion nor the occipital lobes were abnormal at autopsy and the tissue margins adjacent to the empty space shows abnormal ependymal differentiation that would not be seen in ischemic lesions (Fig. 7.1F). 7.3.2. Ectopic expression in rhombomeres The term ectopic is used differently by geneticists from the way it is applied traditionally by morphologically oriented embryologists. In embryology, ectopic cells are abnormally displaced outside the organ of origin; heterotopic cells are displaced within the organ of origin. If the brain is the organ of origin, neurons within the leptomeninges are ectopic, whereas neurons arrested in migration in the subcortical white matter are heterotopic. Geneticists do not use the term heterotopic at all; they speak of ectopic expression of genes to indicate a gene being expressed in a segment in which it normally is not expressed. The upregulation of genes may cause them to be ectopically expressed and to mediate the development of structures that normally do not form in that site. For example, FGF8 can upregulate Gbx-2 in the embryonic mouse brain to transform the forebrain by duplicating hindbrain structures; this gene normally is expressed only in hindbrain (Liu et al., 1999). The HOX genes not only may cause ectopic expression in the hindbrain when upregulated but also are important in the guidance of axonal trajectories in the longitudinal axis, and this ectopic expression can cause reversal of the direction of descending pathways (Kessel, 1993). The classical experiments of Spemann and Mangold in amphibian gastrulas to produce a second, ectopic neural tube in an abnormal site on the body by transplanting the Spemann organizer (homologous with the primitive node of Hensen) is an example of induction (Spemann and Mangold, 1924) but is ectopic expression as we now understand it from a genetic perspective. Genetic experiments in insect larvae produce ectopic expression of upregulated genes that induce the formation of another leg in the place of an antenna or an eye in the place of a leg. Some of these anomalies are the result of disruption of the normal anteroposterior or posteroanterior gradients (Wolpert, 1998). Ectopic expression also accounts for duplication of structures within a segment. Only insects and angels acquire wings without sacrificing legs or arms. Ectopic expression accounts for the wing and leg appearing in the same segment in insects; angels have not been studied from an embryological perspective.

Ectopic expression is rare in the human neural tube and must be distinguished from abnormal or incomplete neuroblast migration. An example of the latter is the nodules of inferior olivary nuclei that may appear along the migratory trajectory from the rhombic lip of His. An example of probable true ectopic expression is the Chiari malformation, discussed in Chapter 6. Retinoic acid (the alcohol of which is vitamin A) causes upregulation of many genes, particularly those of the HOX family, ectopic expression and morphological dysgenesis in the neural tube of animals, by disrupting polarity gradients and segmentation (Durston et al., 1989; Momoi et al., 1990; Morriss-Kay et al., 1991; Ruberte et al., 1991). Infants born to women who take an excess of vitamin A early in gestation have a high incidence of neural tube defects and other CNS malformations, and these are at least partly related to induced ectopic expression. 7.3.3. Agenesis of selective prosomeres Different genes are expressed in the various forebrain neuromeres. For example, EMX1 is strongly expressed in the basal telencephalic neuromere that forms the caudate nucleus, putamen and part of the globus pallidus, but only weakly in the cerebral cortex. EMX2, by contrast, is expressed in the cerebral cortex but not in the ganglionic eminence that gives rise to the basal ganglia; EMX2 is defective in schizencephaly. The family of HOX genes that is so important in hindbrain segmentation is unexpressed in the forebrain. The OTX and PAX families are expressed in both hindbrain and forebrain. Isolated congenital absence of the basal ganglia is a rare malformation that may be due to downregulation of the MASH1 gene in the basal prosencephalic neuromere (Sarnat, 2000). The Distalless family of DLX1, DLX2 and DLX5 are also all focally expressed in the same basal telencephalic prosomere, but redundancy exists amongst them so that underexpression of one may be compensated by the others without clinical effects; DLX genes also are involved in craniofacial development. Children with absence of the basal ganglia do not have dysmorphic facies (Sarnat, 2000). The DLX genes are, therefore, unlikely candidates for the mutations or deletions causing this malformation.

7.4. Neural crest tissue during agenesis of neuromeres The mesencephalic neural crest tissue arises from the dorsal paramedian structures of the neural tube soon after its closure in the mesencephalic neuromere. It

DISORDERS OF SEGMENTATION OF THE NEURAL TUBE migrates forward into the head to form not only peripheral neural structures, such as the ciliary ganglion and nerve sheaths, but also many mesodermal structures of the membranous bone of the face, orbits and cranial vault, cartilage, blood vessels, connective tissue, most of the ocular globe (except the retina, choroid, lens and cornea) and melanocytes. Involvement of mesencephalic neural crest by the rostrocaudal gradient reaching the midbrain in holoprosencephaly results in midfacial hypoplasia (see Ch. 1). Infants with agenesis of the mesencephalon, as discussed above, should theoretically have a defect of mesencephalic neural crest and severe craniofacial dysmorphism. In fact, they do have minor abnormalities of their facies (Sarnat et al., 2002) but not major craniofacial malformations. The reason is probably that premigratory primordial neural crest cells have the ability to move to adjacent neuromeres to survive, and the neuromeric junctions that form barriers to cellular migration within the neural tube seem not to restrict the privileged neural crest cells. In normal development, no neural crest migrates from rhombomeres r3 or r5 because of the expression in these two rhombomeres of the gene EGR2 (human) or Krox-20 (mouse), which impedes neural crest migration outside the neural tube (Schneider-Manoury et al., 1993). The neural crest cells formed in these two rhombomeres simply move rostrally and caudally to the adjacent rhombomeres to migrate with the neural crest formed in those adjacent segments. It is for this reason that the maxillary and mandibular trunks of the trigeminal nerve (generated in r5 and r6, respectively) emerge from the brainstem fused in their proximal portions, whereas the ophthalmic trunk (generated in r4) emerges from the brainstem alone, its cells migrating from the segment in which they form. In mesometencephalic agenesis, therefore, the neural crest cells are preserved and emerge from a displaced site, probably in the caudal end of a diencephalic neuromere and/or caudally into r2. The ease of movement of mesencephalic primordial neural crest cells into the diencephalons is further facilitated by underexpression of EN2 because this gene helps define the position of the dimesencephalic boundary by suppressing a diencephalic fate of the neuromere in which it is expressed (Araki and Nakamura, 1999). 7.4.1. Secondary neurulation Primary neurulation is the dorsal closure of the neural folds to form the neural tube, beginning in the cervical region and reaching the anterior neuropore at 24 days gestation and the posterior neuropore at 28 days. However, the posterior neuropore is not located at the

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extreme caudal end of the neural tube and the lowest sacral portion of the spinal cord is formed from a solid core of neuroepithelium that extends rostrally from the filum terminale to the site of posterior neuropore closure. The central canal of this segment is a new structure that develops as a hollowing out within this core of tissue to form a cylinder, rather than as the vertical slit on the inner surface of the closing neural folds. This is called secondary neurulation (O’Rahilly and Mu¨ller, 1996). Ependymal cells that develop at the surface of the neuroepithelium of the closing neural tube thus meet the ependyma formed within the caudal core of the posterior spinal cord and, at the point where they meet, two central canals may be seen in the fetus, the caudal one ventral to the rostral one. The central canal of secondary neurulation may be dilated just caudal to the point where it overlaps the central canal of primary neurulation, thus resembling hydromyelia. The section of spinal cord with overlapping central canals of primary and secondary neurulation always show these two canals, one above the other in the vertical axis; they are never side by side, as in the duplication of the central canal from upregulation of genes having a dorsoventral gradient in the vertical axis (see Ch. 1). Secondary neurulation may be regarded as a form of segmentation and is a normal ontogenetic process, not a pathological one. Whether secondary neurulation plays a role in the pathogenesis of lumbosacral meningomyeloceles is speculative and uncertain.

7.5. Conclusion Some rare human malformations of the brain are due to defective expression of genes of segmentation of the neural tube. Human disorders include selective deletion of certain neuromeres. Examples are agenesis of the mesencephalon and metencephalon with cerebellar hypoplasia, probably due to a defect in EN2, and agenesis of the basal telencephalic nuclei (i.e. basal ganglia), possibly due to a defect in MASH1. Other disorders of segmentation may involve ectopic expression in the wrong neuromere: Chiari malformation is a probable example. Disorders of segmentation may impair neural crest formation and migration, particularly from the midbrain, resulting in abnormal craniofacial induction by the neural tube and leading to midfacial hypoplasia. Holoprosencephaly in which the rostrocaudal gradient of genetic expression reaches the mesencephalic neuromere is a prototype example. Secondary neurulation is a normal ontogenetic process posterior to the site of posterior neuropore closure and is a form of segmentation that might play a role in sacral neural tube defects.

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Keynes M, Krumlauf R (1994). Hox genes and regionalization of the nervous system. Annu Rev Neurosci 17: 109–132. Keynes R, Lumsden A (1990). Segmentation and the origin of regional diversity in the vertebrate central nervous system. Neuron 2: 1–9. Klein R (1999). Bidirectional signals establish boundaries. Curr Biol 9: R691–694. Kmita M, Tarchini B, Zakany J, et al. (2005). Early developmental arrest of mammalian limbs lacking HoxA/HoxD gene function. Nature 435: 1113–1116. Krauss S, Maden M, Holder N, et al. (1992). Zebrafish pax [b] is involved in the formation of the midbrain–hindbrain boundary. Nature 360: 87–89. Kuemerle H, Zanjani H, Joyner A, et al. (1997). Pattern deformities and cell loss in Engrailed-2 mutant mice suggest two separate patterning events during cerebellar development. J Neurosci 17: 7881–7889. Leestma JE, Torres JV (2000). Unappreciated agenesis of cerebellum in an adult. Case report of a 38-year-old man. Am Forens Med Pathol 21: 155–161. Liu A, Losos K, Joyner AL (1999). FGF8 can active Gbx2 and transform regions of the rostral mouse brain into a hindbrain fate. Development 126: 4827–4838. McClure CFW (1890). The segmentation of the primitive vertebrate brain. J Morphol 4: 35–56. McMahon AP, Joyner AL, Bradley A, et al. (1992). The midbrain-hindbrain phenotype of Wnt-1-/Wnt-1- mice results from stepwise deletion of engrailed-expressing cells by 9.5 days postcoitum. Cell 69: 581–595. Mai JK, Anderessen C, Ashwell KWS (1998). Demarcation of prosencephalic regions by CD15-positive radial glia. Eur J Neurosci 10: 746–751. Mamourian AC, Miller G (1994). Neonatal pontomedullary disconnection with aplasia or destruction of the lower brain stem: a case of pontoneocerebellar hypoplasia? Am J Neuroradiol 15: 1483–1485. Mastick GS, Fan C-M, Tessier-Lavigne M, et al. (1996). Early deletion of neuromeres in Wnt-1(-/-) mutant mice: evaluation by morphological and molecular markers. J Comp Neurol 374: 246–258. Millen KJ, Wurst W, Herrup K, et al. (1994). Abnormal embryonic cerebellar development and patterning of postnatal foliation in two mouse Engrailed-2 mutants. Development 120: 695–706. Millen KJ, Hui C-C, Joyner AL (1995). A role for En-2 and other murine homologues of Drosophila segment polarity genes in regulating positional information in the developing cerebellum. Development 121: 3935–3945. Momoi M, Yamagataa T, Ichihashi K, et al. (1990). Expression of cellular retinoic-acid binding protein in the developing nervous system of mouse embryos. Dev Brain Res 54: 161–167. Morriss-Kay GM, Murphy P, Hill RE, et al. (1991). Effects of retinoic acid excess on expression of Hox-2.9 and Krox-20 and on morphological segmentation in the hindbrain of mouse embryos. EMBO J 10: 2985–2995.

DISORDERS OF SEGMENTATION OF THE NEURAL TUBE Nabi R, Serajee FJ, Zhong H, et al. (2005). Association analyses of EN2 gene in autism. Ann Neurol 58 (suppl 9): S108. Nornes HO, Dressler GR, Knapik EW, et al. (1992). Spatially and temporally restricted expression of Pax-2 during neurogenesis. Development 38: 197–208. O’Rahilly R, Mu¨ller F (1996). In: Human Embryology and Teratology, 2nd edn,Wiley-Liss, New York, pp. 363–365. Puelles L, Rubinstein JL (2003). Forebrain gene expression domains and the evolving prosomeric model. Trends Neurosci 26: 469–476. Puschel AW, Westerfield M, Dressler G (1992). Comparative analysis of Pax-2 protein distributions during neurulation in mice and zebrafish. Mech Dev 38: 197–208. Rao MS, Jacobson M (2005). Developmental Biology, 4th edn, Kluwer Academic/Plenum Publishers, New York, pp. 51–54. Rhinn M, Brand M (2001). The midbrain–hindbrain boundary organizer. Curr Opin Neurobiol 11: 34–42. Robinson RO, Trounce JQ, Janota I, et al. (1993). Late fetal pontine destruction. Pediatr Neurol 9: 213–215. Rowitch DH, Danielian PS, McMahon AP, et al. (1999a). Cystic malformation of the posterior cerebellar vermis in transgenic mice that ectopically express Engrailed-1, a homeodomain transcription factor. Teratology 60: 22–28. Rowitch DH, Kispert A, McMahon AP (1999b). Pax2 regulatory sequences that direct transgene expression in the developing neural plate and external granule cell layer of the cerebellum. Dev Brain Res 117: 99–108. Rowitch DH, McMahon AP (1995). Pax-2 expression in the murine neural plate preceeds and encompasses the expression domains of Wnt-1 and En-1. Mech Dev 52: 3–8. Ruberte E, Dolle P, Campbon P, et al. (1991). Reginoid acid receptors and cellular retinoid binding proteins. Their differential pattern of transcription during early morphogenesis in mouse. Development 111: 45–60. Saint-Jeannet J-P, He X, Varmus HE, et al. (1997). Regulation of dorsal fate in the neuraxis by Wnt-1 and Wnt-3a. Proc Natl Acad Sci USA 94: 13713–13718. Sarnat HB (1992). Cerebral Dysgenesis. Embryology and Clinical Expression, Oxford University Press, NewYork. Sarnat HB (2000). Molecular genetic classification of central nervous system malformations. J Child Neurol 15: 675–687. Sarnat HB, Menkes JH (2000). How to construct a neural tube. J Child Neurol 15: 110–124. Sarnat HB, Benjamin DR, Siebert JR, et al. (2002). Agenesis of the mesencephalon and metencephalon with cerebellar hypoplasia: putative mutation in the EN2 gene – report of 2 cases in early infancy. Pediatr Devel Pathol 5: 54–68. Sasaki H, Hogan BLM (1994). HNF-3b as a regulator of floor plate development. Cell 78: 103–115. Saueressig H, Burrill J, Goulding M (1999). Engrailed-1 and Netrin-1 regulate axon pathfinding by association interneurons that project to motor neurons. Development 126: 4201–4212.

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Schneider-Manoury S, Topilko P, Seitanidou T, et al. (1993). Disruption of Krox-20 results in alteration of rhombomeres 3 and 5 in the developing hindbrain. Cell 75: 1199–1214. Sebahattin C, Zafeiriou DI, Boltshauser E, et al. (2005). Clinical presentation of cerebellar agenesis. Eur J Paediatr Neurol 9: 231(abstract). Song D-L, Joyner AL (2000). Two Pax2/5/8-binding sites in Engrailed-2 are required for proper initiation of endogenous mid-hindbrain expression. Mech Dev 90: 155–165. ¨ ber Induktion von EmbrySpemann H, Mangold H (1924). U onalanlagen durch Implantation aftfremder Organisatoren. Wilhelm Roux Arch Entwick 100: 599–638. Stern CD, Foley AC (1998). Molecular dissection of Hox gene induction and maintenance in the hindbrain. Cell 94: 143–145. ten Donkelaar HJ, Lammens M, Cruysberg JRM, et al. (2007). Development and developmental disorders of the brainstem. In: HJ ten Donkelaar, M Mammens, A Hori (Eds.), Clinical Neuroembryology: Development and Developmental Disorders of the Human Central Nervous System. Springer-Verlag, Berlin. Urbańek P, Fetka I, Meisler MH, et al. (1997). Cooperation of Pax2 and Pax5 in midbrain and cerebellum development. Proc Natl Acad Sci USA 94: 5703–5708. Van Coster RN, De Praeter CM, Vanhaesebrouck PJ, et al. (1998). MRI findings in a neonate with cerebellar agenesis. Pediatr Neurol 19: 139–142. ¨ zmenoglu M (1998). Cerebellar Velioglu SK, Kuzeyli K, O agenesis: a case report with clinical and MR imaging findings and a review of the literature. Eur J Neurol 15: 503–506. Wada H, Satoh N (2001). Patterning the protochordate neural tube. Curr Opin Neurobiol 11: 16–21. Wang P, Maeda Y, Izumi T, et al. (1983). An asssociation of subtotal cerebellar agenesis with organoid nevus: a possible new variety of neurocutaneous syndrome. Brain Dev 5: 506–508. Wassarman KM, Lewandoski M, Campbell K, et al. (1997). Specification of the anterior hindbrain organizer is dependent on Gbx2 gene function. Development 124: 2923–2934. Wassef M, Joyner AL (1997). Early mesencephalon/metencephalon patterning and development of the cerebellum. Perspect Dev Neurobiol 5: 3–16. Wolpert L (1998). Principles of Development, Oxford University Press, Oxford, pp. 117–119142–169. Wurst W, Auerback AB, Joyner AL (1994). Multiple developmental defects in Engrailed-1 mutant mice: an early mid-hindbrain deletion and patterning defects in forelimbs and sternum. Development 120: 2065–2075. Zec N, Rowitch DH, Bitgood MJ, Kinney HC (1997). Expression of the homeobox-containing genes EN1 and EN2 in human fetal midgestational medulla and cerebellum. J Neuropathol Exp Neurol 56: 236–242.



Cerebellar hypoplasias EUGEN BOLTSHAUSER* Division of Pediatric Neurology, University Children’s Hospital, Zurich, Switzerland

8.1. Introduction 8.1.1. Definition The term ‘cerebellar hypoplasia’ is descriptive, denoting reduced cerebellar volume, while cerebellar shape is (near) normal. Additional attributes can clarify (e.g. symmetric/asymmetric; midline/global). Regarding separation of hypoplasia from atrophy, see neuroimaging. 8.1.2. Cerebellar hypoplasias: a heterogeneous group Cerebellar hypoplasias are a heterogeneous group of conditions. As already pointed out by Sarnat (1992) and Barth (1993), cerebellar hypoplasia can result from: prenatal infection (in particular cytomegalovirus); prenatal exposure to teratogens; chromosomal aberrations; metabolic disorders; genetic (isolated) hypoplasias; genetic (complex) malformations; migration disorders; some forms of congenital muscular dystrophies; and pontocerebellar hypoplasias. Therefore, cerebellar hypoplasia can result from primary genetic causes as well as from secondary (i.e. prenatally acquired) events. The distinction between primary (developmental) processes and secondary (disruptive) lesions is of course of utmost importance for pathogenetic considerations and genetic counseling. 8.1.3. Neuroimaging Neuroimaging often does not allow a distinction to be made between primary and secondary cerebellar hypoplasia: a similar MRI pattern may be found in pontocerebellar hypoplasia type 2, congenital disorders of glycosylation and cerebellar injury found in extremely premature infants. In theory the distinction of hypoplasia

from atrophy is not difficult: in hypoplasia the fissures are of normal size compared with the folia, while the fissures (interfolial spaces) are enlarged in atrophy. In practice the distinction is not always straightforward – in patients with genetically determined congenital cerebellar ataxia, enlarged sulci are commonly seen (Fig. 8.1). In addition atrophy can be superimposed on hypoplasia, as documented in congenital disorders of glycosylation. In general the correlation between neuroimaging findings and clinical signs is often poor: for instance, in congenital ataxia the degree of cerebellar signs and/ or cognitive impairment does not correlate to the degree of hypoplasia (Fig. 8.2). In genetic causes of cerebellar hypoplasia there is often considerable variability (often intrafamilial), both in neuroimaging and in clinical phenotype. Similar, if not identical, imaging pattern are known for multiple syndromes (e.g. the cerebellooculo-renal syndromes); patients cannot be separated based on posterior fossa imaging alone. 8.1.4. Clinical manifestations: cerebellum and cognition Different forms of cerebellar hypoplasia differ in their clinical presentation which may even cover a broad phenotypic spectrum. In general children with cerebellar hypoplasia present with early muscle hypotonia, sometimes mimicking a neuromuscular disorder. They have developmental delay, not only in motor skills and at a later stage show classical cerebellar signs. They have a high prevalence of cognitive impairment. In the last 15 years it has become increasingly clear that the cerebellum contributes to nonmotor functions. This was first shown in adults with acquired focal cerebellar infarcts (Schmahmann and Sherman, 1998). The concept of nonmotor cerebellar

*Correspondence to: Eugen Boltshauser MD, Division of Pediatric Neurology, University Children’s Hospital, Steinwiesstrasse 75, CH-8032 Zurich, Switzerland. [email protected] Tel: þ41-44-266-7330, Fax : þ41-44-266-7163.

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Fig. 8.1. MRI ((A) Sagittal T2-weighted, (B) Coronal T2-weighted) in an 18-month-old boy with developmental delay and ataxia, diagnosed as nonprogressive congenital ataxia with static follow-up. Note small dimension of vermis with normal lobulation and increased interfolial spaces of vermis and cerebellar hemispheres.

agenesis, Joubert syndrome (Steinlin et al., 1997, 1998b, 2003; Zafeiriou et al., 2004). Schmahmann and Sherman (1998) coined the term cerebellar cognitive affective syndrome, in view of the following observations:

Disturbance of executive function, including poor planning

Visuospatial disorganization and impaired visualspatial memory

Personality change with blunting of affect or disinhibited and inappropriate behavior

Difficulty with interpreting and producing logical sequences

Language difficulties including dysprosody, mild anomia, and agrammatism. 8.1.5. Development of the cerebellum

Fig. 8.2. MRI (T2-weighted) in a 9-year-old girl with familial (three siblings affected) nonprogressive cerebellar ataxia. Note normal cerebellar anatomy.

functions was also documented in children for acquired (e.g. benign cerebellar tumors) as well as for primary cerebellar disorders, such as congenital ataxia, cerebellar

The morphogenesis and histogenesis of the cerebellum are very complex. A large number of genetic cascades are involved; about 60 spontaneous mice mutations are known to affect the cerebellum and recent insight was gained from the study of mouse knockouts (for reviews see Wang and Zoghbi, 2001; Wingate, 2001; Chizhikov and Millen, 2003; ten Donkelaar et al., 2003). The development of the posterior fossa begins shortly after neural tube closure when the primary brain vesicles (prosencephalon, mesencephalon,rhombencephalon)form along the anterior–posterior axis. Between 3 and 5 weeks gestation, the neural tube bends at the cranial and

CEREBELLAR HYPOPLASIAS cervical flexures and the rhombencephalon subdivides into eight rhombomeres. The cerebellum arises entirely from rhombomere 1. At the midbrain–hindbrain boundary a transverse patterning center, called the isthmus organizer, regulates the early development of the mesencephalon and the rostral part of the rhombencephalon. This organizer plays an important role in establishment of the anterior limit of the cerebellar territory. The isthmus organizer is dependent by the interaction, or reciprocal repression, of otx-2 and gbx-2 (Wang and Zoghbi, 2001). otx-2 and gbx-2 also regulate the expression of fgf-8 (fibroblast growth factor 8). fgf-8 is involved in regulating the various genes expressed in the mid- and hindbrain regions (Reim and Brand, 2000). Mutant mice with a reduced level of fgf-8 expression have a severe patterning defect usually affecting the cerebellum. Across the isthmus other genes are expressed, in particular the homeobox genes en-1 and en-2 and the paired box genes pax-2 and pax-5. McMahon et al. (1992) demonstrated that knockout mice of wnt-1, en-1 or en-2 produced agenesis of the mesencephalic rhombomere and rh1 as well as total cerebellar hypoplasia with wnt-1 and en-1 null affects, or global cerebellar hypoplasia with en2 when this gene was abolished. A nearly identical human malformation was observed in two unrelated infants by Sarnat et al. (2002). The rhombic lip develops between the fourth ventricle and the roof plate in the metencephalon. Along the anterior-posterior axis the rhombic lip extent from the first rhombomere to the eighth. The lower rhombic lip gives rise to the pontine nuclei and the inferior olivary nucleus. Cells from the upper rhombic lip form the external germinal (or granular) layer (Wingate, 2001). Several genes are involved in granule cell generation and migration, and Purkinje cell migration and maintenance (reviewed in Wang and Zoghbi, 2001). A few human mutations are known that involve these processes. Mutation of the reelin gene (localized on human chromosome 7q22) cause autosomal recessive lissencephaly with cerebellar hypoplasia. Fetal and neonatal MRI has allowed cerebellar morphometric normative data to be obtained and the development of the archicerebellum and neocerebellum, appearance of dentate nuclei, cerebellar fissuration and myelination of white matter tracts to be recognized (Triulzi et al., 2005). 8.1.6. Classification of cerebellar malformations Future classifications may be based on improved knowledge of embryonic cerebellar development and progress in molecular genetics and biology. At present morphology (neuroimaging) based classifications are used (Patel and Barkovich, 2002). However this classification includes malformations and (at least in part) disruptions.

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Because there is considerable variability within a given condition, patients may even be assigned to different subclasses. In this context we follow the ‘traditional’ categorization of cerebellar malformations into a) predominantly midline with mainly vermis involvement, b) involvement of both vermis and hemispheres (global hypoplasia) and c) pontocerebellar hypoplasias. Assignment to one of these broad categories is in individual situations and particular syndromes sometimes arbitrary. I briefly discuss first unilateral cerebellar hypoplasia and cerebellar agenesis, highlighting the potential difficulty in separating malformations from disruptions.

8.2. Unilateral cerebellar hypoplasia (aplasia) Unilateral cerebellar aplasia refers to unilateral absence of one cerebellar hemisphere. Patel and Barkovich (2002) used the term ‘one hemisphere hypoplasia’. These are descriptive terms lacking pathogenetic explanation. Strong (1915) was probably the first to report a detailed post-mortem description of an unsteady and retarded 3-year-old girl, who died of measles and was found to have ‘unilateral cerebellar agenesia’. He drew attention to associated secondary changes, namely hypoplasia of ipsilateral middle and superior cerebellar peduncles and superior colliculus, and highly asymmetric pons with absent contralateral olivary body – features recognizable with modern imaging. Subsequently described cases were either incidental findings or associated with developmental delay, mostly reporting single observations (for review see Boltshauser et al., 1996). We have recently seen three further infants with unilateral cerebellar aplasia, presenting with developmental delay. This may well represent a selection bias. Experience with a larger cohort of children followed for prolonged time is not available. Recent experience with prenatal ultrasound (Robins et al., 1998) and fetal magnetic resonance imaging (MRI) have confirmed that unilateral cerebellar aplasia is of prenatal (and not perinatal) origin, it has repeatedly been documented as early as 20–24 weeks of gestation (personal observations, based on imaging provided by colleagues). There is no imaging evidence of hemorrhage but the unilateral absence of one cerebellar hemisphere is also not characteristic of a vascular territory. Hence, the pathogenesis is speculative. It may well be that unilateral cerebellar aplasia represents a prenatally acquired lesion (i.e. a disruption) and not a true malformation. It is conceivable that bilateral unilateral cerebellar aplasia (whatever its underlying mechanism) leads to cerebellar agenesis.

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8.3. Cerebellar agenesis A literature review reveals that the term ‘cerebellar agenesis’ is mostly inappropriate, as the majority of patients had considerable cerebellar tissue left, often asymmetric, as found at post-mortem or by neuroimaging (for recent review and report of one patient see Zafeiriou et al., 2004). The same applies to the five children reported by Gardner et al. (2001) as ‘neartotal absence of the cerebellum’. I consider these findings to be disruptions. Complete (total) cerebellar aplasia is obviously not compatible with life, as it has never been documented in living subjects. We suggested (Zafeiriou et al., 2004) that the designation ‘cerebellar agenesis’ should only be applied to patients with minute cerebellar tissue, usually corresponding to remnants of the lower cerebellar peduncles, anterior vermal lobules and flocculi. Marked pontine hypoplasia is a constant MRI finding. Only few such patients were reported, the best studied patient being described by Richter et al. (2005). All patients were symptomatic but achieved independent walking; all had cognitive impairment of various degrees. So far, all patients with isolated (nonsyndromic) cerebellar aplasia were sporadic, while rare instances of syndromic cerebellar aplasia are on record (Hoveyda et al., 1999). The pathogenesis of cerebellar aplasia is not clear and it is probably not uniform. It is conceivable that cerebellar aplasia is representing one end of the spectrum of cerebellar disruptions. Genetic factors may

be responsible. In a mouse model null mutations for Engrailed-1 led to perinatal lethality accompanied by near-total absence of the cerebellar and caudal midbrain structures (Bilovocky et al., 2003).

8.4. Cerebellar hypoplasias with prominent midline (vermis) involvement 8.4.1. Dandy–Walker malformation There is considerable confusion in the medical literature about terminology, definition, diagnostic criteria and classification of Dandy–Walker malformation (DWM) (Fig. 8.3). In this review I prefer to distinguish ‘classical’ DWM from other related entities (for review see Klein et al., 2003; Parisi and Dobyns, 2003). Classical DWM has the following features: Consistent

Medium posterior fossa cyst widely communicating with the fourth ventricle

Hypoplasia of cerebellar vermis, which is elevated and upwardly rotated

Anterolateral displacement of normal appearing cerebellar hemispheres Inconsistent

Upwards displacement of the tentorium Enlargement of the posterior fossa Lack of patency of the foramina of Luschka and/or Magendie

Hydrocephalus.

Fig. 8.3. MRI ((A) Sagittal T2-weighted, (B) Coronal T2-weighted) in a 5-month-old boy with a congenital heart malformation, revealing Dandy–Walker malformation: ‘Cystic’ midline dilation of fourth ventricle, cerebellar vermis hypoplastic, elevated and rotated. There is no hydrocephalus.

CEREBELLAR HYPOPLASIAS I agree with the recommendation made by Parisi and Dobyns (2003) that the term ‘Dandy–Walker variant’ should be abandoned in view of its variable definition, lack of specificity and confusion with classic DWM. The same applies to the terms ‘Dandy–Walker complex’ and ‘Dandy–Walker continuum’. Any other cystic infratentorial lesion must be separated from DWM, in particular retrocerebellar arachnoid cysts, mega-cisterna magna and Blake’s pouch, although some authors consider that Blake’s pouch is part of the DWM spectrum (Calabro et al., 2002). The often-quoted prevalence of DWM (in the order of 1:30 000 births) is mainly based on earlier neurosurgical series and is likely to be a gross underestimate (Parisi and Dobyns, 2003). There is considerable heterogeneity of DWM. It may occur as part of a defined syndrome, many of these being autosomal recessive in inheritance. DWM has also been reported in a variety of chromosomal anomalies including trisomy 9, 13, 18, triploidy, and several partial duplications and deletions (Bordarier and Aicardi, 1990; Parisi and Dobyns, 2003). Most children with DWM are sporadic observations but the recurrence risk for parents of an affected proband is in the order of 1–5%. The pathogenetic mechanism of DWM is still speculative and poorly understood. Recently, Grinberg et al. (2004) have identified seven children with DWM and de novo interstitial deletions of chromosome 3q24–3q25.33. This region includes two zinc finger genes, ZIC1 and ZIC4. Mice with a heterozygous deletion of these two linked genes have DWM-like hypoplasia, suggesting that heterozygous loss of ZIC1and ZIC4 is causing DWM in individuals with 3q deletion. Patients with DWM have an increased frequency of congenital heart defects, cleft lip and/or palate, and facial hemangiomas. Of greater prognostic importance are associated brain malformations such as occipital encephalocele, polymicrogyria and heterotopia. A significant proportion (30%) of children with DWM have dysgenesis of the corpus callosum. Boddaert et al. (2003) found in a review of 20 patients that children with good intellectual outcome had normal vermian lobulation and normal corpus callosum. It is well known from earlier series that a considerable proportion (40–50%) of children with DWM have impaired cognitive abilities. This is not due to hydrocephalus and/or increased intracranial pressure but is now viewed within the context of ‘cerebellum and cognition’ (Schmahmann and Sherman, 1998). Presentation in about 80% of children with DMW is in infancy or early childhood with macrocephaly/ hydrocephalus. With the increasing frequency of

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prenatal diagnosis a considerable proportion of DMW is now diagnosed prenatally. 8.4.2. Cerebello-oculo-renal and related syndromes A number of syndromes involve cerebellum (vermis), retina, kidneys and may display additional manifestations, in particular coloboma, polydactyly, hepatic fibrosis, other brain abnormalities (polymicrogyria, encephalocele). Various constellations in different expressions with often marked intrafamilial variability occur. Delineation of these syndromes is still debated and has to be reconsidered in the future light of molecular genetic insights. There is considerable overlap clinically and in neuroimaging. In this context I first describe ‘pure Joubert syndrome’ followed by related syndromes. 8.4.2.1. Joubert syndrome Joubert syndrome was first reported in 1969 in a consanguineous sibship with four affected siblings (Joubert et al., 1969). Subsequent observations confirmed autosomal recessive inheritance. An autopsy study revealed extensive brainstem abnormalities and a lack of pyramidal tract crossing (Friede and Boltshauser, 1978). In the 1990s Maria et al. (1997) drew attention to the molar tooth sign on neuroimaging but it later became obvious that the molar tooth sign was not unique to Joubert syndrome (Satran et al., 1999). 8.4.2.1.1. Clinical presentation of ‘pure’ Joubert syndrome Consistent findings are early muscle hypotonia, developmental delay, ataxia and cognitive impairment (rare exceptions); the molar tooth sign is now considered an essential inclusion criterion. Frequent findings are abnormal breathing pattern (tachypnea – apnea), retinal dystrophy (ranging from mild impairment to congenital blindness) and oculomotor apraxia. Rarely observed findings (single cases) are congenital cataract, congenital hydrocephalus and short stature. In view of recent experience it is not yet clear how often nephronophthisis occurs in Joubert syndrome and whether polydactyly, liver fibrosis and colobomas are potential features (detailed description of presentation and outcome in Steinlin et al. 1997; Fennell et al., 1999; Maria et al., 1999). 8.4.2.1.2. Neuroimaging The molar tooth sign is now considered an essential diagnostic feature. Characteristic MRI findings are shown in Fig. 8.4 and Table 8.1.

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Fig. 8.4. MRI ((A) Sagittal T2-weighted, (B, C) Axial T2-weighted) in a 12-year-old boy with Joubert syndrome presenting with cognitive impairment, truncal ataxia and oculomotor apraxia. Note small dysplastic upper vermis, normal pontine prominence. The molar tooth sign on axial views is formed by deep interpeduncular fossa and stretched and thickened superior cerebellar peduncles.

8.4.2.1.3. Molecular genetics A first locus was established to 9q34.3 by linkage analysis in consanguineous Arab families (Saar et al., 1999). A second locus was independently mapped by two groups to 11p11.2–q12.3 (Keeler et al., 2003; Valente

et al., 2003). A third locus was established to 6q23 (Lagier-Tourenne et al., 2004). In a small fraction of patients, deletions in the NPHP1-gene were found (Parisi et al., 2004). The gene on chromosome 6 was identified (AHI1, ‘Abelson helper integration-1’) and first

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Table 8.1

8.4.2.4. Vermis hypoplasia in oculomotor apraxia

Characteristic MRI findings in Joubert syndrome

Isolated vermis hypoplasia has been seen in congenital oculomotor apraxia (Cogan) (COMA) (Fig. 8.5). Harris et al. (1998) reported siblings with COMA, one with, the other without vermis hypoplasia. This observation underlines the intrafamilial variability often seen in cerebellar (but also other) disorders. In our experience, vermis hypoplasia is an inconstant feature of COMA.

Axial view

Coronal view Sagittal view Parasagittal view

Deep interpeduncular fossa Thickened superior cerebellar peduncles Narrow isthmus Batwing-shaped fourth ventricle Vermis clefting Vermis clefting Rudimentary dysplastic vermis Small dysplastic vermis Thin isthmic region Thickened superior cerebellar peduncles

mutations were reported (Dixon-Salazar et al., 2004; Ferland et al., 2004). Remarkably, the AHI1 gene product is particularly expressed in neurons that give rise to the crossing axons of the corticospinal tract and superior cerebellar peduncles – notably tracts known not to cross in Joubert syndrome.

8.4.2.5. Vermis hypoplasia as part of other syndromes and malformations Vermis hypoplasia is a nonspecific finding that can occur in many defined syndromes, e.g. Meckel–Gruber, Aicardi, Smith–Lemli–Opitz. It can occur in many ‘private syndromes’ or associated with cerebral malformations and cerebellar dysplasias (Bordarier and Aicardi, 1990; Takanashi et al., 1999; Soto-Ares et al., 2000). In individual situations the diagnostic significance of vermis hypoplasia is often not conclusive. 8.4.2.6. Vermis hypoplasia and autism

8.4.2.2. Joubert-related syndromes (other syndromes with the molar tooth sign) The molar tooth sign was found in the following syndromes (Gleeson et al., 2004):

Dekaban–Arima syndrome Senior–Loeken syndrome COACH syndrome (cerebellar vermis hypoplasia,

oligophrenia, ataxia, coloboma, hepatic fibrosis) Varadi–Papp syndrome (oro-facial-digital type VI) Malta syndrome Joubert syndrome and retinal dystrophy Joubert syndrome and polymicrogyria.*

There are controversial reports on the relationship between cerebellar (particularly vermal lobules VI and VII) hypoplasia and autism. Based on quantitative MRI measurements, Schaefer et al. (1996) concluded that vermis hypoplasia was a nonspecific finding in many genetic syndromes, many without autistic behavior (e.g. Usher syndrome, mental retardation without autistic behavior). Vermis hypoplasia is not a neuroanatomical marker for autism.

The molar tooth sign has also been observed in Meckel–Gruber syndrome (personal observation). 8.4.2.3. ‘Joubert-like syndromes’ with vermis hypoplasia (but without the molar tooth sign) Janecke et al. (2004) reported two affected children and one fetus within a consanguineous sibship with cerebellar vermis hypoplasia, morning glory disc anomaly, cystic dysplastic kidneys, developmental delay and abnormal breathing pattern but no evidence of hepatic involvement. The molar tooth sign was not present. The authors suggest a distinct syndrome, closely resembling COACH syndrome. Kroes et al. (2005) reported X-linked cerebral, cerebellar and colobomatous anomalies with features overlapping with Joubert syndrome. *

AHI1 mutation established in the meantime.

Fig. 8.5. MRI (coronal T2-weighted) in an 11-year-old boy with oculomotor apraxia (Cogan) and truncal ataxia, revealing cerebellar vermis ‘clefting’ (arrows). No molar tooth sign on axial MRI (not shown).

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8.5. Global cerebellar hypoplasia Global cerebellar hypoplasia refers to involvement of both vermis as well as cerebellar hemispheres. 8.5.1. Nonprogressive congenital ataxia (with or without cerebellar hypoplasia) In our terminology we refer to nonprogressive congenital ataxia for a group of patients without evidence of prenatal infection, obvious perinatal insult, brain malformation other than cerebellar hypoplasia, defined syndromal disorder or postnatally acquired neurological disease (Steinlin et al., 1998b). Even in nonconsanguineous families, more than one sibling may be affected, suggesting a genetic basis. It became obvious that there was considerable intrafamilial variability regarding neuroimaging, with some individuals showing normal MRI findings (Fig. 8.2). Clinical presentation of children with nonprogressive congenital ataxia is characterized by early hypotonia, occasionally even suggesting a neuromuscular disorder, with marked delay of developmental milestones. Formal cerebellar signs (truncal ataxia, intention tremor, dysarthria) become apparent at a later (preschool) stage. In the long term cognitive impairment is seen in a majority of patients, most being incapable of proper vocational training (Steinlin et al., 1998b, 1999). These observations were confirmed in a large cohort studied by Wassmer et al. (2003). Remarkably these patients have a higher frequency of seizures (Parmeggiani et al., 2003), microcephaly, autistic behavior and late-onset dystonia. Cognitive impairment can be interpreted in the context of the cognitive affective cerebellar syndrome (Schmahmann and Sherman, 1998). Imaging in nonprogressive congenital ataxia is variable, as outlined above. In our own experience, as well as in literature reports, not only may the cerebellum be of smaller volume, but markedly enlarged interfoliar spaces can be present (Fig. 8.1), making differentiation from cerebellar atrophy difficult on the basis of a single study (Margari et al., 2004). In one of our families with three similarly affected children with nonprogressive congenital ataxia, MRI was normal (Fig. 8.2). Familial occurrence has been observed repeatedly (see Steinlin et al., 1998b; Margari et al., 2004) but the genes involved are not yet elucidated. In a large, inbred Lebanese family linkage to chromosome 9q (MIM 213200) (Me´garbane´ et al., 1999; Delague et al., 2001) was established. In Cayman Island ataxia, linkage to chromosome 19 (Nystuen et al., 1996) was found. The gene has been subsequently identified (Caytaxin, MIM 608 179). A mouse model (entla) with

ataxia and seizures is associated with calcium channel mutations but no mutations are known in humans (Brill et al., 2004). Autosomal recessive cerebellar hypoplasia in the Hutterite population was found to result from homozygous deletions of the very-lowdensity lipoprotein receptor, VLDLR (Boycott et al., 2005). VLDLR is part of the reelin signaling pathway. 8.5.2. X-linked congenital cerebellar hypoplasia X-linked cerebellar hypoplasia was documented by Renier et al. (1983) and others (Philip et al., 2003). Recently, different mutations in the oligophrenin-1 gene (OPHN1) on Xq12 have been reported in such families (Bergmann et al., 2003; Philip et al., 2003; des Portes et al., 2004). The clinical characteristics consistently mentioned are neonatal hypotonia followed by developmental delay, moderate to severe mental retardation, myoclonic epilepsy and strabismus. Variable features described include mild dysmorphic face, macrocephaly, tall stature, ataxia, and hypogenitalism. Female carriers descriptions vary considerably, ranging from normal appearance and cognition to mild facial dysmorphism and cognitive impairment. Neuroimaging descriptions also suggest some variability. Most patients displayed supratentorial changes: ventricular dilatation, mild atrophy of the caudate nuclei, widened extracerebral spaces. Infratentorial abnormalities described were: various degrees of vermis and hemisphere hypoplasias, vermis dysgenesis and enlarged cisterna magna. OPHN1 gene mutations usually result in nonsyndromic mental retardation. This gene obviously plays a role during cerebellar development. In two families with X-linked cerebellar hypoplasia no linkage to the OPHN1 gene region was found, suggesting genetic heterogeneity (Philip et al., 2003). 8.5.3. Facial hemangioma and cerebellar hypoplasia Pascual-Castroviejo (1978) first recognized the association of facial hemangiomas with vascular and other intracranial malformations. Later, the acronym PHACE was proposed for a neurocutaneous syndrome featuring posterior fossa malformations, hemangiomas, arterial anomalies, coarctation of the aorta and cardiac defects and eye abnormalities (Frieden et al., 1996). PHACE syndrome has many faces (Metry et al., 2001). Many studies confirmed the heterogeneity of the syndrome and the possible absence of one or more components. Facial hemangiomas are considered the identifying feature, while cerebellar abnormalities are the commonest intracranial finding. These include: DWM, cerebellar hypoplasia (often asymmetric with lower vermis

CEREBELLAR HYPOPLASIAS hypoplasia), cerebellar cortical dysplasia (Rossi et al., 2001; Bhattacharya et al., 2004). A personal case was illustrated in Boltshauser (2004). The cognitive development is often not included in imaging focussed reports. One of our patients followed until their teenage years experienced a normal school career.

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global cerebellar hypoplasia, but at least in some cases a superimposed subsequent atrophy can be observed. MRI shows additional brainstem/pontine hypoplasia, potentially mimicking pontocerebellar hypoplasia type 2. It remains to be seen whether DWM, seen in the first patient with congenital disorder of glycosylation type IId, is a constant feature (Peters et al., 2002).

8.5.4. Ritscher–Schinzel syndrome 8.5.6.2. Smith–Lemli–Opitz syndrome I was involved in the initial description of this syndrome in two sisters (Ritscher et al., 1987). One female died in infancy, probably as a result of shunt dysfunction, while the younger sister (not having hydrocephalus) is still followed. She has moderate cognitive impairment, short stature and was subsequently found to have secondary immunodeficiency (Zankl et al., 2003). Her neuroimaging revealed cerebellar hypoplasia. This syndrome was later named 3C syndrome (cranio-cerebello-cardiac). Occasional features observed were unilateral hearing loss and growth hormone deficiency (Wheeler et al., 1999). 8.5.5. Hoyeraal–Hreidarsson syndrome Hoyeraal–Hreidarsson syndrome is a multisystem disorder characterized by prenatal onset of growth failure, secondary microcephaly, developmental delay, ataxia, immunodeficiency and progressive bone marrow failure. Life expectancy is markedly reduced. The syndrome is X-linked, caused by mutations of the DKC1 gene at Xq28. Mutations in the same gene are associated with dyskeratosis congenita (Sznajer et al., 2003). Neuroimaging in Hoyeraal–Hreidarsson syndrome shows cerebellar hypoplasia. Neuropathological findings in a personal case revealed irregularly formed and abnormally pleated cerebellar folia with hypoplasia of the granular layer (Berthet et al., 1994). 8.5.6. Cerebellar hypoplasia in metabolic disorders Many metabolic disorders lead to cerebellar atrophy, cerebellar white matter and/or cerebellar nuclei involvement but cerebellar hypoplasia is only rarely encountered (Steinlin et al., 1998a). Selected examples are listed below. 8.5.6.1. Congenital disorders of glycosylation Congenital disorders of glycosylation are a group of autosomal recessive disorders resulting from several gene defects of the N-glycosylation pathway. The most common type Ia is often associated with marked

Smith–Lemli–Opitz syndrome (a disorder of cholesterol synthesis pathway) may be associated with variable degrees of hypoplasia. Smith–Lemli–Opitz syndrome is added to the growing list of metabolic disorders that may be associated with structural (mostly supratentorial) brain abnormalities (Kelley and Hennekam, 2000). 8.5.6.3. Adenylosuccinase deficiency This extremely rare disorder is reported to be associated with mild cerebellar hypoplasia (Kohler et al., 1999). The clinical manifestation includes severe psychomotor delay, seizures and autistic features. 8.5.6.4. Mitochondrial disease and cerebellar hypoplasia Cerebellar atrophy is not unusual in the context of mitochondrial disorders but cerebellar hypoplasia is rather unusual, although it has been reported in respiratory chain deficiency (Lincke et al., 1996) and in some patients with pyruvate dehydrogenase deficiency. 8.5.7. Cerebellar hypoplasia in neurofibromatosis 1 Cerebellar hypoplasia in neurofibromatosis 1 has been occasionally reported. This aspect was not systematically studied in large cohorts but a chance association seems unlikely. I have seen few patients with this association.

8.6. Pontocerebellar hypoplasias Early descriptions of what is now called pontocerebellar hypoplasia (PCH) date back to the beginning of the 20th century. Based on observations on a Dutch genetic isolate, Barth put forward the concept of pontocerebellar hypoplasias, representing a group of autosomal recessive neurodegenerative disorders with prenatal onset (Barth, 1993; Barth et al., 1995). He suggested a classification into two main types:

Type 1 (PCH1), associated with spinal anterior horn cell degeneration, usually presenting with polyhydramnios, congenital contractures, gross motor

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Fig. 8.6. MRI ((A) Midsagittal T2-weighted, (B) Parasagittal T2-weighted) in a 2-week-old newborn with pontocerebellar hypoplasia type 2. Note marked pontine flattening, reduced size of vermis, remnants of cerebellar hemispheres below the tentorium (arrow).

impairment and microcephaly, resulting in death within a few months of life Type 2 (PCH2), without anterior horn cell involvement, presenting with feeding difficulties, gross developmental delay, secondary microcephaly, often seizures and extrapyramidal dyskinesia. Patients may survive into their teens.

PCH2 is more prevalent than PCH1. Further types or variants may exist, e.g. cases with neonatal lethality or a milder course. Autosomal recessive inheritance is assumed, but linkage to a chromosomal locus is not yet established. Neuroimaging reveals pontine hypoplasia and cerebellar hypoplasia; the hemispheres are usually more affected than the vermis. The vermis may reveal preserved lobulation but the remnants of the hemispheres are often below the tentorium in the manner of wings (Fig. 8.6). On MRI, PCH1 and PCH2 are indistinguishable but PCH2 usually has additional cerebral gliotic white matter involvement. Individual and intrafamilial variability has been observed, with preserved pontine prominence in some cases (Muntoni et al., 1999). Similar MRI findings can be observed in several other conditions: congenital disorders of glycosylation, congenital muscular dystrophy types (Walker– Warburg syndrome, Finnish type of muscle-eye-brain disease), mitochondrial disorders, subtotal cerebellar agenesis and cerebellar disruption in extremely premature infants (Johnsen et al., 2005). Similar MRI findings were observed in two siblings with a mild

phenotype: they had microcephaly, motor and mental retardation, lower limb spasticity but no epilepsy or dyskinesia, and they achieved free ambulation (Dilber et al., 2002). Definitive classification of the spectrum of PCH awaits genetic elucidation. In cattle arthrogryposis, hydranencephaly and cerebellar hypoplasia could be induced by intrauterine injection with Aino virus (Tsuda et al., 2004).

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CEREBELLAR HYPOPLASIAS Boddaert N, Klein O, Ferguson N, et al. (2003). Intellectual prognosis of the Dandy–Walker malformation in children: the importance of vermian lobulation. Neuroradiology 45: 320–324. Boltshauser E (2004). Cerebellum-small brain but large confusion: a review of selected cerebellar malformations and disruptions. Am J Med Genet A 126: 376–385. Boltshauser E, Steinlin M, Martin E, et al. (1996). Unilateral cerebellar aplasia. Neuropediatrics 27: 50–53. Bordarier C, Aicardi J (1990). Dandy–Walker syndrome and agenesis of the cerebellar vermis: diagnostic problems and genetic counselling. Dev Med Child Neurol 32: 285–294. Boycott KM, Flavelle S, Bureau A, et al. (2005). Homozygous deletion of the very low density lipoprotein receptor gene causes autosomal recessive cerebellar hypoplasia with cerebral gyral simplification. Am J Hum Genet 77: 477–483. Brill J, Klocke R, Paul D, et al. (2004). entla, a novel epileptic and ataxic Cacna2d2 mutant of the mouse. J Biol Chem 279: 7322–7330. Calabro F, Arcuri T, Jinkins JR (2000). Blake’s pouch cyst: an entity within the Dandy–Walker continuum. Neuroradiology 42: 290–295. Chizhikov V, Millen KJ (2003). Development and malformations of the cerebellum in mice. Mol Genet Metab 80: 54–65. Delague V, Bareil C, Bouvagnet P, et al. (2001). Nonprogressive autosomal recessive ataxia maps to chromosome 9q34–9qter in a large consanguineous Lebanese family. Ann Neurol 50: 250–253. Des Portes V, Boddaert N, Sacco S, et al. (2004). Specific clinical and brain MRI features in mentally retarded patients with mutations in the oligophrenin-1 gene. Am J Med Genet A 124: 364–371. Dilber E, Aynaci FM, Ahmetoglu A (2002). Pontocerebellar hypoplasia in two siblings with dysmorphic features. J Child Neurol 17: 64–66. Dixon-Salazar T, Silhavy JL, Marsh E, et al. (2004). Mutations in the AHI1 gene, encoding jouberin, cause Joubert syndrome with cortical polymicrogyria. Am J Hum Genet 75: 979–987. Fennell EB, Gitten JC, Dede DE, et al. (1999). Cognition, behavior, and development in Joubert syndrome. J Child Neurol 14: 592–596. Ferland RJ, Eyaid W, Collura RV, et al. (2004). Abnormal cerebellar development and axonal decussation due to mutations in AHI1 in Joubert syndrome. Nat Genet 36: 1008–1013. Friede RL, Boltshauser E (1978). Uncommon syndromes of cerebellar vermis aplasia. I: Joubert syndrome. Dev Med Child Neurol 20: 758–763. Frieden IJ, Reese V, Cohen D (1996). PHACE syndrome. The association of posterior fossa brain malformations, hemangiomas, arterial anomalies, coarctation of the aorta and cardiac defects, and eye abnormalities. Arch Dermatol 132: 307–311. Gardner RJM, Coleman LT, Mitchell LA (2001). Near-total absence of the cerebellum. Neuropediatrics 32: 62–68. Gleeson JG, Keeler LC, Parisi MA, et al. (2004). Molar tooth sign of the midbrain–hindbrain junction: occurrence in

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multiple distinct syndromes. Am J Med Genet A 125: 125–134. Grinberg I, Northrup H, Ardinger H, et al. (2004). Heterozygous deletion of the linked genes ZIC1 and ZIC4 is involved in Dandy–Walker malformation. Nat Genet 36: 1053–1055. Harris CM, Hodgkins PR, Kirss A, et al. (1998). Familial congenital saccade initiation failure and isolated cerebellar vermis hypoplasia. Dev Med Child Neurol 40: 775–779. Hoveyda N, Shiled JPH, Garrett C, et al. (1999). Neonatal diabetes mellitus and cerebellar hypoplasia/agenesis: report of a new recessive syndrome. J Med Genet 36: 700–704. Janecke AR, Muller T, Gassner I, et al. (2004). Joubert-like syndrome unlinked to known candidate loci. J Pediatr 144: 264–269. Johnsen SD, Bodensteiner JB, Lotze TE (2005). Frequency and nature of cerebellar injury in the extremely premature survivor with cerebral palsy. J Child Neurol 20: 60–64. Joubert M, Eisenring JJ, Robb JP, et al. (1969). Familial agenesis of the cerebellar vermis. A syndrome of episodic hyperpnea, abnormal eye movements, ataxia, and retardation. Neurology 19: 813–825. Keeler LC, Marsh SE, Leeflang EP, et al. (2003). Linkage analysis in families with Joubert syndrome plus oculorenal involvement identifies the CORS2 locus on chromosome 11p12–q13. 3. Am J Hum Genet 73: 656–662. Kelley RI, Hennekam RC (2000). The Smith–Lemli–Opitz syndrome. J Med Genet 37: 321–335. Klein O, Pierre-Kahn A, Boddaert N, et al. (2003). Dandy– Walker malformation: prenatal diagnosis and prognosis. Childs Nerv Syst 19: 484–489. Kohler M, Assmann B, Brautigam C, et al. (1999). Adenylosuccinase deficiency: possibly underdiagnosed encephalopathy with variable clinical features. Eur J Paediatr Neurol 3: 3–6. Kroes HY, Nievelstein RJAJ, Barth PG, et al. (2005). Cerebral, cerebellar, and colobomatous anomalies in three related males: sex-linked inheritance in a newly recognized syndrome with features overlapping with joubert syndrome. Am J Med Genet 135A: 297–301. Lagier-Tourenne C, Boltshauser E, Breivik N, et al. (2004). Homozygosity mapping of a third Joubert syndrome locus to 6q23. J Med Genet 41: 273–277. Lincke CR, van den Bogert C, Nijtmans LG, et al. (1996). Cerebellar hypoplasia in respiratory chain dysfunction. Neuropediatrics 27: 216–218. McMahon AP, Joyner AL, Bradley A, et al. (1992). The midbrain–hindbrain phenotype of Wnt-1/Wnt-1: mice results from stepwise deletion of engrailed-expressing cells by 9.5 days postcoitum. Cell 69: 581–595. Margari L, Ventura P, Presicci A, et al. (2004). Congenital ataxia and mental retardation in three brothers. Pediatr Neurol 31: 59–63. Maria BL, Hoang KB, Tusa RJ, et al. (1997). ‘Joubert syndrome’ revisited: key ocular motor signs with magnetic resonance imaging correlation. J Child Neurol 12: 423–430. Maria BL, Boltshauser E, Palmer SC, et al. (1999). Clinical features and revised diagnostic criteria in Joubert syndrome. J Child Neurol 14: 583–590.

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Me´garbane´ A, Delague V, Salem N, et al. (1999). Autosomal recessive congenital cerebellar hypoplasia and short stature in a large inbred family. Am J Med Genet 87: 88–90. Metry DW, Dowd CF, Barkovich AJ, et al. (2001). The many faces of PHACE syndrome. J Pediatr 139: 117–123. Muntoni F, Goodwin F, Sewy C, et al. (1999). Clinical spectrum and diagnostic difficulties of infantile pontocerebellar hypoplasia type 1. Neuropediatrics 30: 243–248. Nystuen A, Benke PJ, Merren J, et al. (1996). A cerebellar ataxia locus identified by DNA pooling to search for linkage disequilibrium in an isolated population from the Cayman Islands. Hum Mol Genet 5: 525–531. Parisi MA, Dobyns WB (2003). Human malformations of the midbrain and hindbrain: review and proposed classification scheme. Mol Genet Metab 80: 36–53. Parisi MA, Bennett CL, Eckert ML, et al. (2004). The NPHP1 gene deletion associated with juvenile nephronophthisis is present in a subset of individuals with Joubert syndrome. Am J Hum Genet 75: 82–91. Parmeggiani A, Posar A, Scaduto MC, et al. (2003). Epilepsy, intelligence, and psychiatric disorders in patients with cerebellar hypoplasia. J Child Neurol 18: 1–4. Pascual-Castroviejo I (1978). Vascular and nonvascular intracranial malformation associated with external capillary hemangiomas. Neuroradiology 16: 82–84. Patel S, Barkovich AJ (2002). Analysis and classification of cerebellar malformations. AJNR Am J Neuroradiol 23: 1074–1087. Peters V, Penzien JM, Reiter G, et al. (2002). Congenital disorder of glycosylation IId (CDG-IId) – a new entity: clinical presentation with Dandy–Walker malformation and myopathy. Neuropediatrics 33: 27–32. Philip N, Chabrol B, Lossi AM, et al. (2003). Mutations in the oligophrenin-1 gene (OPHN1) cause X-linked congenital cerebellar hypoplasia. J Med Genet 40: 441–446. Reim G, Brand M (2002). Spiel-ohne-Grenzen/pou2 mediates regional competence to respond to Fgf8 during zebrafish early neural development. Development 129: 917–933. Renier WO, Gabreels FJ, Hustinx TW, et al. (1983). Cerebellar hypoplasia, communicating hydrocephalus and mental retardation in two brothers and a maternal uncle. Brain Dev S: 41–45. Richter S, Dimitrova A, Hein-Kropp C, et al. (2005). Cerebellar agenesis II: motor and language functions. Neurocase 11: 103–113. Ritscher D, Schinzel A, Boltshauser E, et al. (1987). Dandy– Walker(like) malformation, atrio-ventricular septal defect and a similar pattern of minor anomalies in 2 sisters: a new syndrome? Am J Med Genet 26: 481–491. Robins JB, Mason GC, Watters J, et al. (1998). Case report: cerebellar hemi-hypoplasia. Prenat Diagn 181: 73–77. Rossi A, Bava GL, Biancheri R, et al. (2001). Posterior fossa and arterial abnormalities in patients with facial capillary haemangioma: presumed incomplete phenotypic expression of PHACES syndrome. Neuroradiology 43: 934–940. Saar K, Al-Fazali L, Sztriha L, et al. (1999). Homozygosity mapping in families with Joubert syndrome identifies a

locus on chromosome 9q34. 3 and evidence for genetic heterogeneity. Am J Hum Genet 65: 1666–1671. Sarnat HB (1992). Cerebreal dysgenesis: embryology and clinical expression. Oxford University Press, New York. Sarnat HB, Benjamin DR, Siebert JR, et al. (2002). Agenesis of the mesencephalon and metencephalon with cerebellar hypoplasia: putative mutation in the EN2 gene. Report of 2 cases in early infancy. Pediatr Dev Pathol 5: 54–68. Satran D, Pierpont ME, Dobyns WB (1999). Cerebellooculo-renal syndromes including Arima, Senior–Loken and COACH syndromes: more than just variants of Joubert syndrome. Am J Med Genet 86: 459–469. Schaefer GB, Thompson JN, Bodensteiner JB, et al. (1996). Hypoplasia of the cerebellar vermis in neurogenetic syndromes. Ann Neurol 39: 382–385. Schmahmann JD, Sherman JC (1998). The cerebellar cognitive affective syndrome. Brain 121: 561–579. Soto-Ares G, Delmaire C, Deries B, et al. (2000). Cerebellar cortical dysplasia: MR findings in a complex entity. Am J Neuroradiol 21: 1511–1519. Steinlin M, Schmid M, Landau K, et al. (1997). Follow-up in children with Joubert syndrome. Neuropediatrics 28: 204–211. Steinlin M, Blaser S, Boltshauser E (1998a). Cerebellar involvement in metabolic disorders: a pattern-recognition approach. Neuroradiology 40: 347–354. Steinlin M, Zangger B, Boltshauser E (1998b). Nonprogressive congenital ataxia with or without cerebellar hypoplasia: a review of 34 subjects. Dev Med Child Neurol 40: 148–154. Steinlin M, Styger M, Boltshauser E (1999). Cognitive impairments in patients with congenital nonprogressive cerebellar ataxia. Neurology 53: 966–973. Steinlin M, Imfeld S, Zulauf P, et al. (2003). Neuropsychological long-term sequelae after posterior fossa tumour resection during childhood. Brain 126: 1998–2008. Strong OS (1915). A case of unilateral cerebellar agenesia. J Comp Neurol 25: 361–391. Sznajer Y, Baumann C, David A, et al. (2003). Further delineation of the congenital form of X-linked dyskeratosis congenita (Hoyeraal–Hreidarsson syndrome). Eur J Pediatr 162: 863–867. Takanashi J, Sugita K, Barkovich AJ, et al. (1999). Partial midline fusion of the cerebellar hemispheres with vertical folia: a new cerebellar malformation? Am J Neuroradiol 20: 1151–1153. Ten Donkelaar HJ, Lammens M, Wesseling P, et al. (2003). Development and developmental disorders of the human cerebellum. J Neurol 250: 1025–1036. Triulzi F, Parazzini C, Righini A (2005). MRI of fetal and neonatal cerebellar development. Semin Fetal Neonatal Med 10: 411–420. Tsuda T, Yoshida K, Ohashi S, et al. (2004). Arthrogryposis, hydranencephaly and cerebellar hypoplasia syndrome in neonatal calves resulting from intrauterine infection with Aino virus. Vet Res 35: 531–538. Valente EM, Salpietro DC, Brancati F, et al. (2003). Description, nomenclature, and mapping of a novel cerebello-

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Wingate RJT (2001). The rhombic lip and early cerebellar development. Curr Opin Neurobiol 11: 82–88. Zafeiriou DI, Vargiami E, Boltshauser E (2004). Cerebellar agenesis and diabetes insipidus. Neuropediatrics 35: 364–367. Zankl A, Guengoer T, Schinzel A (2003). Cranio-cerebellocardiac (3C) syndrome: follow-up study of the original patient. Am J Med Genet A 118: 55–59.


Hamartomatous disorders of cellular lineage Chapter 9

Tuberous sclerosis PAOLO CURATOLO* AND ROBERTA BOMBARDIERI Pediatric Neurology Unit, Tor Vergata University of Rome, Rome, Italy

9.1. Overview 9.1.1. Definition Tuberous sclerosis complex (TSC) is a genetically determined, variably expressed, multisystem disorder that may affect any human organ with well circumscribed, benign, noninvasive lesions. The skin, brain, retina, heart, kidney and lung are the organs most often involved. This devastating disorder, resulting from mutations in one of two genes (TSC1 and TSC2), affects as many as 1:5800 live births (Curatolo, 2003). The importance of CNS involvement in TSC is emphasized by the fact that the condition has retained its name for over a century. ‘Tuberous sclerosis of the cerebral convolutions’ was the term used by Bourneville (1880) to describe the unique and distinctive cerebral pathology he found in a patient with seizures and mental subnormality. The term ‘tuberous sclerosis complex’, proposed in 1942 by Moolten, persists as a designation for all forms and variants of the disease. The majority of patients identified as having the disorder experience symptoms referable to the CNS. Even in subjects without neurological symptoms, CNS lesions are present in all brains studied. CNS abnormalities therefore remain the hallmark of TSC and its most common and clinically serious manifestations. 9.1.2. Historical background Friedrich Daniel von Recklinghausen (1862) presented to the Obstetrical Society of Berlin the pathological findings in a newborn who had ‘died after taking a few breaths’. The heart had several tumors protruding on the cardiac surface and the brain contained a ‘great

number of scleroses’. Von Recklinghausen’s brief report contains the first description of the two pathological lesions most often observed in newborns with TSC: cardiac rhabdomyomas and cortical tubers. Bourneville (1880) gave the first detailed report of its neurological symptoms and gross cerebral pathology. On postmortem examination, Bourneville found that many cerebral convolutions had hard, raised, whitish areas of greater density than the surrounding cortex. He concluded that the seizures had a focal origin and progressed to generalized attacks, attributing their partial onset to the conspicuous sclerotic lesions in the ascending frontal and parietal convolutions of the left cerebral hemisphere (Fig. 9.1). One year later Bourneville and Brissaud (1881) made a new clinicopathological observation, emphasizing the association between the cerebral lesions and the kidney tumors. In 1901 Pellizzi reported a precise microscopic description of the cortical tubers, finely illustrated with ink drawings of atypical neurons, subcortical areas of hypomyelination and subependymal nodules. (Fig. 9.2). He also reported the frequent association of the cerebral, renal and cardiac lesions with the ‘cutaneous adenomas’ in TSC patients. In 1908 Vogt described the association between the cerebral sclerosis of the circumvolutions and the facial adenoma sebaceum. Through Vogt’s work, the syndrome consisting of seizures, mental retardation and ‘adenoma sebaceum’ was established as the classic clinical triad of features for the diagnosis of TSC. The dominant inheritance of TSC and its high mutation rate were first described by Gunther and Penrose (1935). Lagos and Gomez (1967) documented a family in which five generations were affected by TSC, reporting that all the mentally retarded patients had previously had seizures.

*Correspondence to: Dr Paolo Curatolo, Paediatric Neurosciences, Tor Vergata University of Rome, Via Montpellier 1, 1–00133, Rome, Italy. [email protected].

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9.2. Neuropathology 9.2.1. Brain abnormalities

Fig. 9.1. Cerebral tuberous sclerosis showing sclerotic, hypertrophic circumvolutions. (From Bourneville and Brissaud, 1881.)

Fig. 9.2. Ink drawings of atypical neurons and cortical cytoarchitectural disorganization. (From Pelizzi, 1901.)

In 1979 Gomez observed in a large series that only 29% of all patients presented all three of Vogt’s triad characteristics. In 6% of the patients none of the classic features was diagnosed. These data determined the weakening of the classic triad of Vogt and gave rise to the revision of the diagnostic criteria that followed. Until the late 1980s, very little real progress was made toward uncovering the molecular basis of tuberous sclerosis. The first report of genetic linkage analysis identifying a probable TSC gene on chromosome 9q34 appeared in 1987. This gene was given the designation TSC1 (van Slegtenhorst et al., 1997). Subsequent studies indicated that not all TSC families demonstrated linkage to the 9q34 region. Later, chromosome 16p13 was identified as the site of a second TSC locus denoted TSC2. (Kandt et al., 1992, European Chromosome 16 Tuberous Sclerosis Consortium, 1993).

A number of brain lesions can be associated with TSC, and more than 95% of patients demonstrate at least one of the abnormalities listed in Table 9.1 (DiMario, 2004). The most commonly identified are cortical tubers, subependymal nodules and subependymal giant cell astrocytomas. Cortical tubers are variable in size, occur most often at the gray–white matter junction and are usually multiple. Tubers are typically observed in the cerebrum but can be seen in the cerebellum as well. Less frequently, tubers can degenerate into cystic lesions associated with subcortical component adjacent to the cortical lesion (Griffiths and Martland, 1997). Tubers themselves can be limited to the cortex or the subcortical white matter and occasionally can have a more ‘wedge-shaped’ appearance and a focal area of calcification. The overlying cortex of a tuber can also be dysplastic and suggestive of pachygyria or polymicrogyria. On gross pathological examination, tubers are firm, well circumscribed nodules that span flattened gyri and sulci (Fig. 9.3). On microscopic examination, the normal hexalaminar structure of neocortex is lost within the tuber and the gray–white junction is blurred (Fig. 9.4). Interestingly, the cytoarchitecture of cerebral cortex surrounding tubers is typically normal, suggesting that tubers result from a developmental defect affecting a restricted population of neuronal precursor cells during corticogenesis. The most prominent abnormal cell types in tubers are large dysplastic neurons and giant cells as well as bizarrely shaped astrocytes (Fig. 9.5). Dysplastic neurons exhibit disrupted radial orientation in cortex and abnormal dendritic arborization. Heterotopic neurons scattered in the deep white matter are a frequent finding as well. Tubers are identified as low-signal lesions on magnetic resonance imaging (MRI) T1-weighted sequences and as high-signal lesions on T2-weighted and Table 9.1 Brain lesions in TSC (modified by DiMario, 2004) Cortical/subcortical tubers Subependymal nodules Subependymal giant cell astrocytomas White matter (30%): linear migration lines, ‘cystlike’ lesions Corpus callosum agenesis/dysplasia Transmantle cortical dysplasia Association with hemimegalencephaly, schizencephaly, intracranial arterial aneurysms (60%)

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Fig. 9.3. View of a cortical tuber.

Fig. 9.4. Cerebral cortex showing disorganization of the normal cortical pattern. Abnormal neurons have lost their normal orientation.

Fig. 9.5. Cortical tubers showing architectural disarray with large, abnormal neurons.

fluid-attenuated inversion recovery (FLAIR) sequences (Fig. 9.6). In very young patients with immature white matter, the MRI appearance of tubers varies. In early infancy, many tubers are not detectable. When detected the tubers do not show the characteristic findings noted

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in the myelinated brain. Instead the tuber may appear relatively hypointense compared to the adjacent unmyelinated white matter on T2-weighted sequences (Baron and Barkovich, 1999). Tubers may be detected by MRI in the neonatal period and have been identified in fetal life as early as 26 weeks of gestation (Fig. 9.7). Subependymal nodules (SENs) are hamartomatous lesions that are typically located along the walls of the lateral ventricles (Fig. 9.8); they have never been observed in the third ventricle. SENs, found in the vast majority of patients, are often multiple and generally small (1 cm or less). Although SENs can occur anywhere along the ventricular surface, they have a particular predilection for the region around the foramen of Monro. The biological behavior of SENs in this region is to enlarge (Inoe et al. 1998). SENs must develop during fetal life and grow in proportion to the remainder of the brain. Calcification can be present in infancy or evolve more gradually over childhood into puberty. Growth tends to stop after the first decade (Hosoya et al., 1999). SENs are predominantly composed of dysplastic astrocytes and mixed-lineage astrocytic or neuronal cell components. MRI of SENs shows these lesions to have a signal isointense to white matter on T1-weighted sequences and iso- or hypointense on T2-weighted sequences. Large calcifications are seen with T2-weighted MRI as a signal void, whereas smaller SENs are more readily imaged with computed tomography (CT), especially if calcified. Some SENs enhance following intravenous gadolinium administration. Any SENs located near the foramen of Monro that enhance must be carefully evaluated by serial imaging studies. SENs are largely asymptomatic and seem to be unrelated to neurological symptoms (Fig. 9.9). Subependymal giant cell astrocytomas (SEGAs) enlarge over a long period of time and are typically identifiable by the end of the first decade of life, but may be detected in infancy (Tien et al., 1990). MRI of a EGA often shows an heterogeneous mass with lower signal than normal white matter on T1 and higher than the adjacent white and gray matter on T2. Hypointense areas within these tumors may be secondary to calcifications, tumor vessels or intratumoral hemorrhage. All SEGAs enhance following gadolinium administration (Nabbout et al., 1999). Many of the cellular constituents of features of SENs and SEGAs, such as giant cells, are similar to those in tubers sharing a similar profile of protein expression (Jozwiak et al., 2006). However, the cellular packing density in SENs/SEGAs is greater than in tubers and these lesions have the cytopathological appearance of a tumor. For example, in SEGAs, multinucleated cells may be seen with rare mitotic figures and cellular

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Fig. 9.6. Subcortical tubers (arrows) shown on (A) FLAIR and (B) T2-weighted images. Small tubers are much more evident on FLAIR images.

Fig. 9.7. Axial fetal MRI at 27 weeks of gestation showing multiple SENs and tubers.

pleomorphism. In addition to giant cells, a heterogeneous array of astrocytic, polygonal, epithelioid and spindle cell populations may also be seen. Vascular endothelial proliferation and necrosis may be observed (Scheithauer and Reagan, 1999). Other brain abnormalities in patients with TSC are also detectable on neuroimaging (Bozzao et al., 2003). These include a number of white-matter linear lesions, referred to as migration lines. White matter lesions appears as radial bands, wedge-shaped and nodular foci mostly affecting the supratentorial white matter. Radial white matter lesions run from the ventricles through the cerebral mantle to the normal cortex or to the cortical tuber (Fig. 9.10). From an histological point of view these anomalies are almost identical to those observed in the cortical tubers. Radial bands are composed of clusters of heterotopic

cells and are indicative of a disorder of migration associated with abnormal cell differentiation. Wedgeshaped lesions are triangular in configuration and have their apex near the ventricle and their base at the cortex. This pattern is usually evident in the T2-weighted imaging. The site, shape and histopathological findings of white matter lesions confirm that TSC is a disorder of both histogenesis and cell migration. In particular, the different location between the lateral ventricles and the cerebral cortex could be the result of different timing of neuronal and glial migration arrest (Houser et al., 1991). Linear migration streaks can be observed in up to 30% of patients when examined very carefully on T2-weighted MRI sequences. Further evidence of brain dysplasia can be observed on neuroimaging, including partial agenesis of the corpus callosum, cortical dysplasia with focal gyriform abnormalities and heterotopias, transmantle cortical dysplasia and hemimegalencephaly. Additional, though rarer, CNS manifestations within the infratentorial compartment include cerebellar folia atrophy, cerebellar nodular white matter calcifications and tuber-like hamartomas in the cerebellum. Therefore, TSC should be considered not merely as collections of isolated CNS abnormalities but more cohesively as a neuronal migration disorder. The essential features of a neuronal migration disorder include 1) abnormal number, size, and thickness of cortical gyri; 2) heterotopic neuronal aggregates; and 3) variable degrees of cortical cytoarchitectural disorganization and aberrant columnar and laminar arrangement. The fundamental pathogenesis of TSC must incorporate a logical explanation for the lesions detectable both outside and within the central nervous system.

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Fig. 9.8. Cortical tubers and subependymal nodules (SENs). Tubers are more evident on FLAIR images (B) than on T2weighted (A) and T1-weighted (C) images. SENs are shown as hypointense nodules in the ependymal layer on T2-weighted sequences (A), because of calcification, and protrude inside the ventricle on T1-weighted images (C). Calcification is shown by CT both in the tubers (arrows) and in the SENs in the same patient (D).

The brain lesions seen in TSC are, histologically, tissue hamartomas. These are focal ‘tumor-like’ malformations composed of abnormal proportions of cellular elements normally present in the brain. These elements have an abnormal cellular morphology, are in abnormal quantities and locations, and are in a disorganized cytoarchitectural arrangement (dysplasia). Tubers, subependymal nodules and subependymal giant cell astrocytomas represent focal tissue dysplasia. Transmantle cortical dysplasia, hemimegalencephaly and cerebellar hemisphere megalencephaly represent more regional tissue dysplasia. Linear migration streaks represent trails of abnormal cell migration (DiMario, 2004). 9.2.2. Molecular pathogenesis Present evidence suggests that the CNS lesions of TSC are due to a developmental disorder of neurogenesis

and neuronal migration. In TSC two populations of neuroepithelial cells are generated by the germinal matrix. The first consists of normal neuroblasts that form normal neurons and astroglia, which migrate to the cortical plate to form histologically normal cerebral cortex. The second is an abnormal cell population that forms primitive cells, which often fail to show clear neuronal and glial differentiation. Some of these cells, named ‘neuroastrocytes’, remain in the germinal matrix zone where they form subependymal nodules and giant cell tumors. Some neuroastrocytes show partial migration, forming heterotopias in the subcortical white matter. More differentiated cells migrate to the cortical plate, where they form aggregates of dysplastic cortex, the cortical tubers (Fig. 9.11). Two tumor-suppressor genes have been associated with the disorder. TSC1 , located on chromosome 9q34, and TSC2, on chromosome 16p13.3, respectively

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Fig. 9.9. Progressive growth of an enhancing lesion at the foramen of Monro in a TSC patient. (A) Coronal T1-weighted image with gadolinium shows a small enhancing lesion (arrow). This has grown markedly at the 3-year follow-up (B).

Fig. 9.10. Axial MRI demonstrating white matter bands in both hemispheres.

encode the proteins tuberin and hamartin. At the moment, about 500 different mutations in the two TSC genes are known. The mechanism of how mutations in these two separate genes lead to tuber formation is not clear. It is likely that hamartin and tuberin participate in the same pathways of cellular growth control and share common biochemical properties that are important in early development and maturation of the brain. The proteins appear to colocalize at the cellular level and recent evidence suggests a direct binding between tuberin and hamartin. These two proteins form a cytosolic complex interacting at the N-terminal ends. This interaction is abolished in some TSC-associated mutations (Nellist et al., 1999).

Major advances in the understanding of the functions of tuberin and hamartin have shown that in normal cells the tuberin–hamartin complex acts as an inhibitor of the kinase mTOR, an important element in a signaling pathway involved in the control of cell growth and proliferation, controlled by the protein-kinase Akt. When stimulated by growth factors or other agents, the activation of the oncoprotein Akt leads to phosphorylation of TSC2 and the consequent inactivation of the inhibitory activity of the tuberin–hamartin complex. In cells lacking both TSC1 and TSC2, mTOR/S6 kinase activity is increased severalfold and is no longer dependent upon signaling, which normally regulates its

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Fig. 9.11. Inactivation of the tuberin–hamartin complex (THC) alters cellular differentiation in the central nervous system. Neurons and astrocytes arise from neuroglial precursors during development. Central nervous system lesions associated with TSC contain giant cells that express a variety of cellular markers, including those characteristic of immature nervous system cells. This suggests that disruption of the THC by tuberin or hamartin loss interferes with the normal maturation of precursor cells and impairs their ability to differentiate into more developmentally mature neurons or astrocytes. (Modified from Scheidenhelm and Gutmann, 2004.)

activity, including through the PI3 kinase pathway. This fundamental biochemical defect in cells lacking TSC1 or TSC2 seems to contribute to the growth of TSC hamartoma (Kwiatkowski et al., 2004) (Fig. 9.12). Giant cells are the hallmark histological cell type within tubers, SENs, and SEGAs that are unique to TSC. They are large (80–150 mm in diameter), polygonal or ovoid eosinophilic cells that extend short, thickened processes of unclear identity (i.e. axons or dendrites). Giant cells are distributed from the pial surface to the subcortical white matter without clear radial or laminar orientation, and may appear in clusters. They do not exhibit preference for superficial or deep parts of cortex. The cell lineage and phenotype of giant cells has been a matter of some debate since giant cells have been shown to express both glial and astrocytic markers. A fundamental issue regarding the pathogenesis of CNS lesions in TSC is which cell precursors, i.e. glial or neuronal, give rise to giant cells in tubers, SENs and SEGAs during early brain development. Early evidence argued independently for neuronal or glial lineage of giant cells based on identification of neuronal or glial structural features using electron microscopy (Trombley and Mirra, 1981; Yamanouchi et al., 1997). For example, the identification of rough endoplasmic reticulum, intermediate filaments extending into processes of ambiguous morphology, prominent paranuclear Golgi zones and dense-core granules (suggestive of secretory vesicles) in giant cells suggested neuronal features. In contrast, a subpopulation of giant cells in SENs and SEGAs is immunoreactive for GFAP, vimentin or S-100, which suggests a glial phenotype. The

predominant cellular phenotype of the subependymal nodules in the Eker rat model of TSC is astrocytic (Yeung et al., 1997). Immunohistochemical and molecular analyses have identified neuronal mRNAs and proteins such as neuron-specific enolase, tubulin, microtubule associated proteins (MAPs) and intermediate filaments in giant cells within tubers (Hirose et al., 1995; Crino et al., 1996; Lopes et al., 1996). In addition, a small proportion of giant cells in SEGAs express metenkephalin, b-endorphin, serotonin and neuropeptide Y, suggesting a neural lineage (Hirose et al., 1995). CD44, a cell adhesion molecule, mediates cell-cell and cellmatrix interactions is expressed near giant cells in both tubers and SEGAs (Arai et al., 2000). CD44 is expressed in astrocytic processes, predominantly in white matter and subpial regions, suggesting its involvement in the maintenance of a stable central nervous system cytoarchitecture. Tuber sections probed with antibodies recognizing the neural marker NeuN, a DNA binding protein present in mature neurons, exhibit a heterogeneous staining pattern (Crino, 2003). Some of the giant cells and most of the dysplastic neurons are labeled but a small proportion are unlabeled, suggesting either a mixed neuroglial phenotype or that these cell types are phenotypically immature. Dysmorphic neurons express a variety of neuronal markers, e.g. cytoskeletal elements, neurotransmitter receptor subunits, which suggest they are indeed of neuronal phenotype. These neural markers have been identified within a subpopulation of giant cells in SEGAs and SENs, suggesting that giant cells in tubers are more closely akin to neurons whereas those in SENs/SEGAs may reflect a more mixed population. Synaptophysin immunoreactivity has been

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Fig. 9.12. Functions of the tuberin–hamartin complex (THC). The THC negatively regulates Rheb, a small guanosine triphosphatase that activates the mammalian target in the rapamycin (mTOR)/S6 kinase (S6K)/factor 4E binding protein (4E-BP) 1 signaling cascade, which is important for the regulation of cell size and proliferation. In addition, the THC may regulate expression of the cyclin D kinase inhibitor p27kip1, which is involved in both cell growth and size control. To relieve the THC-mediated inhibition of cell size and proliferation, phosphatidylinositol-3 kinase (PI3K) and Akt are activated following stimulation of the insulin-like growth factor (IGF) receptor. Akt phosphorylates tuberin, inactivating the complex and resulting in increased cell size and proliferation. (Modified from Scheidenhelm and Gutmann, 2004.)

reported along the cell membrane of giant cells (Lippa et al., 1993) and single-cell mRNA analysis from microdissected giant cell has shown that these cells express NMDA, GluR and GABAA receptor subunit mRNAs, suggesting the possibility of synaptic connectivity between giant cells and surrounding neurons. 9.2.3. Developmental pathogenesis Tubers probably result from the effects of TSC1 or TSC2 mutations on one of several steps during cortical development, including cell proliferation, differentiation and migration. The cellular effects of the TSC

gene mutations in developing cortex occur at least as early as mid- to late corticogenesis, since nascent cortical tubers have been identified in embryonic brains as early as 20 weeks gestation (Park et al., 1997). Similarly, SENs may be formed during embryogenesis or, like SEGAs, may appear in childhood or adulthood. The link between altered function of tuberin and hamartin and the genesis of tubers, SENs and SEGAs, however, remains to be defined. Tubers probably derive from early progenitor cells in the ventricular zone while SENs and SEGAs may derive from neuroepithelial cell precursors in the subventricular zone or ventricular zone. Current data suggest that these proteins probably regulate cellular proliferation and potentially cellular movement, and thus the morphological appearance of tubers and SEGAs suggests alterations in these cellular domains. For example, antisense inhibition of tuberin expression in cultured cells fosters cell proliferation (Soucek et al., 1997) and hamartin probably contributes to the formation of focal adhesions necessary for cellular migration (Lamb et al., 2000). Further understanding of the downstream cellular events resulting from TSC1 or TSC2 mutations may highlight specific cellular pathways or select cell types that account for the histological manifestations of these lesions. It remains to be shown that the same processes are altered in tubers, SENs and SEGAs. There have been several theories proposed to explain how tubers, SENs and SEGAs may form during cortical development. For example, it is believed that all neuroepithelial progenitor cells carry a single allelic TSC gene mutation. In view of the focal nature of these lesions, one hypothesis is that a ‘second-hit’ mutation in the existing normal TSC allele occurs within a single neuroepithelial cell in the ventricular zone or subventricular zone as a result of inherent susceptibility or random mutation. As a consequence of the two mutations within this precursor cell, there is loss of hamartin or tuberin function and the ensuing downstream events may alter one of several steps involved in cellular proliferation or differentiation. This model suggests that all the cells within a tuber, SEN or SEGA derive from the initial cell sustaining a ‘second-hit’ TSC mutation. A problem with this hypothesis is that while loss of heterozygosity may account for SEN and SEGA formation, allelic loss occurs rarely, if ever, in tubers (Henske et al., 1996; Niida et al., 2001). In fact, it is difficult to account for the overt cellular heterogeneity observed in tubers if all cells are derived from a common precursor cell. One possibility is that the precursor cell sustaining the ‘second hit’ can yield progeny of disparate cell phenotypes via intrinsic programs or stochastic events. Similarly, loss of heterozygosity at

TUBEROUS SCLEROSIS either TSC1 or TSC2 loci was not identified in glioneuronal malformations with cellular morphology that was similar to tubers (Norman et al., 1997). The failure to detect loss of heterozygosity in tubers suggests either an experimental artifact that disrupts the allelic loss or that tubers form by a distinct mechanism from other lesions in TSC. Variable mutational events occurring in disparate precursor cell populations or at different developmental timepoints could result in cells populating tubers or SEGAs with remarkably distinct phenotypes. Thus, cellular heterogeneity in tubers and SEGAs may also reflect TSC mutations occurring at distinct timepoints. If a mutation were to occur in an early progenitor cell, then the progeny of that cell might be more diverse than if such a mutation were to occur in a more mature precursor cell, where the progeny would be a more restricted phenotype. Recent studies using retroviral tagging strategies have demonstrated that clonally derived cells may give rise to quite diverse cell populations, including pyramidal and multipolar cells and even astrocytes (Reid et al., 1995). An alternative possible explanation for the observed heterogeneity of protein expression and cell morphology of dysplastic neurons and giant cells may be that in some cases a second-hit mutation occurs in a cell destined to become a pyramidal cell while in others it occurs within a cell destined to become an astrocyte. These disparities may explain why giant cells have been reported to express a variety of often conflicting neural and glial protein lineage markers. In contrast, only specific precursor cells may be capable of becoming a giant cell or dysplastic neuron and hence a requirement might be that second-hit mutations occur in specific precursor cells. Thus, the phenotypic pathological variability reported in tubers and SEGAs may suggest that, in some patients, giant cells in tubers are largely derived from an astrocytic precursor cell whereas, in other patients, tubers are populated by giant cells that derive from neuronal precursors. In fact, these differences might be observed within the same patient, so that a proportion of tubers may be neuronal and a portion astrocytic. These possibilities have important theoretical functional implications: for example, tubers derived from neural precursors might be more epileptogenic than those derived from astrocytes, which would explain why in some TSC patients only one of several tubers is epileptogenic (Crino, 2004). An important additional issue is whether all the cellular constituents of tubers or SEGAs directly result from TSC gene mutations. According to Crino (2003), all cells within these lesions may necessarily reflect downstream effects of TSC gene mutations, or alternatively some cell types, e.g. astrocytes or dys-

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plastic neurons, may be merely ‘innocent bystanders’ whose functional attributes, such as locating the appropriate laminar destination, have been altered by the presence of aberrant giant cells. In this view, the giant cells would be the ‘two-hit’ cells that migrate albeit abnormally into the cortical plate and disrupt migration of adjacent ‘single-hit’ neurons. The cortical dysplasia observed in tubers would reflect a secondary or bystander effect in which ‘normal’ (single mutation) neurons fail to migrate appropriately because of the disruption of migratory pathways by giant cells. Conversely, dysplastic neurons may reflect the ‘two-hit’ cells that cannot migrate correctly and that induce cytomegaly in giant cells through the effects of a released growth factor. Indeed, the mutational status of other cells within tubers or SEGAs, i.e. endothelial cells or macrophages, remains unknown. These considerations are vital towards understanding the pathogenesis of tubers and SEGAs they warrant a clear view of how tuberin or hamartin dysfunction results in abnormal cytoarchitecture and potentially epileptogenesis. In contrast, in the latter scenario, the effects of altered tuberin or hamartin function would be evident in both neurons and giant cells and thus TSC1 and TSC2 may serve as epilepsy susceptibility genes.

9.3. Molecular genetics Tuberous sclerosis results from mutations in TSC1, the gene on chromosome 9q34, and TSC2, the gene on chromosome 16p13 (European Chromosome 16 Tuberous Sclerosis Consortium, 1993; van Slegtenhorst et al., 1997). Frequent loss of heterozygosity for alleles in 16p13.3 and rare loss in 9q34 has been found in hamartomas from TSC patients, indicating that a second somatic mutation may be required to produce the TSC phenotype at the cellular level (Green et al., 1994a, 1994b). These findings are consistent with TSC1 and TSC2 acting as growth suppressor genes (Carbonara et al., 1994). The TSC2 gene maps to a gene-rich region of 16p13.3, approximately 2.25 Mb from the telomere and immediately adjacent to the PKD1 gene. The 5.5 kb transcript spans an estimated 43 kb of genomic sequence and comprises 42 known exons, of which 41 are coding, and encodes an 1807 amino-acid protein, called tuberin, with little similarity to other known genes. 163 amino acids near the COOH terminus are homologous to the catalytic domain of a guanosine triphosphatase (GTPase) activating protein, GAP3 (rap1GAP). GAPs are regulators of the GTP binding and hydrolysing activity of the Ras superfamily of proteins, which help to regulate cell growth, proliferation and differentiation.

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The TSC1 gene consists of 23 exons, of which the last 21 contain coding sequence. The TSC1 protein, which is called hamartin, consists of 1164 amino acids with a calculated mass of 130 kDa. The protein is generally hydrophilic and has a single potential transmembrane domain at amino acids 127–144 as well as a probable 266-aminoacid coiled-coil region beginning at position 730 (van Slegtenhorst et al., 1997; Vinters et al., 1998). The characterization of the TSC1 and TSC2 genes is summarized in Table 9.2. The mutations observed in TSC1 consist of small deletions, small insertions and point mutations. The majority of mutations are likely to inactivate protein function and these findings support the hypothesis that TSC1 functions as a tumor suppressor gene. At the moment, about 500 different mutations in both TSC genes are known. There is an equal distribution of mutations between TSC1 and TSC2 among familial cases, while among sporadic cases TSC2 mutations are much more frequent than TSC1 mutations. The wide range of tissues in which TSC associated hamartomas develop implies a fundamental role for both TSC genes in regulating cell proliferation and differentiation.

9.4. Clinical features Clinical features of TSC are most commonly observed in the brain, skin, kidneys, heart, eyes and lungs. The physical findings can vary greatly, since TSC can affect different organ systems in different ways at different times in the individual’s life. The frequency of clinical signs in different age groups is summarized

in Table 9.3. Most of the findings traditionally regarded as among the most specific for TSC become apparent only in late childhood, limiting the usefulness for early diagnosis in infants. 9.4.1. Neurological manifestations 9.4.1.1. Epilepsy Seizures are the most common neurological symptom of TSC, occurring in about 90% of patients (Curatolo et al., 2002). Epilepsy in TSC often begins during the first year of life and, in most cases, in the very first months. The high incidence of epileptic spasms and hypsarrhythmia has long been emphasized but it is now clear that epileptic spasms in infants with TSC are clinically and electroencephalographically different from the classic epileptic spasms and hypsarrhythmia of West syndrome (Curatolo et al., 2001a). In the same child focal seizures may precede, coexist with or evolve into epileptic spasms. Subtle focal seizures, such as unilateral tonic or clonic phenomena mainly localized in the face or limbs, and other seizures with subtle lateralizing features, such as tonic eye deviation, head turning and unilateral grimacing, can occur but may be missed by the parents until the third or fourth month of life. The EEG at onset usually shows focal or multifocal spike discharges and irregular focal slow activity. Video-EEG monitoring and polygraphic recordings of the epileptic spasms have shown that each spasm consists of a combination of both focal and bilateral manifestations. Although the pathophysiological mechanisms responsible for the

Table 9.2 Characterization of the TSC1 and TSC2 genes TSC1

TSC2

Mutations

9q34 23 exons – 8.6 kb transcript alternate splicing in the 5 UTR Small truncating mutations

Occurrence Phenotype

10–15% of sporadic cases ?less mental impairment

Loss of heterozygosity in hamartomas Product Function(s)

Rare

16p13.3 41 exons – 5.5 kb transcript exons 25, 26 and 31 alternately spliced Large deletions/rearrangementsSmall truncating mutationsMissense mutations 70% of sporadic cases ?more likely to be mentally retarded Contiguous gene deletion syndrome with PKD1 Frequent

Localization Structure

Subcellular localization Animal models

Hamartin ?Regulates cell adhesion through interaction with ezrin and rho Regulator/modulator of tuberin activity Cytoplasmic, ?cortical Knockout mice under development

Tuberin ?GTPase activating proteinRole in the cell cycle

Cytoplasmic, ?Golgi-associated Eker rat, knockout mice, Drosophila (gigas)

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Table 9.3 Frequency of clinical signs in children with definite tuberous sclerosis in different age groups Less than 2 years

2–5 years

5–9 years

9–14 years

Criterion

%

Criterion

%

Criterion

%

Criterion

%

Hypomelanotic macules Cardiac rhabdomyoma Epilepsy, usually IS Renal AMLs Facial angiofibromas

89 83 83 16 10

Retinal hamartoma

8

Hypomelanotic macules Epilepsy Facial angiofibromas Renal AMLs Cardiac rhabdomyoma Shagreen patch Retinal hamartoma

95 91 63 41 21 20 17

Forehead plaque

9

Hypomelanotic macules Epilepsy Facial angiofibromas Renal AMLs Shagreen patch Cardiac rhabdomyoma Retinal hamartoma Forehead plaque

97 94 73 63 34 21 20 16

Liver AMLs

11

Hypomelanotic macules Epilepsy Facial angiofibromas Renal AMLs Shagreen patch Cardiac rhabdomyoma Liver AMLs Forehead plaque Retinal hamartoma Periungual fibromas

97 96 77 64 47 41 23 18 17 11

AML, angiomyolipoma. Modified from Jozwiak et al., 2000.

coexistence of spasms and focal motor seizures are still uncertain, epileptic spasms associated with TSC may show focal features at onset, followed by a rapid secondary generalization. The age at seizure onset and the age when epileptiform activity becomes apparent on the EEG is largely dependent on the location of the cortical tubers detected by MRI and may coincide with functional maturation of the cortex, with an earlier expression for temporo-occipital regions than for frontal ones (Curatolo et al., 1991). A number of young children with TSC, who present with focal seizures or epileptic spasms at onset, later develop intractable seizures with multifocal EEG abnormalities associated with bilateral and more synchronous slow spike-wave complexes and an electroclinical pattern that resembles Lennox–Gastaut syndrome. In patients with TSC, the differential diagnosis between Lennox–Gastaut syndrome and localization related symptomatic epilepsy originating in the frontal lobe may be extremely difficult and only in few cases longterm video-EEG monitoring can reveal subtle electroclinical manifestations suggestive of a focal seizure onset. In these patients, high time-resolution topographic EEG analysis and dipole localization methods may detect secondary bilateral synchrony, often originating in frontal regions and corresponding to prominent cortical tubers detected by MRI in the mesial surface of the frontal or the anterior temporal lobes (Seri et al., 1998) (Fig. 9.13). The natural history of epilepsy in patients with TSC from infancy into childhood tends to be one of increasing seizure frequency and severity, with poor response to antiepileptic drug treatment (Curatolo et al., 2005). The proportion of children with TSC

and epilepsy referred to tuberous sclerosis clinics who achieve prolonged seizure remission is small. Usually, seizure remission is associated with mild neurological deficits, and sustained remission is more likely to be associated with normal intelligence, a greater likelihood of having a normal finding on electroencephalogram at the time of discontinuation, and fewer cortical and subcortical tubers on neuroimaging (Sparagana et al., 2003). Unfavorable prognostic factors include onset before 1 year of age, presence of multiple seizure types (spasms and focal motor or complex partial seizure, drop attacks and atypical absences), multifocal discharges in the awake state that tend to bilateralize in sleep (Fig. 9.11) and/or secondary bilateral synchrony and occurrence of new electroencephalographic (EEG) foci during evolution (Curatolo and Verdecchia, 2004). In TSC, seizures have a focal or multifocal origin with a topographic correspondence between EEG foci and MRI high-signal lesions, demonstrating the preponderant role of cortical tubers as epileptogenic foci. Immunohistochemical and molecular analysis have indicated that the neuronal populations within cortical tubers might have intrinsic epileptogenicity and actively participate in the generation of partial seizures, through the release of neurotransmitters or neuromodulators into the adjacent brain tissue (Wolf et al., 1995). Giant cells in tubers express neurotransmitter-producing enzymes and neurotransmitter receptors, such as NMDA receptor subunit 1 and GABAA receptor subunits (White et al., 2001). It has recently been suggested that changes in the properties of GABAA receptors, possibly related to plastic changes in subunit combinations, may result in an altered regulation of inhibitory function. On the basis

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Fig. 9.13. MRI demonstrates multiple cortical tubers in both hemispheres and subependymal nodules in a 7-year-old girl with intractable seizures and severe learning disability. Interictal EEG showing apparently generalized spike and wave discharges. A lateralized onset from the right frontal tuber was detected by EEG mapping.

of these findings it is possible to hypothesize that epileptogenesis in TSC may result from ‘aberrant plasticity’, that may involve a subunit switch of GABAA receptors and changes in a regulatory system. Concas et al. (1999) reported that the changes in the plasticity of GABAA receptors are related to the physiological changes in plasma and brain concentration of neurosteroids, which may act as endogenous modulators of GABAA receptors. TSC patients with epilepsy may present a dysregulation of enzymes deputed to the synthesis of these neuroactive steroids, possibly resulting in a decreased GABAergic transmission. These modifications could be related to a genetically determined mechanism that regulates 3b-tetrahydroprogesterone synthesis or metabolism, and/or to a functional regulation linked to the expression of receptors to which these neuroactive steroids bind (Di Michele et al., 2003). The mechanism involved in drug resistance in TSC is unknown. Recently, Lazarowski et al. (2004) investigated the expression of the proteins MDR-1 (multidrug resistance gene) and MRP-1 (multidrug resistance-associated protein-1) in epileptogenic cortical tubers of three pediatric patients with TSC and refractory epilepsy obtained after surgical resection. In these specimens, MDR-1 and MRP-1 proteins were strongly immunoreactive in abnormal balloon cells, dysplastic neurons, astrocytes, microglial cells and blood–brain barrier vessels. A more extensive MDR-1 immunoreactivity was also observed, suggesting that refractory epilepsy phenotype in TSC may be associated with the expression of both multi-drug-resistance MDR-1 and MRP-1 transporters in epileptogenic cortical tubers. 9.4.1.2. Cognitive and behavioral disorders Tuberous sclerosis is associated with a wide range of cognitive, behavioral and psychiatric manifestations (Curatolo et al., 1991). About half of individuals with

TSC have normal intelligence, while the other half have mental disabilities ranging from mild learning disabilities to severe mental retardation (Prather and de Vries, 2004). It seems that the majority of individuals with TSC either fall into a severely disabled group or have normal intelligence. This ‘bimodal distribution’ is important, because the behavioral and psychiatric problems associated with ‘able’ and the ‘severely disabled’ group are different. The likelihood of learning disability developing appears to be associated with the nature of the genetic mutation, the extent of brain abnormality and the age of onset and type of seizure disorder (Goodman et al., 1997; Jones et al., 1997; Dabora et al., 2001; Joinson et al., 2003). However, the mechanisms involved are likely to be different according to the nature of the cognitive impairment, with perhaps structural and functional disruptions to the development of different neural networks resulting in distinct, partly overlapping cognitive impairments (de Vries et al., 2005). Individuals with normal overall intellectual abilities are at increased risk of specific cognitive deficits (Harrison et al., 1999; de Vries et al., 2001). These include attention deficits (e.g. difficulties sustaining attention, switching of tasks), executive control problems (e.g. planning, sequencing), language (e.g. expressive or receptive language delay), learning and memory problems, and visual-spatial difficulties. Children with TSC who have such neuropsychological deficits often present with specific scholastic difficulties in reading, writing, spelling and arithmetic. Because of these difficulties, a number of children with TSC are often diagnosed with dyspraxia or dyslexia (Bolton, 2003). A very significant proportion of children and adults with TSC will present with a range of behavioral and psychiatric difficulties. In children with severe global

TUBEROUS SCLEROSIS disabilities (or mental retardation), infantile autism and other autism spectrum disorders are seen in more than 50% of cases (Curatolo and Verdecchia, 2004). Despite the great deal of progress in the last few years, the neural basis of autism in TSC is still largely unknown and represents a major challenge for child neurologists (Wiznitzer, 2004). The epidemiological studies of autism, despite differing tuberous sclerosis populations and varying diagnostic criteria, all arrive at a surprisingly high incidence of autism, between 17% and 61%. The incidence of autism may be significantly higher than the rates of cardiac and renal abnormalities, for which screening is routinely conducted in this population. It is to be hoped that early diagnosis of autism will allow for earlier treatment and the potential for better outcome. From the current evidence, no one factor (learning disability, tuber localization, occurrence of infantile spasms, focal EEG abnormalities or seizure) can be causally linked with the abnormal behavior. Rather, it may be that all the etiological categories detailed above may be causal for autism in TSC. Some patients may exhibit autistic behavior secondary to specifically located cortical tubers, others may be affected by specific epileptic phenomena, with others perhaps harboring a deletion in an adjacent but otherwise independent gene that is critical in the genesis of autism. The better identification of patients, especially the identification of less severely affected patients, presumably with a lower cortical lesion burden, combined with improved imaging with MRI or functional imaging techniques, may aid in studies of the localization of behavior in the developing brain. Similarly, the improved treatments for epilepsy in TSC patients, including the remarkable response of infantile spasms to vigabatrin, as well as the continuing success of epilepsy surgery, may allow for the elimination or reduction of epilepsy as a variable in attempts to distinguish between the various possible etiologies of autism in TSC. Improved seizure control may allow for a more independent analysis of the effects of lesion localization, learning disability or genotype on behavior. Finally, as the underlying genetic mutations in more and more patients with tuberous sclerosis are characterized, genotype/ phenotype correlations with autistic behavior can be made. Autism may also reflect a direct effect of the abnormal genetic program. The terminal 2 megabases of the short arm of human 16 contain the TSC2 locus and there is linkage evidence for bipolar affective disorder and epilepsy, as well as for autism, in this same area (Daniels et al., 2001). A locus of susceptibility to autism has been identified in the 16p13 chromosomal region (Lucarelli et al.,

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2003). The TSC2 gene product tuberin is highly expressed in brain regions involved in the behavioral phenotypes of the autistic disorder. The genetic dissection of the short arm of chromosome 16 in autism could help to localize more precisely a susceptibility gene and clarify its position with respect to the TSC2 locus. Several factors support role for the dopamine b-hydroxylase (DBH) gene, which has been mapped to 9q34 in the etiology of autism. Some families with autistic children have a low level of serum dopamine DBH, which catalyzes the conversion of dopamine to norepinephrine (Robinson et al., 2001). The DBH gene is closely linked to one of the loci for TSC. Attention deficit hyperactivity disorder (ADHD) and related behaviors (such as impulsivity, overactivity and attention problems in daily life) are also seen in about half of children with TSC. ADHD-related behaviors are more likely to be seen in those with severe disabilities but are highly over-represented in those with normal ability as well. Self-injurious behaviors, aggressive outbursts, difficult temper tantrums and chronic sleep problems are often seen in children with TSC with severe disabilities. In older children, adolescents and adults, anxiety and mood-related disorders become increasingly prevalent. Anxiety and mood symptoms are seen in individuals with and without global cognitive difficulties and can be very debilitating to higher-functioning young people and adults. Sleep disorders, such as night waking, prolonged sleep latency and seizure-related sleep problems, are considered one of the most common behavioral manifestations in children with TSC. Prolonged sleep latency and frequent awakenings due to epileptic seizures need to be differentiated using polysomnography (Bruni et al., 1995). 9.4.2. Non neurologic manifestations 9.4.2.1. Cutaneous manifestations The most well-known cutaneous manifestations of TSC are facial angiofibromas, which don’t tend to appear until late childhood or early adolescence. They start out as flat, reddish macular lesions that, at an early stage, may seem to be freckles. As time passes, angiofibromas become increasingly erythematous and nodular and sometimes present with a friable surface that may bleed easily. Facial angiofibromas are usually noted first in childhood and progress during puberty and adolescence. Other skin lesions consist of hypomelanotic macules (i.e. ash leaf), ungula or gingival fibromas, and thickened, firm areas of subcutaneous tissue often on the lower back or on the buttocks or torso (shagreen patch) or forehead and face (fibrous

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plaques). The hypomelanotic macules tend to be round or oval in shape and range from a few millimeters to as much as 5 cm in length. Occasionally they have an irregular, reticulated appearance, as if white confetti paper had been strewn over the skin (confetti lesions). Whenever the scalp is involved, an area of poliosis can result. Hypomelanotic macules may be present at birth or not show up until later in life. Their location and number varies extensively from person to person. Once the criterion of ‘three or more hypomelanotic macules’ has been met, the number or size is not significant. A widespread technique for enhancing their visualization involves an examination of the skin under ultraviolet light using a Wood’s lamp. Fibromas of the skin arise in multiple locations. When present in the lumbar region, they are called a ‘shagreen patch’. These are connective tissue hamartomas consisting of various amounts of vascular structures, fat, collagen, elastic tissue, smooth muscle and skin. Seldom found in infants, they become more common after the first decade of life and persist throughout adult life. Fibromas can also occur in the periungual regions, gingivae or potentially anywhere in cutaneous or mucosal tissues. The underlying tissue may be hypertrophic or hamartomatous. 9.4.2.2. Renal manifestations Renal manifestations of TSC are the third most common clinical feature. Four types of lesion can occur: angiomyolipomas (AMLs), isolated renal cyst(s), autosomal dominant polycystic kidney disease (PKD) and renal cell carcinoma. AMLs are observed in as many as 80% of individuals with TSC, usually detected after the third year of age. AMLs tend to grow very slowly over several years. Growth spurts can be observed in preadolescent boys and postmenarche girls. AMLs consist of abnormal blood vessels, smooth muscle cells and fat, with each present in varying degrees. Individuals with TSC may have either multiple small AMLs studding the surface of the kidney, multiple small AMLs throughout the kidney or one or more larger lesions. These larger lesions are more apt to be symptomatic, particularly when greater than 4–6 cm in their largest diameter. They frequently produce nonspecific complaints such as flank pain. Of greater concern are potentially life-threatening retroperitoneal hemorrhages from rupture of dysplastic, aneurysmal blood vessels that feed the AMLs. These hemorrhages also can destroy adjacent normal renal parenchyma or produce abdominal distention and obstruction due to mass effects. Some studies indicate that as many as 75% of AMLs will increase in size over time. Very large AMLs (> 6–8 cm in diameter) are likely to progress

and often result in hemorrhage, particularly if prominent abnormal vasculature is present. AMLs with fewer dysplastic vessels may have a smaller risk of catastrophic hemorrhage but can present problems from their sheer size. If there are too many AMLs to count, they are not surgically respectable, which can lead to hypertension and renal failure if too much normal kidney tissue is destroyed between the large AMLs. Renal ultrasounds are performed to assess changes in the size of AMLs or cysts, in the hope that this will allow intervention prior to development of renal failure. Small renal cysts and AMLs may not grow significantly until after puberty, but excessive growth has been sometimes observed in young children. Renal cysts are found in 20% of males and 9% of females with TSC. They are rarely if ever symptomatic. Simple renal cysts often occur with AMLs and this combination should suggest the diagnosis of TSC. Sometimes multiple renal cysts can be confused with true PKD. PKD occurs in 2–3% of persons with TSC and usually presents early in life with hypertension, hematuria or renal failure. This occurs as the result of a genetic abnormality (usually a single large deletion) affecting both the TSC2 gene and the PKD1 gene immediately adjacent to it. Renal cell carcinoma seems to occur more frequently in individuals with TSC than in the general population, and at an earlier age. 9.4.2.3. Cardiac manifestations Cardiac involvement is typically maximal at birth or early in life and it may be the presenting sign of TSC, particularly in early infancy. Some 50–60% of individuals with TSC show evidence of cardiac involvement, usually in the form of rhabdomyomas. On the other hand, anywhere from 50–85% of infants with isolated cardiac rhabdomyomas have been observed to later show definite evidence of TSC. Rhabdomyomas are benign tumors that may be focal or diffuse and infiltrating in character. They produce symptoms predominantly through outflow tract obstruction or by interfering with valvular function. Additionally, they can disrupt electrical conductivity and cause arrhythmias. Rhabdomyomas develop during intrauterine life (usually between weeks 22 and 26 of gestation). The lesions usually undergo spontaneous regression in the first few years of life, although residual areas of histologically abnormal myocardium may persist. These lesions can involve the cardiac conducting system and thus may predispose an affected individual to ventricular pre-excitation or other arrhythmias both in infancy and later in life.

TUBEROUS SCLEROSIS 9.4.2.4. Ophthalmic manifestations At least 50% of patients have ocular abnormalities; some studies have reported the prevalence as high as 80%. These lesions are again characterized as hamartomas, specifically retinal astrocytomas, which typically become calcified over time. Initially appearing as rounded, nodular or lobulated areas on funduscopic examination, they become whitish in color as they calcify. They tend to be indolent and rarely produce symptoms or require intervention. On the rare occasion that visual acuity is affected, a retinal hamartoma may be found impinging upon and compromising the retinal fovea and/or optic nerve. Hypopigmented areas of the retina, iris and even eyelashes have also been reported (Jozwiak, 2003). 9.4.2.5. Pulmonary manifestations Three forms of symptomatic pulmonary involvement in TSC have been described: multifocal micronodular pneumocytic hyperplasia, pulmonary cysts and lymphangioleiomyomatosis (LAM) (Franz, 2004). Involvement occurs mainly in adult women, commonly aged 30 or older. It was long thought to be distinctly uncommon, affecting 1% or less of women with TSC. However, recent prospective and retrospective studies have found cystic pulmonary abnormalities in as many as 40% of women with TSC, although most of those women remain asymptomatic. About 60% of women with sporadic LAM also have renal AMLs but do not have other characteristics of TSC. Smooth muscle cells undergo abnormal proliferation with secondary compromise of bronchioles, venules and lymphatic structures. Slowly, normal pulmonary elasticity is lost, with the resultant decrease in vital capacity and increase in residual volume. Pulmonary hypertension and worsening hypoxia and hypercapnia eventually supervene. When LAM is suspected clinically, high-resolution CT of the chest is the most sensitive diagnostic modality. Because of the overwhelming predominance of LAM in women, it is possible that estrogen accelerates the progression of the condition. LAM may be progressive in a small percentage of individuals with TSC, particularly women. Recent studies have shown that around 40% of women with TSC have subtle signs of LAM; a baseline CT of the lungs should be carried out. In cases where LAM is progressive, a lung transplant may be necessary. Interestingly, LAM has occasionally recurred in transplanted lungs. Karbowniczek et al. (2004) demonstrated the migration of cells from renal AMLs to the lungs of women with LAM regardless of whether they had TSC, and the cells almost certainly cause the disorder. These cells frequently present abnormalities of

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either TSC1 or TSC2, which produce the characteristic smooth muscle hypertrophy and destruction of normal lung. 9.4.3. Genotype/phenotype correlations Although TSC2 mutations are about five times more common than TSC1 mutations in sporadic TSC patients, the two genes appear to account for an equal proportion of mutations in large TSC families suitable for linkage analysis (Povey et al., 1994; Jones et al., 1997). Smaller TSC families with only two or three affected individuals are intermediate in that TSC1 mutation frequency is 15–50% and TSC2 accounts for the majority of the remainder (Niida et al., 1999; van Slegtenhorst et al., 1999; Dabora et al., 2001). These observations suggest that, on average, patients with TSC1 mutations may be less severely affected than those with TSC2 mutations, so that each new sporadic TSC1 patient has a better chance of founding a family than a new TSC2 patient. Jones et al. (1997, 1999) assessed the frequency of intellectual disability in sporadic cases as a reliable and important aspect of disease severity. Unlike many other components of the TSC phenotype, which show age-dependent penetrance, intellectual disability is almost invariably present from early childhood if it develops at all, and rarely escapes detection. Moderate to severe intellectual disability also clearly limits reproductive capacity. Intellectual disability was significantly more frequent among sporadic cases with TSC2 than TSC1 mutations (Jones et al., 1999) However, other series have not replicated this finding (Kwiatkowska et al., 1998; Young et al., 1998; Niida et al., 1999; van Slegtenhorst et al., 1999). Sporadic patients with TSC1 mutations have, on average, a milder phenotype than patients with TSC2 mutations. They have a lower seizure frequency, moderate to severe mental retardation, fewer subependymal nodules and cortical tubers, less severe kidney involvements, no retinal hamartomas and less severe facial angiofibroma (Dabora et al., 2001). Moreover, TSC2 mutations are associated with a significantly earlier epilepsy presentation than TSC1 mutations, which results in frequent epileptic spasms (Jozwiak et al., 2001). In this patient set, those patients without an identified mutation had clinical features that were on average significantly milder than TSC2 patients and generally similar to those of TSC1 mutations, apart from kidney disease, which was similar to the TSC2 patient set (Sancak et al., 2005). These observations suggest that a significant proportion of these patients have mosaicism for a TSC2 mutation, causing a generally milder phenotype. Some of these patients might possibly have

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mutations in a third TSC gene, accounting for a small proportion of all cases.

9.5. Diagnostic criteria At the time of initial diagnosis diagnostic studies are commonly performed, either to confirm the presence of TSC or to evaluate presenting symptoms such as seizures or cardiac dysfunction. In addition, it is often useful to establish a baseline assessment in areas that could develop complications later. Unless specific areas of concern are identified by these initial studies, later tests are aimed towards areas with a high risk of dysfunction and towards lesions that might be possibly treated (Roach and Sparagana, 2004). Moreover, at the time of initial presentation, the majority of the patients with TSC undergo MRI to search for additional evidence of TSC already suspected on the basis of other signs (Roach et al., 1998). The number, size and perhaps the location of the dysplastic cortical lesions detected by MRI tend to correlate with the severity of the clinical neurological dysfunction (Curatolo et al., 1991; Shepherd et al., 1995; Goodman et al., 1997). EEG is useful when the initial presentation includes epileptic seizures. Nevertheless, children who never manifested seizures and are not suspected of having epileptic seizures generally do not need to undergo baseline EEG. Usually, adolescents and adults have a greater chance of developing symptomatic renal angiomyolipomas than children, but it sometimes happens even in childhood (Jozwiak et al., 1998). For each patient renal ultrasonography is carried out at the time of diagnosis. Furthermore, cardiac arrhythmias occasionally occur even in patients with TSC who do not have a demonstrable cardiac rhabdomyoma. An arrhythmia can be present at birth or develop later. Wolff–Parkinson–White syndrome appears to be the most commonly noted arrhythmia in patients with TSC. Therefore, a baseline study must be performed at the time of diagnosis. Echocardiography may reveal one or more cardiac rhabdomyomas in more than half of the younger individuals with TSC (Gibbs, 1985; Jozwiak et al., 1994). However, these cardiac tumors are more innocuous than they might seem, as they tend to involute over the years, often disappearing completely by adulthood. Moreover, the most rapid reduction in lesion size occurs during the first 3 years of life, a period after which rhabdomyomas tend to change less dramatically (DiMario et al., 1996). Each patient affected by TSC should have an accurate ophthalmic examination at the time of diagnosis. Children usually do not suffer from facial angiofibromas or ungual fibromas at the time of initial diagnosis,

and typical hypomelanotic macules can be early recognized by most clinicians who are knowledgeable about TSC. Generally, a dermatological examination might be important when the skin lesions are atypical or when the diagnosis of TSC is uncertain. A scrupulous age-appropriate screening for behavioral, cognitive and neurodevelopmental dysfunction should also be performed at the time of diagnosis. Unfortunately, children with apparently normal initial testing and developmental milestones can still suffer milder deficits that interfere with their learning. It is advisable that each child is reassessed around the time school begins, even if no abnormalities were detected by the previous screenings. Children with abnormal behavior or cognitive function should be periodically retested, and re-evaluation is also appropriate when there is a significant change in behavioral or cognitive function. Newly diagnosed adolescents or young adults with a well established pattern of completely normal social and cognitive function as determined by educational achievement sometimes do not require formal testing. Gene characterization could ultimately identify patients at a greater or lesser risk of particular complications. Moreover, further diagnostic studies could be performed selectively on individuals at greatest risk for certain specific complications, thereby decreasing the number of useless investigations. It is possible that, in the near future, gene typing at the time of initial diagnosis will reveal its usefulness once molecular testing becomes readily available and there are sufficient data to determine which clinical phenotypes correlate with which. The diagnostic criteria for TSC are summarized in Table 9.4. 9.5.1. Evaluation of family members Since a number of affected individuals show only subtle clinical features of TSC, diagnosis of family members may be difficult. Such a problem is crucial for accurate genetic counseling in this autosomal dominant disorder. The majority of affected individuals who are subject to a thorough physical examination, including a skin examination with ultraviolet light and a retinal examination through dilated pupils, have at least subtle physical findings of TSC. Furthermore, a completely normal physical examination and extensive radiographic testing, including high definition MRI, still cannot definitely exclude TSC, since there is always a chance of germline mosaicism. Occasionally, MRI may be helpful, in parents with few physical findings and other normal diagnostic studies. The usefulness of MRI often lies in its ability to help confirm a diagnosis that is already suspected.

TUBEROUS SCLEROSIS Table 9.4 Revised clinical diagnostic criteria for tuberous sclerosis complex Major features

Minor features

Facial angiofibromas or forehead plaque

Multiple, randomly distributed pits in dental enamel Hamartomatous rectal polyps Bone cysts

Nontraumatic ungueal or periungual fibroma Hypomelanotic macules (three or more) Shagreen patch (connective tissue nevus) Multiple retinal nodular hamartomas Cortical tuber Subependymal nodule Subependymal giant cell astrocytoma Cardiac rhabdomyoma, single or multiple Lymphangiomyomatosis and or renal angiomyolipoma

Cerebral white matter radial migration lines Gingival fibromas Nonrenal hamartoma Retinal achromic patch ‘Confetti’ skin lesions Multiple renal cysts

Modified from Roach et al., 1998.

No systematic studies have addressed the advantage of renal ultrasonography in identifying minimally affected individuals with TSC. Nonetheless, ultrasonography clearly demonstrates renal angiomyolipomas and is broadly available and certainly cheaper than other studies. This is why renal ultrasonography is recommended where diagnostic studies are carried out on family members who might potentially be affected. Molecular diagnosis will be used increasingly to differentiate TSC patients from clinically normal family members. Molecular testing for TSC can identify certain individuals with TSC who do not satisfy the clinical diagnostic criteria (Roach et al., 1998). Occasionally, couples have more than one child with TSC, in spite of the fact that neither patient has either physical or radiological evidence of the disease. In these families, TSC probably results from germline mosaicism. Unfortunately, neither routine diagnostic studies nor DNA-based testing is likely to detect germline mosaicism in these individuals, because the parent who carries the mutation will not have a detectable mutation in DNA extracted from leukocytes (Rose et al., 1999; Yates et al., 1997). Therefore, genetic counseling for families with one affected child should include a small (1–2%) possibility of recurrence, even in parents who have no evidence of TSC after a thorough diagnostic evaluation (Roach et al., 1998).

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Testing recommendations at the time of diagnosis and recommended frequency of follow-up investigation are summarized in Table 9.5. Confirmatory testing by DNA analysis for TSC is helpful in individuals who fail to meet the criteria for definite TSC and to improve genetic counseling. Prenatal genetic testing for TSC is also possible when there is a defined TSC mutation in a specific family. Preimplantation genetic diagnosis is a method of determining the genetic characteristics of an embryo created by in vitro fertilization. Although preimplantation genetic diagnosis is feasible for TSC, its use is not widespread. Several issues limit the usefulness of confirmatory testing for TSC, beginning with the fact that most patients develop the disorder via a spontaneous mutation. And, although there are few false-positive tests, as much as 15–20% of the time the test fails to demonstrate a disease-causing mutation. Nevertheless, confirmatory testing in an individual who already fulfills the diagnostic criteria for defining disease can help to identify a mutation that can then be sought in other family members or for subsequent prenatal diagnosis. Somatic and germline mosaicism also complicate confirmatory testing for TSC (Au et al., 2004). A conservative recurrence risk for seemingly unaffected Table 9.5 Testing recommendations Assessment

Initial testing

Repeat testing

Neurodevelopmental testing

At diagnosis and at school entry At diagnosis

As clinically indicated

Ophthalmic examination Electroencephalography

At diagnosis

Electrocardiography

At diagnosis

Echocardiography

If cardiac symptoms occur At diagnosis At adulthood (women only) At diagnosis

Renal ultrasonography Chest CT

Cranial MRI

Modified from Roach et al., 1999.

As clinically indicated As clinically indicated As clinically indicated If cardiac dysfunction occurs Every 1–3 years If pulmonary dysfunction occurs Children/ adolescents: every 1–3 years

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couples with a single affected child, even when they have no demonstrable TSC mutation, is 2% (Rose et al., 1999).

9.6. Clinical management Long-term surveillance testing should be directed toward lesions that are frequent, lesions that can be treated if early identified, and lesions that carry a significant risk of dysfunction or death. A surveillance protocol based on the natural history of TSC provides some practical basis for driving follow-up tests. Any effort should be made to minimize costly testing of asymptomatic patients and to maximize the likelihood of early identification of a treatable lesion. The guidelines that follow are designed for long-term clinical management of an asymptomatic patient whose diagnosis is perfectly assured (Roach et al., 1998). 9.6.1. Central nervous system Subependymal giant cell astrocytomas are locally invasive and can cause hydrocephalus because of their typical occurrence in the anterior lateral ventricle. Enlarging giant cell tumors can be removed through early identification, before symptoms develop and before they become locally invasive. Children should undergo periodic cranial imaging with either CT or MRI scans every 1–3 years, depending on the level of clinical suspicion in each child. In general, there is a greater likelihood for children to develop subependymal giant cell astrocytomas than for adults. Usually the need for EEG is dictated by the clinical features and response of seizures to antiepileptic drugs. As a rule, EEG is not required for adults with TSC who do not have epileptic seizures. However, since seizures are not regularly clinically obvious, EEG should always be considered in the evaluation of a patient with an unexplained decline of cognitive or behavioral function in whom epileptic seizures are suspected. During early infancy, the seizure pattern can change rapidly and it may sometimes be necessary to repeat the studies at frequent intervals. A number of neurodevelopmental and behavioral dysfunction patterns occur as a result of TSC. In contrast to mental retardation, which is a classic and common feature of TSC, learning disabilities, autism, attentional deficits and other difficulties are probably under-recognized (Hunt and Dennis, 1987; Curatolo et al., 1991). The children who suffer such problems can benefit from early recognition, specific education and treatment plans. Formal cognitive testing is not necessary for adolescents and adults with well established patterns of normal social and cognitive function.

When a patient is diagnosed with TSC in infancy or early childhood, testing should be repeated around the time the child enters school. Older children, instead, should be reassessed periodically in response to educational or behavioral concerns. 9.6.2. Kidney By the age of 10 years, almost 75% of children with TSC have sonographic evidence of one or more renal angiomyolipomas (Ewalt et al., 1998; Weiner et al., 1998). During the first decade of life, the number and size of the renal angiomyolipomas tend to increase but large renal angiomyolipomas are more likely to cause symptoms than smaller lesions (Steiner et al., 1993). It is therefore wise to monitor patients with large tumors more closely. Renal ultrasonography should be generally undertaken every 1–3 years. How frequently patients are tested depends mostly on the degree of concern for them and on the results of previous examinations. Regardless of age, patients who have large renal lesions or lesions that seem to have grown substantially should have more frequent follow-up examinations. MRI might be necessary for these patients to define the extent of the kidney disease with greater precision. 9.6.3. Heart Although about two-thirds of infants with TSC have echocardiographic evidence of one or more cardiac rhabdomyomas, these tumors tend to regress over time and can disappear altogether by adulthood (Alkalay et al., 1987; Smythe et al., 1990; Webb et al., 1993). The majority of the patients with TSC who have a cardiac rhabdomyoma remain asymptomatic (Jozwiak et al., 1994). Continuous cardiac evaluations are not required and are even unnecessary for most asymptomatic TSC patients. However some patients may occasionally develop arrhythmias during adolescence or adulthood. A cardiologist should follow all those patients who have new symptoms that might indicate cardiac dysfunction and those with previous symptoms, benefiting from periodic studies to evaluate heart function. 9.6.4. Lungs Pulmonary disease (lymphangiomyomatosis) due to TSC is uncommon. In the rare cases in which it occurs, it is suffered almost exclusively by women (Smolarek et al., 1998). The average age of onset is 32–34 years. The best method to use to investigate the pulmonary abnormalities of TSC is the chest CT scan. The panel

TUBEROUS SCLEROSIS recommended no routine testing in either asymptomatic children or adolescents. Women should undergo chest CT scans at least once on reaching adulthood. If pulmonary symptoms develop, this test should be repeated. 9.6.5. Retina Around 75% of patients with TSC have retinal lesions (Kiribuchi et al., 1986). In a severely impaired child, ophthalmic examinations are sometimes difficult to perform without sedation, and are unlikely to identify impending visual loss from a treatable lesion. Repeated ophthalmological evaluations are usually unnecessary, unless there is some specific reason for concern. 9.6.6. Skin Facial angiofibromas are benign skin tumors that can have major consequences for some patients. Laser therapy is one way in which skin tumor growth can be limited, although the treatments often need to be repeated periodically as the lesions tend to regrow gradually after the treatment is over. Ungual fibromas sometimes cause severe problems, which can be effectively treated.

9.7. Treatment The pathological manifestations of TSC can cause variable symptoms based on the size and location of the hamartomas. Therefore, a variety of management considerations are necessary. Table 9.6 describes the various treatment considerations for each feature. The goals of treatment for individuals with TSC are the same as for all individuals with a multisystem chronic condition: providing the best possible quality of life with the fewest complications from the underlying disease process, fewest adverse treatment effects and fewest medications. Appropriate and effective therapy is not only aggressive but also relies upon recognition of the natural history of the various lesions of TSC. Unfortunately, no one medical treatment usually results in satisfactory relief for all or even most individuals with TSC. A combination of medical treatment modalities is then frequently required. The choice of specific antiepileptic drugs for treating seizures in individuals with TSC is based on the seizure type(s), epilepsy syndrome(s), other involved organ system, age of the individual, and antiepileptic drug side effect profiles and formulations available. Treatment of children with TSC and infantile spasms with vigabatrin is very effective (Chiron et al., 1991, 1997; Jambaque et al., 2000; Elterman

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et al., 2001; Curatolo et al., 2006). The major advantages of vigabatrin are the ability to rapidly escalate the dosage at the initiation of treatment, rapid efficacy, suitability for outpatient treatment and particularly good tolerability with generally only minor adverse effects, with the exception of visual field loss. Vigabatrin is a novel drug that works by preventing the breakdown of the neurotransmitter GABA. The safety of vigabatrin has caused concern since a specific visual field loss has been documented in treated adults and some children (Harding et al., 2002). Visual field loss is usually asymptomatic and can be detected only by perimetric visual field studies. In very young children it is difficult if not impossible to detect the visual field loss. Until a clear answer about the occurrence of this adverse effect in children has been establish through randomized studies, VGB may still be considered first-line therapy in epileptic spasms. We recommend a 10-day course of VGB as initial therapy Adrenocorticotropic hormone (ACTH) should be the first drug of choice if VGB is not available (Curatolo et al., 2001b). When VGB or ACTH fails to control spasms, then topiramate can be prescribed. Recently, some authors reported that the use of levetiracetam as adjunctive antiepileptic therapy in patients with TSC can reduce seizure frequency (Collins et al., 2006). Alternative treatments to antiepileptic drugs should be considered in patients with TSC whose seizures cannot be effectively controlled. Current nonpharmacological treatments included vagus nerve stimulation, ketogenic diet and resective epilepsy surgery (Thiele, 2004). Epilepsy surgery has a very important role in the management of children when an epileptogenic tuber or epileptogenic zone is identified (Romanelli et al., 2004). Converging information between videoEEG monitoring and extracranial localization with MRI fusioning can improve our ability to select candidates who could benefit from surgical treatment. There is little doubt that the treatment of TSC will benefit from present and future research on the molecular mechanisms involved in the pathogenesis of this devastating disease. Knowledge regarding the function of the tuberin–hamartin complex has led to therapeutic intervention trials (Lee et al., 2005). Rapamycin, a commercially available immunosuppressant, inhibits the ability of mTOR to phosphorylate downstream substrates, such as the S6Ks and 4EBPs. Rapamycin is generally a well tolerated medication. Common, typically self-limiting side effects include aphthous ulcers, acneiform rash, diarrhea and arthralgias, as well as potentially dramatic elevation of serum cholesterol and lipoproteins. Oral rapamycin therapy can induce regression of astrocytomas associated with

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Table 9.6 Recommendation for treatment

Dermatological Neurological

Neuropsychological and cognitive Pulmonary

Pathological manifestations

Recommendations for management

Facial angiofibromas Ungular fibromas/shagreen patch Cortical tubers Subependymal giant cell astrocytomas Learning disabilities, affective disorders, autism spectrum disorders Lymphangioleiomyomatosis

Repeated laser treatment, dermal abrasion and surgical removal Surgical or laser removal Anticonvulsants, resective epilepsy surgery Surgical removal with or without ventriculoperitoneal shunt

Renal

Angiomyolipomas Renal cell carcinoma Polycystic kidney disease

Cardiac

Rhabdomyoma

TSC and might offer an alternative option to surgical treatment of these lesions (Franz et al., 2006). Current research in the use of inhibiting factors will probably help control several different forms of benign and malignant tumoral growth, and could offer new hope for a better quality of life for patients.

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Early childhood programs and ongoing evaluations and treatment Pulmonary decorticationHormone therapy (medroxyprogesterone acetate, surgical estrogen ablation, tamoxifen)Lung transplantation Aggressive approach to AMLs > 3.5 cm with either arterial embolization or surgical resection, exploration with renal conserving surgery, treatment of hypertension with medication, renal transplant Antiarrhythmic agents and diureticsSurgical intervention

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Lopes MBS, Altermatt HJ, Scheitauer BW, et al. (1996). Immunohistochemical characterization of subependymal giant cell astrocytomas. Acta Neuropath 91: 368–375. Lucarelli P, Palminiello S, Curatolo P, et al. (2003). Association study of the autistic disorder and chromosome 16p. Am J Med Genet 119A: 24: 2–246. Moolten SE (1942). Hamartial nature of the tuberous sclerosis complex and its bearing on the tumour problem: report of one case with tumour anomaly of the kidney and adenoma sebaceum. Arch Intern Med 69: 589–623. Nabbout R, Santos M, Rolland Y, et al. (1999). Early diagnosis of subependymal giant cell astrocytoma in children with tuberous sclerosis. J Neurol Neurosurg Psychiatry 66: 370–375. Nellist M, van Slegtenhorst MA, Goedbloed M, et al. (1999). Characterization of the cytosolic tuberin–hamartin complex. Tuberin is a cytosolic chaperone for hamartin. J Biol Chem 274: 35647–35652. Niida Y, Lawrence-Smith N, Banwell A, et al. (1999). Analysis of both TSC1 and TSC2 for germiline mutations in 126 unrelated patients with tuberous sclerosis. Hum Mutat 14: 412–422. Niida Y, Lawrence-Smith N, Banwell A, et al. (2001). Survey of somatic mutations in tuberous sclerosis complex (TSC) hamartomas suggests different genetic mechanisms for pathogenesis of TSC lesions. Am J Hum Genet 69: 493–503. Normann S, Green AJ, von Bakel I, et al. (1997). Tuberous sclerosis-like lesions in epileptogenic human neocortex lack allelic loss at the TSC1 and TSC2 regions. Acta Neuropathol (Berl) 93: 93–96. Park SH, Pepkowitz SH, Kerfoot C, et al. (1997). Tuberous sclerosis in a 20-week gestation fetus: immunohistochemical study. Acta Neuropathol (Berl) 94: 180–186. Pellizzi GB (1901). Contributo allo studio dell’idiozia. Riv Sper Freniatr Med Leg Alien Ment 27: 265–269. Povey S, Burley MW, Attwood J, et al. (1994). Two loci for tuberous sclerosis: one on 9q34 and one on 16p13. Ann Hum Genet 58: 107–127. Prather P, de Vries PJ (2004). Behavioural and cognitive aspects of tuberous sclerosis complex. J Child Neurol 19: 666–674. Reid CB, Liang I, Walsh C (1995). Systematic widespread clonal organization in cerebral cortex. Neuron 15: 299–310. Roach ES, Sparagana SP (2004). Diagnosis of tuberous sclerosis complex. J Child Neurol 19: 643–649. Roach ES, Gomez MR, Northrup H (1998). Tuberous sclerosis complex consensus conference: revised clinical diagnostic criteria. J Child Neurol 13: 624–628. Robinson PD, Schutz CK, Macciardi F, et al. (2001). Genetically determined low maternal serum dopamine betahydroxilase levels and the etiology of autism spectrum disorders. Am J Med Genet 100: 30–36. Romanelli P, Verdecchia M, Rodas R, et al. (2004). Epilepsy surgery for tuberous sclerosis. Pediatr Neurol 31: 239–247. Rose VM, Au K-S, Pollon G, et al. (1999). Germline mosaicism in tuberous sclerosis: how common. Am J Hum Genet 64: 986–992. Sancak O, Nellist M, Goedbloed M, et al. (2005). Mutational analysis of the TSC1 and TSC2 genes in a diagnostic

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Chapter 10

Hemimegalencephaly syndrome LAURA FLORES-SARNAT* Alberta Children’s Hospital, Calgary, Alberta, Canada

10.1. Introduction Hemimegalencephaly (HME) or unilateral megalencephaly is an uncommon but major congenital malformation of the brain, characterized by hamartomatous overgrowth limited to one cerebral hemisphere. (Hamartoma: tissue composed of dysplastic and disorganized cells situated within their organ of origin. The tissue architecture (arrangement of neurons) and also the morphology of the cells themselves are abnormal.) The term hemimegaloencephaly is rarely used but refers to the same brain malformation (Sugiyama et al., 1994; Taha et al., 1994; Ahmed et al., 2002). HME differs from all other cerebral dysgeneses because of its extreme asymmetry not corresponding to any normal stage of human brain development (Sarnat, 1992). The etiology of hemimegalencephaly is unknown but, even when no chromosomal abnormalities or causative gene(s) have been identified and great variation in the clinical, neuroimaging and pathological features exists, HME represents a unique clinicopathological entity. In this context, HME should be considered a syndrome because it is manifested by numerous symptoms and signs, although the etiology is unknown. The neurocutaneous syndromes are the most important condition associated with hemimegalencephaly and it is crucial to recognize this association (Flores-Sarnat, 2002, 2006). There are several examples of normal asymmetry in the evolution of the nervous system. The myotomes of amphioxus, an extant chordate regarded as a prevertebrate, are not matched pairs but alternate on the two sides of the body wall (Sarnat and Netsky, 1981). In many fishes and amphibians, the left or right habenula, depending upon the species, is significantly larger and better organized into distinct nuclei in the dorsal

diencephalon than its smaller pair (Sarnat and Netsky, 1981). In humans, however, both habenulae are symmetrically small and poorly developed. In the zebrafish the pineal gland does not straddle the midline but shows a left-sided bias (Liang et al., 2000; Fekete, 2001). In humans, functional cerebral dominance is accompanied by subtle anatomical asymmetry (Wada et al., 1975; Kopp et al., 1977; Hering-Hanit et al., 2001). A study with prenatal ultrasound demonstrated a larger left hemisphere in normal fetuses at 20–22 weeks gestation, irrespective of gender (Hering-Hanit et al., 2001). HME is neither secondary to, nor associated with, metabolic or neurodegenerative disorders. 10.1.1. Historical background The earliest mentions of HME in the literature are four autopsy reports from the 19th century. In 1835, in a review of 253 autopsies, Sims mentioned the finding of an enlarged left cerebral hemisphere in an adult woman, but he did not provide further clinical or pathological information (Sims, 1835). The second case was reported by Martin, who described the brain of a man with hypertrophy of the right cerebral hemisphere and deviation of the medial line to the left (Martin, 1844). The third report provided unequivocal and detailed clinicopathological information to conclude the presence of HME. It was made by J. Batty Tuke in 1873 in a man with mental retardation and epilepsy since the neonatal period (the mother attributed it to: ‘when he was 10 days old a drunken man stumbled against the child’s head and immediately afterwards epileptic convulsions set in’). The title of his report was ‘A case of hypertrophy of the right cerebral hemisphere, with coexistent atrophy of the left side of the

*Correspondence to: Laura Flores-Sarnat MD, Alberta Children’s Hospital, Division of Paediatric Neurology, 2888 Shaganappi Trail NW, Calgary, Alberta T3B 6A8, Canada. E-mail: [email protected], Tel: 1-403-955-3013, Fax: 1-403-955-2922.

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body’. His patient also showed ipsilateral enlargement of the right cerebellum and medulla oblongata. This report was the first with histological description (Tuke, 1873). The fourth report was a 24-year-old woman, probably with Proteus syndrome, who died after amputation of her enlarged left leg. ‘The same asymmetrical enlargement affected all parts of the left side of the brain’, but no further description was provided and histological studies were not performed (Eve, 1883). The first clinicopathological report of hemimegalencephaly in the 20th century, by Webster, was in a child and remains one of the most thorough and meticulous reports to date; his mentor was Professor Hans Chiari (Webster, 1908).

10.2. Pathogenesis of hemimegalencephaly Until recently, hemimegalencephaly was considered a primary disturbance of neuroblast migration initiated around the third month of gestation. However, from studies with neuropathology using neuronal and glial markers, it appears more likely to be an earlier, genetically programmed developmental disorder of symmetry and cellular lineage that occurs around the third week of gestation (Flores-Sarnat, 2002; Flores-Sarnat et al., 2003). The establishment of symmetry of the body and the cellular lineage also occurs at the end of the third week of gestation, just after gastrulation. The neuroblast migratory abnormalities observed in HME are secondary to both abnormal radial glial cells and to abnormal neuroblasts, occurring after the eighth week of gestation. The neuropathology of excessive growth in HME also suggests a disturbance in proliferation that has been supported by many authors. It has been postulated that the excessive proliferation occurs early and possibly continues beyond the normal proliferative period (Ronnett et al., 1990). A relationship between epidermal growth factor and excessive proliferation has been considered in the pathogenesis of HME (Takashima et al., 1991; Kato et al., 1996). In a recent study, increased cerebral tissue levels of nerve growth factor and brain-derived neurotrophic factor in children with HME suggest another mechanism that contributes to the development of this malformation (Antonelli et al., 2004). 10.2.1. Genetic theories of pathogenesis of hemimegalencephaly The recent knowledge of molecular genetic programming of brain development has provided an insight into and perspective of ontogenesis and dysgenesis of the nervous system not previously possible. Organizer

genes are now recognized that are responsible for left–right symmetry of the neuraxis and indeed of the basic body plan of bilateral symmetry of all vertebrates. Examples include PITX2, which is expressed as early as in the primitive (Hensen) node (Ryan et al., 1998), and lefty-1 and lefty-2, which are expressed only on the left side of mouse embryos (Saijoh et al., 1999). It was shown that lefty 1 is switched on in the future head region of mouse embryos at 4 days and that a leftward flow of extraembryonic fluid in the node cavity starts the symmetry-breaking event (Shiratori and Hamada, 2006). Other nodal-related genes, squint (sqt) and cyclops (cyc), are expressed at the time of gastrulation and first function in tissue differentiation and patterning of the midline; later cyclops and molecules of downstream genes such as PITX2 and lefty are expressed transiently, only on the left side of the forebrain (Liang et al., 2000; Fekete, 2001). Asymmetrical gene expression in the brain requires an intact midline and nodal related factors (Liang et al., 2000). Positional cloning has identified another gene on the X-chromosome, ZIC3, that is responsible for some cases of human heterotaxy, failure of the normal left–right asymmetry of the body with situs solitus, situs ambiguus or, in extreme form, situs inversus in the positions of visceral organs in particular (Casey, 1998). A report found that an excess of retinoic acid suggests an influence in the early steps in establishment of the left–right embryonic axis by causing perturbations of heart looping (Wasiak and Lohnes, 1999). Continuous advances in the study of the genetic mechanisms that underlie brain asymmetry involve a larger list of genes (Geschwind and Miller, 2001). In a recent study of gene expression levels between the left and right embryonic cerebral hemispheres, the authors identified 27 differentially expressed genes; LMO4 was more highly expressed in the right perisylvian human cerebral cortex than in the left (Sun et al., 2005). Many genes are now recognized as mitogens and regulate ratios of neuronal populations that are synaptically related. The forebrain overgrowth gene (fog) in the homozygous mouse produces, among other CNS defects, an excessive cellular proliferation and growth in the forebrain that result in symmetrical megalencephaly (Harris et al., 1997). If this or another similar mitogenic gene interacted with a gene of symmetry or lateralization, as mentioned above, it might explain some cases of human HME in which the enlarged cerebral hemisphere is excessively cellular. These genes may or may not play a pathogenetic role in human HME but at least they provide a theoretical genetic basis for a variety of asymmetrical brain malformations (Flores-Sarnat, 2002; Table 10.1).

HEMIMEGALENCEPHALY SYNDROME Table 10.1 Asymmetrical brain disorders possibly related to genes of symmetry Sturge-Weber syndrome Unilateral familial pachygyria Unilateral familial schizencephaly Familial porencephaly Unilateral familial nodular periventricular heterotopia Dysplastic gangliocytoma of cerebellum (Lhermitte-Duclos disease) Progressive hemifacial atrophy (Parry-Romberg syndrome)

10.3. Pathogenesis of neurocutaneous syndromes The traditional explanation that the common origin of skin and central nervous system from ectoderm is the cause of neurocutaneous syndromes started at the beginning of the 20th century with Van der Hoeve’s concept of ‘phakomatosis’ (Van der Hoeve, 1920). However, it was soon noted that mesodermal and endodermal tissues also were involved (Yakovlev and Guthrie, 1931). Furthermore, with the advent of molecular genetics, the traditional concept of three germ layers has been challenged because the expression of many developmental genes is not restricted to one germinal layer. At present, there are many clues, both clinical and molecular, that support the new concept that an abnormality in the formation, migration or differentiation of neural crest cells is the common pathogenetic factor for most, if not all, primary neurocutaneous syndromes (Sarnat and Flores-Sarnat, 2005). The role of the neural crest in neurocutaneous syndromes is manifested in common features shared by most of these disorders, which include vascular malformations of the skin and other organs, hypo- or hyperpigmented cutaneous lesions and peripheral nerve lesions. The pattern of skin lesions observed in several neurocutaneous syndromes that follow the Blaschko lines (incontinentia pigmenti, hypomelanosis of Ito, epidermal nevus syndrome), also can be attributed to abnormalities in neural crest (Sarnat and FloresSarnat, 2005). Lipomas, characteristic in several neurocutaneous syndromes, are terminal overgrowths due to dysregulation of pluripotential neural crest cells (ValBernal et al., 2002; Sarnat and Flores-Sarnat, 2005). Some examples of neurocutaneous syndromes coexisting with lipomas (hemifacial, corporal, intracranial) are epidermal nevus syndrome (ENS), Proteus syndrome, Klippel–Trenaunay syndrome (KTS), Cowden syndrome, encephalocraniocutaneous lipomatosis and

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Bannayan–Riley–Ruvalcaba syndrome. Some patients develop a more severe condition of a unilateral facial fatty mass named by Slavin ‘congenital infiltrating lipomatosis of the face’ (CILF) (Slavin et al., 1983). This is a nonencapsulated lesion of mature lipocytes typically located in the cheek, that infiltrates into adjacent tissues such as dermis, tongue, masticatory muscles, lower lip, and the parotid gland, with a tendency to recur after resection (Slavin et al., 1983; Donati et al., 1990; de Lone et al., 1999; Unal et al., 2000; Aydingo¨z, 2002). There are many cases in the literature associated with hemimegalencephaly in which this lesion was not recognized. In the patient reported by Donati, CILF was confirmed, concomitant with cytomegalovirus infection, but the HME was not diagnosed (Donati et al., 1990). These authors reported several patients presenting the hemifacial lesion associated with hemimegalencephaly; some had ENS that was unrecognized. Infiltrating lipomatosis is larger in size than the usual facial lipoma and has a more aggressive behavior with infiltration of surrounding tissues. In earlier literature, this condition had been named ‘unilateral progressive facial hypertrophy’ (Updegraff, 1930), or ‘congenital hypertrophy of the face’ (Stafne and Lovestedt, 1962; Furnas et al., 1970). The hemifacial lipoma or infiltrating lipomatosis may be observed in epidermal nevus, Proteus and KTSs. 10.3.1. Classification As mentioned above, hemimegalencephaly can be considered a syndrome, therefore, I consider it necessary to change the terminology I proposed previously (Flores-Sarnat, 2002). There are three forms of hemimegalencephaly (Table 10.2):

Table 10.2 Classification of hemimegalencephaly 1. Isolated 2. Associated Neurocutaneous syndromes: Epidermal nevus Proteus Klippel–Trenaunay–Weber Hypomelanosis of Ito Neurofibromatosis 1 Tuberous sclerosis Encephalocraniocutaneous lipomatosis Others: Aicardi syndrome, Hirschsprung’s disease 3. Total

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1. Isolated hemimegalencephaly occurs as a sporadic disorder without associated hemicorporal hypertrophy, cutaneous or systemic involvement 2. Associated hemimegalencephaly is accompanied by other conditions, mainly neurocutaneous syndromes; consequently, many of these forms follow a mendelian pattern of inheritance 3. Total hemimegalencephaly, first described by Hallervorden (1923), is the least frequent form; it also involves ipsilateral enlargement of cerebellum and brainstem (Sener, 1997). Total hemimegalencephaly may be isolated or associated. It is very important to distinguish the isolated form of HME from that associated with other conditions, particularly with neurocutaneous syndromes, because investigation, management and prognosis are different.

10.4. Neuropathology of hemimegalencephaly There is a great diversity of gross and microscopic lesions, as well as degrees of severity; however, there are no differences between isolated HME and associated HME. Neither are there distinctive features in the neuropathological findings among the group of neurocutaneous syndromes associated with HME (Flores-Sarnat et al., 2003), similar to the lack of distinction of clinical, electroencephalography (EEG) and neuroimaging features between isolated and syndromic hemimegalencephaly (Flores-Sarnat, 2002). Only moderate and severe cases of HME have been studied neuropathologically. There are no reports in the literature of post-mortem or surgical resection of mild HME. Pathological examination does not, therefore, support the concept of a diversity of etiologies with different mechanisms of pathogenesis, despite the association of HME with various syndromes, but rather suggests a common end-result that might be produced in several genetic disorders. In this context, it is analogous to holoprosencephaly. 10.4.1. Neuropathological findings reported in the literature 10.4.1.1. Macroscopic (gross) findings in published cases The first feature that calls attention is the overt evident asymmetry in size. The cortical surface of the enlarged hemisphere shows pachygyria predominantly, mixed with zones of lissencephaly and polymicrogyria (Bignami et al., 1968; Yasha et al., 1997; Abdelhalim et al., 2003; Flores-Sarnat et al., 2003). The cortical gray matter is thickened as much as three times normal but the subcortical white matter also has a larger than

Fig. 10.1. Gross coronal section of posterior part of resected hemisphere, after formalin fixation, of a 4-month-old girl with left hemimegalencephaly (same girl as Fig. 10.9). The occipital horn of the lateral ventricle is nearly occluded. Lissencephaly, pachygyria and polymicrogyria characterize the cerebral cortex. The border between gray and white matter is indistinct and serrated. The occipital lobe (bottom) was shifted across the midline.

normal volume (Kato et al., 1996) and the gray–white matter junction is poorly demarcated (Fig. 10.1). The lateral ventricle of the enlarged hemisphere usually is straightened (similar to callosal agenesis) and mildly or moderately dilated but is seldom massively expanded and is sometimes narrow or even compressed. Few previous pathological studies of HME have described the corpus callosum. A short and thin corpus callosum is one of the reported anomalies (Yasha et al., 1997). Occasionally small nodules of heterotopic gray matter are demonstrated in the centrum semiovale, particularly in the temporal lobe, but it is rare to find confluence of periventricular nodular heterotopia (Hannan et al., 1999; Broumandi et al., 2004). Other cases exhibit no macroscopic heterotopia, although they do have scattered single heterotopic neurons (see below). The cerebellum shows no hemispheric asymmetry in most cases but, in a few patients, ‘total hemimegalencephaly’ can be found; the cerebellar hemisphere ipsilateral to the side of the cerebral enlargement is also enlarged with abnormal foliation (Hallervorden, 1923; Reardon et al., 1996; Abdelhalim et al., 2003). The brainstem and spinal cord are usually normal, although the corticospinal tracts forming the cerebral

HEMIMEGALENCEPHALY SYNDROME peduncles in the midbrain and the pyramids at the base of the medulla oblongata may be mildly asymmetrical, enlarged ipsilateral to the large cerebral hemisphere (Reardon et al., 1996), but more frequently they are equal in size. In total HME, not only are the peduncles asymmetrical but the midbrain, tegmentum, basis pontis and medulla oblongata are similarly unequal in size. 10.4.1.2. Microscopic findings in published cases Cerebral cortical lamination is indistinct or lacking in the more severe forms, including poor demarcation of even the molecular layer in some areas; large and small neurons are mixed randomly rather than separated into distinct laminae and the architecture of the cortex often is more columnar than layered. In milder form, the layers of the cortex may indeed be distinguished but are not as distinctive as normal age-matched controls, regardless of which lobe of the cortex is examined microscopically, and many neurons are disoriented with axons and dendritic trunks extending in the wrong direction. Extensive single heterotopic neurons are demonstrated throughout the subcortical white matter and usually appear more ‘isolated’ than grouped or clustered. Small leptomeningeal glioneuronal heterotopia also may be found (Takashima et al., 1991; Kato et al., 1996). One of the most distinctive features of HME that distinguishes it from most other cerebral malformations is that many neurons are greatly enlarged, with extensive cytoplasmic Nissl bodies (Manz et al., 1968; Bignami et al., 1968; Takashima et al., 1991; De Rosa et al., 1992; Flores-Sarnat et al., 2003), confirmed ultrastructurally as granular endoplasmic reticulum (Robain et al., 1989) and by biochemical analysis, flow cytometry and quantitative histochemistry as an increase of both DNA and RNA in these enlarged cells (Bignami et al., 1968). The hypertrophic neurons are mainly found in the deep layers of the cortex and in many cases giant neurons are more numerous than the small granule cells that normally populate layers 2 and 4 (Tjiam et al., 1978; Adamsbaum et al., 1998). The small neurons, as well as the enlarged ones, exhibit bizarre shapes and are disoriented (Dambska et al., 1984). Many, though not all, large pyramidal cells in the deep layers of cortex are shown to be disoriented in Golgi impregnations (Takashima et al., 1991). Many cases exhibit ‘balloon neurons’ similar to those seen in focal cortical dysplasias of the Taylor type (Adamsbaum et al., 1998). Increased dendritic ramifications and synaptic boutons of enlarged neurons are observed histochemically and confirmed with standard silver and Golgi impregnations as well as ultrastructurally (Robain et al., 1989; Takashima et al., 1991; Tagawa

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et al., 1997). The distribution of spines on basal dendrites is abnormal, particularly at the depths of shallow sulci, and some neurons are fusiform and have synaptic spines on the soma as well as on their dendrites, reminiscent of an immature state (Takashima et al., 1991). Because of the increased total thickness of the cortex, the number of synapses in a column of cerebral cortex is increased but the number of synapses per neurons and the proportions of axospinous, axodendritic and axosomatic synapses in hemimegalencephalic cortex do not differ from controls (O’Kusky et al., 1996). It cannot, therefore, be regarded strictly as a ‘synaptic dysgenesis’. In one case with hemicerebellar as well as hemicerebral enlargement, the histological architecture of the cerebellar cortex remained preserved (Reardon et al., 1996). The histopathological details of the internal architecture of the brainstem and cerebellum in most cases of asymmetrical infratentorial structures are poorly documented, particularly in isolated HME. Many axons are excessively thick but may show disproportionately thin myelin sheaths, confirmed with silver impregnations and electron microscopy. Dysmyelination, a feature demonstrated by special techniques of magnetic resonance imaging (MRI) in living patients, is confirmed histopathologically in the white matter of at least some hemimegalencephalic hemispheres (Adamsbaum et al., 1998); myelin basic protein is poorly expressed (Takashima et al., 1991). In some cases, the myelination appears to remain normal. Few or no binucleated or multinucleated neurons are demonstrated in most cases, as may occur in tuberous sclerosis, but occasional cases do exhibit this cytological aberration (Takashima et al., 1991). No storage material accumulates in neurons or glia and no concentric lamellar bodies or membranous debris are seen ultrastructurally. Viral inclusions are not reported. No mitotic activity is demonstrated, although some authors have raised the question of neoplasia (Dom and Brucher, 1969; Townsend et al., 1975; Choi and Kudo, 1981). Proliferation of glia, especially astrocytes, is evident in both gray and white matter; some binucleated astrocytes, but no giant multinucleated cells, are found (Robain et al., 1989; Tagawa et al., 1997). Most astrocytes are excessively large with bizarre processes but are not gemistocytic; this unique gliosis is confirmed by immunocytochemical demonstration of glial fibrillary acidic protein (Tagawa et al., 1997). Takashima et al. (1991) confirmed abnormal astrocytic forms and processes but found only borderline hypertrophy in four of seven cases and normal sized astrocytes in the others.

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The ependyma is discontinuous and subventricular ependymal rosettes and heterotopic sheets of ependymal cells are seen, particularly in regions underlying a ventricular surface not covered by ependyma; ependymal cells have a normal morphology and are not enlarged (Sarnat et al., 1993a). The parenchymal and meningeal vasculature is normal, without endothelial proliferation or telangiectasia. Small areas of dystrophic calcifications are sometimes found in the parenchyma. These microscopic foci are associated with tissue necrosis or infarction but extensive zones of cerebral necrosis or hemorrhages as with major cerebral arterial occlusions do not occur and there are no inflammatory cell infiltrates except for reactive macrophages. They frequently follow a perivascular distribution around arterioles (Takashima et al., 1991). No remarkable changes are reported in the leptomeninges, except for the occasional glioneuronal heterotopia. Rarely, cytological changes are demonstrated in the opposite hemisphere in cases of isolated HME, of a milder but similar nature.

or abnormally proliferated. Mitotic spindles were not demonstrated in any cells. This study included histochemistry and immunocytochemistry with neuronal and astrocytic markers. 10.4.2.1. Histochemical stains

Luxol fast blue showed normal a myelination pat

10.4.2. Results in our cases In the neuropathological study of three patients, most of the macroscopic and histological findings described above were reconfirmed (Flores-Sarnat et al., 2003). Numerous enlarged cells with bizarre shapes and processes were demonstrated in both the gray and white matter. Balloon cells were scattered throughout but were distinct from the other enlarged cells with either predominantly neuronal or glial morphology (Fig. 10.2). Oligodendrocyte nuclei were seen throughout the white matter but these cells were not enlarged

10.4.2.2. Immunocytochemical reactivities

MIB1 (Ki67marker of G1, G2 and S phases of

Fig. 10.2. & eosin). orange–red not of glial

Balloon cells in white matter (hematoxylin Acridine orange fluorochrome showed weak RNA fluorescence characteristic of neurons but cells (not shown).

tern in the white matter, consistent with the age of the patient Periodic-acid–Schiff (PAS) showed abundant, but not excessive, glycogen in most of the enlarged cells with little distinction between them. The PAS-positive material was digested by diastase Acridine orange fluorochrome demonstrated orange-red RNA fluorescence in neurons of the cerebral cortex (normal) and also in most of the enlarged cells both in the cortex and in the subcortical white matter. Those heterotopic cells with neuronal morphology, in terms of shape and nuclear detail, had strong cytoplasmic AORNA fluorescence; balloon cells also showed a similar fluorescence but it was distributed centrifugally, similar to chromatolytic neurons.

mitotic cell cycle) showed only a few, widely scattered, reactive nuclei in either gray or white matter. In particular, the enlarged heterotopic cells and balloon cells were not immunoreactive MAP2 (neuronal and dendritic marker) showed variable but positive reactivity in nearly all the subcortical heterotopic enlarged cells, including balloon cells, regardless of cytological morphology (Fig. 10.3). The small oligodendrocytes were nonreactive, as were ependymal cells, endothelial cells and leptomeningeal cells. Cortical neurons were uniformly reactive CgrA (neuronal marker) was reactive in about half of the cortical neurons, with no special predilection for layer or cellular type, and also was seen in many heterotopic cells in the white matter, although not in balloon cells. The intensity of immunoreactivity was extremely variable NeuN (neuronal marker) showed strong nuclear reactivity in all cortical neurons and in a majority of cells in the subcortical white matter, regardless of size or shape and including balloon cells. Cytoplasmic reactivity also was seen and was variable NFP (neuronal and axonal marker) immunoreactivity was demonstrated in all cortical neurons and in a majority of subcortical heterotopic cells

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Fig. 10.3. Neuronal marker. Microtubule-associated protein2 (MAP 2) demonstrates that nearly all of the large cells in the subcortical white matter are immunoreactive but with variable degrees of intensity. The balloon cells (arrow) show weaker reactivity than more characteristic neurons, but some small neurons also show weak reactivity. The reactivity of the majority of cells with glial markers, such as GFAP, indicates mixed lineage. 250.

Synaptophysin (marker of axonal terminals) was

demonstrated diffusely in the neuropil of the cortical gray matter but was most striking in the subcortical white matter: it clearly demonstrated axons and their terminals in relation to many subcortical heterotopic neurons and a minority of balloon cells. Some of the terminals on heterotopic neurons were asymmetrical, confined mainly to one side of the cell and some terminals appeared distorted, such as spiraled GFAP (astrocytic marker) – reactive and nonreactive cells were demonstrated in the subcortical white matter but about 80% of these cells were immunoreactive, particularly the large cells (Fig. 10.4). Astrocytic end-feet terminating upon parenchymal capillaries were demonstrated by GFAP (normal). Balloon cells generally were reactive. Some cortical neurons also showed weak GFAP reactivity. Ependymal cells showed a persistent fetal expression of GFAP S-100b protein (glial marker) was also demonstrated in 80% or more of subcortical cells, both large and small, and in scattered cortical cells, most with glial but some with neuronal morphology. Nearly all balloon cells were reactive. Ependymal cells, both at the ventricular surface and in subventricular rosettes, were reactive, as is characteristic of the fetus but not the term neonate Vimentin (glial marker, also expressed in immature neurons and radial glia) was demonstrated not only in the cells of glial morphology but also in many cells that clearly were neurons in both

Fig. 10.4. Glial marker (GFAP): about 80% of large cells in the subcortical white matter are immunoreactive for glial fibrillary acidic protein (GFAP), though some show stronger reactivity than others. A few cells show no reactivity. Many cells are closely adherent to others and appear fused. 250.

the cerebral cortex and in the subcortical white matter. This reactivity was seen both with and without thermal intensification of the incubation. 10.4.2.3. Ultrastructural findings

Many large, heterotopic cells in the subcortical

white matter have neuronal ultrastructure, including extensive granular endoplasmic reticulum in the cytoplasm and coarse chromatin and large, prominent nucleoli in the nuclei. Furthermore, axonal terminals surround the cell body and proximal dendrites Many giant axons are seen, surrounded by excessively thin myelin sheaths for the diameter of the axon, although the concentric whorls of myelin have normal, regular interperiodic distances. The axoplasm contained the expected mixture of neurofilaments, microtubules and mitochondria and show no degenerative features Some subcortical cells have ultrastructural features that are somewhat ambiguous, both neuronal and glial in character, in terms of cell shape, the appearance of processes, the presence and

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L. FLORES-SARNAT distribution of Nissl substance and the morphology of the nuclei No vascular abnormalities, such as capillary proliferation, abnormal branching, endothelial proliferation or large, thin-walled vascular channels are seen. No metabolic storage materials, inclusions or mitochondrial alterations are demonstrated in any cells.

These results have shown that there is a mixed cellular lineage in the same cells with both neuronal and glial markers in HME and that this is one of the mechanisms that contribute to the pathogenesis of this singular malformation (Flores-Sarnat et al., 2003). Two of these patients had isolated HME and the other had ENS; however, they did not show any distinctive features to separate them on neuropathological grounds.

10.5. Epidemiology Hemimegalencephaly affects all ethnic groups and both genders; several reports show a male predominance (Vigevano et al., 1989; Rintahaka et al., 1993; Griffiths et al., 1994; Battaglia et al., 1999; Sasaki et al., 2005). All reported cases of HME thus far, are sporadic (Sasaki et al., 2005). The real frequency of HME is unknown. Most reports consist of single cases with no figures of the population studied. In the past, it was considered a very rare malformation, occasionally diagnosed by pathology. However, the wide use of neuroimaging has shown that its frequency is greater than once thought. It is not a rare condition: more than 120 cases have been reported. Nevertheless, there are many cases in the literature that go unrecognized; some, even, with neuroimaging or pathological evidence. For the same reason it is difficult to specify the proportion of isolated and associated cases. In particular, in those cases associated with neurocutaneous syndromes, the authors sometimes recognize the hemimegalencephaly but not the skin lesions or other features characteristic of a determined neurocutaneous syndrome (Trounce et al., 1991; Cristaldi et al., 1995; Calzolari et al., 1996; Aydingo¨z, 2002). In other reports the authors recognize the neurocutaneous syndrome or another associated condition but not the hemimegalencephaly (de Jong et al., 1976; Choi and Kudo, 1981; Hall-Craggs et al., 1990; El-Shanti et al., 1992; Pelayo et al., 1994; Torregrosa et al., 2000; Cruz et al., 2002). A third source of confusion is reports of ‘hemihypertrophy’ or ‘hemihyperplasia’, with or without a neurocutaneous disorder. In some cases they have also a neurocutaneous syndrome with neurological

manifestations of epilepsy and mental retardation; however, hemimegalencephaly is not considered and may therefore be unrecognized (Riyaz et al., 2004). An international multicenter report on isolated hemihyperplasia (hemihypertrophy) included the study of 168 patients with this condition in search of neoplasms; the presence of hemimegalencephaly was not investigated, raising doubt that some of these patients might have this condition (Hoyme et al., 1998). In another asymmetric entity, congenital hemifacial infiltrating lipomatosis, the presence of HME may go unrecognized (Donati et al., 1990; de Lone, 1999). From a review of the literature and my own clinical experience, there appears to be a slight predominance of associated hemimegalencephaly over the isolated form.

10.6. Clinical features of isolated and associated hemimegalencephaly The age at which HME is manifested is in childhood, usually in infancy and often in the neonatal period. More rarely, this malformation is not diagnosed until adult life (Cheruy and Heller, 1987; Fusco et al., 1992; Hoffmann et al., 2000; Hommet et al., 2002). In general, it is considered a serious malformation with a high and early mortality; however, there are milder cases (Flores-Sarnat, 2002). The neurological picture between isolated and associated forms of HME does not differ (Table 10.3). In isolated HME, neurological manifestations dominate the clinical picture. Three different degrees of severity can be identified. Mild, moderate and severe neurological manifestations correspond with the anatomical involvement of the affected hemisphere. In severe cases, epilepsy is often intractable; severe psychomotor retardation and unilateral motor deficits are the typical clinical manifestations of these patients. Table 10.3 Neurological clinical picture in hemimegalencephaly Macrocephaly Colpocephaly Asymmetry of the face/head Epilepsy Rapid enlargement of the head Global developmental delay Mental retardation Hemiparesis Hemianopia Learning disorder The first 4 features may be noted from the prenatal or neonatal period.

HEMIMEGALENCEPHALY SYNDROME Usually, the parents of affected children are healthy and of usual reproductive age. Pregnancy and birth are generally uncomplicated but sometimes cesarean section is required for cephalopelvic disproportion (Rintahaka et al., 1993; Calzolari et al., 1996; Prayson et al., 1999; Herman and Siegel, 2001). Usually, the first neurological manifestation at the time of birth is macrocrania with cranial asymmetry due to greater volume of the affected side (Hung and Wang, 2005). In some instances in isolated HME, the external appearance of the head is normal in both size and shape, without dysmorphic features, skin lesions or lipomas, and the only abnormality is facial asymmetry due to central facial paralysis (Flores-Sarnat, 2002). Many reports mention a rapid and progressive enlargement of the head in the first months of life (Tuke, 1873; Laurence, 1964; Townsend et al., 1975; Yasha et al., 1997; Broumandi et al., 2004), sometimes misdiagnosed as progressive hydrocephalus and leading to unnecessary surgical procedures (Griffiths et al., 1994). In other instances, this early and progressive enlargement of the head may suggest the presence of a tumor (Townsend et al., 1975; Yasha et al., 1997; Broumandi et al., 2004). Even in these cases, bulging of the fontanelles, separation of sutures, ‘setting-sun sign’ of the eyes, or other clinical features encountered in intracranial hypertension are absent. An unusual course of hemimegalencephaly disclosed in the newborn period with arrested growth in later infancy or even atrophy of the affected hemisphere has been reported (Wolpert et al., 1994; Parmar et al., 2003; Sakuma et al., 2005). Global developmental delay is noted early. Language is affected in all moderate and severe cases; however, some children with moderate involvement may learn to speak a few words or even simple sentences. Different degrees of hemiparesis contralateral to the HME are seen. Another neurological manifestation is hemianopsia ipsilateral to the hemiparesis, often detected in early infancy (Vigevano et al., 1989; Rintahaka et al., 1993). Refractory epilepsy has an early onset and is characterized by many different types of seizure, including generalized clonic or tonic–clonic, partial motor or partial complex, partial with secondary generalization, myoclonic, tonic, atonic, infantile spasms and epilepsia partialis continua. Seizures of early onset and those that are difficult to control pharmacologically correlate with poor outcome. In the neonatal period infantile spasms or West syndrome are the most common presentation, followed by tonic seizures or Otahara syndrome (Vigevano et al., 1989; Martıńez-Bermejo et al., 1992; Ohtsuka et al., 1999; Flores-Sarnat, 2002) and partial motor seizures that require multiple

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antiepileptic drugs or surgical treatment, particularly hemispherectomy. A newborn with Ohtahara syndrome and hemimegalencephaly presented epileptic negative myoclonus with a constant and nonreactive pattern of burst suppression (Guzzetta et al., 2002). Status epilepticus is a serious and frequent complication in children with HME (Sakuta et al., 1989; El-Shanti et al., 1992; Calzolari et al., 1996), sometimes leading to death (Taha et al., 1994). Mild cases without developmental delay are infrequently recognized. In such patients, the only manifestations may be pathological left-handedness, febrile seizures, learning disabilities and clumsiness (Lo´pezPisoń et al., 1998). Children with mild HME may have normal intellectual development with partial epilepsy (Fusco et al., 1992).

10.7. Associated hemimegalencephaly 10.7.1. With neurocutaneous syndromes The distinctive skin lesions of neurocutaneous syndromes and their systemic involvement distinguish them from the isolated form of HME. The association of hemimegalencephaly and neurocutaneous syndromes has been reported for a century, initially as a coincidence. Webster (1908) reported a patient with hemimegalencephaly and diverse cutaneous lesions, hemifacial lipoma and other features suggestive of Proteus syndrome (‘acromegalia of the feet’). Hallervorden (1923) reported a patient with total hemimegalencephaly and skin lesions suggesting KTS (Hallervorden, 1923). An early report of a patient with a typical picture of epidermal nevus syndrome, hemifacial lipoma and ipsilateral HME included confirmation by autopsy (Gross and Uiberrak, 1955). There are many overlapping features among many neurocutaneous syndromes that sometimes make the diagnosis difficult (Table 10.4). They include hyperand hypopigmented cutaneous lesions: cafe´ au lait (NF1, Proteus); white maculae (tuberous sclerosis complexs; epidermal nevi (ENS, Proteus); hypo or hyperpigmented whorls (hypomelanosis of Ito, incontinentia pigmenti); cutaneous, subcutaneous and internal vascular lesions (KTS, Parkes–Weber syndrome, Proteus); and lipomas (ENS, Proteus syndrome, KTS). All these features and some malignancies, such as neuroblastoma and pheochromocytoma, can be attributed to defective neural crest. Other abnormalities that involve overgrowth in many tissues, including hemihypertrophy, are common to many neurocutaneous syndromes (Cohen et al., 2002). The following are the most important neurocutaneous syndromes associated with HME.

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Table 10.4 Overlapping features in neurocutaneous syndromes associated with hemimegalencephaly Hemicorporal hypertrophy Overgrowth of hands and feet Epidermal nevi Hemifacial lipoma or infiltrating lipomatosis; systemic lipomas Intracranial calcifications Vascular malformations Systemic involvement Tumours, neoplasia

Proteus, KTS, PWS, ENS, hypomelanosis of Ito Proteus, KTS ENS, Proteus ENS, Proteus, KTS ENS, Proteus, TS KTS, PWS, Proteus Tuberous sclerosis, ENS, Proteus ENS, tuberous sclerosis, neurofibromatosis 1

ENS, epidermal nevus syndrome; KTS, Klippel–Trenaunay syndrome; PWS, Parkes Weber syndrome.

10.7.1.1. Epidermal nevus syndrome Epidermal nevus syndrome is defined by an association of epidermal nevi with abnormalities in other organ systems (Solomon et al., 1968; Rogers et al., 1989). When there is involvement of the brain, neurological manifestations are present. Several types of nevus can be found (Solomon and Esterly, 1975; Rogers et al., 1989; Happle, 1995); one of the most common is the linear nevus sebaceous of Jadassohn, usually located in the midline of the face (Fig. 10.5). Verrucous nevi are common and in some cases focal areas of alopecia (Pelayo et al., 1994) or cutis aplasia

(Mimouni et al., 1986) can be found (Fig. 10.6). Another type of nevus, the inflammatory linear verrucous epidermal nevus (ILVEN) shows a particular pattern following the lines of Blaschko. Hemimegalencephaly is a characteristic of this syndrome (Pavone et al., 1991; El-Shanti et al., 1992; Herman and Siegel, 2001; Flores-Sarnat, 2002). Approximately 50% of cases of ENS are associated with HME. A subcutaneous hemifacial lipoma is a common feature when both conditions are associated (Fig. 10.7). A characteristic triad, consisting of epidermal nevus, HME and hemifacial lipoma, was first reported by Gross and Uiberrak

Fig. 10.5. (A) 3-year-old girl with a midline nevus of Jadassohn on the forehead extending to the tip of the nose. She had mild epilepsy and mild left hemiparesis. Her CT scan (B) demonstrates right moderate hemimegalencephaly and agenesis of the corpus callosum; the right frontal horn is straightened and the frontal white matter is increased. The callosal plate and the prosencephalic neural crest both arise in the lamina terminalis: the association of this particular nevus and the callosal agenesis is, therefore, not a simple coincidence.


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invasive condition known as ‘congenital infiltrating lipomatosis of the face’ (Unal et al., 2000; Di Rocco et al., 2001; Aydingo¨z et al., 2002). In some patients, the MRI shows an ipsilateral expansion of the diploic space overlying the dysplastic cerebral hemisphere (Dhamecha and Edwards-Brown, 2001; Flores-Sarnat, 2005). Hemimegalencephaly and ENS may occur without facial asymmetry (Bonioli et al., 1997; Flores-Sarnat, 2002). In ENS epilepsy varies from mild to severe; however, when associated to hemimegalencephaly, it is usually more severe. 10.7.1.2. Proteus syndrome

Fig. 10.6. Adolescent boy with epidermal nevus syndrome, left hemimegalencephaly, intractable epilepsy since infancy and mental retardation. He has extensive verrucous nevi on the left side of the forehead, face and neck. Note the area of alopecia in the left parietal scalp.

in 1955 and later confirmed by many authors (Pavone et al., 1991; El-Shanti et al., 1992; Egan et al., 2001; Flores-Sarnat, 2002). This lipoma is ipsilateral to the HME. Some patients develop the more severe and

This is a sporadic complex hamartomatous disorder manifesting polymorphic features, many of them overlapping with other neurocutaneous syndromes, particularly ENS (Griffiths et al., 1994; Ahmetoglu et al., 2003), Klippel–Trenaunay–Weber syndrome (Plo¨tz et al., 1998), encephalocraniocutaneous lipomatosis (McCall et al., 1992) and neurofibromatosis type 1 (Cohen, 1987). These patients may develop unilateral or generalized progressive hypertrophy of the body, usually of fingers and toes. They have thick, hyperpigmented skin with focal or extensive lipomatosis, lymphangiomata and hemangiomata. Neurological manifestations are common and, if they are present, hemimegalencephaly has to be ruled out (Cristaldi et al., 1995; Sierra-Cardona et al., 1997). The first report of Proteus syndrome associated with HME and hemifacial lipoma was published by Webster in 1908. The systemic involvement includes pulmonary, urogenital and congenital heart abnormalities. Premature eruption of teeth may be seen, in association with congenital infiltrating lipomatosis of the face (Furnas et al., 1970). Severe infiltrating lipomatosis of the face, ipsilateral to the HME, can be defined by MRI (de Lone et al., 1999). In one severe case HME was associated with subependymal calcific nodules and

Fig. 10.7. Two children with epidermal nevus syndrome, hemimegalencephaly and ipsilateral nevus with hemifacial lipoma.

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developed dural sinus thrombosis (Dietrich et al., 1998). HME with open lip schizencephaly and severe epilepsy was reported in another case (Griffiths et al., 1994). A fatal case in infancy with total HME had polysyndactyly and Chiari I malformation (Reardon et al., 1996). A 2.5-year-old girl with congenital hemihypertrophy and liver hemangioma has been reported (Alpay et al., 1996). Patients with Proteus syndrome may also present a rare myopathy characterized as a ‘muscular dysgenesis’ (Sarnat et al., 1993b). Muscle biopsy may help to distinguish Proteus syndrome from ENS, KTS and encephalocraniocutaneous lipomatosis. 10.7.1.3. Klippel–Trenaunay syndrome, Parkes Weber syndrome These terms are used interchangeably; however, Cohen et al. (2002) distinguish between the syndrome described by Klippel and Trenaunay in 1900 and that described by Parkes Weber in 1907. Even when they are similar conditions, usually sporadic and characterized by developmental vascular abnormalities, there are some important clinical differences. In Parkes Weber syndrome (PWS) arteriovenous fistulas predominate. The classical triad of KTS consists of: 1) vascular malformations of the capillary, venous and lymphatic vessels; 2) varicosities of unusual distribution, particularly the lateral venous anomaly; and 3) unilateral soft and skeletal tissue hypertrophy, usually of the lower extremity. The neurological picture includes epilepsy and mental retardation. Hallervorden described one of the first cases of KTS associated with hemimegalencephaly (1923). Other cases of KTS and total HME have been reported (Torregrosa et al., 2000). A patient with a unilateral facial port wine stain and HME in the same side without involvement of extremities was diagnosed as Klippel–Trenaunay–Weber syndrome; however, this presentation suggests ENS (Dhamecha and EdwardsBrown, 2001). A report detailed a newborn with facial port-wine stain, cutaneous telangiectasia, left facial hemihypertrophy and left hemimegalencephaly at birth who subsequently developed hypertrophy of left limb. Despite these abnormalities she had normal neurological development and no epilepsy until 2 years of age (Chen and Shu, 1996). Her facial ‘hemihypertrophy’ probably corresponded to a lipoma. A patient diagnosed with KTS and HME in adult life also showed hypertrophy of the entire right side and her neurological symptoms consisted only of a mild cognitive deficit (Cheruy and Heller, 1987). 10.7.1.4. Hypomelanosis of Ito The characteristic skin lesions consist of hypopigmented whorls, streaks, and patches that follow the lines of

Blaschko. There are multiple neurological manifestations, including macrocephaly; HME is less frequent. It has been observed with both contralateral (Peserico et al., 1988) and ipsilateral (Tagawa et al., 1997) skin lesions. Children with ipsilateral skin lesions, HME and hemicorporal hypertrophy have been reported occasionally (Ardinger and Bell, 1986; Singh et al., 2004). In the first case, HME was well described but not recognized. A newborn with hemimegalencephaly and hemihypertrophy was diagnosed by ultrasound and confirmed by MRI with identification of the characteristic hypomelanotic skin lesions (Auriemma et al., 2000). One report described a girl with multiple congenital anomalies and right hemihypertrophy who was diagnosed with hypomelanosis of Ito at 12 years and right HME at 13 years (Williams and Elster, 1990). Her neurological manifestations were mild. 10.7.1.5. Neurofibromatosis type 1 This is the most common neurocutaneous syndrome, heritable as an autosomal dominant trait. Megalencephaly is recognized as a feature of neurofibromatosis type 1, ranging from 45–75% (Gutmann, 1999). By contrast, hemimegalencephaly is very rare in patients with neurofibromatosis type 1. Two children with this association were reported with mild neurological symptoms; one of them without history of epilepsy (Cusmai et al., 1990). In an adult woman with neurofibromatosis type 1, epilepsy and mental retardation, HME was diagnosed at autopsy (Ross et al., 1989). 10.7.1.6. Tuberous sclerosis This autosomal dominant systemic disease is associated with a number of characteristic and distinctive cutaneous lesions and involvement of almost every organ (Go´mez, 1991; Curatolo, 2003). Hemifacial or hemicorporal hypertrophy is not observed in patients with tuberous sclerosis. The classic neuropathological abnormalities of tuberous sclerosis does not include HME. Eight cases with an association between tuberous sclerosis and HME have been reported (Davis and Nelson, 1961; Maloof et al., 1994; Griffiths et al., 1998; Galluzi et al., 2002; Parmar et al., 2003; Sakuma et al., 2005; Cartwright et al., 2005; Area et al., 2006). In the case of Davis and Nelson, HME was not recognized. A special case was described in a clinicopathological report by Robain et al. of four patients with HME (Robain et al., 1988). One case was considered to be tuberous sclerosis; however, the cutaneous lesions corresponded to ENS, not to tuberous sclerosis, the brain did not show tubers, the protruding nodules in the lateral ventricle probably corresponded to periventricular

HEMIMEGALENCEPHALY SYNDROME Table 10.5 Histopathological comparison of tuberous sclerosis and hemimegalencephaly

Criterion Architecture of tissue Cellular pleomorphism Nuclear pleomorphism Mitotic spindles Gliosis Calcifications Immature cell markers Mixed cellular markers Balloon cells Binucleated neurons Localization of lesions

Tuberous sclerosis

Hemimegalencephaly

Disorganized

Disorganized

þ

þ

þ

þ

No No þ þ

No þ þ þ

þ

þ

Many Many

Few Few

Bilateral

Unilateral

Aicardi syndrome – agenesis of the corpus callo-

heterotopia and the authors considered the possibility of Solomon syndrome. Despite the infrequent association, tuberous sclerosis is the only neurocutaneous syndrome that shares a similar histological appearance to HME (Table 10.5). Some of the cytological similarities between tuberous sclerosis and HME include: disorganization in the architecture of tissue, cellular pleomorphism, calcifications, expression of mixed cellular markers in the same cell (FloresSarnat et al., 2003). A longitudinal study of a patient with both conditions showed progressive reduction and actually atrophy of the hemimegalencephalic side (Parmar et al., 2003). 10.7.1.7. Encephalocraniocutaneous lipomatosis This is a rare condition characterized by unilateral subcutaneous and intracranial lipomas, alopecia, unilateral porencephalic cyst, epibulbar choristoma and other ophthalmic abnormalities, first described by Haberland and Perou (1970). It is occasionally associated with HME, which may be unrecognized (Cruz et al., 2002; Jozwiak and Janniger, 2005). A great overlap exists with Proteus syndrome (McCall et al., 1992). An infant with the association of encephalocraniocutaneous lipomatosis and neurocutaneous melanosis showed, in addition, HME. The authors correlated the occurrence of both neurocutaneous syndromes to defective neural crest (Ahmed et al., 2002).

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10.7.2. Other uncommon forms of associated hemimegalencephaly

sum, including cortical dysgenesis and periventricular heterotopia have been reported (Dulac et al., 1994). The association with HME is uncommon and might go unrecognized (de Jong et al., 1976). In one report of ‘atrophy of the left hemisphere’ (Hall-Craggs et al., 1990), the illustrated MRI shows that the right side is actually hemimegalencephalic Hirschsprung’s disease is rarely associated with HME; in the single report in the literature it was accompanied by severe epilepsy (Tu¨rkdoganSo¨zuër et al., 1998) Adams–Olivier syndrome – aplasia cutis congenita, acrania partial and abnormalities of the limbs, including distal amputations (Gomes et al., 2001) Giant congenital melanocytic nevi – a report of a child with giant melanocytic nevi covering 80% of his body surface also showed HME and multiple lipomatosis, both subcutaneous and intracranial (Wieselthaler et al., 2002) Frontonasal dysplasia – one report of this malformation associated with HME and a frontal meningocele was diagnosed by prenatal ultrasound (Martinelli et al., 2002) Macrocefalia–cutis marmorata telangiectatica congenita – a multiple congenital anomaly associated with developmental delay/mental retardation; cutis marmorata may be absent in some cases (Giuliano et al., 2004) Gorlin syndrome – this case was confirmed by genetic study (Garcıá-Oguiza et al 2006).

10.8. Diagnosis Hemimegalencephaly is symptomatic even in mild cases and can be suspected from the clinical presentation, but always requires confirmation by neuroimaging. By inspection, the most orienting findings at birth are macrocephaly and asymmetry of the head. Several conditions that may cause macrocephaly in the newborn and infancy, and unilateral intracranial lesions such as hematomas, cysts, congenital tumors, etc., are easily ruled out from the first imaging. The presence of skin lesions such as epidermal nevi, hemangiomas, lipomas, etc. that suggest a neurocutaneous syndrome, raise the possibility of HME and requires investigation of cerebral involvement by neuroimaging. Other important features that should raise suspicion of a neurocutaneous syndrome and/or HME is hemicorporal hypertrophy and hemifacial

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lipoma or lipomatosis. In many cases skin biopsy will confirm or support the diagnosis and other studies such as cardiac and abdominal ultrasound are necessary to evaluate the presence and extension of systemic involvement. 10.8.1. Imaging features 10.8.1.1. Ultrasound Prenatal ultrasound may suggest HME and may show macrocephaly (Calzolari et al., 1996) or ventricular asymmetry with enlargement of lateral ventricle interpreted as hydrocephalus (Paladin et al., 1989; Hager et al., 1991; Malinger et al., 2004; Hung and Wang, 2005) corresponding to colpocephaly. HME can be confused with a tumor by prenatal ultrasound (Nishimaki et al., 2004). HME associated with frontonasal dysplasia has also been diagnosed in utero by ultrasound (Martinelli et al., 2002). Postnatal cranial ultrasound also may show unilateral ventricular dilation (Griffiths et al., 1994) with hemispheric enlargement (Calzolari et al., 1996), or extensive calcifications with unilateral absence of lateral ventricle resembling compression (Griffiths et al., 1994). 10.8.1.2. Computed tomography and magnetic resonance imaging MRI and CT confirm the presence and degree of severity of HME. Just as there is no difference in the clinical and neuropathological findings in isolated or associated HME, neither are there differences in the neuroimaging findings between these two forms of HME. The less common type, total hemimegalencephaly, can be identified better by MRI. Constant features in HME, observed in both modalities, are the gross asymmetry with enlargement of one hemisphere, dysplastic cortex and asymmetry and deformity of the ventricular system. Four main abnormalities in the affected ventricle may be observed: 1. Straightening of the frontal horn is seen in both mild and severe forms 2. Mild to extreme dilatation of the lateral ventricle 3. The reverse may be seen, with reduced size of the frontal horn which appears collapsed or occluded, suggesting a mass effect in some cases 4. Colpocephaly (selective disproportionately developmental dilatation of the occipital horn of the lateral ventricles), is seen in all grades of HME (Fig. 10.8). In massive dilatation there is an apparent lack of tissue, or a thin band of parenchyma (El-Shanti et al., 1992; Yuh et al., 1994; de Lone et al., 1999; FloresSarnat, 2002), whereas in cases with a reduced lateral

Fig. 10.8. Axial CT scan without contrast of a newborn boy with moderate isolated right hemimegalencephaly. Note the frontal calcification, the straightening of the frontal horn and colpocephaly.

ventricle the brain parenchyma appears excessive. The midline may be straight or bowed; usually it is displaced contralaterally, totally or only in the posterior region (Kalifa et al., 1987; Barkovich, 2005); in a few cases it remains undisplaced. Several neuroblast migratory disorders, such as lissencephaly, may be demonstrated by CT and MRI; pachygyria, polymicrogyria, heterotopia and other focal dysplasias are better delineated by MRI. The most severe cases affect the whole hemisphere, a condition termed ‘hemilissencephaly’ (De Rosa et al., 1992), as seen with CT and MRI. A distinctive ‘occipital sign’ is a marked shift and displacement across the midline of an enlarged, lissencephalic/pachygyric occipital lobe to the contralateral side (George et al., 1990; Flores-Sarnat, 2002) (Fig. 10.9). The accompanying deviation of the straight sinus is characteristic (Ardinger and Bell, 1986). In an extreme case the repeated bending of the occipital lobe lead to a bizarre, ‘kiwi slice’ shape (Barkovich, 2005, Fig. 5.65). This phenomenon is explained in part because a brain without convolutions occupies more space and the falx cerebri is thinner posteriorly and has less resistance than frontally. Schizencephaly is very rare (Griffiths et al., 1994). The


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and sometimes it goes unrecognized (Clancy et al., 1985; Ardinger and Bell, 1986; El-Shanti et al., 1992) or is confused with other dysgenesis as typical diffuse agyria–pachygyria (Kalifa et al., 1987). Most reports refer to moderate and severe cases; mild cases are usually not recognized. 10.8.1.2. Magnetic resonance imaging

Fig. 10.9. CT scan with contrast of a newborn girl with epidermal nevus syndrome, severe left hemimegalencephaly and intractable epilepsy. Note ‘occipital sign’ with contralateral displacement of the occipital pole. The shift of the midline and hypodensity of the enlarged occipital white matter were initially interpreted as a congenital tumor.

unaffected hemisphere usually appears smaller than normal and distorted due to displacement; often it seems normal. There is no true compression, since the ventricle and subarachnoid space usually are normal. Cases with vascular abnormalities are rarely reported with CT and MRI (George et al., 1990; Di Rocco and Iannelli, 2000) or with cerebral angiography. Sometimes CT is interpreted as normal. Asymmetry of the cranium is evident. A common finding in HME is focal, small or extensive calcifications in white and gray matter, which may be observed in different grades of HME. Diagnosis of HME by neuroimaging is not always easy

MRI is a method of choice for the study of brain malformations, providing more detail in both white matter and the brain surface. Calcifications are less obvious than with CT. An increase in the volume of white matter with an abnormal high signal has been reported in many patients with HME (Hoffmann et al., 2000; Herman and Siegel, 2001; Flores-Sarnat, 2002) (Fig. 10.10) and advanced myelination has been suggested (Tagawa et al., 1997), after observation of precocious myelination of the internal capsule and corpus callosum (Yagishita et al., 1998). A thick cortex with poor gray/white matter differentiation in the affected hemisphere is a common finding documented by many authors. Cortical dysplasia is better delineated than with CT and includes lissencephaly/pachygyria, polymicrogyria and schizencephaly. The lateral ventricle may appear small or effaced, but more frequently it is enlarged, all or only the body, the atrium or the occipital horn (colpocephaly) (FloresSarnat, 2002). Neuroblast migratory anomalies are usual findings resulting in multiple gyral abnormalities; the most frequent are pachygyria and lissencephaly. Also, heterotopia in the white matter are common; however, the association of periventricular

Fig. 10.10. Axial MRI-T2 (A) and T1(B) of two different children with severe and moderate hemimegalencephaly. There is increased volume of white matter in the enlarged hemisphere.

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Fig. 10.11. Axial MRI-T1 (A, B) and coronal (C) of a 2-year-old boy with moderate right hemimegalencephaly. There is a moderate cortical dysplasia, increase in white matter (see Fig. 10.10B) and a mild occipital sign. The corpus callosum shows asymmetry with mild thickening in the right side; straightening on the right frontal horn also is present.

heterotopia with HME is rare (Sugiyama et al., 1994). They have been demonstrated by MRI and confirmed pathologically in two patients (Hannan et al., 1999). MRI clearly shows the characteristic finding is the marked shift of an enlarged lissencephalic occipital lobe with deviation across the midline to the contralateral side (‘occipital sign’). The corpus callosum is almost always asymmetric; the affected side is usually enlarged and dysplastic, but may be bilaterally hypoplastic or absent (Griffiths et al., 1994) (Fig. 10.11). Often, it appears severely distorted (Renowden and Squier, 1994; Broumandi et al., 2004) and more heavily myelinated than its contralateral half (Yagishita et al., 1998); in mild cases it may appear normal (Cusmai et al., 1990; Fusco et al., 1992; Lo´pez-Pisoń et al., 1998). Dysplasia of the basal ganglia may suggest a tumor with mass effect (Carrenõ et al., 2001; Broumandi et al., 2004). Total HME may be documented by MRI (Calzolari et al. 1996; Sener, 1997). In some cases, the ipsilateral cerebellar hemisphere is displaced downward, resembling compression; often it is also enlarged (El-Shanti et al., 1992; Calzolari et al., 1996; Battaglia et al., 1999). An unusual finding is Chiari I (Meow-Keong Thong et al., 1999; Torregrosa et al., 2000). In rare instances hypoplasia of the cerebellar hemisphere ipsilateral to the HME may be observed (Ahmed et al., 2002). HME may be diagnosed prenatally by MRI (Yuh et al., 1994; Malinger et al., 2004; Agid et al., 2006). There are three degrees of severity of hemimegalencephaly; they are nevertheless difficult to classify because there is overlap of the different grades. The following is a suggested anatomical grading of severity (modified from Battaglia et al., 1999); in

general, this scheme is correlated with the clinical picture and macroscopic pathology (Flores-Sarnat, 2002):

Grade I: mild asymmetry and hypertrophy of the

affected hemisphere; slight abnormality of the ventricles with straightening of the frontal horn; hyperintensity of white matter; the midline is nondisplaced; there is not apparent cortical dysplasia Grade II: moderate hemispheric hypertrophy with slight or moderate displacement of midline; slight or moderate ventricular dilatation; the frontal lateral ventricle may appear occluded (either side), colpocephaly; there may be calcifications; focal or moderate cortical dysplasia, and the ‘occipital sign’ Grade III: severe, with marked hemispheric hypertrophy, bowing and displacement of the midline contralaterally; ‘occipital sign’; marked dilatation or distortion of the lateral ventricle (often colpocephaly); abnormal white matter with high signal in T2; calcifications; possible abnormalities in the contralateral hemisphere; severe, extensive cortical dysplasia (lissencephaly, pachygyria, schizencephaly, polimicrogyria, etc), with marked shift of the enlarged, occipital lobe to the contralateral side. The most severe cases show ‘hemilissencephaly’ (De Rosa et al., 1992).

Most reports of hemimegalencephaly, including neuroimaging, refer to moderate and severe cases; mild cases usually are not detected. MRI is also the method of choice for the study of the hemifacial infiltrative lipomatosis, because it permits a distinction from lipoma and provides information about the extent of infiltration in surrounding

HEMIMEGALENCEPHALY SYNDROME tissues that facilitates planning for surgery (Unal et al., 2000; Aydingo¨z et al., 2002). Functional neuroimaging also is an important aid to demonstrate abnormalities in the affected hemisphere that contribute with specific additional information to the diagnosis of hemimegalencephaly. 10.8.1.4. Proton magnetic resonance spectroscopy Examination with MRI and magnetic resonance spectroscopy (MRS) show the structural changes of the hemimegalencephalic hemisphere and also the associated metabolic disturbances with marked reduction of glutamate and N-acetylaspartate (NAA) in white matter. A mild decrease of NAA in the white matter of the contralateral hemisphere was observed (Hanefeld et al., 1995). 10.8.1.5. Single-photon emission computed tomography A good correlation between CT/MRI and iofetamine (IMP)–single-photon emission computed tomography (SPECT) was observed in two patients with severe epilepsy, with different seizures and EEG patterns; both patients showed similar decrease in tracer uptake (Konkol et al., 1990). An ictal and interictal brain SPECT in two neonates showed similar focal hyperperfusion, corresponding to the EEG seizure foci in the hemimegalencephalic hemisphere (Alfonso et al., 1998). Studies with SPECT provide valuable information in both the preoperative and postsurgical evaluation of patients, including the involvement of the nonmalformed hemisphere (Soufflet et al., 2004). 10.8.1.6. Positron emission tomography Functional imaging has disclosed cortical hypometabolism through visual analysis comparing the enlarged with the apparently unaffected hemisphere (Rintahaka et al., 1993; Ohta et al., 1994; Prayson et al., 1999). A good correlation between CT/MRI and positron emission tomography (PET) in delineating the structurally abnormal areas on the hemimegalencephalic side was found, however. PET also disclosed functionally abnormal brain regions in the non-HME, which appeared structurally normal on CT/MRI (Rintahaka et al., 1993). In an adult patient, HME was confirmed with PET, excluding the presence of a tumor (Hoffmann et al., 2000). 10.8.1.7. Magnetoencephalography Somatosensory evoked fields measured by magnetoencephalography may differentiate the severity or HME; they were absent in two patients with severe cortical dysplasia and present in a patient with relatively preserved cortical lamination (Isibashi et al., 2002).

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10.8.1.8. Electroencephalography Electroencephalography is an obligatory investigation in these patients because of a high frequency of epilepsy. The EEG is nonspecific in neurocutaneous syndromes but is characteristic in hemimegalencephaly. It is always abnormal in hemimegalencephaly, both isolated or associated. Frequently, asymmetrical, continuous paroxysmal activity is encountered, sometimes without clinical expression. In general, the presence of a defined epileptic syndrome can be identified. An uncommon, but one of the earliest (neonatal period) EEG patterns observed in patients with hemimegalencephaly is the asymmetrical suppression-burst pattern, which coincides with the clinical picture of Otahara syndrome (Martıńez-Bermejo et al., 1992). Infantile spasms are a frequent mode of onset, often with a pattern of asymmetrical hypsarrhythmia, originating on the affected side (Tjiam et al., 1978; Tu¨rkdogan-So¨zuër et al., 1998; Ohtsuka et al., 1999). A suppression-burst pattern can be recorded until adult life in patients with epilepsia partialis continua (Ohtsuka et al., 1999). Paladin et al. (1989) described three types of EEG abnormalities in HME: 1. Triphasic complexes of very large amplitude, predominating on the affected side. This pattern was observed in patients with partial seizures 2. Asymmetrical suppression-burst pattern, present at birth or after a few months 3. Asymmetrical alpha-like activity with high amplitude and little modified in the waking state. The authors found a better outcome with this pattern than with the other two. Video EEG is recommended in all patients with HME, particularly in those with subclinical paroxysmal activity. Visual and median somatosensory evoked potentials provide useful information. In patients studied with somatosensory evoked potentials, a distinction can be made from HME and other brain malformations such as lissencephaly (DiCapua et al., 1993). 10.8.2. Diagnostic criteria Hemimegalencephaly is a cerebral dysgenesis with multiple anatomical forms. By contrast with other malformations that are very characteristic and similar in most cases, in HME there are no two brains that are identical; there is no single ‘typical’ imaging. Furthermore, the anomalies encountered are sometimes completely opposed, such as the abnormalities in the ventricular system. In some cases the lateral ventricles appear collapsed and in others they are hugely dilated.

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The parenchymal abnormalities also are diverse and bizarre; neuroblast migratory disorders are frequent but they are nonspecific for HME. The abundance of abnormalities observed has led to confusion and misdiagnosis in many cases. Therefore, it is important to delineate the characteristic features to enable an accurate diagnosis. During life all cases can be diagnosed by neuroimaging. In many cases, a single picture can demonstrate unequivocal HME; however, several cases reported in the literature only include a single plane that does not show the characteristic features of HME. In such cases, it is indispensable to include at least one axial and one coronal section showing the frontal and occipital horns, to confirm the diagnosis. The first two features are the most constant findings in neuroimaging: 1. Asymmetry, with unilateral enlargement of one hemisphere, and 2. Abnormal ventricles: straightening of the frontal horn and/or unilateral colpocephaly 3. ‘Occipital sign’, (displacement of the affected occipital lobe to the contralateral side) is less frequent than the previous two but, if present, is very characteristic of hemimegalencephaly 4. Asymmetrical or distorted corpus callosum, (in mild HME may be normal) 5. Cortical dysplasia and poor distinction of gray/ white matter boundary 6. Increased white matter. If surgical resection or autopsy are available, the five previous features can be corroborated in the macroscopic neuropathological study. Histologically, there are many abnormalities; however, the most characteristic should be demonstrated: 1. Abnormal cortical architecture with indistinct lamination 2. Large neurons, displaced and disoriented with bizarre shapes, cytomorphology and coarse processes 3. Balloon cells, less numerous than in tuberous sclerosis 4. Proliferation of glia, especially astrocytes, (demonstrated with GFAP) that are enlarged and with bizarre processes. Immunocytochemical studies must be performed to determine the presence of 1) mixed cellular lineage; 2) individual heterotopic neurons in the white matter (with synaptophysin); 3) excessive immature neural cells expressing vimentin, nestin, as well as neuronal and glial markers. The clinical features are not specific; however, the following manifestations support the diagnosis of HME:

1. Macrocephaly and cranial asymmetry at birth 2. A neurocutaneous syndrome, particularly ENS, Proteus, KTS 3. Early epilepsy, partial or unilateral; EEG asymmetrical 4. Hemicorporal hypertrophy 5. Hemifacial lipoma or infiltrative lipomatosis 6. Developmental delay or mental retardation 7. Motor deficit: hemiparesis.

10.9. Management Seizure control is one of the principal goals of therapy. In the pharmacological treatment, all old and new antiepileptic drugs, depending on the type of epilepsy, have been used. Usually multiple antiepileptic drugs are required and this polypharmacy may result in complications of toxicity and multiple adverse side-effects. Steroids, adrenocorticotropic hormone (ACTH), valproic acid, benzodiazepines and vigabatrin have been used for infantile spasms and myoclonic seizures; for partial seizures, phenobarbital, phenytoin, carbamazepine, lamotrigine or topiramate have been tried. There are very few reports of the use of ketogenic diet in HME but the results are unsatisfactory: one infant received the diet only for 1 day because he developed brain death (Abdelhalim et al., 2003). There should be careful monitoring of serum levels for the antiepileptic drug used, and hepatic and hematological toxicity should be monitored. For those patients with frequent, refractory, intractable seizures, surgical treatment should be regarded as a therapeutic method and should not be postponed. Evaluation with electroencephalography, including video EEG monitoring, is recommended for all patients with HME, and is especially important for surgical evaluation and for postoperative follow-up. Multiple antiepileptic medications are usually required and also introduce complications of multiple adverse side-effects. Serum levels should be followed for the drugs used, and hepatic and hematological toxicity should be monitored. Hemispherectomy, functional or anatomical, is advocated as the best choice of treatment for cases with refractory epilepsy, after adequate trial with anticonvulsant medications (Di Rocco et al., 2006). In general, after hemispherectomy there is control of or reduction in seizures (George et al., 1990; Taha et al., 1994; Carrenõ et al., 2001) and children show improvement in global development, particularly in speech and in quality of life. Nevertheless, hemispherectomy/hemispherotomy carries a high morbidity and risk of mortality and should be performed by an experienced pediatric neurosurgeon (Di Rocco et al., 2006).

HEMIMEGALENCEPHALY SYNDROME Careful preoperative evaluation should be performed and close intraoperative anesthetic supervision by a skilled pediatric anesthesiologist is mandatory for optimal results. Many authors recommend early hemispherectomy in order to control severe epilepsy and preserve the development of higher cortical functions in the more normal contralateral hemisphere (King et al., 1985; Vigevano and Di Rocco, 1990; Di Rocco et al., 2006). A recent study showed that the function of the nonmalformed hemisphere is impaired from the first months in children with HME but can be restored after surgery (hemispherotomy in most of their cases), and they support the recommendation to operate as early as possible (Soufflet et al., 2004). Unnecessary shunting for ventriculomegaly or colpocephaly should be avoided. When a patient presents with a hemifacial lipoma, surgical excision is recommended, mainly for cosmetic reasons, with generally good results (Egan et al., 2001). On the other hand, the surgical resection of facial lipomatosis is necessary because this is a more aggressive lesion that causes severe disfigurement and functional alteration. Its resection is more difficult because of the infiltrative behavior of this nonencapsulated lesion, with a high rate of recurrence often requiring reintervention (de Lone et al., 1999; Unal et al., 2000). Physiotherapy may be helpful for those children with spastic hemiplegia due to HME, which may worsen after hemispherectomy. Formal psychometric testing is required to place school-age children in an educational setting most likely to meet their individualized learning needs associated with cognitive deficits. Speech therapy may maximize the child’s ability to communicate effectively. Attention to special dental needs should not be neglected. It is important to keep parents informed about the nature, prognosis and individual treatment of their child with this malformation, in terms they can understand.

10.10. Prognosis The prognosis of HME will depend on the grade of severity of the hemimegalencephaly and consequently on the severity of the neurological clinical manifestations (see degrees of severity in Diagnosis by MRI). Epilepsy in particular is associated with high morbidity if it presents early and is severe and refractory to medical treatment. In the most severe cases, epilepsy usually starts in the neonatal period. It is important to emphasize that the grade of severity of the hemimegalencephaly does not relate to being an isolated or associated form; it is related to the morphological involvement, which may be determined from anatomical and

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functional neuroimaging and by neuropathology if available. In all cases of HME, a neurocutaneous disorder must be excluded, seeking systemic involvement, which will change the prognosis. The risk for systemic involvement and development of malignancies is major in the presence of a neurocutaneous syndrome. The tendency to develop malignant tumors is related to the fact that the etiological genes in several neurocutaneous diseases are also tumor suppressor genes. There are no differences in neurological clinical manifestations, neuroimaging features and neuropathological findings between isolated and associated HME or between the different neurocutaneous syndromes. In any patient with a neurocutaneous syndrome with presence of asymmetry in the form of hemifacial lipoma, hemicorporal hypertrophy, hemiparesis and severe epilepsy, hemimegalencephaly should be excluded by neuroimaging. On the other hand, in all patients with hemimegalencephaly, even those considered with an isolated form, a careful examination of the skin (including biopsy if necessary) should be performed in order to search for a neurocutaneous syndrome, to anticipate possible systemic complications and provide better genetic counseling and prognosis. It is important that studies or reports on hemicorporal hypertrophy or hyperplasia (hemihypertrophy, hemihyperplasia) investigate the presence of hemimegalencephaly by CT or MRI of the brain. Also, the earliest diagnosis of HME should be established prenatally. In case of suspected HME by ultrasound during pregnancy, an MRI to confirm or rule out this diagnosis is indicated, which will permit to anticipate and planning of management of obstetrical and neonatal complications, and further investigations.

10.11. Conclusions Hemimegalencephaly is a hamartomatous malformation of the brain, involving excessive growth in one cerebral hemisphere. It differs from all other cerebral dysgeneses because of its extreme asymmetry, not corresponding to any normal stage of human brain development. Hemimegalencephaly is a syndrome because it presents a recognizable constellation of multiple symptoms and signs, although the etiology is unknown. Immunocytochemical studies have shown that hemimegalencephaly is an early disorder of cellular lineage, beginning at the time of differentiation of neuroepithelium but long before the initiation of neuroblast migration. HME can occur as an isolated disorder but is more often associated with neurocutaneous syndromes. The three most commonly involved are ENS, Proteus syndrome and KTS. A common pathogenesis for neurocutaneous syndromes

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can be related to abnormalities in the migration and differentiation of the neural crest cells. This new concept may explain many features of neurocutaneous syndromes and also the problem of overlapping manifestations. There are three grades of severity in HME, mild, moderate and severe. The grade of severity correlates with clinical, neuroimaging and neuropathological evaluation and is unrelated to the type of neurocutaneous syndrome. The prognosis will depend upon the severity of HME and whether it is associated with a systemic disease.

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Disorders of radial neuroblast migration and cerebral cortical architecture Chapter 11

Periventricular nodular heterotopia GIORGIO BATTAGLIA* AND TIZIANA GRANATA Neurological Institute ‘C. Besta’, Milan, Italy

11.1. Introduction The existence of collections of gray matter located in abnormal position within the cerebral hemispheres was recognized in early neuropathology reports dating back to the 19th century (Meschede, 1864; Virchow, 1867; Matell, 1893; Jacob, 1936). According to those classic neuropathological studies, two types of cerebral heterotopia were distinguished: the laminar heterotopia, which are separated from the overlying cortex by a thick layer of white matter, and the more common nodular heterotopia, which are formed by nodular masses of neurons and glial cells in close proximity to the ventricular walls (reviewed in Friede, 1989). The large-scale use of magnetic resonance imaging (MRI) since the mid-1980s has modified the classification of heterotopia: laminar heterotopia are now termed ‘band heterotopia’ (Barkovich et al., 1994; Gleeson et al., 1998), whereas nodular heterotopia are usually referred to as ‘periventricular nodular heterotopia’ for the periventricular location of the heterotopic neurons, close to the subependymal germinal matrix (Barth et al., 1987). Periventricular nodular heterotopia or PNH is classified as a malformation of cortical development (MCD) due to impaired neuronal migration (Barkovich et al., 2005). It can be diagnosed in vivo by means of neuroimaging and is, together with focal cortical dysplasia, the most frequent human brain dysgenesis in most clinical studies addressing MCD features (Raymond et al., 1995). In the last two decades, several papers have described the clinical and imaging features of PNH, demonstrating its clinical and genetic heterogeneity.

PNH may be bilateral or unilateral, sporadic or, less frequently, familial and genetically determined. In the vast majority of all PNH cases the most common and prominent clinical feature is the presence of focal epilepsy, frequently refractory to antiepileptic treatment.

11.2. Neuroimaging features Periventricular nodules may be recognized on computed tomography (CT) scan (Zimmerman et al., 1983) but MRI is the method of choice to detect the presence of PNH. The heterotopic nodules are isointense to cortical gray matter in all imaging sequences, and they frequently bulge into the walls of the lateral ventricles. On the basis of the MRI appearance, PNH cases have been classified in various ways in an attempt to relate different PNH types to specific etiologies (Barkovich and Kjos, 1992; Raymond et al., 1994b; Dubeau et al., 1995; Battaglia et al., 1997, 2006; d’Orsi et al., 2004). According to the more recent classification (Battaglia et al., 2006), three types of bilateral and two types of unilateral PNH can be distinguished (Fig. 11.1). The first type is bilateral and symmetrical PNH, which is the best known and most studied PNH form for the frequent relation to FLN1 gene mutations (see below). In bilateral and symmetrical PNH, multiple and contiguous nodules symmetrically line the entire extension of the ventricular walls (Fig 11.1A,B). Mega cisterna magna is frequently present, associated with cerebellar hypoplasia in some patients. The morphology of the overlying cortical areas is usually normal. This PNH type has been observed in first-degree relatives from several families (DiMario et al., 1993; Kamuro

*Correspondence to: Giorgio Battaglia MD, Molecular Neuroanatomy Laboratory, Experimental Neurophysiology and Epileptology Department, Istituto Neurologico ‘C. Besta’, Via Celoria 11, 20133 Milan, Italy. E-mail:[email protected], Tel: þ39-02-23942606, Fax: þ39-02-23942619.

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PERIVENTRICULAR NODULAR HETEROTOPIA and Tenokuchi, 1993; Huttenlocher et al., 1994; Oda et al., 1994), but it is also found in sporadic patients (Sheen et al., 2001). In the second type of bilateral PNH, i.e. bilateral and asymmetrical PNH, the heterotopia are made up by bilateral but clearly asymmetrical nodules in all cases (Fig. 11.1C). In both symmetrical and asymmetrical bilateral PNH there is a striking female predominance, and thin curvilinear gray matter bands located around the lateral ventricles may be associated (Tassi et al., 2005; Battaglia et al., 2006). However, the rationale for distinguishing the two types of PNH depends on the fact that in bilateral and asymmetrical PNH the nodules may not be confined in the subependymal periventricular region but frequently extend to the overlying cortex, and that FLN1 mutations are rarely observed in this latter type of PNH (personal observations). The third type of bilateral PNH is bilateral single-nodule PNH (Fig. 11.1C), in which the nodules are isolated, nonconfluent, small and scattered along the lateral ventricular walls. This type of PNH is much more common in males, it is not related to FLN1 gene abnormalities and it is frequently associated to focal or generalized enlargements of the lateral ventricles (Fig. 11.1D). Cases with unilateral PNH can be subdivided on the basis of the presence or absence of cortical involvement (Fig 11.1E,F). In unilateral PNH, the nodules extend from the subependymal region into the overlying white matter (Fig. 11.1E). The neocortical areas overlying the nodules may be malformed and linear clusters of gray matter may extend from the nodules toward the malformed cortex, but no anatomical continuity between nodules and cortex is evident (Fig. 11.1E). By contrast, in unilateral PNH with cortical extension, the heterotopic nodules may be very large and extend from the periventricular region to involve the adjacent neocortical and/or archicortical areas (Fig.11. 1F). These latter cases have been referred to by some authors as subcortical nodular heterotopia (Barkovich and Kjos, 1992; Dubeau et al., 1995; Barkovich, 2000) and are more frequently associated with midline abnormalities and with neurological and mental deficits due to the massive cortical involvement (Batta-

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glia et al., 2006). However, hippocampal hypoplasia ipsilateral to the PNH side is present in the majority of patients of both groups, neuropathology features are very similar in unilaterally affected PNH patients with or without cortical involvement (Battaglia et al., 1996; Spreafico et al., 1998) and the epileptic outcome is similar in both groups (Battaglia et al., 2006). This evidence suggests that these two groups are determined by similar pathogenic mechanisms, leading to unilateral periventricular nodules in milder cases and to periventricular and subcortical nodules extending to the neocortex in more severe cases. In all the abovementioned cases, the heterotopic nodules are the only or the primary abnormalities, which alter the brain structure and determine the clinical features of affected patients. However, PNH may be also part of more complex brain malformations: in particular, single periventricular nodules are not infrequently found in schizencephaly (see also Ch. 15) or polymicrogyria. The latter cases are most probably due to abnormalities of factors governing the proper organization of the cortex and not only to the impairment of neuronal migration (Barkovich et al., 2005). Since their pathogenic mechanisms are different, it is our opinion that these cases should be kept separated from PNH. In addition, bilateral PNH has been observed in rare patients with different malformation syndromes: 1) male sporadic patients with gyral abnormalities, mental retardation and frontonasal dysplasia (Guerrini and Dobyns, 1998), or cerebellar hypoplasia, cortical abnormalities, severe mental retardation and syndactyly (Dobyns et al., 1997); 2) familial male patients with gastrointestinal malformations (Nezelof et al., 1976) or congenital nephrosis (Palm et al., 1986); 3) three pedigrees and nine sporadic cases (all females except one) with Ehlers–Danlos syndrome, a connective tissue disorder (Cupo et al., 1981; Thomas et al., 1996; Sheen et al., 2005; Gomez-Garre et al., 2006); iv) familial cases with hydrocephalus (Sheen et al., 2004a). In some of these ‘variant’ syndromes of bilateral PNH a causal relation with FLN1 mutations or Xq28 abnormalities has been demonstrated or suspected (Fink et al., 1997; Sheen et al., 2005; Gomez-Garre et al., 2006).

Fig. 11.1. MRI features in the different PNH types. (A, B) Bilateral symmetrical PNH. Note the similarities between the neuropathological (A) and the MRI (B, axial IR image) images showing multiple nodules lining the entire extension of the lateral wall of the lateral ventricles. (C) Bilateral single-nodule PNH. The axial IR image demonstrates small, non-confluent, single nodules (arrowheads) adjacent to the posterior trigones of the lateral ventricles and the associated posterior ventriculomegaly. (D) Bilateral asymmetrical PNH. The coronal T1-weighted image shows multiple, coalescent heterotopic nodules along the trigones; note that the nodules extend to a malformed right parietal cortex. (E) Unilateral PNH. The coronal IR image shows coalescent nodules extending through the white matter toward the overlying left fronto-parietal cortex (arrows). (F) Unilateral PNH extended to the neocortex. The axial IR image shows a very large right PNH involving the frontal, parietal and temporal lobes. Note that the right hemisphere is slightly smaller than the left. (A) Reproduced from Okazaki and Scheithauer, 1988, with permission from Lippincott Publishing; (B–F) reproduced from Battaglia et al., 2006, with permission from Blackwell Publishing.

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11.3. Neuropathology features The neuropathology features of gray matter heterotopia were originally reported in autopsy studies not only of patients affected by periventricular nodular heterotopia but also of more complex cases with different clinicopathologic diagnoses, including peroxisomal disorders and chromosomic and dysmorphic syndromes (Harding, 1996). More recently, given the marked decline in autopsy investigations in most industrialized countries, heterotopic nodules have mainly been analyzed in cerebral samples of surgically treated epileptic patients (Battaglia et al., 1996; Spreafico et al., 1998; Tassi et al., 2005) or in autopsy samples of selected patients with known FLN1 mutations (Kakita et al., 2002). In both autopsy and surgery specimens and in the clinically diverse patients, however, heterotopic nodules display common neuropathologic features. Macroscopically, PNH appears as nodular masses adjacent to and frequently protruding into the walls of the lateral ventricles (Fig. 11.2A), but never in the walls of the third or fourth ventricles (Harding, 1996). Microscopically, the PNH is made up by rounded and irregular nodules of gray matter separated by layers of myelinated fibers. Nodules of different size may be grouped or fused together to form larger macronodules (Fig. 11.2A). Around the borders of the nodules, thin layers of tangentially arranged

myelinated fibers are commonly found, sometimes penetrating into the core of the nodule (Fig. 11.2B) (Battaglia et al., 1996; Joseph, 1997). Neurons and glia of normal size and morphology, without evidence of cortical lamination, are present within the nodules. Immunocytochemistry for neuronal markers, such as antibodies against microtubule-associated protein 2 (MAP2) or neurofilaments, reveals normal pyramidal neurons with randomly oriented apical dendrites within the nodules’ core (Fig. 11.2C) and laminar fusiform neurons located at the borders of the heterotopia (Fig. 11.2D) (Battaglia et al., 1996; Spreafico et al., 1998; Thom et al., 2004). GABAergic interneurons are also present within the nodules. Although the density of GABAergic cell bodies and neuropil is similar to that of the overlying cortical areas, they are less organized and show more immature features (in terms of cell size and complexity of dendritic and axonal processes) compared with the neocortex (Hannan et al., 1999; Thom et al., 2004). The presence within the nodules of cell sparse areas surrounded by concentric areas of increased density of GABAergic interneurons and then larger cells has been taken by some authors as evidence of rudimentary cortical lamination (Harding, 1996; Thom et al., 2004). By contrast, the neocortical areas close to periventricular nodular heterotopia are frequently reported to be normal. However, some neuropathology data sug-

Fig. 11.2. Neuropathology features of periventricular nodular heterotopia. (A, B) Photomicrographs from a surgery specimen of a patient affected by bilateral asymmetrical PNH. Note the multiple coalescent nodules close to the lateral ventricle (arrows in A), and the myelinated fibers entering the nodules (arrows in B). Luxol Fast Blue staining. (Courtesy of Roberto Spreafico.) (C, D) Photomicrographs from a surgery specimen of a patient affected by unilateral PNH extended to the neocortex. Note the randomly oriented pyramidal neurons within the core of the nodules (arrowheads in C), and the fusiform marginal neurons located around the nodule borders (arrowheads in D). Immunolabeling with antibodies against neuronal cytoskeletal elements. (Reproduced from Battaglia et al., 1996, with permission from Elsevier.) Calibration bars: 0.5 cm in A, 20 mm in B, 50 mm in C, D.

PERIVENTRICULAR NODULAR HETEROTOPIA gest that brain abnormalities may also involve the neocortex. Slight architectural abnormalities have been reported in unilateral PNH cases (Battaglia et al., 1996) and diffuse microvascular abnormalities have been found in the neocortex overlying bilateral nodules of a single patient with FLN1 mutation (Kakita et al., 2002). In addition, in unilateral cases with large PNH, cytological abnormalities have been reported in some neurons in both the heterotopic nodules and adjacent cortical areas (Tassi et al., 2005). In two patients with PNH and clinically evident dementia, Alzheimer-type degenerative lesions have been found in both nodules and neocortex (Joseph, 1997). The similarity of neuropathological abnormalities between nodules and overlying neocortex supports the concept that in PNH heterotopic nodules are formed by neurons originally committed to the neocortex (Battaglia et al., 2003a). Regarding the possible mechanisms of epileptogenesis, the evidence from human neuropathology supports the existence and possible contribution of diverse mechanisms. The presence of immature GABAergic interneurons may underscore a possible aberrant excitatory activity of these interneurons (Hannan et al., 1999). On the other hand, alterations of the a subunit of the Ca2þ/calmodulin-dependent kinase II and the NMDA receptor complex have been demonstrated in samples of human patients with unilateral nodules (Battaglia et al., 2002). These findings suggest the possible existence of an imbalance between excitatory and inhibitory mechanisms that may account for the intrinsic hyperexcitability of heterotopic nodules. In addition, reciprocal anatomical connections between the periventricular nodules and the overlying neocortex have been demonstrated in different studies (Hannan et al., 1999; Kakita et al., 2002). Thus, the intrinsic hyperexcitability of the heterotopic nodules and the existence of functional circuits connecting nodules and cortex may together explain the onset and propagation of the epileptic phenomena that are almost invariably associated with human PNH.

11.4. Etiopathogenesis According to the more recent classification of human malformations of cortical development (Barkovich et al., 2005), all types of PNH are determined by impairment of the ontogenetic mechanisms that govern the migration of postmitotic young neurons from the ventricular zone to the developing cortical plate. However, different etiological factors are involved in the genesis of PNH. They may be either acquired or genetic, and they may eventually lead to the genesis of different PNH types.

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It is not easy to relate a particular PNH patient to a specific etiology. In most sporadic cases, no mutations of known causative genes are found (Sheen et al., 2001) and prenatal risk factors for brain damage cannot be demonstrated (Battaglia et al., 1997). Even in familial cases, the same PNH phenotype may be determined by different genes, which most probably code for proteins acting in the same molecular pathway. On the contrary, different mutations of the same gene may lead to completely different clinical phenotypes (see below). The first clear evidence of the etiology of PNH came from the analysis of families with bilateral and symmetrical PNH. In these families, only female members are affected and male cases are characterized by prenatal or perinatal lethality (Huttenlocher et al., 1994), thus suggesting an X-linked mode of inheritance (Eksioglu et al., 1996). This hypothesis was shortly later revealed as correct by the demonstration of mutations of the FLN1 gene on chromosome Xq28 in affected members (Fox et al., 1998). The FLN1 gene encodes filamin 1 (or filamin A), a member of a family of actin-binding proteins. Filamin 1 is a high-molecular-weight (2647 amino acid residues) protein, with three major functional domains allowing its homodimerization and heterodimerization with actin and possibly with filamin B (Sheen et al., 2002). Filamin 1 has a number of essential functions in hemostasis, in vascular remodeling and in the migration of different cell types (Fox et al., 1998). The role of filamin 1 in neuronal migration is probably in the motor system that allows the migration of young neurons or in the system that makes neurons competent for migration. This would explain why, in patients with loss of function of the filamin 1 protein, some neurons fail to migrate but still retain the capacity for normal cellular differentiation. In all families with bilateral and symmetrical PNH that have been so far reported, only females were affected, no surviving male offspring were described and FLN1 mutations were inherited from female to female (Sheen et al., 2001). This relevant gender difference in genotype–phenotype correlation is related to the residual functional activity of filamin 1. In affected females, the random X inactivation in individual neurons creates a cell autonomous mosaic phenotype, in which neurons expressing mutant X chromosome fail to migrate, whereas neurons expressing normal X chromosome migrate properly. Affected males, who harbor a single mutant X chromosome, are unable to survive gestation because of the severe loss of protein function (Sheen et al., 2001). More recent studies have revealed a greater complexity of the pathogenic role of FLN1 mutations.

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Mosaic mutations (Parrini et al., 2004) and point mutations compatible with the preservation of residual filamin 1 functions (Sheen et al., 2001) have been described in males, associated with survival and mild clinical phenotype in the affected patients. In a recently described Japanese family, a point mutation led to an unexplained milder phenotype in the hemizygous father than in the heterozygous daughter (both unilaterally affected), and to asymptomatic phenotypes in two additional carrier females (Guerrini et al., 2004). Mutations not interfering with the production of full-length protein but rather determining gain-offunction of filamin 1 lead to a broad range of congenital malformations (otopalatodigital syndrome types 1 and 2; frontometaphyseal dysplasia and Melnick– Needles syndrome) collectively termed otopalatodigital spectrum disorders (Robertson et al., 2003). The diverse biological role of different FLN1 mutations has recently been confirmed by the association of otopalatodigital spectrum disorders and PNH in a male patient with a single mutation determining the expression of two mRNAs, the first coding for a truncated and the second for a mutated filamin 1 protein (Zenker et al., 2004). Recent studies have also revealed that not all examined familial cases carry filamin 1 mutations (seven out of eight families, according to Sheen et al., 2001 and Moro et al., 2002). Sheen and co-workers have recently reported three families with recessive mode of inheritance, and demonstrated in two of them mutations of the gene ADP-ribosylation factor guanine nucleotide-exchange factor-2 (ARFGEF2) on chromosome 20, which codes for a protein (BIG2) involved in vesicle trafficking from the trans-Golgi network (Sheen et al., 2003a, 2004b). They also reported two sporadic patients, with no FLN1 mutations, carrying duplications of the distal region of chromosome 5p (Sheen et al., 2003b). Finally, balanced translocations involving chromosomes 1 and 6, and 2p24 and 9q32, respectively, were reported in two patients with PNH associated with complex brain and somatic malformations (Leeflang et al., 2003; Ramocki et al., 2003). Taken together, these data indicate that genes other than FLN1 may determine bilateral and symmetrical PNH. This idea is further supported by the fact that only a minority (about 20% of females and 8–9% of males) of sporadic patients with bilateral symmetrical periventricular nodular heterotopia carry FLN1 mutations (Sheen et al., 2001; Guerrini et al., 2004). If we consider that bilateral asymmetrical and single-nodule PNH are rarely linked to FLN1 disruptions (personal observation), it is clear that the genetics of bilateral PNH is far from being fully elucidated. The similarity of the brain malformation may be explained, as

already indicated, by the fact that the putative novel genes may code for proteins involved in the same or similar cellular pathways relevant for the migration of cortical neurons. By contrast, acquired mechanisms could be involved in the genesis of unilateral PNH. This is suggested by the following: (1) the presence of prenatal risk factors for brain damage in the history of some unilaterally affected patients; (2) the possible association of single periventricular heterotopic nodules and polymicrogyria, a condition for which a pathogenic vascular mechanism is postulated (Barkovich and Kjos, 1992); (3) the frequent location in the posterior paratrigonal region, which is a vascular watershed area (Volpe, 1995); and (4) the anatomical continuity with structural abnormalities of the hippocampus (Raymond et al., 1994a). In unilateral cases, therefore, early acquired insults, possibly of an ischemic nature (Sarnat, 1992), may determine the failure of neuronal migration and the structural abnormalities of a limited region of the brain. It is possible, however, that combined genetic and epigenetic factors may contribute to determine unilateral periventricular nodular heterotopia. Regarding the mechanisms underlying the genesis of the malformation in unilateral PNH, the presence of disorganized radial glial fibers around periventricular nodules of autopsy fetal brains suggested that disruption of radial glia could contribute to failure of migration from the ventricular zone (Santi and Golden, 2001). However, radial glia abnormalities could be the consequence rather than the origin of impaired neuronal migration. Neurogenetic studies with the thymidine analog bromodeoxyuridine have been conducted in an available experimental model of human PNH, i.e. rats treated with methylazoxymethanol acetate (Cattabeni and Di Luca, 1997; Colacitti et al., 1999; Gardoni et al., 2003). These birthdating studies have indicated that the failure of the migration of early generated cortical neurons is sufficient to set the base for the formation of cerebral heterotopia (Battaglia et al., 2003b). Therefore, it is conceivable that, in humans also, a lesion event, for instance ischemic, in a limited region of the germinative neuroepithelium may eventually lead to nodular heterotopia (Battaglia et al., 2003a).

11.5. Prevalence As for all other malformations of cortical development, precise information on the prevalence of periventricular nodular heterotopia is lacking. The reported cases in the last decade indicate that the condition is equally distributed geographically and among ethnic groups. In selected clinical series, periventricular nodular heterotopia was found in 2% of patients with refractory

PERIVENTRICULAR NODULAR HETEROTOPIA epilepsy analyzed with MRI (Li et al., 1995) and accounted for about 20% of all cortical dysgeneses (Raymond et al., 1995). Among the different types of PNH, bilateral and unilateral cases are approximately equally common, and bilateral and symmetrical PNH account for about a third of bilateral cases. Approximately half of the unilateral cases extend to adjacent cortical areas (Barkovich, 2000; Battaglia et al., 2006). Males and females are not equally affected. Indeed, a remarkable gender prevalence has been recently reported in all the different types of PNH, suggesting the relevance of the genetic background in all PNH cases (Battaglia et al., 2006).

11.6. Clinic and epileptological features In general, patients affected by periventricular nodular heterotopia are individuals capable of conducting a normal social and working life. As in other types of cerebral malformation, the clinical picture, particularly for neurological and mental deficits, is strictly related to the amount of the heterotopic tissue, the distribution of nodules and extension to the overlying cortex. Therefore, the clinical presentation may vary in the different types of PNH. In a few cases, the presence of PNH is an incidental finding in asymptomatic patients who undergo MRI examination for unspecific complains such as migraine attacks, acute facial palsy or head trauma. In the majority of cases of all PNH types, however, epilepsy is the most common symptom and usually the presenting one. 11.6.1. Clinical features In most patients affected by bilateral and symmetrical PNH, no neurological deficits or mental retardation are present. However, IQ scores may be within the lower limits of normality (Battaglia et al., 1997). In addition, deficits in reading skills despite normal intelligence have been recently reported in patients with bilateral PNH (Chang et al., 2005). Patients with FLN1 mutations have an increased risk of cardiovascular complications, such as coagulopathies, aortic aneurysms and cardiac defects, including patent ductus arteriosus and bicuspid aortic valve (Fox et al., 1998). They are probably related to the role played by filamin 1 in vascular development and blood coagulation. Failure of these functions is probably responsible for defects in embryogenesis and severe perinatal hemorrhages leading to miscarriages and lethality in affected males (Huttenlocher et al., 1994; Fox et al., 1998). The clinical picture may be more complex in the so-called ‘variant’ syndromes of bilateral PNH, for

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the presence of multiple brain and somatic malformations. In particular, male patients with such bilateral PNH are characterized by diffuse cortical abnormalities and therefore by severe mental retardation and motor impairment (Dobyns et al., 1997; Guerrini and Dobyns, 1998). The patients affected by the Ehlers– Danlos ‘variant’ form of bilateral PNH are usually characterized by hypotonia, hyperflexible joints with frequent spontaneous dislocations, hyperextensible skin and variable expression of multiple facial dysmorphisms (Sheen et al., 2005). In patients with bilateral asymmetrical and singlenodule PNH, mild mental retardation and mild neurological deficits may be present, particularly when the nodules extend to the cortex or are associated with ventricular enlargement (Battaglia et al., 2006). A broader spectrum of clinical presentation is found in patients with unilateral PNH, in whom the clinical outcome is related to the size of the nodules, the extension to the overlying cortex and the presence of midline brain abnormalities, such as thinning or agenesis of the corpus callosum or septum pellucidum. Patients with small unilateral nodules may be asymptomatic until the onset of focal epilepsy, whereas, in patients with large nodules involving the adjacent neocortical areas, developmental delay may be evident since childhood and motor deficits, cortical visual defects and mental retardation of varying severity may be present (Battaglia et al., 2006). 11.6.2. Epilepsy features As already indicated, epilepsy is the main clinical symptom in all PNH patients. In patients with bilateral PNH, epilepsy onset is in the second decade of life, preceded by infantile febrile convulsions in some cases. Generalized tonic–clonic seizures may mark the onset of epilepsy but they are fairly rare and easily controlled by treatment. By contrast, focal seizures are observed in all epileptic patients and the semeiology of seizures is suggestive of multifocal onset in quite a number of cases. In bilateral symmetrical and asymmetrical PNH, focal seizures are frequently refractory to antiepileptic drugs, even if high-frequency seizures are rare and epileptic status is never observed. Epilepsy outcome is much more favorable in patients with single-nodule PNH, because they usually present sporadic seizures easily controlled by antiepileptic treatment. In patients with unilateral PNH, epilepsy may be a major clinical problem, given the high percentage of drug-resistant cases with highly frequent focal seizures. The mean age of onset is still in the second decade but seizures may present as early as in the first 2 years of life. However, no consistent relationship

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exists between earlier age of onset and worse epileptic outcome (Battaglia et al., 2006). In addition, the extension of PNH to the cortex is positively related to the presence of neurological and/or mental deficits but not to the severity of epilepsy. As in bilateral PNH, the semeiology of focal seizures suggests a multifocal onset in many unilaterally affected patients. Focal seizures are frequently drug-resistant and may require epilepsy surgery (see below), but malignant seizures and epileptic status are usually not reported. Epilepsy surgery outcome may be good, providing that accurate neurophysiological evaluation by means of stereoelectroencephalographic (SEEG) analysis is performed prior to the surgery (Francione et al., 1994; Aghakhani et al., 2005; Tassi et al., 2005). 11.6.3. EEG features Given the presence of drug-resistant seizures, a careful EEG assessment of epilepsy is critical in PNH patients. In addition, neurophysiology data can not only provide interesting clues to the circuitry organization of the malformed PNH brain but may also guide epilepsy surgery in patients with frequent and disabling seizures. In general, the EEG tracings in both wakefulness and slow sleep mainly reveal focal interictal EEG abnormalities consistent with the anatomical location of the periventricular nodular heterotopia (Fig. 11.3).

In slow sleep, focal activities frequently change in bilaterally diffused bursts of polyspikes (Fig. 11.3A). In addition, EEG recordings frequently demonstrate the presence of multifocal interictal EEG abnormalities. Indeed, interictal spiking activity may be asynchronously (and most probably independently) recorded not only in the bilaterally affected patients but also on different leads exploring the PNH in the unilaterally affected patients (Battaglia et al., 2005, 2006). This finding and the presence of multiple types of focal seizure suggest that PNH patients are affected by multifocal epilepsy, even if in the course of the epilepsy history a particular brain region may become predominant in generating clinical seizures. In a minority of patients generalized discharges of 3–4 Hz spike-and-wave activity have been reported, which may mimic primary generalized epilepsy (Raymond et al., 1994b; Giza et al., 1999). The analysis of the EEG tracings is also relevant in demonstrating the modality of seizure onset in the PNH brain (Fig. 11.4). There has been a long debate on whether or not the heterotopic nodules participate in the genesis of the epileptic discharges (Francione et al., 1994; Kothare et al., 1998). Recently, a number of papers have addressed this issue and provided relevant new data. The analysis of EEG ictal patterns from the surface (Battaglia et al., 2005) or from SEEG recordings (Aghakhani et al 2005; Tassi et al., 2005), or the

Right unilateral PNH, extended to neocortex

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Fig. 11.3. Interictal EEG features in PNH. (A) The interictal EEG abnormalities are consistent with the anatomical location of the PNH (left panel, sharp and slow wave complexes on the right temporal leads). In the same patient, the sharp and slow wave complexes change in diffuse bursts of polyspikes during slow sleep (right panel). (B) Photic driving of the background EEG activity during intermittent photic stimulation (IPS). In this patient with bilateral asymmetrical PNH the photic driving is bilateral but more evident on the right side, where the nodules are more pronounced (upper panel). The spectral representation illustrates the 1:1 response to the 5 Hz stimulus and the higher harmonic frequencies on both sides (lower panel).

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Right paratrigonal PNH with extension to the cortex

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Fig. 11.4. Brief focal seizure in a patient with right paratrigonal PNH extending to the parieto-occipital cortex. After a burst of sharp and slow wave complexes on the right parieto-occipital and posterior temporal regions, the seizure is marked by a tonic discharge of fast activity diffused to the right hemisphere but more evident on the parieto-occipital and posterior temporal regions (left panel). Note the increase of the interictal sharp waves on the occipital and posterior temporal leads immediately after the seizure (right panel). Clinically, the seizure was characterized by brief unresponsiveness and then delayed response to simple commands. Thereafter, the patient was unaware of having had a seizure.

combined analysis of both surface EEG and SEEG in the same patient (Battaglia et al., 2006), have revealed the simultaneous origin of epileptic discharges in neocortical areas, mesial temporal structures and adjacent heterotopic nodules. These neurophysiology studies altogether support the idea that epilepsy in PNH patients is generated by abnormal anatomical circuitries, including the heterotopic nodules and adjacent archicortical and neocortical areas. This finding is of particular relevance for a correct planning of epilepsy surgery in affected patients (see below). An additional and peculiar EEG finding in PNH patients is the frequent photic driving of the background activity after intermittent light stimulation (in 81% of the patients, according to Battaglia et al., 2006). The photic driving is always related to the PNH side (Fig. 11.3B) and may be also revealed in heterotopic nodules by means of intracerebral electrodes (Tassi et al., 2005). This finding most probably reflects the existence of reciprocal anatomical connections between the neocortical areas normally responsive to the visual inputs and the underlying heterotopic nodules. The activation of heterotopic nodules by peripheric visual stimuli is consistent with data from positron emission

tomography (PET) and functional MRI studies (see below) demonstrating the activation of PNH by simple stimuli or functional paradigms (Richardson et al., 1998; Janszky et al., 2003; Lange et al., 2004).

11.7. Diagnostic assessment and differential diagnosis As indicated above, the diagnosis is based on neuroimaging and is easier when last-generation MR images of appropriate thickness are obtained. Heterotopic nodules are round or ovoid, with smooth surface, never calcified and almost always isointense to gray matter on all imaging sequences. With the exception of rare cases associated with malformation syndromes, the cerebral cortex has a normal gyral pattern, providing that nodules do not extend into cortical areas. The nodules may be fused together to form macronodules of larger size, particularly in the unilateral cases, but they are usually separated from each other by thin fascicles of white matter. When macronodules are located in the paratrigonal region, posterior cranioencephalic hypoplasia may be present (Battaglia et al., 1997).

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Functional imaging have been also employed to evaluate the functional activity of periventricular nodules. Fluoro-2-deoxy-d-glucose positron emission tomography (FGD-PET) and single photon emission computed tomography (SPECT) have revealed that the metabolic activity and blood flow of bilateral and symmetrical PNH are identical to those of the normal cerebral cortex (Morioka et al., 1999). Oxygen-15labeled water PET studies have demonstrated increased blood flow in unilateral PNH during a unilateral finger tapping task (Richardson et al., 1998), and, in keeping with that, functional MRI (fMRI) data have revealed equal blood oxygen level dependent (BOLD) signal in the activated motor cortex and periventricular nodules of a bilateral and symmetrical PNH patient (with FLN1 mutation) during bilateral finger tapping (Lange et al., 2004). Finally, fMRI have reported visual, sensorimotor and speech-related activation of heterotopic nodules in PNH patients; interestingly, nodules extending to the overlying neocortex were more easily activated (Janszky et al., 2003). Taken together, all these data underscore the fact that heterotopic nodules are integrated in functional circuits and may participate, at least to some extent, in normal brain function. Recent studies have also demonstrated the possibility of diagnosing PNH during pregnancy. Both ultrasonography and MRI are capable to detect heterotopic nodules in utero at early gestational stages (Mitchell et al., 2000). However, as in other malformations of cortical development, small PNH nodules may be below the limits of resolution of these neuroimaging techniques. The PNH nodules should be easily differentiated from subependymal lesions commonly found in tuberous sclerosis. These lesions are often calcified, irregular in shape, iso- or hypo-intense to white matter and enhance after gadolinium administration. The differential diagnosis is also facilitated by the absence in PNH patients of family history and the typical features of tuberous sclerosis at the physical examination (see Ch. 9).

11.8. Management and prognosis Given the high incidence of epilepsy, seizure control is the most relevant clinical aspect. With few exceptions (Preul et al., 1997), epilepsy is not necessarily characterized by high seizure frequency and epileptic status is a very rare occurrence. However, in most patients, focal seizures may require the use of polytherapy with different antiepileptic drugs and still remain difficult to control (Battaglia et al., 2006). For the presence of drug-resistant seizures, PNH patients are candidates for epilepsy surgery. In the first reported series, surgical outcome was reported as poor

(Li et al., 1997; Sisodiya, 2000). However, epilepsy surgery was not guided by SEEG analysis of seizure semeiology and was not aimed at removing the heterotopic nodules. More recent series have indicated that the results of surgery can be very favorable, providing that the abnormal circuitry generating seizures is carefully assessed prior to, and then removed with, epilepsy surgery (Francione et al., 1994; Aghakhani et al., 2005; Tassi et al., 2005). Aside from the issue of drug-resistant seizures, the prognosis is favorable. Anaplastic ganglioglioma and b-amyloid plaques have been rarely reported in the heterotopic nodules of patients affected by PNH (Demaerel et al., 1996; Joseph 1997). It should again be remembered that the risk for stroke and other vascular and coagulopathic complications is increased in FLN1-linked PNH patients (Fox et al., 1998; Sheen et al., 2001). Therefore, careful cardiological and hematological assessment should be considered in bilaterally affected patients, particularly when FLN1 mutations are present or suspected.

Acknowledgments The authors wish to thank Dr Adele Finardi for her contribution in preparing the manuscript.

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Chapter 12

Subcortical laminar (band) heterotopia TERUYUKI TANAKA1 AND JOSEPH G. GLEESON*,2 1

Graduate School of Medicine and Faculty of Medicine, University of Tokyo, Tokyo, Japan and 2 University of California San Diego, La Jolla, CA, USA

12.1. Historical background A first report of subcortical band (laminar) heterotopia (SBH) dates back to 1893, which revealed a symmetrical subcortical neuronal layer in an autopsied brain specimen from a woman with epilepsy and psychomotor retardation (Matell, 1893). Developmental neuropathological studies in following decades demonstrated various types of generalized cortical malformations; however, it was not until the late 1980s that SBH was rediscovered and appreciated in epileptic patients by the advent of high-resolution brain magnetic resonance imaging (MRI) (Barkovich et al., 1989; Friede, 1989; Livingston and Aicardi, 1990; Palmini et al., 1991). SBH thus became diagnosable during life, and subsequent series of clinicoimaging studies from various facilities revealed its clinical heterogeneity, significant female predominance and occasional familial occurrence in which affected females have SBH and affected males have lissencephaly, suggestive of X-linked dominant inheritance (Pinard et al., 1994; Berg et al., 1998). In the late 1990s, a genetic locus for SBH/ X-linked lissencephaly was mapped to Xq22.3–q23, and subsequently a causative gene, doublecortin (DCX) was positionally cloned (des Portes et al., 1997, 1998b; Ross et al., 1997; Gleeson et al., 1998). In rare male cases with SBH, mosaic mutations were identified in DCX or another causative gene for lissencephaly, LIS1 on chromosome 17p13.3 (Reiner et al., 1993, 1995; Pilz et al., 1999; Sicca et al., 2003a). Identification of causative genes for SBH/ lissencephaly has expanded molecular biological studies of neuronal migration and enabled creation of mouse models, which have further advanced our understanding of the molecular basis of these disorders (Hirotsune et al., 1998; Corbo et al., 2002). By the year 2006, molec-

ular interaction of DCX with LIS1, CDK5, dynein, AP-1, AP-2, JNK, JIP, DFFRX, neurofascin, neurabin II/spinophilin and PP1 were identified, and roles for DCX in nuclear-centrosome coupling and formation of leading process and growth cone were demonstrated (Caspi et al., 2000; Friocourt et al., 2001; Kizhatil et al., 2002; Tsukada et al., 2003; Gdalyahu et al., 2004; Schaar et al., 2004; Tanaka et al., 2004a, 2004b; Friocourt et al., 2005; Kappeler et al., 2006; Shmueli et al., 2006). Still, molecular mechanism of neuronal migration defects in SBH/lissencephaly is not fully elucidated and development of effective treatments will be the focus of the future.

12.2. Clinical features 12.2.1. Imaging findings 12.2.1.1. General description Brain MRI demonstrates three major features: subcortical band, variably affected cortex and ventriculomegaly (Fig. 12.1). Occasional features include cerebellar hypoplasia and T2 prolongation in the white matter. The subcortical band is displayed as a bilateral, well defined and well marginated band of tissue isointense with gray matter underlying the cortical mantle and separated from it by a thin rim of white matter (Kuzniecky, 1994; Barkovich and Kuzniecky, 2000). Overlying cortex is either apparently normal or displays variable abnormality ranging from mildly simplified gyri and sulci to more severe pachygyria with broad gyri and shallow sulci. The striate, cingulate, fusiform gyri and medial temporal areas are relatively spared. The subcortical band varies in thickness among patients. The thickness of the band reflects

*Correspondence to: Joseph G. Gleeson MD, Department of Neurosciences, Leichtag 3A16, University of California San Diego, 9500 Gliman Drive, La Jolla, CA 92093–0691, USA. E-mail: [email protected], Tel: þ1-858-822-2856; Fax: þ1-858-822-1021.

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Fig. 12.1. (A) MRI and (B) gross specimen of subcortical band heterotopia.

the degree of migration arrest and therefore correlates with the degree of overlying cortical pachygyria and ventriculomegaly, such that patients with thicker subcortical band have significantly more severe pachygyria and ventriculomegaly, and these MRI parameters further correlate with clinical severity (Barkovich et al., 1994). 12.2.1.2. Anteroposterior gradient The subcortical band is usually present throughout the neocortex and the thickness of the band and the degree of pachygyria show regional predominance in individual patients. The dominant patterns of subcortical band/pachygyria can be classified into three groups: 1) anterior-biased or global, 2) posterior-biased and 3) limited or unilateral. The anterior-biased or global pattern is the most common of the three. There is a genotypic–phenotypic correlation between these gradient patterns and the genes mutated: anterior-biased or global SBH is generally associated with DCX mutations, whereas relatively uncommon posterior-biased SBH is associated with mosaic LIS1 mutations (Pilz et al., 1999; Gleeson et al., 2000a; Matsumoto et al., 2001; Sicca et al., 2003a; Leventer, 2005). 12.2.2. Clinical findings The majority of patients are female: of 241 patients reported from 1989–2005, 191 cases were female and 50 were male (Livingston and Aicardi, 1990; Chen et al., 1991; Palmini et al., 1991; Ricci et al., 1992;

Soucek et al., 1992; Hashimoto et al., 1993; Iannetti et al., 1993; Landy et al., 1993; Miura et al., 1993; Palmini et al., 1993; Barkovich et al., 1994; De Volder et al., 1994; Granata et al., 1994; Parmeggiani et al., 1994; Harding, 1996; Mardesic et al., 1996; Ono et al., 1997; Berg et al., 1998; des Portes et al., 1998a; Richardson et al., 1998; Kato et al., 1999, 2001; van der Valk et al., 1999; Vossler et al., 1999; Yoshikawa et al., 1999; Gleeson et al., 2000a; Pinard et al., 2000; Sasaki et al., 2000a; Bernasconi et al., 2001; Jacobs et al., 2001; Matsumoto et al., 2001; Spreer et al., 2001; Akanuma et al., 2002; D’Agostino et al., 2002; Eriksson et al., 2002; Grant and Rho, 2002; Poolos et al., 2002; Guerrini et al., 2003; Mai et al., 2003; Munakata et al., 2003; Sicca et al., 2003a, 2003b; Toulouse et al., 2003; Draganski et al., 2004; Janzen et al., 2004; Tai et al., 2004; Torres et al., 2004; Jirsch et al., 2006.). The male:female ratio, as well as the likelihood of specific features reported here, is subject to selection bias in reporting, so the frequency of associated features should be interpreted cautiously. The main clinical features were epilepsy, mental retardation, and cognitive/behavior problems. Barkovich et al. analyzed 27 patients with SBH and established the following correlations: 1) severity of T2 prolongation in the brain with motor delay; 2) degree of ventricular enlargement with the age of seizure onset and with development and intelligence; 3) severity of pachygyria with the age of seizure onset, seizure type, and an abnormal neurological examination; 4) parietal involvement with delayed speech development; 5) occipital involvement

SUBCORTICAL LAMINAR (BAND) HETEROTOPIA with age of seizure onset; 6) age of seizure onset with development and intelligence and with an abnormal neurological examination; and 7) severity of pachygyria and thickness of band with development of symptomatic generalized epilepsy and Lennox–Gastaut syndrome (Barkovich et al., 1994). 12.2.2.1. Epilepsy Of 178 reported patients for whom clinical histories were presented, 171 patients (96%) had epilepsy. Seizure onset ranged from neonates to teenagers, averaging 5 years of age (Tanaka, unpublished data) (Fig. 12.2). Seizure types included both partial and generalized seizures; simple/complex partial seizures (68% prevalence in reported cases), tonic/tonic–clonic seizures (57%), myoclonic seizures (16%), astatic/drop attacks (30%), absence (23%) and epileptic spasms (6%). Of 34 patients who started seizures before 1 year of age, 10 cases developed West syndrome. At least 16% of patients were reported to have developed Lennox– Gastaut syndrome. Of 133 patients for whom seizure severity was described, 104 cases (78%) were refractory to antiepileptic drugs. Electroencephalographic (EEG) findings are not disease-specific and demonstrate variably abnormal patterns of symptomatic generalized epilepsies with a propensity for focalization (Palmini et al., 1991, 1993; Ricci et al., 1992; Landy et al., 1993; Iannetti et al., 1993; De Volder et al., 1994; Parmeggiani et al., 1994; Vossler et al., 1999; Bernasconi et al., 2001; D’Agostino et al., 2002; Grant and Rho, 2002; Poolos et al., 2002). Interictal EEG abnormalities include diffuse bilateral synchronous, irregular theta background activity frequently interrupted by bursts of generalized high-amplitude slow waves of delta and slow theta frequencies. Multifocal or diffuse slow spike

20

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and waves or polyspike-wave discharges often appear with fronto-central predominancy, whereas, in cases with posterior dominant SBH, abnormal discharges tend to predominate posteriorly (Fig. 12.3A). During nonrapid-eye-movement (NREM) sleep, physiological sleep discharges are often replaced by diffuse slow spike and waves and bursts of rhythmic spikes at 10–11 Hz (Fig. 12.3B). In the rapid-eye-movement (REM) stage multifocal abnormalities prevail. Ictal EEG patterns consist of generalized spike-slow waves or diffuse attenuation, which often arise independently from both hemispheres. Intracranial stereo-EEG (SEEG) recording from the subcortical band has been reported on five patients, giving rise to variable results (Bernasconi et al., 2001; Mai et al., 2003; Russo et al., 2003). Two patients displayed no epileptiform activity from the subcortical bands while one of them featured nonepileptic, monomorphic, synchronous, rhythmic theta waves (Bernasconi et al., 2001). Three other patients displayed epileptiform activity from the subcortical heterotopia in different manners: (case 1) electrical discharges started on the mesial temporal lobe and subsequently propagated to the normotopic and heterotopic cortices (Russo et al., 2003); (case 2) both normotopic and heterotopic cortices independently generated high-voltage spikes and slow waves and electrographic seizures, but only seizure activity arising in the normotopic cortex resulted in clinical seizures (Bernasconi et al., 2001); (case 3) both normotopic and heterotopic cortices generated interictal spikes or sharp waves, which were often synchronous in the neocortex but not in the mesial temporal lobe cortex, and ictal discharges associated with loss of contact began as low-voltage fast activity in the heterotopic and outer cortices and spread to the mesial temporal structures (Mai et al., 2003).

6 7 8 9 10 11 12 13 14 15 16 Age (y) of seizure onset

Fig. 12.2. Age of seizure onset in 171 patients with SBH. Average: 5 years. (Source: Tanaka, unpublished data).

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B

Fig. 12.3. Interictal awake and sleep EEGs from an 8-year-old girl with SBH. See text for details.

A magnetoencephalographic (MEG) study of two cases similarly demonstrated involvement of both the normotopic and heterotopic cortices in the generation of interictal spikes, with occasional sequential activation of two cortices (Toulouse et al., 2003). While these data demonstrate considerable heterogeneity in epileptogenicity and neural connectivity among individuals with SBH, they indicate that the subcortical heterotopia is capable of generating ictal discharges and is reciprocally wired with the outer cortex. 12.2.2.2. Mental retardation and neuropsychological problems Patients with SBH have mental retardation with variable degree. Of 157 reported patients for whom developmental data were available, 106 (68%) had mental retardation to a mild to profound degree (Tanaka, unpublished data) (Fig. 12.4A). There is a correlation between the severity of mental retardation and the age of seizure onset, such that patients with severe– profound mental retardation have earlier seizure onset whereas those with normal intelligence have significantly later seizure onset (Fig. 12.4B). Psychomotor development slows after onset of seizures. Patients who developed Lennox–Gastaut syndrome displayed more severe mental retardation.

Because of the scantiness of detailed neuropsychological reports on patients with SBH, our understanding of cognitive and psychological features of SBH is fragmentary. Two patients displayed significantly lower nonverbal IQ, particularly on measures of perceptual organization index (POI), than verbal IQ (WISC-III non-verbal IQ/POI/verbal IQ ¼ 66/67/84 (Jacobs et al., 2001), 59/59/82 (Keene et al., 2004)). One of them demonstrated reduced speed of processing both for visuomotor and oral output, and reduced rapid input of information (Jacobs et al., 2001). The other patient demonstrated that, despite stronger verbal ability with relatively spared receptive vocabulary, complex aspects of language comprehension and expression, such as processing sentences with multiple meanings, understanding figurative language and making inferences, were impaired (Keene et al., 2004). These conditions are reminiscent of white matter abnormalities by which the flow of information is interrupted, and it is possible that disruption in cortical-subcortical connections by subcortical heterotopia is responsible for the characteristic neuropsychological deficits in these patients. Further comprehensive neuropsychological studies are crucial to better understand and treat these patients. Another study on four patients with SBH demonstrated relatively intact episodic memory despite intellectual impairment and other severe cognitive deficits. This is suggestive

SUBCORTICAL LAMINAR (BAND) HETEROTOPIA

195

30

Percentage (%)

25 20 15 10 5 0

A

Severe-profound Moderate 35-50 85

Severe-profound Moderate 35-50 85

10

Mean age seizure onset

9 8 7 6 5 4 3 2 1 0

B

Fig. 12.4. (A) IQ/DQ profile of 157 patients for whom developmental data were available. (B) Correlation between IQ/DQ range and the age of seizure onset in 157 patients. Patients with severe–profound mental retardation have earlier seizure onset, whereas those with normal intelligence have significantly later seizure onset. Error bars: standard error of the means. (Source: Tanaka, unpublished data.)

of intact functioning of the mesial temporal lobe and is consistent with neuroimaging and pathological evidence of intact medial temporal structures in patients with SBH (Janzen et al., 2004). 12.2.2.3. Metabolism and functionality Cellular metabolism, function and connectivity of the subcortical heterotopic band have been investigated in only a small number of patients and thus have not been fully clarified, but several lines of evidence support involvement of the heterotopia in physiological activities. Interictal 99mTc-HMPAO single photon emission computed tomography (SPECT) imaging performed in two patients demonstrated identical or increased perfusion of the subcortical heterotopia com-

pared with the normal cortex (Matsuda et al., 1995; Iannetti et al., 1996). Interictal positron emission tomography (PET) scanning with [18F]-fluorodeoxyglucose (FDG) in three cases similarly demonstrated normal or even higher glucose uptake in the subcortical heterotopia compared to the normal cortex, suggestive of synaptic activity in the heterotopic neurons (Miura et al., 1993; De Volder et al., 1994). Proton magnetic resonance spectroscopic (MRS) imaging studies on ten patients demonstrated a normal Nacetylaspartate/creatine (NAA/Cr) ratio in the subcortical heterotopia in six patients, whereas relative decrease of the ratio indicative of abnormal metabolism and dysfunction in the other four suggesting heterogeneity in cellular metabolism and the function of

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the heterotopia (Li et al., 1998; Iannetti et al., 2001; Munakata et al., 2003). Roles of subcortical heterotopia in voluntary motor and sensory functions were tested using functional MRI in 12 cases (Pinard et al., 2000; Spreer et al., 2001; Draganski et al., 2004; Keene et al., 2004). Finger tapping fMRI paradigm produced activation of both the contralateral motor cortex and the underlying subcortical heterotopia in all cases. Neither visual stimulation or language activation paradigm elicited activation of the heterotopic band (Spreer et al., 2001; Keene et al., 2004). Sensory stimulus, consisting of manual brushing over the skin of the palm and fingers, elicited activation of contralateral primary sensory cortex and the underlying heterotopia in three out of six patients (Jirsch et al., 2006). An MEG study revealed that the somatosensory evoked response exhibited an early activation of normotopic neurons in the primary sensory cortex and a later activation of nearby neurons in the subcortical heterotopia, and the auditory response similarly exhibited an early activation in the auditory normotopic cortex and a later activation in the heterotopia (Toulouse et al., 2003). A diffusion tractographic study on five patients demonstrated that white matter traversed or ended within the band heterotopia (Eriksson et al., 2002). Together, these data suggest that subcortical heterotopia has a function in the physiological signal generation and transduction, as well as epileptogenesis, with considerable heterogeneity among individuals, and emphasize the crucial importance of comprehensive neuropsychological and functional evaluation of each patient.

12.3. Pathology Histological studies of autopsied brains demonstrated that the subcortical heterotopia ranged in shape from a thin ribbon through clusters of elongated islands to thick, solid sheets and was separated from the cortex by a variable layer of white matter that included U-fibers. The outer part of the heterotopia contained haphazardly oriented neurons and a somewhat columnar arrangement was evident in the deeper part, highlighted by radially oriented bundles of myelinated fibers. The bottom of the heterotopia broke up into nodules in some areas, separated by thicker bands of white matter (Friede, 1989; Harding, 1996) (Fig. 12.5). An immunohistochemical study on resected specimens obtained from epilepsy surgery demonstrated that the white matter between the cortex and the heterotopia was intensely stained with GFAP, and contained abundant heterotopic neurons of both inhibitory GABAergic (calretinin-, calbindinand paralbumin-positive) and excitatory glutamatergic

Fig. 12.5. Hematoxylin–eosin–Luxol Fast Blue-stained section ( 10) of SBH heterotopia. Note islands of lighter-stained heterotopic gray matter within darker-stained white matter tracts.

(GluR2/3-positive) types arranged in columns. There were no residual radial glial fibers within the white matter. The subcortical heterotopia was unlayered and consisted of clustered small or medium-sized rounded or pyramidal neurons haphazardly oriented and separated by bundles of fibers. Both excitatory and inhibitory neurons were distributed in the subcortical heterotopia (Mai et al., 2003). An immunohistochemical study on a brain from a 22-week aborted female fetus demonstrated deficient immunoreactivity of the subcortical heterotopia to doublecortin, whereas the cortical plate and ventricular zone were immunopositive (Mizuguchi et al., 2002), consistent with the model of neuronal segregation due to lyonization.

12.4. Molecular genetics of subcortical band heterotopia 12.4.1. DCX (doublecortin) 12.4.1.1. The DCX gene An X-linked locus for SBH was suspected following early reports because of the great preponderance in females. In 1994, two families were described in which the mothers and their two daughters had SBH whereas their two sons had classical lissencephaly (Pinard et al., 1994). These observations suggested that a single X-linked gene mutation could lead to both classical lissencephaly and SBH. By genetic linkage studies on these and similar families the X-linked locus was mapped to Xq22.3–q23, and subsequently a novel 9.5 kb brain-specific gene was positionally

SUBCORTICAL LAMINAR (BAND) HETEROTOPIA cloned and named doublecortin (DCX) (NCBI GeneID: 1641, HGNC ID: 2714) (des Portes et al., 1997, 1998b; Ross et al., 1997; Gleeson et al., 1998). The DCX gene spans 118 kb on the X chromosome and consists of nine exons. A coding region of 1080 bases lies between exons 4 and 9 and encodes for a 40 kDa, 360 amino acid protein, doublecortin (DCX). 12.4.1.2. Subcortical band heterotopia and X-linked lissencephaly Females have two X chromosomes and randomly inactivate one of them in every cell early in embryonic development (lyonization). Therefore, females with a mutation in DCX have a mosaic state with two populations of neurons: one expressing a normal copy of DCX that presumably migrates normally and one expressing a mutant copy of DCX that presumably has defective migration and gives rise to the band of heterotopia. Immunohistochemistry study on human fetuses supports this (Mizuguchi et al., 2002). In males with a mutation in the DCX gene, all neurons lack functional DCX protein and thus fail to migrate normally, giving rise to the more severe lissencephaly cortex. Another mechanism by which DCX mutations give rise to SBH is somatic or germline mosaicism, which simulates a functional mosaic due to the lyonization phenomenon in females and accounts for the rare SBH phenotype in males (Pilz et al., 1999; Kato et al., 2001; Matsumoto et al., 2001; D’Agostino et al., 2002; Gleeson et al., 2000b; Poolos et al., 2002; Aigner et al., 2003). 12.4.1.3. DCX mutation analysis in subcortical band heterotopia patients From 1998–2005, 43 missense mutations and 24 truncation mutations in DCX were reported (des Portes et al., 1998a; Gleeson et al., 1998, 1999b, 2000b; Pilz et al., 1998, 1999; Kato et al., 1999, 2001; Demelas et al., 2001; Matsumoto et al., 2001; D’Agostino et al., 2002; Poolos et al., 2002; Aigner et al., 2003; Forman et al., 2005). Truncation mutations occur randomly throughout the protein; in contrast, missense mutations are tightly clustered in two regions that correspond to amino acids 42–129 and 178–253, suggesting the functional significance of these regions (Fig. 12.6). There is a significant difference in the genotypes of DCX mutation between familial and sporadic patients: in familial cases, a majority displayed missense mutations or mosaicism of truncation mutations, whereas sporadic cases exhibited missense and truncation mutations equally. A correlation between the genotype and SBH phenotype was also demonstrated. Matsumoto et al. (2001) graded SBH into four groups in order of

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severity according to the thickness and location of the band, and showed that the prevalence of truncation mutations was significantly high in the more severe two groups compared with the other, less severe, two groups. These data suggest that the missense mutations give rise to partial loss of DCX function and lead to a milder phenotype, whereas truncation mutations are associated with near-complete loss of function and a more severe phenotype, which may hinder affected patients from conceiving and bearing children and thus explain the low prevalence of truncation mutations in familial cases. Mutations in the DCX gene were identified in 100% of familial SBH/X-linked lissencephaly cases, 38–90% of sporadic SBH cases, 20% of males with classic lissencephaly and 27% of sporadic male cases of SBH (des Portes et al., 1998a; Pilz et al., 1998; Gleeson et al., 1999b; Matsumoto et al., 2001; D’Agostino et al., 2002). These data suggest that other genetic mechanisms or loci may exist for sporadic SBH. 12.4.1.4. The DCX protein product 12.4.1.4.1. Expression DCX is expressed exclusively in postmitotic neurons during periods of neuronal migration. In postmortem human cerebral cortices, DCX is highly expressed at 22 weeks gestation and at lower levels during the early childhood developmental time points at 2 years and 4 years of age. Very little is expressed in adult brain. The expression profile has been studied in detail in mice, in which it is highly expressed in the developing cerebral cortex, ganglionic eminences, thalamus, hippocampus, cerebellum, spinal cord, retina and peripheral nervous system. Expression begins at embryonic day 10.5, which precedes the beginning of neuronal migration, and is sustained at high levels throughout the embryonic period. At the completion of migration there is downregulation of expression (Francis et al., 1999; Tanaka et al., 2004b). Within the cortex, it is expressed at low levels in neurons in the proliferative ventricular zone and rapidly increases in the intermediate zone and the cortical plate. It is highly expressed in tangentially migrating neurons from the ganglionic eminence, and throughout adulthood in migrating neurons along the rostral migratory stream from the subventricular zone toward the olfactory bulb (Gleeson et al., 1999a). 12.4.1.4.2. Structure and function DCX is a microtubule-associated protein. DCX binds and directly polymerizes purified tubulin into microtubules, and misexpression in heterologous cells leads to the formation of depolymerization-resistant

Fig. 12.6. (A) Missense mutations in DCX reported from 1998–2005. Each DNA mutation is numbered with reference to the ATG. The predicted DCX protein alteration is numbered with reference to the amino acid residue number. Amino acid substitution mutations are referenced by the wild-type amino acid and position, followed by the mutant amino acid. In the sporadic/ familial category: F, familial; S, sporadic. (B) Schematic representation of the mutations in the table 6A. Most missense mutations cluster in two regions of the DCX protein. (C) Truncation mutations in DCX reported from 1998–2005. Numbering and abbreviations follow (A). (D) Schematic representation of the truncation mutations in (C). In contrast to missense mutations, truncation mutations occur randomly throughout the protein.

SUBCORTICAL LAMINAR (BAND) HETEROTOPIA microtubules. Tight clustering of missense mutations in two regions of N-terminal DCX led to identification of an evolutionarily conserved tandem of two homologous domains, i.e. R1 (amino acid 42–140) and R2 (amino acid 170–260) (Fig. 12.7). These two regions have 27% amino acid identity and 47% amino acid conservation. Each domain alone is capable of binding tubulin, whereas neither is sufficient for coassembly with microtubules, and two intact tubulin-binding domains are necessary for microtubule polymerization and stabilization. On the C-terminal of DCX lies a serine/proline-rich domain that functions to negatively regulate the interaction of DCX with microtubules (Taylor et al., 2000; Tanaka et al., 2004b). The molecular function of DCX in neuronal migration has begun to be elucidated. DCX has been shown to play pivotal roles in cytoskeletal regulation and signal transduction during migration (Coquelle et al., 2006). Through its microtubule-bundling and polymerization activity, it provides a major cytoskeleton for nuclearcentrosome coupling and growth cone formation in the leading process via interaction with LIS1, motor protein dynein or kinesin. These interactions are dynamically regulated by phosphorylation of DCX by various kinases such as Cdk5, protein kinase A (PKA), the MARK/PAR-1 family of protein kinases and JNK (Gdalyahu et al., 2004; Schaar et al., 2004; Tanaka et al., 2004a). DCX interacts with an F-actin binding protein, neurabin II/spinophilin, and induces bundling and cross-linking of microtubules and F-actin. DCX phosphorylation and dephosphorylation mediated by neurabin II and protein phosphatase 1 (PP1) dynamically regulate DCX binding to F-actin (Tsukada et al., 2003, 2005, 2006). DCX interacts with phospho-FIGQY tyrosine in the cytoplasmic domain of the L1 cell adhesion molecule neurofascin, suggesting its role as a phosphotyrosine adaptor at the plasma membrane (Kizhatil et al., 2002).

R1

199

12.4.2. LIS1 (lissencephaly-1 or PAFAH1B1) 12.4.2.1. The LIS1 gene and its protein product The LIS1 gene on chromosome 17p13.3 was identified as a causative gene for classical lissencephaly (NCBI GeneID: 5048, HGNC: 8574) (Reiner et al., 1993; Chong et al., 1997; Lo Nigro et al., 1997). It spans 92 kb on the genome and consists of 11 exons. A coding region of 1233 bp lies between exons 2 and 11 and encodes for a 45 kDa, 410 amino acid protein, LIS1, or PAFAH1B1 (platelet-activating factor acetylhydrolase, isoform Ib, alpha subunit) (Hattori et al., 1994). The LIS1 protein consists of an N-terminal coiled-coil domain and C-terminal seven WD40 repeats predicted to form a circular, propeller-like structure with seven blades, each made up of four beta strands, and to interact with various protein motifs (Fig. 12.8). Like DCX, LIS1 is a microtubule-associated protein and has been shown to interact with dynein heavy chain, dynein intermediate chain, dynactin, DCX, NDEL1, NDE1, CLIP-170 and NUDC (Morris et al., 1998; Caspi et al., 2000; Efimov and Morris, 2000; Feng et al., 2000; Niethammer et al., 2000; Sasaki et al., 2000b; Smith et al., 2000; Coquelle et al., 2002; Tanaka et al., 2004a). 12.4.2.2. Lissencephaly/subcortical band heterotopia and the LIS1 gene mutation Heterozygous inactivating mutations (haploinsufficiency) in the LIS1 gene lead to classical lissencephaly with pseudoautosomal dominant inheritance. Of 42 intragenic LIS1 mutations identified, 36 were truncation mutations and six were missense (Cardoso et al., 2002; Torres et al., 2004). Patients with missense mutations in LIS1 have a significantly milder phenotype than those with truncation mutations, and LIS1 dose-dependent defects in neuronal migration have been demonstrated on lis-1 knockout mice, suggesting

R2

S/P rich a.a. 360

Fig. 12.7. Schematic representation of the DCX protein. R1 (amino acids 42–140) and R2 (amino acids 170–260) represent an evolutionarily conserved tandem of two homologous domains. All the reported missense mutations cluster in these regions. On the C-terminal lies a serine/proline-rich domain.

Coiled-coil domain

WD40 repeats

a.a. 410

Fig. 12.8. Schematic representation of the LIS1 protein, consisting of an N-terminal coiled-coil domain followed by seven WD40 repeats.

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that the neuronal migration is sensitive to functional LIS1 dosage (Hirotsune et al., 1998; Cardoso et al., 2000; Gambello et al., 2003). Lissencephaly due to LIS1 mutations exhibits posterior dominant pachygyria/agyria, whereas DCX hemizygous mutations give rise to anterior dominant lissencephaly (Pilz et al., 1998; Dobyns et al., 1999). These observations led to the hypothesis that the rare posterior dominant SBH could derive from milder mutations in LIS1, and indeed missense mutations or mosaic mutations were identified in males, as well as females, with posterior SBH (Pilz et al., 1999; Sicca et al., 2003a; Torres et al., 2004).

12.5. Genetic testing In all patients with SBH, direct sequencing of the DCX gene should be considered. In patients with atypical posterior dominant SBH, sequencing of LIS1 should be considered. If mosaicism is suspected, DNA from hair root or buccal mucosa can be tested using single-stranded conformational polymorphism, restriction enzyme analysis or denaturing high-pressure liquid chromatography (Gleeson et al., 2000b; Kato et al., 2001; Sicca et al., 2003a).

12.6. Management 12.6.1. Medical Antiepileptic medication corresponding to the symptomatic generalized epilepsy with focalization is the mainstay of treatment for SBH. Antiepileptic drugs should be selected according to each patient’s seizure types. Two cases were reported who responded well to lamotrigine (Vossler et al., 1999). However, seizures associated with SBH are often refractory to current antiepileptic drugs and in such cases it is sometimes difficult to overcome severity of the epileptogenesis and stop neuropsychological impairment. Development of new medication is a critical outstanding issue. 12.6.2. Surgical Corpus callosotomy has been performed in five cases with frequent atonic seizures or drop attacks, and yielded significant to considerable reduction of the seizure frequency in three of them, slight improvement in one case and no significant reduction in the other (Palmini et al., 1991; Landy et al., 1993; Vossler et al., 1999). Callosotomy is not capable of eliminating seizures, because of its palliative nature; nevertheless, it would be worth considering for patients with frequent drop attacks. Of ten reported cases who under-

went focal resection of epileptogenic tissue, only three patients had significantly improved outcome. Considering diffuse nature and heterogeneous connectivity of the heterotopia, focal resection is not a treatment of choice for a majority of cases (Bernasconi et al., 2001; Russo et al., 2003; Tai et al., 2004). 12.6.3. Supportive Comprehensive neuropsychological assessment should be performed on each patient to reveal cognitive strength and weakness. Based on each patient’s neuropsychological profile, special care and intervention should be directed to enhance their ability to learn and acquire skills in the classroom and society and to minimize functional deficits.

12.7. Genetic counseling Genetic counseling of families constitutes an essential part of treating patients with SBH. Since the counseling varies in its process among individual cases, we will just outline the principle and associated unique issues such as mosaicism and incomplete penetrance. An essential prerequisite is correct diagnosis and mutation analysis. If SBH/lissencephaly is diagnosed and a DCX mutation is identified in a proband, brain MRI and mutation analysis can be performed on the mother to determine whether the DCX mutation occurred de novo in the proband or was inherited from the mother. Normal brain MRI cannot rule out the absence of a mutant allele, because of possible incomplete penetrance (Demelas et al., 2001). Since a DCX mutation in males gives rise to lissencephaly, the father is usually not tested. If the mother carries the same mutation in the DCX gene, the risk of recurrence of SBH in females and lissencephaly in males is 50%. If a mutation is not detected in the mother’s DNA prepared from lymphocytes, the mutation of the proband must have occurred de novo. However, according to the published reports, there is still about a 10% chance of somatic or germline mosaicism in the mother, which could be passed down to posterity, and to clarify this, DNA from hair root or buccal mucosa can be tested (Gleeson et al., 2000b; Kato et al., 2001; Aigner et al., 2003; Sicca et al., 2003a). LIS1 mutations in SBH are rare and theoretically either missense or mosaic mutations. If such a condition is identified in a proband, brain MRI and mutation analysis can be performed on both parents. If the same missense mutation is detected in either parent, the recurrence risk is 50%. If a mutation is not detected in either parent’s DNA prepared from lymphocytes, the mutation of the proband must have occurred de

SUBCORTICAL LAMINAR (BAND) HETEROTOPIA novo. In contrast to DCX mutations, only two cases of somatic mosaicism in LIS1 have been reported so far, and thus the recurrence risk in such a situation may be lower, but this has not yet been confirmed.

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Kappeler C, Saillour Y, Baudoin JP, et al. (2006). Branching and nucleokinesis defects in migrating interneurons derived from doublecortin knockout mice. Hum Mol Genet 15: 1387–1400. Kato M, Kimura T, Lin C, et al. (1999). A novel mutation of the doublecortin gene in Japanese patients with X-linked lissencephaly and subcortical band heterotopia. Hum Genet 104: 341–344. Kato M, Kanai M, Soma O, et al. (2001). Mutation of the doublecortin gene in male patients with double cortex syndrome: somatic mosaicism detected by hair root analysis. Ann Neurol 50: 547–551. Keene DL, Olds J, Logan WJ (2004). Functional MRI study of verbal fluency in a patient with subcortical laminar heterotopia. Can J Neurol Sci 31: 261–264. Kim MK, Park MS, Kim BC, et al. (2005). A novel missense mutation of doublecortin: mutation analysis of Korean patients with subcortical band heterotopia. J Korean Med Sci 20: 670–673. Kizhatil K, Wu YX, Sen A, Bennett V (2002). A new activity of doublecortin in recognition of the phospho-FIGQY tyrosine in the cytoplasmic domain of neurofascin. J Neurosci 22: 7948–7958. Kuzniecky RI (1994). Magnetic resonance imaging in developmental disorders of the cerebral cortex. Epilepsia 35 (suppl. 6): S44–S56. Landy HJ, Curless RG, Ramsay RE, et al. (1993). Corpus callosotomy for seizures associated with band heterotopia. Epilepsia 34: 79–83. Leventer RJ (2005). Genotype-phenotype correlation in lissencephaly and subcortical band heterotopia: the key questions answered. J Child Neurol 20: 307–312. Li LM, Cendes F, Bastos AC, et al. (1998). Neuronal metabolic dysfunction in patients with cortical developmental malformations: a proton magnetic resonance spectroscopic imaging study. Neurology 50: 755–759. Livingston JH, Aicardi J (1990). Unusual MRI appearance of diffuse subcortical heterotopia or ‘double cortex’ in two children. J Neurol Neurosurg Psychiatry 53: 617–620. Lo Nigro C, Chong CS, Smith AC, et al. (1997). Point mutations and an intragenic deletion in LIS1, the lissencephaly causative gene in isolated lissencephaly sequence and Miller–Dieker syndrome. Hum Mol Genet 6: 157–164. Mai R, Tassi L, Cossu M, et al. (2003). A neuropathological, stereo-EEG, and MRI study of subcortical band heterotopia. Neurology 60: 1834–1838. Mardesic D, Sucic Z, Papa J, et al. (1996). Complex disorder of neuronal migration in an infant with possible congenital cytomegalovirus infection. Acta Med Croatica 50: 151–155. Matell M (1893). Ein Fall von Heterotopie der grauen Substanz in den beiden Hemispha¨ren des Grosshirns. Arch Psychiatr Nervenkr 25: 124–136. Matsuda H, Onuma T, Yagishita A (1995). Brain SPECT imaging for laminar heterotopia. J Nucl Med 36: 238–240. Matsumoto N, Leventer RJ, Kuc JA, et al. (2001). Mutation analysis of the DCX gene and genotype/phenotype correlation in subcortical band heterotopia. Eur J Hum Genet 9: 5–12.

SUBCORTICAL LAMINAR (BAND) HETEROTOPIA Miura K, Watanabe K, Maeda N, et al. (1993). Magnetic resonance imaging and positron emission tomography of band heterotopia. Brain Dev 15: 288–290. Mizuguchi M, Takashima S, Ikeda K, et al. (2002). Loss of doublecortin in heterotopic gray matter of a fetus with subcortical laminar heterotopia. Neurology 59: 143–144. Morris SM, Albrecht U, Reiner O, et al. (1998). The lissencephaly gene product Lis1, a protein involved in neuronal migration, interacts with a nuclear movement protein, NudC. Curr Biol 8: 603–606. Munakata M, Haginoya K, Soga T, et al. (2003). Metabolic properties of band heterotopia differ from those of other cortical dysplasias: a proton magnetic resonance spectroscopy study. Epilepsia 44: 366–371. Niethammer M, Smith DS, Ayala R, et al. (2000). NUDEL is a novel Cdk5 substrate that associates with LIS1 and cytoplasmic dynein. Neuron 28: 697–711. Ono J, Mano T, Andermann E, et al. (1997). Band heterotopia or double cortex in a male: bridging structures suggest abnormality of the radial glial guide system. Neurology 48: 1701–1703. Palmini A, Andermann F, Aicardi J, et al. (1991). Diffuse cortical dysplasia, or the ‘double cortex’ syndrome: the clinical and epileptic spectrum in 10 patients. Neurology 41: 1656–1662. Palmini A, Andermann F, de Grissac H, et al. (1993). Stages and patterns of centrifugal arrest of diffuse neuronal migration disorders. Dev Med Child Neurol 35: 331–339. Parmeggiani A, Santucci M, Ambrosetto P, et al. (1994). Interictal EEG findings in two cases with ‘double cortex’ syndrome. Brain Dev 16: 320–324. Pilz DT, Matsumoto N, Minnerath S, et al. (1998). LIS1 and XLIS (DCX) mutations cause most classical lissencephaly, but different patterns of malformation. Hum Mol Genet 7: 2029–2037. Pilz DT, Kuc J, Matsumoto N, et al. (1999). Subcortical band heterotopia in rare affected males can be caused by missense mutations in DCX (XLIS) or LIS1. Hum Mol Genet 8: 1757–1760. Pinard JM, Motte J, Chiron C, et al. (1994). Subcortical laminar heterotopia and lissencephaly in two families: a single X linked dominant gene. J Neurol Neurosurg Psychiatry 57: 914–920. Pinard J, Feydy A, Carlier R, et al. (2000). Functional MRI in double cortex: functionality of heterotopia. Neurology 54: 1531–1533. Poolos NP, Das S, Clark GD, et al. (2002). Males with epilepsy, complete subcortical band heterotopia, and somatic mosaicism for DCX. Neurology 58: 1559–1562. Reiner O, Carrozzo R, Shen Y, et al. (1993). Isolation of a Miller–Dieker lissencephaly gene containing G protein beta-subunit-like repeats. Nature 364: 717–721. Reiner O, Albrecht U, Gordon M, et al. (1995). Lissencephaly gene (LIS1) expression in the CNS suggests a role in neuronal migration. J Neurosci 15: 3730–3738. Ricci S, Cusmai R, Fariello G, et al. (1992). Double cortex A neuronal migration anomaly as a possible cause of Lennox–Gastaut syndrome. Arch Neurol 49: 61–64.

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Chapter 13

Lissencephaly type I RICHARD LEVENTER* Royal Children’s Hospital, Parkville, Victoria, Australia

13.1. Definitions and terminology When translated from Greek, ‘lissencephaly’ literally means ‘smooth in head’ (lissos ¼ smooth; enkephali ¼ in head). The term lissencephaly has generally been used to describe disorders in which the mature brain is deficient in gyration. In practice, a completely smooth brain devoid of sulcation and gyration is rarely seen. Most patients with lissencephaly have a combination of areas of agyria (absent gyri) and areas of pachygyria (broad gyri). In 1983 Dambska first used the terms ‘type I’ and ‘type II’ lissencephaly (Dambska et al., 1983). Type I lissencephaly was used to refer to the ‘classical agyria syndrome’ in which the cortex was abnormally thickened but showed lamination (albeit abnormal). Type II lissencephaly was used to refer to cortical malformations in which the cortex was severely disorganized, and the patients also had abnormalities of muscle and eyes, as part of a ‘cerebro–oculo–muscular syndrome’. Both type I and type II lissencephaly had been observed to occur as familial and sporadic cases. Type I lissencephaly is now generally referred to as ‘classical lissencephaly’ and type II lissencephaly as ‘cobblestone lissencephaly’ (or ‘cobblestone dysplasia’ or ‘cobblestone complex’). The terms type I lissencephaly, classical lissencephaly and agyria/pachygyria all appear in current literature and refer to the same type of cortical malformation. In this chapter, the term ‘classical lissencephaly’ will be used. Classical lissencephaly is primarily a disorder of neuroblast migration, and the first of the human cortical malformations for which the genetic basis, and subsequently the molecular mechanism, was identified. Classical lissencephaly is however a somewhat heterogeneous group, containing subtypes with variable

pathological, imaging and clinical features and differing etiologies and genetic bases. This chapter will describe the features of the main subtypes of classical lissencephaly in humans, emphasizing those features of greatest importance to the practicing clinician, including the clinical and imaging characteristics and the known genetic causes.

13.2. Epidemiology As with most other cortical malformations, the precise incidence of lissencephaly is not known. A study of lissencephaly in the Netherlands showed a prevalence of 11.7 per million births (de Rijk-van Andel et al., 1991), while data from metropolitan Atlanta between 1968 and 1993 showed rates of 4–11 per million births (Dr William Dobyns, personal communication). At the Royal Children’s Hospital in Melbourne (which provides tertiary pediatric care for a population of approximately 4 million people from south-eastern Australia), we see two or three new patients with classical lissencephaly per year, which equates to an incidence of approximately 1:25 000 live births (Leventer, unpublished data). As with other cortical malformations, the diagnosis of lissencephaly has increased with improved imaging techniques (magnetic resonance imaging, MRI), greater use of such techniques and an improved classification and recognition of different cortical malformations (Fig. 13.1). Although lissencephaly is probably the best known of the cortical malformations, imaging-based studies have shown it to be less common than other malformations such as polymicrogyria, focal cortical dysplasia and gray matter heterotopia (Leventer et al., 1999).

*Correspondence to: Dr Rick Leventer MBBS, BMedSci, FRACP, Paediatric Neurologist, Children’s Neuroscience Centre and Murdoch Children’s Research Institute, Royal Children’s Hospital, Flemington Road Parkville, Victoria, Australia 3052. E-mail: [email protected], Tel: þ613-9345-5661, Fax: þ613-9345-5977.

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Fig. 13.1. Main features of classical lissencephaly. (A) Axial T2-weighted MRI, (B) coronal T1-weighted MRI and (C) threedimensional surface reconstruction MRI showing agyria in the occipito-parietal regions transitioning to pachygyria in the fronto-temporal regions. The maximum cortical thickness in the occipital lobes is 20 mm (compared to a normal cortical thickness of 4 mm). This posterior-to-anterior gradient of severity is typical of lissencephaly secondary to abnormalities in the LIS1 gene, the most common cause of classical lissencephaly.

13.3. Pathology The macroscopic hallmarks of classical lissencephaly are reduced or absent gyration combined with thickening of the cerebral cortex (Fig. 13.2A). On macroscopic inspection the brain shows poorly developed Sylvian and Rolandic fissures and failure of opercularization of the insular areas (Harding and Copp, 2002). In

Fig 13.2. Pathological features of classical lissencephaly. (A) Macroscopic photograph of the brain of a patient with Miller– Dieker syndrome. There is near-complete agyria, with shallow Rolandic and Sylvian fissures visible. (Courtesy of William Dobyns, University of Chicago (patient LP89–002).) (B) Photomicrograph of a patient with lissencephaly showing a thickened, four-layered cortex. Layer I is a poorly formed marginal zone, layer II is a superficial gray zone of poorly organized neurons, layer III is a relatively cell-sparse zone and layer IV is a deep gray zone of poorly compacted neurons. (Courtesy of Jeffrey Golden, University of Pennsylvania.)

the most severe cases, there is a failure of development of even the primary sulci (Harding and Copp, 2002). The brain size and weight are usually at the lower range of normal but not generally micrencephalic. On both macroscopic inspection and MRI, some areas of the brain may appear more severely affected than others, with ‘gradients of severity’ now apparent for different types of lissencephaly associated with mutations in different genes (Dobyns et al., 1999b). For example, abnormalities of the LIS1 gene tend to result in a form of lissencephaly that is more severe in the occipital and parietal lobes (Pilz et al., 1998; Dobyns et al., 1999b). Associated abnormalities of classical lissencephaly may include enlarged lateral ventricles, absence of the claustra and external capsules, abnormalities of the corpus callosum, persistent cavum septum pellucidum, hypoplasia of the pyramidal tracts, heterotopia of the inferior olives and abnormalities of the cerebellum such as vermis hypoplasia. Microscopic examination of the most common type of classical lissencephaly shows a thick and poorly organized cortex with four rather than the normal six layers (Crome, 1956; Norman et al., 1995; Harding and Copp, 2002). These are shown in Figure 13.2B and consist of: 1) a poorly defined marginal zone with increased cellularity; 2) a superficial cortical gray zone with diffusely scattered neurons; 3) a relatively neuron-sparse zone; and 4) a deep cortical gray zone with neurons often oriented in columns. The superficial cortical gray zone corresponds to the true cortex but lacks the normal layering and organization. The deep cortical gray zone is much thicker than the superficial

LISSENCEPHALY TYPE I cellular layer and consists of large numbers of neurons presumed to have arrested their migration prematurely. The underlying periventricular white matter is generally thin. On microscopic inspection, classical lissencephaly associated with DCX mutations may show subtle differences compared to lissencephaly associated with LIS1 mutations, with DCX-associated lissencephaly occasionally showing a six-layered cortex (Viot et al., 2004). Recently, other forms of lissencephaly have been described including lissencephaly associated with cerebellar hypoplasia and hippocampal abnormalities secondary to RELN mutations (Hong et al., 2000), lissencephaly associated with agenesis of the corpus callosum and ARX mutations (Kitamura et al., 2002) and lissencephaly associated with extreme congenital microcephaly (microlissencephaly or MLIS). The pathological findings in these rarer lissencephaly syndromes are likely somewhat different to those described above, but neuropathological studies of these forms of lissencephaly are lacking.

13.4. Clinical features 13.4.1. General features The clinical manifestations of lissencephaly are variable depending on 1) the severity and topography of the malformation, 2) associated congenital brain abnormalities and 3) abnormalities in other organ systems. The common clinical features of classical lissencephaly include severe to profound mental retardation, early hypotonia, which may persist or evolve to mixed axial hypotonia and limb spasticity, epileptic seizures and feeding problems (de Rijk-van Andel et al., 1990; Barkovich et al., 1991; Dobyns et al., 1991, 1992, 1993). Intractable epilepsy may be an independent factor contributing to mental retardation and delayed development. Seizures will often begin in the first year of life with infantile spasms, later progressing to an intractable mixed seizure disorder. Fetal onset of seizures has also been reported (Patane and Ghidini, 2001). Electroencephalography often shows a hypsarrhythmic pattern, with high-amplitude generalized and bisynchronous sharp and slow waves as well as periods of bursts of sharp activity alternating with periods of suppression (Hakamada et al., 1979; de Rijk-van Andel et al., 1992). The background often shows high-voltage fast activity, lack of a posterior predominance and absence of sleep architecture (Worle et al., 1990). Some forms of lissencephaly have a much more severe neurological phenotype, usually with a markedly reduced lifespan. These include Miller–Dieker syndrome (MDS), X-linked lissencephaly with abnormal

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genitalia (XLAG) (Berry-Kravis and Israel, 1994; Dobyns et al., 1999a), lissencephaly with complete agenesis of the corpus callosum (Sztriha et al., 1998) and lissencephaly associated with extreme cerebellar hypoplasia and mutations in the RELN gene (Hong et al., 2000; Ross et al., 2001). Rare patients have been described with partial lissencephaly and consequently less severe neurological abnormalities (Leventer et al., 2001a). 13.4.2. Syndromes with lissencephaly The clinical, imaging and genetic features of the named lissencephaly syndromes are shown in Table 13.1. 13.4.2.1. Miller–Dieker syndrome Lissencephaly associated with characteristic facial dysmorphism was described by Miller in 1963 and Dieker et al. in 1969. In 1983 Dobyns et al. described two patients with lissencephaly, a characteristic facial appearance and monosomy of chromosome 17p and first named this combination as the ‘Miller–Dieker syndrome’ (Dobyns et al., 1983; Stratton et al., 1984). MDS consists of severe classical lissencephaly with a posteriorto-anterior gradient combined with distinctive facial features (Fig. 13.3) consisting of a prominent forehead, bitemporal hollowing, short, upturned nose, flat midface, usually with a prominent skin fold from the nose to the cheeks, protuberant upper lip with downturned vermilion borders and a small jaw (Dobyns et al., 1991; Allanson et al., 1998). Additional anomalies such as heart malformations and omphaloceles have been described in a minority of patients (Dobyns et al., 1991; Chitayat et al., 1997). MDS is a contiguous gene deletion syndrome due to deletions of multiple genes at 17p13.3, including the LIS1 and 14–3-3E genes (Cardoso et al., 2003). It is the deletion of these two genes that accounts for the severe grade of lissencephaly seen in these children (Cardoso et al., 2003; Toyo-Oka et al., 2003). Deletions of other genes in the region are thought to account for the facial dysmorphism and other congenital anomalies (Allanson et al., 1998). 13.4.2.2. Isolated lissencephaly sequence Isolated lissencephaly sequence (ILS) consists of classical lissencephaly of a generally milder grade than that seen in MDS, mild craniofacial abnormalities (bitemporal hollowing and mild micrognathia) and the neurological sequelae of the malformation as outlined above (Dobyns et al., 1984, 1992; Pavone et al., 1990, 1993). ILS is seen in patients with lissencephaly secondary to mutations of the DCX gene, and patients with small deletions of 17p13.3 including LIS1 and intragenic deletions and mutations of LIS1 (Cardoso et al., 2002).

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Table 13.1 Clinical, imaging and genetic features of the main lissencephaly syndromes Main clinical features

Imaging features

Inheritance

Gene defect

MDS

Facial dysmorphism Profound global developmental delay Hypotonia Intractable epilepsy Feeding problems Death within first 2 years Global developmental delay (usually severe) Intractable epilepsy Evolving spasticity Global developmental delay (usually severe) Intractable epilepsy Evolving spasticity Variable clinical features, however most have severe disability with epilepsy, spasticity and often neonatal death RELN LCH – severe global developmental delay, hypotonia, epilepsy Abnormal or ambiguous genitalia (male) Temperature instability Neonatal seizures Death in neonatal period or infancy Congenital microcephaly A gradient Moderately dilated lateral ventricles

AD (sporadic or related to parental balanced translocation involving 17p13.3)

Deletions of 17p13.3 including LIS1 and 14–3–3E

Mild to severe LIS (grades 2–4) with a P>A gradient Mildly dilated lateral ventricles

AD (sporadic or related to parental balanced translocation involving 17p13.3)

Deletions or intragenic mutations of LIS1

Moderate to severe LIS (grades 1–3) with an A>P gradient Mildly dilated lateral ventricles Mild cerebellar vermis hypoplasia (20%) Cerebellar vermis and hemisphere hypoplasia Variable lissencephaly grade and gradient Occasional brainstem hypoplasia RELN mutations: A>P gradient and abnormal hippocampi Complete agenesis of the corpus callosum Occasional interhemispheric cyst Mild LIS with a P>A gradient

X-linked – sporadic or familial*

Intragenic mutations of DCX

AD, AR or X-linked – sporadic and familial forms

Most causes unknown LIS1, DCX or RELN intragenic mutations reported

X-linked – sporadic or familialy

Intragenic mutation of ARX

Variable grade and gradient of LIS Occasional periventricular gray matter heterotopia, corpus callosum, brainstem or cerebellar abnormalities

AR sporadic

Unknown

ILS (LIS1)

ILS (DCX)

LCH

XLAG

MLIS

*

Carrier mothers may have subcortical band heterotopia (SBH). Carrier mothers may have agenesis of corpus callosum. MDS¼Miller–Dieker syndrome, ILS¼isolated lissencephaly sequence, LCH¼lissencephaly with cerebellar hypoplasia, XLAG¼X-linked lissencephaly with abnormal genitalia, MLIS¼microlissencephaly, LIS¼lissencephaly, P>A¼posterior greater than anterior severity, A>P¼anterior greater than posterior severity, AD¼autosomal dominant, AR¼autosomal recessive, SD¼standard deviations.

y

R. LEVENTER

Syndrome

LISSENCEPHALY TYPE I

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Fig 13.3. Facial features of Miller–Dieker syndrome. This 6-week-old infant with severe lissencephaly, dysmorphic features and a deletion of 17p13.3 visible by routine karyotype analysis and confirmed using LIS1-specific FISH shows many of the typical facial features of MDS, including tall, square forehead, frontal bossing, bitemporal hollowing, small chin, ears pinned back, prominent upper lip, thin vermilion border, short, upturned nose, lateral nasal folds and a flattened midface. A vertical forehead furrow was seen when the child cried. Patient permission held by author.

13.4.2.3. X-Linked lissencephaly with abnormal genitalia XLAG is characterized by mild lissencephaly with a posterior-to-anterior gradient, agenesis of the corpus callosum, severe neurological abnormalities from birth and abnormal or ambiguous genitalia in genotypic males (Berry-Kravis and Israel, 1994; Dobyns et al., 1999a). The corpus callosum is always completely absent and some patients have interhemispheric cysts. All patients have profound developmental delay, mixed axial hypotonia and limb spasticity, poor temperature regulation from birth and neonatal or even prenatal seizures. Most die within the first few months of life. This disorder has been found to be secondary to mutations of the ARX gene (Kitamura et al., 2002; Uyanik et al., 2003). A number of carrier mothers have been found to have isolated agenesis of the corpus callosum (Kato et al., 2004). (d) Lissencephaly and cerebellar hypoplasia Six groups of syndromes with the combination of lissencephaly and cerebellar hypoplasia have been described (Ross et al., 2001). These are a heterogeneous group of malformation syndromes with differing brain phenotypes, clinical sequelae and inheritance patterns. The majority of patients have microcephaly, epilepsy and variable degrees of neurological impairment. Four follow an autosomal recessive inheritance pattern. Mutations in three genes (LIS1, DCX and RELN) have been found in some patients with lissencephaly and cerebellar hypoplasia (LCH), although in the majority of patients the genetic abnormality has not been identified.

(e) Microlissencephaly Mlissencephaly is defined as a lissencephaly in the presence of a birth head circumference equal to or less than 3 SD below the mean (Barkovich et al., 1998; Dobyns and Barkovich, 1999). These are a heterogeneous group of malformation syndromes, with three groups having been described. Mlissencephaly group a (or Norman– Roberts syndrome) consists of extreme microcephaly, severe lissencephaly (grade 1) and normal brainstem and cerebellum (Norman et al., 1976; Dobyns et al., 1984; Dobyns and Barkovich, 1999). Mlissencephaly group b (or Barth type) consists of extreme microcephaly, severe lissencephaly (grade 1) and brainstem and cerebellum hypoplasia (Barth et al., 1982; Dobyns and Barkovich, 1999; Kroon et al., 1996). Mlissencephaly type c consists of extreme microcephaly, moderate lissencephaly (grade 3) with a sudden transition from anterior agyria to posterior simple sulcation and normal brainstem and cerebellum (Dobyns and Barkovich, 1999). These children all generally have severe neurological sequelae, often with neonatal death. Most cases are presumed to be autosomal recessive based on reports of recurrence in consanguineous families, although no genes have yet been identified. 13.4.3. Imaging features The imaging features of the main types of lissencephaly are shown in Fig. 13.4. Moderate and severe forms of lissencephaly can usually be diagnosed on computed tomography (CT) scanning. The cerebral surface

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Fig 13.4. Patients with the P>A gradient of Lissencephaly typical of LIS1 mutations. All images are T1-weighted or T2-weighted MRI scans. The top row shows axial scans and the bottom row coronal scans. Grade 1 is near complete agyria, grade 2 is posterior agyria and rudimentary shallow gyri anteriorly, grade 3 is posterior agyria and anterior pachygyria and grade 4 is generalized pachygyria.

appears smooth with absent opercularization and a characteristic ‘figure of eight’ appearance (Dobyns and McCluggage, 1985). Milder forms of lissencephaly and accompanying brain malformations such as cerebellar abnormalities may be missed on CT scanning, while other cortical malformations may be misdiagnosed as lissencephaly. This is especially true for polymicrogyria, in which the overfolded cortex can appear thickened. Occasionally, a paucity of gyration can be detected by ultrasound after 23 weeks gestation (Greco et al., 1998; Fong et al., 2004), however antenatal diagnosis is usually not accurate until towards the end of gestation. Using 1.5 Tesla MRI the gyral pattern (agyria or pachygyria), thickened cortex and other brain abnormalities can readily be appreciated (Barkovich et al., 1991). In addition, it may be possible to identify a ‘cell-sparse zone’ of high signal on T2-weighted and low signal on T1-weighted sequences between an outer cellular layer of disorganized cortex and a thick layer of heterotopic neurons deep to this (Barkovich, 2000). Several different patterns of lissencephaly have been recognized using MRI, which led to development of a detailed grading system (Pilz et al., 1998; Dobyns et al., 1999b). The grading system considers both the severity of the lissencephaly as reflected by cortical thickness and degree of agyria, and the pattern or gradient along the anterior to posterior axis. Most patients

have a posterior-to-anterior (P>A) or ‘a’ gradient in which the gyral malformation is more severe posteriorly than anteriorly (Fig. 13.4). Other patients have the reverse anterior to posterior (A>P) or ‘b’ gradient (Fig. 13.5). In patients with severe grade 1 lissencephaly, the gradient may be difficult to determine. The P>A gradient is associated with mutations in the LIS1 ( 14-3–3E) and ARX genes, whilst the A>P gradient is associated with mutations in the DCX and RELN genes. There are many variants or atypical forms of classical lissencephaly. These forms include those with associated microcephaly (the Mlissencephaly syndromes), those with cerebellar hypoplasia (the LCH syndromes) and those with varying patterns or gradients of severity. A few patients have a gradient in which the posterior frontal and central regions are more severely involved than the anterior frontal region, with both more severe than posterior regions (C>A>P), resulting in a complex ‘c’ gradient. Examples of some of these atypical forms are shown in Fig. 13.6.

13.5. Etiology, molecular and genetic basis The genetic and molecular basis of the main forms of lissencephaly is summarized in Table 13.2. No environmental causes for lissencephaly have been identified. There are occasional reports of lissencephaly in

LISSENCEPHALY TYPE I

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Fig 13.5. Patients with the A>P pattern of lissencephaly typical of DCX mutations. All images are axial T1-weighted or T2weighted MRI scans. (A) Male, showing near complete agyria; a cell-sparse zone of low signal (arrows), maximal in the frontal regions, is the only clue suggesting a DCX mutation. (B) Female, showing frontal pachygyria only. (C) Female, showing frontal pachygyria transitioning to a thick posterior subcortical band heterotopia (arrows). (D) Female, showing bilateral thick subcortical band heterotopia (arrows).

Fig 13.6. All images are T1-weighted or T2-weighted MRI scans. The top row are axial scans and the bottom row midline sagittal scans. (A) Subtle Lissencephaly with an A>P gradient and severe hypoplasia of the cerebellar vermis and hemispheres, consistent with LCH in a patient with a RELN mutation. (Courtesy of Christopher Walsh, Harvard University.) (B) Subtle Lissencephaly with a P>A gradient and complete agenesis of the corpus callosum. This patient was a genotypical male with ambiguous genitalia and carried a mutation in the ARX gene. (Courtesy of William Dobyns, University of Chicago (patient LR00–185).) (C) Patient with congenital microcephaly and Lissencephaly with a P>A gradient consistent with MLIS. (D) Patient with congenital microcephaly, severe Lissencephaly with an A>P gradient, cerebellar vermis hypoplasia and complete agenesis of the corpus callosum, another variant of MLIS.

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Table 13.2 Genes implicated in lissencephaly Involved in the following lissencephaly syndromes

Protein

Chromosome

Protein function in brain

LIS1

LIS1 protein or plateletactivating factor acetylhydrolase, isoform Ib, alpha subunit

17p13.3

1. Hydrolyzes PAF 2. Microtubule associated protein/dynein regulator

MDS ILS LCH (SBH posterior)

14–3-3E

17p13.3

Mediates signal transduction by binding to phosphoserinecontaining proteins

MDS

DCX

Tyrosine 3-monooxygenase/ tryptophan 5monooxygenase activation protein, epsilon polypeptide DCX or doublecortin

Xq22.3–q23

Binds microtubules

RELN

Reelin

7q22

Control cell-cell interactions by affecting signal transduction (?)

ILS LCH (SBH generalized and anterior) LCH

ARX

Aristaless-related homeobox protein

Xp22.1–p21.3

Homeobox protein acting as a transcriptional regulator (?)

XLAG

Presumed mechanism of lissencephaly 1. Impaired neurite and growth cone structure 2. Impaired neuroblast proliferation 3. Impaired neuroblast migration (radial, somal and tangential) by altered dynein-mediated nucleokinesis Impaired neuroblast migration by altered dynein-mediated nucleokinesis in combination with LIS1 and NDEL1

Failure of optimal bundling of microtubules, impairing radial neuroblast migration Altered interaction with ApoER2 and VLDLR affecting DAB1 signaling, resulting in abnormal splitting of preplate leading to inverted cortical layering Impaired proliferation, differentiation and tangential migration of GABAergic neurons

MDS=Miller–Dieker syndrome, ILS=isolated lissencephaly sequence, LCH=lissencephaly with cerebellar hypoplasia, XLAG=X-linked lissencephaly with abnormal genitalia, SBH=subcortical band heterotopia, PAF=platelet activating factor.

R. LEVENTER

Gene

LISSENCEPHALY TYPE I association with cytomegalovirus infection (Hayward et al., 1991) but it is likely that these cases are actually polymicrogyria and not lissencephaly. Five genes associated with lissencephaly syndromes have been identified, and in about 80% of cases of typical classical lissencephaly, a genetic cause can be found, usually an abnormality of the LIS1 or DCX genes (Pilz et al., 1998; Cardoso et al., 2002). The first genotype–phenotype correlation for lissencephaly or subcortical band heterotopia (SBH) came in 1983 with the description by Dobyns et al. of an association between lissencephaly and abnormalities of chromosome 17p in the Miller–Dieker contiguous gene deletion syndrome (Dobyns et al., 1983). Identification of LIS1 as the causative gene for lissencephaly in these and other nonsyndromic cases of lissencephaly did not come until 1993 (Reiner et al., 1993; Lo Nigro et al., 1997), and the role of DCX in both lissencephaly and SBH (or double cortex) was not determined until 1998 (des Portes et al., 1998b; Gleeson et al., 1998). There is a wide spectrum of severity and phenotypes evident for lissencephaly (and the related disorder subcortical band heterotopia) and the basis of this spectrum is recognized in many cases to be due to specific variations in the types of mutation of either LIS1 or DCX (Cardoso et al., 2000; Matsumoto et al., 2001; Leventer, 2005). 13.5.1. The LIS1 gene LIS1 was cloned from the lissencephaly critical region in chromosome band 17p13.3, as defined by physical mapping of small deletions in a series of patients with MDS or ILS (Reiner et al., 1993; Chong et al., 1997). LIS1 consists of 11 exons and encodes a 410 amino acid and 46 kDa protein known as LIS1 or PAFAH1B1, which is expressed in the forebrain ventricular zone and cortical plate (Clark et al., 1997). The protein is highly conserved as there is only a single amino acid difference between mouse and human (Peterfy et al., 1994). It contains a putative microtubule binding region and seven WD40 repeats, the latter common to beta subunits of G proteins (Reiner et al., 1993; Garcia-Higuera et al., 1996; Reiner and Sapir, 1998). LIS1 is the noncatalytic subunit of heterotrimeric platelet-activating factor acetylhydrolase brain isoform 1b (PAFAH), an enzyme that hydrolyzes platelet activating factor (PAF) in the brain to inactive lyso-PAF (Bix et al., 1999; McNeil et al., 1999). MDS is caused by large deletions of chromosome 17p13.3, which involve LIS1 and several other nearby genes including 14–3-3E (Dobyns et al., 1991, 1993; Chong et al., 1997; Cardoso et al., 2003). Several different cytogenetic mechanisms have been observed in MDS including de novo telomeric and interstitial

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deletions, rings and unbalanced derivatives of reciprocal translocations or pericentric inversions. About 60% of these are visible under the microscope, while the rest are submicroscopic but can be detected by fluorescence in situ hybridization (FISH) (Cardoso et al., 2002). ILS is caused by smaller deletions of 17p13.3 involving LIS1 or intragenic abnormalities including deletions, nonsense and missense mutations (Ledbetter et al., 1992; Lo Nigro et al., 1997; Cardoso et al., 2000, 2002, 2003). There is a robust genotype– phenotype association for patients with LIS1 mutations showing increasing severity of lissencephaly phenotype with mutations within the gene predicted to result in the greatest functional impairment of the LIS1 protein (Fogli et al., 1999; Cardoso et al., 2000; Leventer et al., 2001a; Caspi et al., 2003). LIS1 has been suggested to affect neuronal migration by two proposed mechanisms. The first of these is as a component of PAFAH. Evidence for a role of PAF and PAFAH in neuronal migration comes from studies showing that addition of PAF or inhibition of PAFAH decreases migration of cerebellar granule cells and addition of PAF to cultured hippocampal neurites produces growth cone collapse, neurite retraction and neurite varicosity formation (Bix et al., 1999). In addition, the three subunits of PAFAH are coexpressed in the developing brain with highest levels of expression coinciding with peak times of neuronal migration (Albrecht et al., 1996). The other proposed role of LIS1 in neuronal migration is as a microtubule-associated protein. In this role, it acts separately to the other subunits of PAFAH. Evidence for this role comes from studies that demonstrate the colocalization and interaction of LIS1 with tubulin, the major component of microtubules (Sapir et al., 1997, 1999). The coassembly of LIS1 with microtubules results in a reduction of microtubule catastrophic events and thus an increase in the length of microtubules that would be predicted to enhance and maintain microtubule elongation, a process necessary for optimal neuronal migration. Further support for the role of LIS1 in microtubule function comes from fungi. LIS1 has a 42% homology to the NudF protein in Aspergillus nidulans, which is a nuclear distribution protein that mediates nuclear migration through a microtubuledependent process involving dynein and dynactin (Morris et al., 1998; Morris, 2000). There is extensive evidence now showing that the LIS1 protein forms a component of multiprotein complexes essential for cellular motility during development. These multiprotein complexes include dynein, dynactin, NUDe and NDEL1. Not only does the LIS1 protein function in neuroblast migration but it is also required for cellular proliferation and intracellular transport (reviewed in

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Leventer et al., 2001b). Furthermore, LIS1 appears to have a role in neural stem cell division, morphogenesis and early neuroblast motility (Tsai et al., 2005). LIS1 may also cooperate with both DCX and reelin during cortical development (Caspi et al., 2000; Assadi et al., 2003). Haploinsufficiency of the LIS1 gene results in lissencephaly primarily by affecting radial migration but may also affect other modes of neuroblast migration including somal translocation and tangential migration of interneurons (McManus et al., 2004; Pancoast et al., 2005; Tsai et al., 2005). Confirmation of the importance of LIS1 comes from a mouse model created by the targeted knockout of the LIS1 gene. Homozygous null mice die early in embryogenesis while heterozygous and compound heterozygous mice survive, showing variable evidence of delayed neuronal migration (cortical, hippocampal, cerebellar and olfactory bulb disorganization) confirmed by in vitro and in vivo cell migration assays (Hirotsune et al., 1998; Gambello et al., 1999). 13.5.2. The 14–3-3e gene Patients with MDS generally have a more severe form of lissencephaly than patients with ILS, including those with complete deletion of one copy of the LIS1 gene (Cardoso et al., 2000; Cardoso at al., 2002; Cardoso et al., 2003). Until recently, the reason for this phenotypic difference was unknown. Evidence now has shown that MDS patients with the most severe grade of lissencephaly (grade1) have large deletions which involve the 14–3-3E gene (Cardoso et al., 2003). MDS patients without deletion of this gene, or ILS patients, never have grade 1 lissencephaly (Cardoso et al., 2003). The 14–3-3E protein has been shown to participate in normal neuroblast migration by interaction with the same multiprotein complex as LIS1 and mice heterozygous for both LIS1 and 14–3-3E have more severe neuronal migration defects than single heterozygotes (Toyo-Oka et al., 2003). Thus the more severe lissencephaly phenotype in patients with MDS and grade 1 lissencephaly is most probably due to deletion of two genes (LIS1 and 14–3-3E), both required for normal neuroblast migration. 13.5.3. The DCX gene Mutations of the DCX gene generally cause lissencephaly in males and subcortical band heterotopia in females. The DCX gene was cloned by linkage analysis in several multiplex families and by physical mapping of the Xq22.3–q23 breakpoint in a girl with ILS and a de novo X; 2 translocation (Ross et al., 1997; des Portes et al., 1998a, 1998b; Gleeson et al., 1998). DCX consists

of seven exons and encodes a 360 amino acid 40 kDa protein expressed in the soma and leading processes of migrating neurons (Francis et al., 1999). This protein is known as DCX or doublecortin. DCX most likely functions as a microtubule-associated protein, binding and stabilizing microtubules causing bundling (Francis et al., 1999; Gleeson et al., 1999; Horesh et al., 1999). It is expressed in the soma and leading processes of migrating neurons in both the central and peripheral nervous systems (Gleeson et al., 1999; Sapir et al., 1999; Yoshiura et al., 2000). Without optimal bundling of microtubules, neuronal locomotion and migration may be impaired. The vast majority of DCX mutations described thus far cluster in two evolutionarily conserved tandem repeat functional domains named pep1 and pep2 (Sapir et al., 2000; Taylor et al., 2000; Matsumoto et al., 2001). Both the pep1 and pep2 domains must be intact for optimal function of doublecortin and most missense mutations described thus far affect these domains (Sapir et al., 2000; Taylor et al., 2000). Females are hypothesized to be functional DCX mosaics in which the heterotopic band is composed of neurons with the mutant DCX gene expressed while the normal overlying cortex is composed of neurons with the normal DCX gene expressed. Males, being hemizygous for the X-chromosome are presumably unable to express DCX appropriately in migrating neurons and therefore develop Lissencephaly. 13.5.4. The RELN gene Mutations in the RELN gene on 7q22 have been found to cause a form of lissencephaly with extreme cerebellar hypoplasia and hippocampal abnormalities in six children with LCH from two families with consanguineous parents (Hong et al., 2000). The RELN gene was originally identified from the study of the naturally occurring ataxic reeler mouse (Caviness et al., 1972). The autosomal recessive mouse mutant reeler has tremors, impaired motor coordination, and ataxia (Falconer, 1951). Pathological studies have shown an inverted cortex in which the normal inside-out pattern is lost due to inability of later arriving neurons to migrate past earlier arriving neurons (Caviness and Rakic, 1978; Rakic and Caviness, 1995). The gene responsible for the reeler phenotype was isolated and the protein product was named reelin (reln in mouse and RELN in human) (Hirotsune et al., 1995; D’Arcangelo and Curran, 1998). RELN consists of 65 exons extending over more than 400 kb of genomic DNA. It encodes a very large 388 kDa protein with more than 4000 amino acids, which contains domains of homology to epidermal growth factor and some extracellular matrix proteins

LISSENCEPHALY TYPE I involved with cell adhesion (Hong et al., 2000). In the brain, it is secreted into the extracellular matrix by Cajal–Retzius cells of the marginal zone and other early postmigratory neurons and acts upon migrating cortical neurons by binding to the VLDL and ApoER2 receptors, as well as to a3b1 integrin (Curran and D’Arcangelo, 1998; D’Arcangelo et al., 1999; Hiesberger et al., 1999; Trommsdorff et al., 1999; D’Arcangelo, 2001). These observations suggest that reelin serves as a neuron-to-matrix adhesion molecule which provides an extracellular cue to migrating neuroblasts (Goffinet, 1995). 13.5.5. The ARX gene Mutations in ARX have been found in nine boys with XLAG (Kitamura et al., 2002). The possible role of ARX in human lissencephaly was suspected after similarities were recognized between the mouse Arx knockout and humans with XLAG. ARX is located on Xp22.13 and contains five exons that encode a protein of 562 amino acids. It is a homeobox gene which is expressed in the forebrain, floor plate and testis of mouse embryos (Miura et al., 1997). Mouse Arx mutants have both brain and testis abnormalities (Kitamura et al., 2002). The cortical abnormalities suggest decreased neuronal proliferation as well as abnormalities of the thalami and migration of GABAergic neurons from the ganglionic eminence (Kitamura et al., 2002). XLAG is thus the first cortical malformation in humans that arises from abnormalities of tangential neuroblast migration (Kato and Dobyns, 2005).

13.6. Conclusion Classical or type I lissencephaly are a group of cortical malformations with the common features of cortical thickening and a reduction in gyration. A number of lissencephaly syndromes have been described, each with distinguishing clinical, imaging or genetic features. Lissencephaly is primarily a disorder of migration of neuroblasts in the developing brain, although some forms may also involve abnormal neuroblast proliferation and neuronal organization. In general, the clinical consequences are severe and tragic. The recent elucidation of the genetic basis of the majority of these lissencephaly syndromes has allowed clinicians to provide accurate prognostic and genetic counseling to affected families. In addition, these rare disorders have provided molecular geneticists and developmental neurobiologists with a unique opportunity to gain insight into the normal processes required for cortical development in humans and other species.

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Chapter 14

Lissencephaly type II ˘ LU* AND BERIL TALIM HALUK TOPALOG Hacettepe University Children’s Hospital, Ankara, Turkey

14.1. Introduction Lissencephaly type II is a group of complex brain malformations that anatomically consists of ‘cobblestone’ cortex, abnormal white matter, enlarged ventricles, small brainstem, hypoplastic vermis and cerebellar polymicrogyria. The spectrum varies from a mild disorganization of the layers to severely dysplastic brains. All these conditions are usually associated with eye malformations and congenital muscular dystrophy (MDC) (Dobyns and Truwit, 1995). The first description of type II lissencephaly was published by Walker in 1942 and MDC was first reported in patients with type II lissencephaly by Krijgsman (Krijgsman et al., 1980). The descriptive term ‘cobblestone cortex’ was originally proposed by Haltia (Dubowitz, 1994; Dubowitz and Fardeau, 1995). The cortical changes consist of mixed agyria, pachygyria and polymicrogyria with a pebbled surface. Leptomeningeal neuronal and glial heterotopia may partly obstruct the subarachnoid space. The white matter changes vary from simple myelin pallor to severe edema and cystic degeneration and appear as bright signals on T2-weighted magnetic resonance imaging (MRI). If severe, the expression may be progressive hydrocephalus, apparent hydranencephaly and occipital cephaloceles. Grossly there is massive glial and neuronal ectopia, which results from a failure of arrest of neuronal migration due to defects in the integrity of the pial–glial barrier (Squier, 1993). Isolated lissencephaly type II in the absence of muscular dystrophy is a very rare condition and some cases have additional cerebellar hypoplasia, for which we do not yet know the responsible genes (Hughes et al., 1983; Hourihane et al., 1993). For practical purposes we shall exclusively review syndromes that are associated with muscle pathology, as these are widely

known and there have been tremendous cumulative scientific advances over the past decade in this field. Our aim is by no means to review MDC in general, although we shall cover the necessary sections. Neuromuscular disorders with cerebral malformations is the topic of a different chapter in this book. Thus, we shall mostly concentrate on the association between the genes/ proteins and the brain in lissencephaly type II. Defects in glycosylation have been linked to all these conditions and the common mechanism is the defective posttranslational processing of a-dystroglycan, a heavily glycosylated peripheral membrane component of the dystrophin-associated glycoprotein complex. Mutations in the enzymes involved in the synthesis of O-linked glycans on a-dystroglycan indicate that these diseases are in fact all congenital disorders of glycosylation in the form of ‘O-mannosylation defects’. (Table 14.1). The first form of MDC with severe mental retardation and structural brain involvement was described by Fukuyama (Fukuyama et al., 1960). This was followed by the description of muscle–eye–brain disease (MEB) from Finland in 1977 (Santavuori et al., 1977). Walker– Warburg syndrome (WWS) was first delineated by the observations of Walker in 1942 and Warburg in 1978. The very first descriptions of now so-called ‘merosindeficient MDC’ goes back to the early 1980s. In the past 10 years, a significant input to the field has come from the activities of the European Neuromuscular Centre (ENMC) Congenital Muscular Dystrophy Consortium, which convened nine dedicated workshops (Dubowitz, 1994, 1996, 1997, 1999; Dubowitz and Fardeau, 1995; Muntoni and Guicheney, 2002; Muntoni et al., 2002, 2003; Muntoni and Voit, 2005). We shall review the related disorders in chronological order of the resolving of the biochemistry, cellular aspects and molecular

*Correspondence to: Professor Haluk Topalog˘lu, Department of Child Neurology, Hacettepe Children’s Hospital, 06100 Ankara, Turkey. E-mail: [email protected], Tel: þ90-533-273-2731, Fax: þ90-312-467-46-56.

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Table 14.1 Clinical conditions with cobblestone lissencephaly and glycosylation defects Congenital muscular dystrophies with brain involvement

Primary mutation in the gene

Merosin-deficient congenital muscular dystrophy (MDC1A)* Fukuyama congenital muscular dystrophy (FCMD) Muscle–eye–brain disease (MEB) Walker–Warburg syndrome (WWS)

LAMA2

MDC1C with brain involvement MDC1D *

Target protein

MIM 156225

fukutin

Laminin a2 chain of merosin a-dystroglycan

253800

POMGnT1 POMT1 POMT2 FKRP LARGE

a-dystroglycan a-dystroglycan a-dystroglycan a-dystroglycan a-dystroglycan

253280 236670 236670 606612 603590

Cobblestone lissencephaly is rarely present in this condition.

pathology, simply because consecutive discoveries of the responsible mechanisms one after the other have given us a better understanding of the field in general.

14.2. Merosin-deficient congenital muscular dystrophy Although cobblestone lissencephaly is not a classical feature of merosin-deficient congenital muscular dystrophy (muscular dystrophy congenital type 1A, MDC1A), it will be mentioned here briefly since some cases also have structural changes of the brain (Fig. 14.1). Intelligence is typically normal or subnormal in MDC1A. Brain MRI, however, invariably shows white matter changes. The changes are diffuse, resembling a leukodystrophy, and affect both hemispheres but spare the internal capsule, corpus callosum, basal ganglia, thalami and cerebellum (Philpot et al., 1999). A small proportion of patients also have a neuronal migration defect that characteristically affects the occipital lobes (Sunada et al., 1995). In addition, children with MDC1A have a demyelinating neuropathy, which can be demonstrated by reduced peripheral motor nerve conduction velocity (Shorer et al., 1995). This disorder is due to recessive mutations in the laminin a2 gene (LAMA2), mapped to chromosome 6q22-23 (Tome et al., 1994; Helbling-Leclerc et al., 1995). Several mutations have now been found, most of which are nucleotide substitutions, small deletions or insertions, resulting in nonsense or splice site changes (Guicheney et al., 1998). The laminins are glycoproteins that form the backbone of basement membranes in almost every human or animal tissue. Each laminin is a heterotrimer

composed of one heavy (a) and two light chains (b and g). They form polymers that bind to a number of other macromolecules, such as nidogen, agrin and collagen IV in the extracellular matrix and to the two main transmembrane laminin receptors, dystroglycan and various integrins. Through their interactions, laminins contribute to cell-cell recognition, differentiation, cell shape, movement and tissue survival (Vachon et al., 1996; Colognato and Yurchenco, 2000). Laminin a2 is strongly expressed not only in the striated muscle basement membrane but also in the basal lamina of the cerebral blood vessels and Schwann cells, and this pattern of tissue expression gives rise to the muscle and central/peripheral nervous system pathology seen in MDC1A. In the central nervous system (CNS), laminin a2 is expressed in the basement membrane of blood vessels, including capillaries that form the blood–brain barrier, but it is absent from meningeal and choroid blood vessels, which do not form part of the blood–brain barrier (Villanova et al., 1997). On the brain surface, laminin a2 is expressed in the glia limitans, suggesting a role in the guidance of neuronal migration. In addition, nontraditional patterns of laminin a2 expression have been observed along developing axon tracts, in neuronal fibers and in punctate, potentially synaptic structures of limbic brain regions (Morissette and Carbonetto, 1995; Hagg et al., 1997), which might correspond to distinct roles for distinct neuronal populations especially during development. Involvement of the CNS in laminin a2 deficiency was first detected as abnormal white matter signal on MRI or computed tomography (CT) scans (Topalog˘lu et al., 1991). The white matter abnormalities are usually widespread and diffuse in complete laminin a2 deficiency and appear most marked in the periventricular

LISSENCEPHALY TYPE II

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Fig. 14.1. 12-month-old girl with merosin-negative MDC. Diffuse white matter changes on T2; left occipital cortical dysplasia is evident. MRI, 1.5 T

white matter and the frontal U-fibers. These changes have been attributed to a disturbance of the blood–brain barrier (Caro et al., 1999). In addition to the white matter abnormalities, structural brain changes have been

reported in some patients with complete or mutation proven partial laminin a2 deficiency. These include focal cortical dysplasia, occipital polymicrogyria and/ or agyria, hypoplasia of pons and/or cerebellum and

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mild enlargement of the lateral ventricles (Sunada et al., 1995; Pini et al., 1996; van der Knaap et al., 1997; Tsao et al., 1998; Pegoraro et al., 2000). In the only available brain autopsy study of a MDC1A patient with abnormal cortical gyration, focal folding abnormalities of the gray matter with partial fusion of gyri, focally irregular cortical lamination and multifocal leptomeningeal heterotopias were reported in a 4-month-old boy, while there was a normal pattern of myelination consistent with early age (Taratuto et al., 1999). Such observations would support a role for laminin a2 in neuronal guidance. Subtle cortical changes that are not readily detected by MRI could be common in laminin a2 deficiency and patients with structural changes might be more likely to have cognitive impairment (Mercuri et al., 1999). The eyes appear structurally and functionally normal in patients with laminin a2 deficiency but visual evoke potentials show conduction delay, most probably due to the white matter changes (Mercuri et al., 1998).

14.3. Abnormal glycosylation of the a-dystroglycan Abnormal glycosylation of the a-dystroglycan is a key feature of the cobblestone lissencephalies. adystroglycan is a heavily glycosylated protein located on the extracellular side of the sarcolemmal membrane (Henry and Campbell, 1998; Martin and Freeze, 2003; Michele and Campbell, 2003). It plays an active role in the basement membrane assembly. No primary mutations have yet been identified in human disease. However, recently it has been shown that defective processing of a-dystroglycan in the form of hypoglycosylation is the common underlying mechanism in MDC with brain involvement. Abnormal dystroglycan–ligand interactions result in disorganization of neurons in the cortex due to impaired arrest of migration and cause defects of neuronal migration in the form of cobblestone lissencephaly with characteristic nodularity of the brain surface in these MDC syndromes. Six conditions belonging to this group have been identified thus far; they are characterized by mutations in proven or putative glycosyltransferases and all share an abnormally glycosylated a-dystroglycan. For this reason, these conditions are also known as adystroglycanopathies (Muntoni and Voit, 2004). Dystroglycan is thought to serve as an important link from the cytoskeleton to the basal lamina, where it interacts by calcium-dependent binding with several extracellular matrix proteins, such as laminin a2, but also other laminin G domains, agrin, neurexin and perlecan. a and b-dystroglycan are encoded by a single gene (DAG1), which undergoes post-translational cleavage to give rise to the two glycoproteins, tightly

Fig. 14.2. Causative genes of cobblestone lissencephaly with corresponding congenital muscular dystrophy phenotypes (solid lines indicate major phenotypes while broken lines represent less frequently seen phenotypes).

associated via noncovalent interactions (IbraghimovBeskrovnaya et al., 1993; Holt et al., 2000). adystroglycan is heavily glycosylated and the six conditions described below have all been implicated in this enzymatic glycosylation process. Animal studies have shown that, in mice, complete disruption of dystroglycan is embryonically lethal because it prevents the correct formation of the basement membrane (Reichert’s membrane) that separates the embryo from the maternal circulation (Williamson et al., 1997; Henry and Campbell, 1998). Striated-musclespecific disruption causes loss of the dystrophin–glycoprotein complex and muscular dystrophy (Cohn et al., 2002), while brain-specific disruption leads to discontinuities in the glia limitans, with consequent overmigration of the neurons into the subarachnoid space resulting in abnormal cortical layering and cobblestone lissencephaly-like brain malformations (Michele et al., 2002; Moore et al., 2002). In mouse cerebellum, adystroglycan expression has been observed on late embryonic and early postnatal cerebellar neurons, including Bergman glial scaffolds, showing downregulation after migration is complete (Henion et al., 2003). There is a wide spectrum of severity of phenotypes caused by abnormal glycosylation of a-dystroglycan. The mildest defects of a-dystroglycan affect striated muscle only. With increasing severity, the cerebellum becomes involved, followed by the pons, the eyes and the supratentorial brain. The cerebellum is the most prone or sensitive brain structure affected by aberrant a-dystroglycan glycosylation (Voit and Tome, 2004). As shown in Figure 14.2, there is also a significant overlap between clinical phenotypes and gene defects causing a-dystroglycanopathies. One gene can cause more than a single phenotype and one phenotype can be caused by more than one gene. 14.3.1. Fukuyama congenital muscular dystrophy Historically Fukuyama congenital muscular dystrophy (FCMD) was the very first disorder in which a form

LISSENCEPHALY TYPE II of muscular dystrophy was linked to a congenital disorder of glycosylation condition (Hayashi et al., 2001). The classical picture of FCMD is the combination of generalized muscle weakness, severe brain involvement with mental retardation, frequent occurrence of seizures and abnormal eye function (Fukuyama et al., 1960). FCMD is endemic in Japan, where it is the most common form of muscular dystrophy after Duchenne muscular dystrophy. Most, if not all, previously reported cases of FCMD outside Japan do not appear to have mutations in the fukutin gene. Onset is often in utero with poor fetal movements, although severe arthrogryposis is rare. These patients typically do not speak meaningful words, although most learn to speak short sentences and may even learn to read and write a few characters. Most patients develop seizures before age 3 years. Brain MRI shows frontoparietal polymicrogyria and, more rarely, hemispheric fusion or obstructive hydrocephalus. The cerebellum shows cystic lesions under the cerebellar cortex containing granular cells and mesenchymal tissue. In addition, MRI typically shows a transient delay of myelination that tends to gradually diminish with age (Osawa et al., 1997). About 50% of the classical FCMD cases show signs of ocular involvement ranging from abnormal eye movements, poor visual pursuit and strabismus to severe myopia or hyperopia or cataracts. Striking retinal lesions including retinal detachment and persistent primary vitreous body can be also seen, even in patients with a mild clinical phenotype (Hino et al., 2001). The gene responsible for FCMD is the fukutin gene on chromosome 9q31. A recessive retrotransposal insertion into the 30 UTR of fukutin mRNA accounts for 87% of FCMD chromosomes in Japan, hence explaining the fact that the condition is endemic in that country (Kobayashi et al., 1998). This is considered to be a relatively mild mutation as it only partially reduces the stability of the full-length mRNA. Although FCMD-fukutin mutations were initially described in only Japanese patients or individuals with mixed Japanese–Caucasian parents, two patients from Turkey with WWS phenotype and different fukutin mutations have recently been reported (Silan et al., 2003; de Bernabe et al., 2003). This finding indicates that fukutin mutations probably occur worldwide and may present phenotypic variability, as a much more severe form than classical FCMD. The fukutin protein product has sequence homologies with bacterial glycosyltransferase but its precise function is unknown. As with the remaining a-dystroglycanopathies, FCMD results in marked reduction of glycosylated a-dystroglycan but not b-dystroglycan in skeletal and cardiac muscle (Hayashi et al., 2001). Reduced laminin a2 expression is also present (Hayashi et al., 1993).

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In the FCMD brain, breaches in the glia limitans, a basement membrane covering the cortical neurons, have been observed as early as 18-week fetal brain and are responsible for vertical overmigration of the neurons into the subarachnoid space, resulting in widespread pachygyria/polymicrogyria (Nakano et al., 1996; Yamamoto et al., 1997). A similar pattern of abnormal migration was induced by brain-specific disruption of a-dystroglycan in mice, indicating that the functional knockout of a-dystroglycan in FCMD through aberrant glycosylation is sufficient to cause a lissencephaly type II migration disorder (Moore et al., 2002). In children with FCMD, there is typical ‘cobblestone’ lissencephaly polymicrogyria/pachygyria or even agyria pattern. The regular layering of the cerebral cortex is lost. Dysplasia of the pyramidal tracts has been noted and was linked to ectopic migration of neurons of the pontine nucleus (Saito et al., 2003). Mild to moderate increase of ventricular width is common, and a few patients require shunting. The cerebellum may show numerous pits and caveoles under the cerebellar cortex containing granular cells and mesenchymal tissue. These are cerebellar cysts and are regularly seen on MRI (Aida, 1988; Aida et al., 1994; Takada et al., 1988). After identification of the fukutin gene, immunohistochemical and in situ hybridization studies have revealed expression of fukutin protein in the migrating neurons and at lower levels in the glial cells that form the glia limitans (Saito et al., 2000; Sasaki et al., 2000). Reduced expression of fukutin protein in the developing FCMD cortex suggests a primary role in the settling process of migrating neurons; however, in a recent neuropathological study of the FCMD brainstem, leptomeningeal glioneuronal heterotopia and an ‘aberrant pyramidal tract’ have been identified, demonstrating that certain neuronal structures are selectively vulnerable. Based on these findings, it is hypothesized that the migration anomalies may result not only from physical fragility of the glia limitans but also from disrupted neuroglial interactions in specific populations of migrating neurons (Sasaki et al., 2000). 14.3.2. Muscle–eye–brain disease Almost within months following the discovery that FCMD is actually a congenital disorder of glycosylation syndrome related with loss or modification of glycosylation of a-dystroglycan, the pathophysiological mechanism underlying MEB was revealed by identification of a glycosyltransferase as the responsible gene. MEB is characterized by congenital muscular dystrophy, eye involvement (congenital myopia and glaucoma, retinal hypoplasia), mental retardation and structural brain involvement (pachygyria, flat brainstem and

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cerebellar hypoplasia with cerebellar cysts) (Santavuori et al., 1977). The brain involvement is less severe than in WWS. Although originally described in patients of Finnish descent, MEB has a wider demographical prevalence than originally suspected and this form of congenital muscular dystrophy has now been described in most ethnic groups (Taniguchi et al., 2003). Typically, MEB patients present in the neonatal period with hypotonia and poor visual alertness. Patients at the severe end of the spectrum remain bedridden and never achieve sitting, head control or visual contact. These patients may die during the first years of life. Moderately affected patients usually show high myopia but have some preserved vision, enabling them to establish contact. Their maximum motor ability is to sit unsupported and, occasionally, to speak a few words. Patients at the milder end of the spectrum may acquire ambulation for a number of years. Often their functional abilities are more impaired by the coexistence of spasticity than weakness. Vision is preserved in these patients and limited verbal communication skills are possible. Epilepsy is common in MEB (Santavuori et al., 1989, 1998; Taniguchi et al., 2003). Eye changes in MEB are usually more severe than those in FCMD. Almost all patients develop severe myopia (6 diopters or more) and various degrees of retinal dysplasia, including abnormal pigmentation. A girl followed by us initially had 8 diopters of myopia at the age of 18 months, which then progressed to 13 within a period of 2 years. Progressive myopia may lead to retinal detachment. Optic colobomas, persistent hyperplastic primary vitreous, glaucoma and juvenile cataracts can also be present (Pihko et al., 1995; Haltia et al., 1997). In some cases,

there may not be any overt eye findings visible from the outside (personal observation). The gene responsible for MEB is the glycosyltransferase O-mannose b-1,2-N-acetylglucosaminyltransferase gene (POMGnT1) (Yoshida et al., 2001). This is the first known glycosyltransferase causing muscular dystrophy and neuronal migration disorder. It catalyzes the transfer of N-acetylglucosamine to O-mannose of glycoproteins, including dystroglycan. The reduced glycosylation of a-dystroglycan is, therefore, probably related to this missing enzymatic step. Recently, a biochemical enzyme assay for POMGnT1 has been established (Zhang et al., 2003). Recessive mutations in POMGnT1 have now been reported from at least 30 MEB patients from different ethnic backgrounds. Mutations are scattered throughout the gene and, within a group of missense, nonsense and frameshift mutations, a slight correlation was observed between the location of the mutation and clinical severity in the brain. Patients carrying mutations towards the 50 end of the gene have a more severe brain phenotype compared to those located towards the 30 end, which may also lead to diagnostic confusion with other severe presentations (Taniguchi et al., 2003). POMGnT1 is known to be a laminin binding ligand of a-dystroglycan and there is reduced a-dystroglycan and laminin a2 chain immunolabeling in the skeletal muscles of MEB patients (Kano et al., 2002). Patients with MEB invariably show structural changes of the CNS in the form of cobblestone lissencephaly and moderate to severe mental retardation. At the milder end of the spectrum of structural changes, only flattening of the brainstem and cerebellar changes, including vermis hypoplasia and cerebellar cysts, are present (Fig. 14.3). Changes in more severely affected

Fig. 14.3. 5-year-old girl with muscle–eye–brain disease. Historically, the first non-Finnish MEB case proven by linkage analysis in 1999. Cerebellar and brainstem hypoplasia, pachygyria, multiple cerebellar cysts, periventricular white matter changes. MRI, 0.5 T

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Fig. 14.4. 9-year-old girl with muscle–eye–brain disease. One of the first proven mutation cases. Brainstem and cerebellar vermis hypoplasia, corpus callosum hypoplasia, diffuse polymicrogyria, increased T2 signal, ventricular dilatation, small cerebellar cysts. MRI, 0.5 T

patients include the whole spectrum of the pachygyria/ polymicrogyria/agyria complex and show a nodular (cobblestone) surface on anatomic inspection (Haltia et al., 1997). This can be associated with transient dysmyelination, absent septum pellucidum, partial absence of the corpus callosum (Fig. 14.4), hypoplasia of the

pyramidal tracts and obstructive hydrocephalus requiring a shunt as was present in some of the Finnish cases (Dubowitz, 1994; Pihko, personal communication). Encephaloceles are practically not present in MEB. Cortical migration defect may not be evident in fetal MRI or in the first few months of age, although cerebellar

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hypoplasia, flattening of the brainstem and ventricular dilatation are readily recognized (Longman et al., 2004). The clinical spectrum of POMGnT1 mutations is significantly expanded with the reports of mild patients with some speech, absent eye involvement in the first years of life, without the typical pachygyric changes on brain MRI, and relative preservation of a-dystroglycan expression (Muntoni et al., 2003). We have recently defined severe autistic features and stereotypical hand movements as a part of the broad MEB spectrum (Halilog˘lu et al., 2004). 14.3.3. Congenital muscular dystrophy 1C Soon after the resolving of the FCMD and MEB background and cellular chemistry in 2001, the first data came from the Hammersmith group in London for congenital muscular dystrophy 1C (MDC1C). This was initially described as a severe form of congenital muscular dystrophy without brain involvement (Brockington et al., 2001a). More recent reports suggest that the spectrum of the condition secondary to mutations of the responsible gene, the fukutin-related protein gene (FKRP), is much wider. It includes, at the milder end of the spectrum, a common form of adult-onset limbgirdle muscular dystrophy (LGMD2I) (Brockington et al., 2001b) and, at the severe end of the spectrum, severe structural brain involvement resembling WWS phenotype (Beltran-Valero de Bernabe et al., 2004). Precise epidemiological figures are not available; however, MDC1C is a rare form of congenital muscular dystrophy. The clinical features of MDC1C without brain involvement as the original description of this

entity are weakness and hypotonia from birth or the first few months of life followed by a marked delay of motor milestones (Brockington et al., 2001a). The gene responsible for MDC1C is FKRP, which has turned out to be the gene giving rise to the largest spectrum of phenotypes among a-dystroglycanopathies. Recessive mutations have been identified in patients ranging from the mildest LGMD2I to the severe WWS-like phenotype. Double nonsense or frameshifting mutations have not been described and are probably not compatible with life. FKRP has sequence homologies with bacterial glycosyltransferase but its precise function is unknown. As with the remaining a-dystroglycanopathies, in MDC1C and LGMD2I there is marked reduction of glycosylated a-dystroglycan and variable reduction of laminin a2 expression in the skeletal muscle (Brown et al., 2004). 14.3.3.1. Patients with MDC1C and brain involvement The severity of brain involvement in this group of patients ranges from mild mental retardation and structural changes of the cerebellum with cerebellar cysts with normal brainstem and eye examination, through patients with features indistinguishable from MEB, to patients with even more severe features that resemble those of WWS. FKRP mutations with CNS involvement were first identified in two unrelated Turkish patients presenting with severe congenital muscular dystrophy (never able to walk), mental retardation, cerebellar cysts, normal eyes, normal brainstem and cerebral cortex and reduction of a-dystroglycan and laminin a2 labelling in skeletal muscle (Topalog˘lu et al., 2003) (Fig. 14.5).

Fig. 14.5. (A) 10-year-old boy and (B) 6-year-old girl with FKRP mutations. Severe white matter abnormality and subcentrimetric cerebellar cysts (A), and multiple cerebellar cysts (B). Cerebellar atrophy in both. The pons is relatively preserved. These were the very first cases of brain involvement in MDC1C. MRI, 1.5 T (A), 0.5 T (B).

LISSENCEPHALY TYPE II They had two different homozygous missense mutations. One of these patients was previously reported as a novel entity with merosin deficiency, mental retardation and cerebellar cysts, unlinked to LAMA2, FCMD and MEB loci (Talim et al., 2000). Following this, two new homozygous missense FKRP mutations were reported in six Tunisian and one Algerian patient with MDC1C associated with mental retardation and cerebellar cysts, hypoplasia of vermis and abnormal white matter with progressive regression (Louhichi et al., 2004). These patients also did not have any gyral malformations or structural eye changes. MDC1C with eye and brain abnormalities resembling MEB and WWS phenotype were recently reported in two patients with new homozygous FKRP mutations (Beltran-Valero de Bernabe et al., 2004). One of these patients had congenital muscular dystrophy, pontocerebellar hypoplasia, cerebellar cysts and supratentorial changes with focal thickening of the frontal cortex, resembling MEB phenotype. This boy also had eye involvement as retinal changes with abnormal pigmentation, progressive severe myopia leading to retinal detachment and blindness. The other patient described in the same report as WWS phenotype presented with cobblestone lissencephaly, microphthalmia, coloboma and retinal pigmentary changes. Based on these cases representing the spectrum of CNS changes caused by a single gene defect, it seems that cerebellar involvement with cysts is the mildest of structural CNS changes, followed by brainstem and retinal involvement, which in turn is followed by derangement of the cortical architecture, as mentioned before. 14.3.4. Walker–Warburg syndrome The molecular peculiarities of the WWS were solved, at least in part, only a couple of years ago. WWS is the most severe of the a-dystroglycanopathies. The characteristic features are congenital muscular dystrophy in combination with type II lissencephaly and retinal malformation (Dobyns et al., 1989). The CNS features typically dominate the clinical presentation. Affected children have virtually absent psychomotor development; encephaloceles and severe hydrocephalus are often detected prenatally. On brain imaging, complete type II lissencephaly or agyria combined with pontocerebellar hypoplasia is visible. An additional feature is blindness, secondary to both anterior and posterior chamber eye malformations. Muscle bulk is very much reduced and contractures may already be present at birth or develop rapidly thereafter. Average survival is around 9 months; it very rarely exceeds 2 years. Unilateral or bilateral microphthalmia occurs in more than half of patients with WWS. The optic nerve can be hypoplastic or even absent. Ocular colobomas, usually

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involving the retina, are observed in 25% of cases. Retinal changes are the rule, consisting of retinal detachment, which may be complete and can be accompanied by persistent primary vitreous. Anterior chamber malformations include cataracts, iris malformation or hypoplasia, and congenital or infantile glaucoma secondary to an abnormal anterior chamber angle. In severe microphthalmos, the anterior chamber may be missing (Chan et al., 1980; Warburg, 1987). Recessive mutations in the protein O-mannosyltransferase 1 gene (POMT1) have been identified in six out of 30 (approximately 20%) WWS cases (Beltran-Valero de Bernabe et al., 2002). In another series of 30 WWS cases, POMT1 mutations were even rarer (two patients, approximately 7%) (Currier et al., 2005) These findings clearly suggest that the genetic background of WWS is heterogeneous: POMT1 mutations account for a minority of cases and there are other genes giving rise to the WWS phenotype. Mannosyltransferase 1 catalyzes the first step in O-mannosyl glycan synthesis. A second putative O-mannosyltransferase, POMT2, shows an expression pattern in adults that overlaps with mannosyltransferase 1, and recent data indicate that both POMT1 and POMT2 form a complex that confers the enzymatic O-mannosyltransferase activity (Manya et al., 2004). The deficient POMT1 activity results in severe reduction of a-dystroglycan glycosylation and, in turn, in the inability of a-dystroglycan to effectively bind to its extracellular ligands, such as laminin a2, agrin and neurexin (Kim et al., 2004). This leads to defects in the basal lamina formation in muscle and brain, which determines the characteristic combination of brain and muscle involvement. In mice, targeted disruption of POMT1 causes defects in the formation of Reichert’s membrane, the first basement membrane to form in the embryo, and results in embryonic lethality (Willer et al., 2004). Recently, a 3.5-year-old Japanese boy with a novel POMT1 mutation has been described. Showing exceptionally long survival for WWS with a milder pattern of cortical dysplasia, this case displays an intermediate phenotype between WWS and MEB in terms of clinical findings (Kim et al., 2004). Therefore, POMT1 mutations may also cause a more benign WWS phenotype. There are also observations of somewhat milder cases, which are not published yet. The clinical features of WWS patients linked and unlinked to POMT1 are indistinguishable (Muntoni et al., 2004). In addition, homozygous null mutations of the fukutin gene were recently discovered in two patients with the WWS phenotype (Silan et al., 2003; de Bernabe et al., 2003). Moreover, a homozygous

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FKRP mutation was recently observed in another patient with the WWS phenotype (Beltran-Valero de Bernabe et al., 2004). a-dystroglycan expression is invariably reduced in skeletal muscle of all these cases. In WWS, the brain MRI shows a complete or nearcomplete absence of gyration and widespread, confluent white matter changes appearing as decreased T1 or increased T2 signal. Corpus callosum is frequently absent of hypoplastic, partial fusion of cerebral hemispheres are common. The cerebral cortical mantle is thin, usually with associated widening of the lateral ventricles (Fig. 14.6). Severe atrophy of the cerebellar vermis and hemispheres and a flattened aspect of the pons and brainstem are the rule. Arachnoid cysts are common, particularly in the posterior fossa. Meningoor encephaloceles, usually from the posterior fossa, are encountered occasionally (Dobyns et al., 1989; Cormand et al., 2001; Beltran-Valero de Bernabe et al., 2002). Neuropathological examination typically shows a cobblestone surface caused by the uncontrolled spillover of neurons that have migrated through breaches of the glia limitans and invaded the leptomeninges. Histological examination shows complete loss of cortical layering accompanied by a markedly abnormal vascular architecture, both on the surface of the brain and in the cortex, where the straight vertical appearance of perforating vessels is replaced by capillaries running parallel to the surface (Squier, 1993; Larroche

and Nessmann, 1993; Stoltenburg-Didinger and Steinbrecher, 2003). Hypoplasia of the pyramidal tracts and malformations of the inferior olivary and dentate nuclei suggest a neuronal role for a-dystroglycan or other O-mannosylated proteins for these structures. The hypoplastic brainstem is mostly surrounded by abundant gliomesenchymal tissue. The cerebellar cortex shows distorted layering of numerous cysts thought to result from remnants of the arachnoid trapped by neurons migrating both from inside out and outside in, as is characteristic for cerebellar development (Stoltenburg-Didinger and Steinbrecher, 2003). A novel member of the protein-O mannosyl transferase family, POMT2, is abundant in maturing spermatids, particularly in the acrosome, and has been studied especially in WWS patients with particular testicular defects (Willer et al., 2002, 2003); one human mutation has been identified (see below). 14.3.5. Muscular dystrophy congenital type 1D This is an exceptionally rare form of congenital muscular dystrophy. Only a single case of muscular dystrophy congenital type 1D (MDC1D) had been reported by 2003. This was a 17-year-old girl who presented with congenital muscular dystrophy, profound mental retardation and white matter changes, hypoplastic brainstem and mild pachygyria on brain MRI (Longman et al., 2003). The reduced a-dystroglycan expression in muscle is similar to what is observed in other a-dystroglycanopathies. This patient has been shown to have a compound heterozygous mutation in the LARGE gene. Although its precise enzymatic function is still unknown, recent studies indicate that, to function as a glycosyltransferase, LARGE directly interacts with a-dystroglycan intracellularly in order to stimulate a-dystroglycan hyperglycosylation (Kanagawa et al., 2004; Brockington et al., 2005). LARGE encodes a putative glycosyltransferase in the mouse, and mutations in this gene give rise to myodystrophy mouse characterized by small size, shuffling gait, thoracic kyphosis, myopathy, cardiomyopathy, abnormal brainstem evoked potentials, a neuronal migration defect affecting the cerebellum and cerebrum, and absence of glycosylated epitopes of a-dystroglycan (Grewal et al., 2001; Holzfeind et al., 2002). 14.3.6. POMT2 mutations causing WWS

Fig. 14.6. CT scan, Walker–Warburg syndrome: 17-day-old neonate with a totally dysplastic brain. The cortical mantle is extremely thin, the brainstem structures are hardly visible and the pons is very small. The patient died at 4 months.

In 2005, van Reeuwijk et al. reported homozygosity for the POMT2 locus at 14q24.3 in four out of 11 consanguineous families with typical features of the WWS. Homozygous POMT2 mutations were identified in

LISSENCEPHALY TYPE II two of these families, along with one other patient from another cohort of six WWS families (van Reeuwijk et al., 2005). Severe reduction of a-dystroglycan in muscle biopsies of these patients is consistent with the postulated role for POMT2 in the O-mannosylation pathway. POMT2 is the fifth gene identified to be responsible for the WWS phenotype and altogether these account for almost one-third of WWS cases. Therefore, other genes related with WWS are still to be identified.

14.4. Unidentified conditions In addition to the cobblestone lissencephaly syndromes discussed above with known gene defects, there are a number of cases collected by the ENMC consortium having MDC with CNS involvement in the form of a-dystroglycanopathy but without known gene defects. Some informative families have been excluded from the known loci and more genes related with abnormal glycosylation of a-dystroglycan are expected to be identified as the responsible genes in this group of patients. There were also some cases documented in the literature, before the molecular era, with muscular dystrophy accompanied by peculiar findings of brain and/or eye involvement. Considering the advances that have taken place in the molecular and physiopathological understanding of cobblestone lissencephaly in the last 5 years, re-evaluation of such cases with up to date information would narrow the unidentified conditions list. Although mostly accompanied by muscular dystrophy, there are few reports of cobblestone lissencephaly without muscle involvement, which will be discussed below. 14.4.1. Cobblestone lissencephaly with muscle involvement In three Italian families, a severe phenotype was described with congenital muscular dystrophy with calf hypertrophy, microcephaly and severe mental retardation (Villanova et al., 2000). In all cases cerebellar vermis hypoplasia and white matter changes were present, while one of the patients also had severe myopia. Further studies have revealed deficiency of a-dystroglycan accompanying partial deficiency of laminin a2 in these cases (Muntoni et al., 2003). Therefore, this condition is a form of a-dystroglycanopathy but the gene causing this phenotype has not been identified yet. There are some other cases reported with MDC and lissencephaly type II of brain malformations but recent

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data are not available about a-dystroglycan expression in skeletal muscle of these. It is possible that most, if not all, of these cases may belong to the group of a-dystroglycanopathies. Such examples include a single patient reported from Belgium, affected by merosin-positive MDC and cobblestone lissencephaly (Belpaire-Dethiou et al., 1999). Interestingly, this patient also had peripheral nerve involvement. A similar combination of features (merosin-positive MDC, mental retardation, focal cortical dysplasia, pontocerebellar hypoplasia, cerebellar cysts and reduced motor nerve conduction velocity) was also reported in three siblings from a family in Argentina (Ruggieri et al., 2001) and abnormal a-dystroglycan expression in muscle has been identified in this family (Muntoni et al., 2003). MRI findings of cortical dysplasia, cerebellar atrophy and cerebellar hypoplasia have occasionally been reported in merosin-positive MDC patients (Trevisan et al., 1996; van der Knaap, 1997; Echenne et al., 1998; Voit et al., 1999; Philpot et al., 2000) and the relation of these conditions with a-dystroglycanopathies remains to be determined. Another phenotype was described as lethal congenital muscular dystrophy in two siblings with arthrogryposis multiplex and cobblestone lissencephaly, dysmorphic features and normal eyes (Seidahmed et al., 1996). The biochemical defect is still unknown. 14.4.2. Cobblestone lissencephaly without muscle involvement This is a very rare and poorly identified condition. Craniotelencephalic dysplasia characterized by craniosynostosis and cobblestone lissencephaly associated with microphthalmia and optic hypoplasia has been described, without adequate information about muscle involvement (Hughes et al., 1983). Another report has identified three children from two families, one with cobblestone lissencephaly, normal eyes, severe developmental delay and mental retardation, normal muscle tone and creatine kinase with a nondystrophic muscle biopsy. The other two were cousins with cobblestone lissencephaly, mental retardation, mild myopia and hypotonia, severe developmental delay and normal creatine kinase. Muscle biopsy was not performed (Dobyns et al., 1996). Similar findings were observed in three siblings who had lymphedema as an associated finding (Hourihane et al., 1993). Another entity to be mentioned is a report of cobblestone lissencephaly with severe ocular malformations and normal muscle histology at necropsy in a 7-year-old boy without clinically apparent weakness (Clark et al., 1997).

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14.5. Spectrum of migrational abnormalities As a result, in this group of cobblestone lissencephaly disorders with varying severity, the common pathway could be outlined as the brain-specific disruption of a-dystroglycan perturbing the glia limitans, with consequent overmigration of the neurons into the subarachnoid space, resulting in loss of cortical layering and ending with lissencephaly type II (Michele et al., 2002). The crucial role of a-dystroglycan for maintaining basement membrane function, particularly in brain and muscle, was highlighted by the discovery that the genes mutated in WWS and MEB catalyze the first and second steps of Ser/Thr O-mannosylation, respectively, and lead to a severe reduction of glycosylated a-dystroglycan in skeletal muscle (Beltran-Valero de Bernabe et al., 2002; Kano et al., 2002). In MEB, Western blot studies using an antibody against a nonglycosylated epitope of the core protein revealed that the a-dystroglycan core protein with a molecular weight of 60 kDa was preserved but had lost its binding activity for the extracellular ligands laminin, agrin and neurexin (Michele et al., 2002). Similar findings were also documented in FCMD and MDC1C (Brockington et al., 2001a; Hayashi et al., 2001). Recently, reduced perlecan-binding activity of dystroglycan and abnormal laminin/perlecan complexes were reported in Largemyd mice, suggesting functional disruption of the trimolecular complex of dystroglycan, laminin and perlecan in the pathogenesis of a-dystroglycanopathies (Kanagawa et al., 2005). The current understanding of the whole process can be summarized as: mutations in known or putative glycosyltransferase enzymes result in abnormal glycosylation of a-dystroglycan in brain; this leads to abnormal dystroglycan–ligand interactions, causing perturbations in the glia limitans and overmigration of neurons. There is a ranking of severity of changes that holds true for all these disorders, as hypothesized recently by Voit and Tome (2004). The mildest defects affect striated muscle only and the molecular shifts of the highest-molecular-weight form of a-dystroglycan in these may be subtle. With increasing severity, the cerebellum becomes involved, followed by the pons, the eyes and the supratentorial brain. Conversely, whenever the brain shows marked pachygyria or complete lissencephaly, the eyes, pons and cerebellum will always show marked changes. The cerebellum is simply the ‘commonest target organ’, the first brain structure affected by aberrant a-dystroglycan glycosylation. This hierarchy of changes is best reflected by phenotypes caused by FKRP mutations, but is also fully supported by the growing spectrum of MEB as discussed in the individual sections above. Thus, the vast phenotypic overlap

could simplistically be explained by the type and severity of the mutations. A recent discovery of mutations within the WWS gene (POMT1) causing a rather benign course of limb-girdle-onset muscular dystrophy and mild mental retardation without any MRI evidence of brain abnormalities has been the last example (Balci et al., 2005).

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Chapter 15

Schizencephaly TIZIANA GRANATA* AND GIORGIO BATTAGLIA Neurological Institute ‘C. Besta’, Milan, Italy

15.1. Introduction Schizencephalies (from the Greek schizein ¼ to divide, to split) are brain malformations characterized by fullthickness holes or clefts in the cerebral mantle. The clefts span the cerebral hemispheres unilaterally or bilaterally, extending from the external pial surface to the lateral ventricles, and they are lined throughout by heterotopic gray matter (Fig. 15.1). The first descriptions of brain defects similar to schizencephalies date back to the early neuropathological literature and were referred to as porencephalies (Kundrat, 1882). The term schizencephaly was first used in 1946 in two seminal papers by Yakovlev and Wadsworth, who described the morphological features of five autopsied patients with severe mental and motor deficits (Yakovlev and Wadsworth, 1946a, 1946b). On the basis of distinctive neuropathology features, i.e. the symmetrical location of the cleft, the extension to the ventricle and the persistence of gray matter along the cleft walls, it was proposed to classify schizencephalies as true brain malformations resulting from agenesis of the cerebral mantle in early stages of development, and to separate them from encephaloclastic porencephalies (Yakovlev and Wadsworth, 1946a, 1946b). This concept is still debated and some authors prefer the term ‘porencephaly’, to emphasize the encephaloclastic rather than the malformative nature of these brain defects. For instance, in a comprehensive review of the issue of developmental neuropathology, Friede stated: ‘the term schizencephaly is not used here since it was coined to emphasize a hypothetical concept of maldevelopment for lesion traditionally described under the terms hydranencephaly or porencephaly’ (Friede, 1989). Nevertheless, the term schizencephaly has been commonly used to define full-thickness

defects of the cortical mantle associated with polymicrogyric or heterotopic gray matter (Barkovich and Kjos, 1992; Hayashi et al., 2002). In our opinion, the term ‘porencephaly’ should be used to indicate late perinatal encephaloclastic lesions and not developmental disorders (Ashwal, 1994).

15.2. Anatomical features as revealed by neuroimaging In the last 15 years several reports have described the magnetic resonance imaging (MRI) features of cases of schizencephaly (Barkovich and Kjos, 1992; Granata et al., 1996a; Packard et al., 1997; Denis et al., 2000; Hayashi et al., 2002). There is general agreement with the original description by Yakovlev and Wadsworth (1946a, 1946b). All the authors classify patients according to the presence of unilateral versus bilateral clefts, and the schizencephalic clefts are divided into closedlipped (type 1) and open-lipped (type 2). The cleft is defined as closed when the walls are in contact with each other and the cerebrospinal fluid space within the cleft is obliterated (Fig. 15.2A). Conversely, in openlipped schizencephaly (type 2) the walls are separated and the cleft is filled with cerebrospinal fluid from the lateral ventricle to the subarachnoid space (Fig. 15.2B). In addition, after the accurate MRI report by Barkovich and Kjos (1992), who tried to relate imaging features with the clinical outcome of schizencephaly patients, additional features have been considered in describing schizencephaly. First, the clefts are described in relation to their size as small (i.e. the cleft involves less than one-third of a single lobe), medium (i.e. the cleft involves more than one-third but less than two-thirds of a single lobe) and large (the cleft involves more than two-thirds of a lobe, or more than

*Correspondence to: Tiziana Granata MD, Department of Child Neurology, Istituto Neurologico ‘C. Besta’, Via Celoria 11, 20133 Milano, Italy. E-mail: [email protected], Tel: þ39-02-23942702, Fax: þ39-02-23942181.

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T. GRANATA AND G. BATTAGLIA

Fig. 15.1. Comparison between neuropathological and MRI features of schizencephaly. (A) The horizontal sections through the cerebral hemispheres (reported in the original paper by Yakovlev and Wadsworth) demonstrates bilateral cleft lined by heterotopic gray matter extending from the leptomeningeal surface to the lateral ventricles. (B) The axial T2-weighted image similarly shows a left prerolandic cleft surrounded by gray matter extending through the cerebral mantle to the lateral ventricle (arrows). He, subcortical gray matter heterotopia; Pes: cortex-lined cleft. Ve: lateral ventricle. (A, Reproduced from Yakovlev and Wadworth, 1946 with permission from the Journal of Neuropathology and Experimental Neurology; B, reproduced from Granata et al., 1996a, with permission from Blackwell Publishing.)

Fig. 15.2. MRI features of schizencephaly. (A) Coronal inversion recovery (IR) T1-weighted image showing closed-lipped schizencephaly located in the left posterior sylvian region. (B) Coronal IR T1-weighted image demonstrating bilateral frontal schizencephaly, closed-lipped on the right side and open-lipped on the left, associated with agenesis of the septum pellucidum. Note the dimpling of the walls of the lateral ventricles and the heterotopic cortex lining the entire extension of the clefts.

one lobe). In addition, particular attention has been paid to the presence and type of associated brain malformations (Barkovich and Kjos, 1992). In all reports, the most frequent anatomical localization is in the frontal and parietal lobes, which are involved in up to 70% of cases, particularly in the areas adjacent to the central fissure (Barkovich and Kjos, 1992; Packard et al., 1997; Granata et al., 1999; Denis et al., 2000; Hayashi et al., 2002). The temporal and occipital lobes may be involved by large clefts extending to more than one cerebral lobe but they are rarely selectively affected. In unilateral schizencephalies, closed-lipped and open-lipped clefts are

observed equally often. In bilateral cases, the clefts are more frequently open-lipped and usually symmetrical. Open- and closed-lip clefts, however, can coexist in a given patient (Fig. 15.2B) and three clefts have been described, albeit rarely, in individual patients (Packard et al., 1997; Hayashi et al., 2002). Both unilateral and bilateral schizencephalies are frequently (i.e. in 50–90% of cases) associated with other brain abnormalities, thus suggesting that in most cases the clefts are part of a widespread developmental disorder (Barkovich and Kjos, 1992; Packard et al., 1997; Granata et al., 1999; Denis et al., 2000). The most frequently associated malformations are partial or

SCHIZENCEPHALY complete agenesis of septum pellucidum (reported in up to 70% of cases), often associated with agenesis or thinning of corpus callosum, and cortical dysgenesis. Areas of polymicrogyria (or, more generally, of dysplastic cortex) may be identified ipsilaterally and even not in direct continuity with the cleft, as well as in locations symmetrical to the cleft in the contralateral hemisphere (Packard et al., 1997; Granata et al., 1999; Denis et al., 2000; Hayashi et al., 2002). Periventricular heterotopic nodules have been also described (Barkovich and Kjos, 1992; Granata et al., 1999), even if their frequency is considerably less than that reported by neuropathological accounts of schizencephaly (see below). The association between midline abnormalities and cortical dysgenesis dates the time of origin of schizencephaly in the late first or early second trimester of gestation. In fact, at this developmental stage, both corpus callosum and septum pellucidum develop in the prosencephalic midline, while neuroblast migration and cortical organization are still taking place. The spectrum of brain malformations associated with schizencephaly also includes hippocampal abnormalities (Hayashi et al., 2002), arachnoid cysts (Barkovich and Kjos, 1992; Packard et al., 1997; Granata et al., 1999; Denis et al., 2000; Hayashi et al., 2002) and multiple and diffuse calcifications (Denis et al., 2000). Abnormalities of the posterior fossa have been also observed, including cerebellar hypoplasia and mega cisterna magna. Finally, white matter abnormalities are almost invariably present. White matter volume is reduced in most cases, the reduction being more severe in patients with large clefts. Signal abnormalities are present in 20% of cases, close to the clefts or in the white matter just beneath the overlying polymicrogyric areas (Hayashi et al., 2002).

15.3. Etiopathogenesis Schizencephaly is thought to be due to impairment of the mechanisms that govern cortical organization during the late phases of embryonic cortical ontogenesis (Barkovich et al., 2001). However, the etiology of schizencephaly is still much debated and it is possible that different etiological factors are involved in different cases. The most widely accepted hypothesis is that schizencephaly is secondary to hypoperfusion or ischemic cortical injury (Barkovich and Kjos, 1992). This assumption is primarily based on the fact that polymicrogyria is present in about half of the affected patients within or adjacent to the schizencephalic clefts (Hayashi et al., 2002). Polymicrogyria consists of small irregular gyri without sulci or with sulci bridged by fusion of the overlying molecular layer (Friede, 1989; Ferrer and

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Catala, 1991; Barkovich and Kjos, 1992). It is believed to arise from cortical ischemic necrosis primarily affecting the fifth cortical layer (Sarnat, 1992). Therefore, it has been hypothesized that both polymicrogyria and schizencephaly result from ischemic damage to the developing cortex (Barth, 1987; Barkovich and Kjos, 1992). According to Barkovich and Kjos, mild ischemic damage could determine polymicrogyria without cortical infolding (i.e. without schizencephaly), whereas severe and earlier ischemic damage deeply involving radial glial fibers could determine a schizencephalic cleft lined by polymicrogyria through the entire thickness of the neocortex (Barkovich and Kjos, 1992). In addition to cortical ischemia, infections and toxic agents or trauma have been considered as causative factors (Dekaban, 1965; Barth et al., 1987; Dominguez et al., 1991; Barkovich and Kjos, 1992). The possibility that transmissible agents may produce schizencephaly is supported by the description of two children affected by schizencephaly in whom cytomegalovirus DNA and antibodies were found (Iannetti et al., 1998), and by data from experimental animals (Takano et al., 1999). The intraplacental inoculation in hamsters of the Kilham strain of mumps virus during prenatal neuronal migration is able to induce cerebral hemorrhage, cortical microsulci and the formation of a cleft through the entire thickness of the cerebral mantle (Takano et al., 1999). These animals provide evidence that a destructive process from exogenous factors may impair neuronal migration and induce brain malformations, and they represent the only animal model for schizencephaly available so far. The alternative theory, which is in line with the original assumption of Yakovlev and Wadsworth, is that schizencephaly is genetically determined. This idea was first suggested by the existence of rare familial cases (Hosley et al., 1992; Hilburger et al., 1993; Granata et al., 1997; Muntaner et al., 1997; Tietjen et al., 2005) and received much stronger support from reports of heterozygous germline mutations of the homeobox gene EMX2 in sporadic and familial cases (Brunelli et al., 1996; Faiella et al., 1997; Granata et al., 1997). These observations are particularly interesting, since EMX2 is one of the homeotic genes expressed in proliferating neuroblasts committed to the neocortex and involved in controlling neuronal migration and structural patterning of the developing forebrain (Gulisano et al., 1996; Cecchi and Boncinelli, 2000; Mallamaci et al., 2000). However, the role of EMX2 in the pathogenesis of schizencephaly still needs to be fully demonstrated. Among the reported EMX2 gene disruptions, very few mutations bear a biological significance, since they determine the truncation of the protein or prevent the

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correct splicing of the transcript in vitro (Brunelli et al., 1996; Faiella et al., 1997). The other reported EMX2 alterations are synonymous mutations without any obvious biological significance. After the original reports, 15 new patients were reported as negative (unpublished study cited in Barkovich et al., 2001) and a new cohort of 14 sporadic and three familial cases did not reveal any mutation but the previously reported Arg156Arg substitution (three cases; Granata et al., 2005). The latter substitution is surprisingly frequent among patients affected by schizencephaly but its presence in unaffected relatives suggests that it should be regarded as a polymorphic variant (Granata et al., 2005). In addition, the phenotypes of experimental mutant mice do not provide evidence to confirm that the EMX2 mutations are responsible for the human disease. The EMX2 mutations described in humans are heterozygous and therefore dominant, but heterozygous Emx2þ/ mice display a normal phenotype (Pellegrini et al., 1996; Yoshida et al., 1997), whereas homozygous Emx2/ knockout mice are defective in urogenital system development and in the structure of the olfactory bulbs and hippocampal dentate gyrus, but they do not display clefts in the cerebral cortex resembling human schizencephaly (Pellegrini et al., 1996; Yoshida et al., 1997; Tole et al., 2000). In addition to EMX2, it is possible that other genes are responsible for schizencephaly. The search for such hypothetical genes has been so far hampered by the lack of sufficiently large pedigrees and by the fact that severely affected patients rarely have children. However, a large family with three affected siblings has been recently reported. In this family, linkage analysis and direct sequencing ruled out any causal relationship with EMX2 and suggested additional candidate regions on chromosomes 8q24.22–24.3 or 5q21.3–23.2 (Tietjen et al., 2005). Taken together, all this evidence suggests that the etiology of schizencephaly is most probably heterogeneous and that both acquired and genetic determinants are likely to play a role. Among genetic factors, the role of EMX2, if any, is restricted to a minority of cases, and other genes are involved. Therefore, the pathogenesis of schizencephaly still remains a matter of study, requiring further investigations to be definitively settled.

15.4. Epidemiology and risk factors Schizencephaly is a rare condition that affects both males and females almost equally. It is usually sporadic, although a number of familial cases have been reported (Hosley et al., 1992; Hilburger et al., 1993; Granata et al., 1997; Muntaner et al., 1997; Tietjen et al., 2005). As for all other malformations of cortical

development, precise information on the prevalence of schizencephaly are lacking and only few studies have addressed this specific issue. Schizencephaly accounted for 5% of all cortical malformations in a population of pediatric patients with malformations of cortical development (Leventer et al., 1999). More recently, an epidemiological study reported a population prevalence of 1.54:100.000 in more than 4 million births from 1985–2001 in California, USA (Curry et al., 2005). Different risk factors have been associated with schizencephaly, such as prenatal exposure to toxic agents, including cocaine and other street drugs, and prenatal viral infections (Dominguez et al., 1991; Iannetti et al., 1998; Sener, 1998). Inappropriate or insufficient levels of assistance during pregnancy may also be a risk factor, since schizencephaly is more frequent in adopted children (Barth et al., 1987) and a relatively high proportion of adopted or abandoned children has been found in schizencephaly series (Dekaban, 1965; Granata et al., 1996a). Finally, genetic risk factors may play a role in schizencephaly, even if conclusive evidence regarding the causative genes is still not available (see above).

15.5. Neuropathological features Detailed descriptions of the anatomical features of schizencephaly are rare and date back to classic neuropathology studies (Yakovlev and Wadsworth, 1946a, 1946b; Dekaban, 1965; Landrieu and Lacroix, 1994). The clefts are more frequently located around the sylvian fissures but they can also involve the prefrontal and, more rarely, the temporal and occipital lobes. Polymicrogyric (or, in more general terms, malformed) cortex is always associated (Fig. 15.3). It is located either immediately adjacent to the pallial defect or in a region symmetrical to the cleft in the opposite hemisphere (Dekaban, 1965). More circumscribed areas of polymicrogyria or heterotopic islands of gray matter located at distance from the schizencephalic cleft are also frequently described (Yakovlev and Wadsworth, 1946a; Dekaban, 1965; Landrieu and Lacroix, 1994). Macroscopically, the close-lipped clefts are usually cone-shaped with the apex tapering towards the lateral ventricles, and covered by slightly thickened arachnoid and pia membranes. The cortical circumvolutions around the clefts are usually malformed but they progressively merge with a normal appearing cortex. Microscopically, the cortical gray matter that forms the seam extending to the lateral ventricle is made up by small neurons oriented parallel to the axis of the seam. Some cytological abnormalities (large cells with increased amount of chromatin) and no evidence of cortical lamination or architectonic pattern have been described (Yakovlev and Wadsworth, 1946a; Landrieu

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Fig. 15.3. Neuropathological features of schizencephaly. (A) Macroscopic appearance of bilateral clefts in the middle frontal convolution. (B) Nissl-stained section passing through the defects revealing the presence of polymicrogyria in the immediate vicinity of the defects and the transition to normal cortex. (Reproduced from Dekaban, 1965, with permission from the Journal of Neuropathology and Experimental Neurology.)

and Lacroix, 1994). However, the most superficial layers of the cleft cortex are in continuity with layers I–III of the normal cortex outside the cleft (Landrieu and Lacroix, 1994). Fiber tract staining suggested the presence of connections between the schizencephalic seam and the surrounding cortical areas. In many cases, reduction of the cerebral peduncles and bulbar pyramids has been reported, suggesting a severe impairment of the descending inputs from the cerebral cortex. In bilateral cases, the majority of cortical circumvolutions may be malformed, or even completely absent in the most severe cases (Yakovlev and Wadsworth, 1946b). Atypical vessels may be present in the clefts (Landrieu and Lacroix, 1994). The thalamus and thalamo-cortical radiations may be hypoplastic, in contrast to the striatum, which may be hyperplastic and project dorsally and rostrally into the enlarged ventricular cavity. Midline abnormalities are common, with partial or even complete agenesis of corpus callosum and septum pellucidum (Dekaban, 1965). The cortical projections, cerebral peduncles and bulbar pyramids may be virtually absent (Yakovlev and Wadsworth, 1946b). Cerebral samples of surgically treated epileptic patients with schizencephaly have also been analyzed (Leblanc et al., 1991; Landy et al., 1992; Maehara et al., 1997). Information from these papers is, however, scarce, because of the limited number of treated patients and because surgical removal was frequently not extended to the schizencephalic clefts. The only pathological abnormality reported in the resected epileptogenic areas outside the clefts was the presence of neuronal loss and gliosis (Leblanc et al., 1991; Landy et al., 1992). Within the schizencephalic clefts, subpial microdysgenesis and the presence of cytologically abnormal and malpositioned neurons were reported (Leblanc et al., 1991; Maehara et al., 1997).

15.6. Clinical features The main clinical manifestations are motor deficits, mental retardation and epileptic seizures. The presence and severity of motor and cognitive deficits is related to location, type and size of the clefts, number of involved lobes, and presence and type of associated brain malformations (Barkovich and Kjos, 1992; Granata et al., 1996a; Packard et al., 1997; Denis et al., 2000; Granata et al., 2005). As a rule, patients with small unilateral closed-lipped clefts display the mildest clinical signs, whereas the more severe clinical pictures are observed in patients with large bilateral open-lipped clefts. Accordingly, the presenting symptoms may vary, ranging from focal seizures in late childhood or even adulthood in unilaterally affected patients to severe hypotonia and developmental delay in patients with bilateral clefts. In patients with large, open clefts, abnormal head enlargement due to progressive hydrocephalus may be the first sign leading to clinical evaluation (Packard et al., 1997). In a study attempting to quantify the clinical outcome in large series of patients (Packard et al., 1997), developmental outcome was categorized as mild (i.e. normal motor skills and normal or minimally affected language), moderate (i.e. moderate hemiparesis, learning disabilities and slow for age language) and severe (i.e. severe mental retardation, spastic tetraparesis, absence of language or total dependency). In unilaterally affected patients, the clinical outcome was judged to be mild in 28%, moderate in 34% and severe in 38%, and it was most favorable in patients with involvement of a single lobe. By contrast, in bilaterally affected patients the outcome was severe in 72% of cases and moderate in the remaining 28%, and no patients fell into the ‘mild’ category (Packard

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et al., 1997). In addition, the same study demonstrated that, in both unilateral and bilateral cases, the prognosis was significantly better in patients with closed clefts than those with open clefts (mild or moderate outcome in 78% versus 31% of patients) and in patients with no associated brain abnormalities. Similar results were found in other studies addressing the clinical features of different cohorts of patients (Barkovich and Kjos, 1992; Granata et al., 1996a; Denis et al., 2000). The location and extent of the anatomical malformation is not, however, the only factor influencing the clinical picture. Functional MRI (fMRI) and transcranial magnetic stimulation in patients with unilateral schizencephaly have demonstrated activation of the unaffected hemisphere, including cortex and corticospinal tract, during motor tasks carried out with the paretic hand (Lee et al., 1999; Vandermeeren et al., 2002; Calistri et al., 2004; Kim et al., 2004). On the other hand, activation of the dysgenetic cortex during covert verb generation was demonstrated in an asymptomatic patient with a left frontal cleft (Spreer et al., 2000). Therefore, since unaffected brain regions may replace the functional abilities of malformed areas and the malformed cortex may contribute to physiological cerebral functions, the functional reorganization of both malformed and unaffected cortex is most probably an additional factor in determining the clinical outcome. 15.6.1. Motor features Motor impairment is almost invariably present, in line with the frequent location of the cleft in the central areas. In unilateral schizencephaly motor deficits are usually contralateral to the malformation. In a minority of cases bilateral deficits are observed, possibly related to subtle cortical dysgenesis contralateral to the cleft and not detectable by MRI. The severity of deficits may vary from mild asymmetry of motor skills, resulting in no or minimal functional impairment, to spastic hemiparesis. In most cases, asymmetrical muscle tone is the first symptom detected in the course of the first year. In patients with small, unilateral closed-lipped cleft, however, motor deficits may be mild and overlooked until a neurological evaluation is required for additional symptoms, such as seizures, presenting in late childhood or even adulthood (Bisgard and Herning, 1993; Cho et al., 1999). In bilateral schizencephaly, motor impairment is, by contrast, more frequently severe and bilateral. The most common presenting symptoms in early infancy are muscle hypotonia and delays in motor skills, which progressively evolve in spastic tetraparesis.

15.6.2. Mental and speech abilities Mental and speech abilities are impaired in about half of cases with unilateral schizencephaly, and in virtually all patients with bilateral malformation. As for motor deficits, the presence and severity of mental deficits depends on the localization of the cleft and the amount of brain tissue involved by the malformation (Aniskiewicz et al., 1990; Barkovich and Kjos, 1992; Granata et al., 1996a; Packard et al., 1997; Denis et al., 2000). Language development is also strictly related to the extension of schizencephaly. In unilateral schizencephaly, speech defects are related to the presence of open-lipped clefts and fronto-parietal location. The side of the cleft, by contrast, appears to be less important in predicting speech development (Packard et al., 1997; Denis et al., 2000). Severe language impairment or even total lack of speech are commonly observed in patients with bilateral open-lipped schizencephaly (Barkovich and Kjos, 1992; Packard et al., 1997; Denis et al., 2000). As mentioned before, a major role in mental and speech outcome is also played by the functional reorganization of the malformed brain. In early determined brain injuries, not only may the unaffected cortex incorporate abilities of the damaged areas but the dysgenetic cortex may also contribute to physiological cerebral function (Spreer et al., 2000). It is thus conceivable that either a complete shift of functions to the unaffected hemisphere or the physiological recruitment of the malformed cortex may lead to completely normal cognitive function. Indeed, detailed neuropsychological investigations have demonstrated that cognitive functions may be completely normal in patients with unilateral schizencephaly (Brown et al., 1993). On the other hand, the incorporation of specific abilities in the unaffected hemisphere may lead to competition between different cortical functions in normal cortical areas (the so called ‘crowding effect’) (Lansdell, 1969; Strauss et al., 1990). This effect may explain why the neuropsychological profile may not be strictly consistent with the anatomical features of the malformation but rather reveal a widespread cortical involvement and specific impairment of unaffected cortical areas (Aniskiewicz et al., 1990; Granata et al., 1996a; Denis et al., 2000). 15.6.3. Epilepsy features Epilepsy is present in about half of patients (Barkovich and Kjos, 1992; Granata et al., 1996a, 1996b; Packard et al., 1997; Denis et al., 2000) and may be the presenting and most relevant symptom in patients with unilateral cleft (Barkovich and Kjos, 1992; Bisgard and Herning, 1993; Granata et al., 1996a; Packard

SCHIZENCEPHALY et al., 1997; Cho et al., 1999; Denis et al., 2000; Caraballo et al., 2004). As a rule, epilepsy does not run a malignant course: it does not affect neurodevelopmental outcome (Packard et al., 1997) nor interfere with the patient’s everyday life (Granata et al., 1996b; Denis et al., 2000). Nevertheless, seizures are refractory to treatment in about one-third of cases and may occur at high frequency in a few, thus physically and socially disabling affected patients (Barkovich and Kjos, 1992; Packard et al., 1997; Granata et al., 1999; Denis et al., 2000; Caraballo et al., 2004). In contrast to neurological and mental deficits, the occurrence and severity of epilepsy is not strictly correlated with the type and extension of clefts (Granata et al., 1996a; Packard et al., 1997; Denis et al., 2000). Indeed, in most clinical series, epilepsy is more frequent in unilaterally than bilaterally affected patients (Granata et al., 1996a; Denis et al., 2000). Age at first seizure is more frequently in childhood and adolescence, although onset in early infancy or in adulthood is observed in quite a number of cases (Landy et al., 1992; Bisgard and Herning, 1993; Granata et al., 1996a; Packard et al.,1997; Cho et al., 1999; Denis et al., 2000; Caraballo et al., 2004). A relationship between early onset and the presence of bilateral or open-lipped clefts has been reported but must not be considered the rule (Barkovich and Kjos, 1992; Granata et al., 1996a; Packard et al., 1997; Denis et al., 2000). Seizures are mostly focal, and single-type seizures are usually observed in a given patient. The semiology

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is mostly related to the site of the clefts and associated malformations. Accordingly, in line with the frequent fronto-parietal location, focal motor or versive seizures are the most frequently reported (Granata et al., 1996a; Caraballo et al., 2004). Complex focal seizures are also frequently described, particularly in surgery series (Leblanc et al., 1991; Silbergeld and Miller, 1994; Maehara et al., 1997; Cascino et al., 2004). This finding may reflect the contribution of temporal areas to the onset and propagation of ictal discharges even in patients with clefts not involving the temporal lobe. Intracranial EEG recordings have indeed demonstrated that ictal discharges may arise from or spread to temporal structures in patients with no evidence of temporal lobe malformations (Leblanc et al., 1991; Silbergeld and Miller, 1994). In contrast to the frequent occurrence of focal seizures, primarily generalized seizures, including tonic, atonic or myoclonic seizures as well as atypical absences, are rarely observed. Infantile spasms are also rarely reported in schizencephaly, in contrast to what is observed in other malformations of cortical development. Even focal seizures are rarely followed by secondary tonic–clonic generalization. These findings have been related to the presence of the cleft, which may inhibit the propagation of epileptic discharges (Granata et al., 1996a; Denis et al., 2000). Interictal electroencephalographic (EEG) recordings (Figs. 15.4, 15.5) are usually characterized by the presence of focal epileptiform abnormalities, which are

Fig. 15.4. EEG recordings from a patient with small, open-lipped unilateral schizencephaly located in the left rolandic cortex. During wakefulness, the EEG interictal discharges of sharp and slow wave complexes mainly involve the left centro-parietal and temporal regions (left panel). Slow sleep increases the occurrence of hemispheric epileptic discharges (right panel).

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Fig. 15.5. EEG recordings from a patient with bilateral schizencephaly. Bilateral sharp and slow wave complexes, with quasiperiodic recurrence on the right hemisphere, are evident during wakefulness (left panel). During drowsiness, asynchronous EEG abnormalities become more evident (right panel).

consistent with the location of the clefts and the semeiology of seizures (Leblanc et al., 1991; Silbergeld and Miller, 1994; Granata et al., 1996a; Maehara et al., 1997; Denis et al., 2000; Caraballo et al., 2004). The focal EEG abnormalities may increase during slow sleep but do not usually spread to the contralateral hemisphere. This finding, together with the rarity of generalized seizures, is probably related to the presence of the cleft and resulting rearrangement of cortico-cortical and cortico-subcortical pathways connecting the two hemispheres, which may inhibit the propagation of epileptic discharges (Granata et al., 1996a; Denis et al., 2000). This hypothesis can also explain the rare occurrence of seizures in patients with large bilateral cleft, in whom the wide extent of the clefts and the frequently associated agenesis of corpus callosum may prevent the onset and diffusion of hyperexcitability phenomena leading to seizures. 15.6.4. Schizencephaly as part of complex syndromes Schizencephaly clefts may be observed in the context of complex syndromic pictures, including multiple congenital anomaly-mental retardation syndromes (MCA/MR), either codified or reported as single observations. The most frequent associated syndrome is septo-optic dysplasia (SOD), which accounts for 6–25% of all reported schizencephaly patients (Barkovich et al., 1989; Kuban et al., 1989; Barkovich and Kjos., 1992; Packard et al., 1997; Denis et al., 2000, Miller et al., 2000). The term

SOD is used to refer to a still loosely defined condition in which absence of septum pellucidum, atrophy of the optic nerve and hypopituitarism are variably associated. In addition, unilateral or bilateral clefts, or, less frequently, other brain dysgenesis, are described in a number of patients. The association between SOD and schizencephaly, termed SOD–SCH (Barkovich et al., 2001), was proposed as a separate entity with distinctive imaging features, different clinical presentation and diverse embryogenetic origin (see below). The most frequently presenting symptoms in SOD patients without clefts are related to hypothalamic–pituitary dysfunction, and seizures and/or visual defects in SOD–SCH patients (Barkovich et al., 1989). In rare cases, schizencephaly has been also associated with arthrogryposis multiplex congenita (Brodtkorb et al., 1994) and reported in rare syndromes, such as Vici, Beckwith–Wiedeman, and triple X syndromes (del Campo et al., 1999; Worth et al., 1999; Ehara and Eda, 2001). Finally, schizencephaly clefts may be part of complex malformative pictures involving extracerebral structures, including limbs, heart and kidney (Yakovlev and Wadsworth 1946a, 1946b; Humbertclaude et al., 1996; Packard et al., 1997).

15.7. Diagnostic assessment and differential diagnosis 15.7.1. Clinical assessment A detailed personal and family history should be collected to detect familial and prenatal risk factors. Clin-

SCHIZENCEPHALY ical evaluation includes the assessment of motor and mental functions, in the view of rehabilitative intervention. A standardized neuropsychological evaluation should ideally be performed in every case, as it can detect subtle deficits in specific neuropsychological functions even in patients whose mental abilities and speech are sufficiently well preserved to appear normal on clinical observation (Aniskiewicz et al., 1990). The assessment of epilepsy includes the detailed description of ictal semeiology and seizure frequency, as well as accurate EEG evaluations. In patients who are candidates for epilepsy surgery for the relief of drug-resistant seizures, intracranial recording of seizures is mandatory as the epileptogenic area may extend beyond the location of the cleft (Leblanc et al., 1991; Landy et al., 1992). 15.7.2. Neuroimaging assessment Given the correlation between anatomical features and clinical outcome, careful evaluation of neuroradiology features is a crucial step in patient assessment to predict the motor and mental outcome. Although schizencephaly may be diagnosed by computed tomography (Miller et al., 1984), MRI is the method of choice for imaging assessment. MRI shows the extension of the clefts, their relation to the lateral ventricles and the resultant dimpling of the ventricular walls. In addition, MRI reveals the presence and extension of the dysplastic cortex lining schizencephaly and the associated brain abnormalities. In addition to MRI, functional studies may be also helpful in predicting the patient outcome. Positron emission tomography (PET), single photon emission computed tomography (SPECT), and fMRI may provide useful information in the evaluation of the physiologic function of affected and unaffected cortical areas, which is particularly relevant in patients candidate to surgical treatment for the relief of drug-resistant seizures (Lee, et al., 1999; Morioka et al., 1999; Jang et al., 2001; Janszky et al., 2003; Cascino et al., 2004). 15.7.3. Prenatal diagnosis Neuroimaging is increasingly used in prenatal diagnosis of brain malformations and schizencephaly has been recognized in utero in a few cases (Lituania et al., 1989; Komarniski et al., 1990; Suchet, 1994; Denis et al., 2001; Oh et al., 2005). Ultrasonographic diagnosis of schizencephaly may be possible in the case of large, open clefts but more frequently it can only be suspected in the presence of ventricular dilatation. In this case, prenatal MRI is indicated as it can visualize the cleft and the dysgenetic cortex as well

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as associated brain malformations such as callosal abnormalities. Prenatal MRI may also differentiate developmental anomalies (e.g. arachnoid cyst, isolated ventriculomegaly, holoprosencephaly) from destructive lesions (e.g. porencephalic cyst) (Oh et al., 2005). On the other hand, closed-lipped or small open-lipped schizencephaly may be overlooked, as prenatal ultrasonography and MRI may detect large, open-lipped clefts only. Indeed, no report is available about prenatal diagnosis of small schizencephalic defects (Denis et al., 2001; Oh et al., 2005). 15.7.4. Differential diagnosis The differential diagnosis is restricted to a few conditions. The existence of full-thickness cleft of cortical mantle associated with heterotopic cortical gray matter is pathognomonic of schizencephaly. The main differential diagnosis is with encephaloclastic porencephaly, which is not lined by heterotopic cortical gray matter, and with polymicrogyria, in which communication with lateral ventricles is absent. Extremely large, bilateral, open-lipped schizencephaly must be differentiated from hydranencephaly. Finally, the differential diagnosis must consider schizencephaly as part of SOD. The different imaging features in SOD patients with and without schizencephaly have been analyzed in detail (Barkovich et al., 1989). Patients with SOD and schizencephaly had normal ventricular size, a remnant of the septum pellucidum and normal-appearing optic radiation. On the other hand, diffuse white matter hypoplasia (including the optic radiation), ventriculomegaly and complete absence of the septum pellucidum characterize the MRI picture of SOD patients without schizencephaly.

15.8. Prognosis and management The management of patients with schizencephaly is that of associated motor and mental deficits and epileptic seizures. The clinical picture in patients with unilateral, small clefts may be very mild and allow a normal social and working life. In some of these patients, however, the presence of focal epilepsy is a relevant clinical problem. By contrast, the neurological deficits in patients with bilateral clefts are usually severe, impairing most patients’ everyday life, and making them, in the most severe cases, unable to walk unassisted or talk. The management is that of a patient with cerebral palsy and must be planned according to the severity of the clinical picture. Different medical and rehabilitative specialists should be involved to provide careful evaluation and rehabilitation to achieve best improvement in all

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areas of development. Rehabilitative motor interventions, including kinesitherapy, and the use of orthesis and mobility devices, may facilitate the acquisition of motor skills and prevent orthopedic deformities. In more severe cases, medical or surgical treatment of spasticity may be required. In addition, speech therapy and individualized educational programs should be planned for patients with language or mental deficits. Progressive obstructive hydrocephalus, which may be observed in patients with open-lipped schizencephaly, may require ventriculoperitoneal shunt. In some cases shunting has been demonstrated to induce expansion of the cortex and decrease cleft size (Packard et al., 1997). In patients with epilepsy, treatment has to be chosen on the basis of the electroclinical picture. Although epilepsy is not usually characterized by a malignant course, in a third of patients the seizures, mostly focal, are refractory to antiepileptic drugs. Epilepsy surgery should therefore be considered in selected cases. As for other cortical malformations, the best outcome is expected in patients for whom the clinical, electrophysiological, radiological and neuropsychological data are consistent in identifying the resectable epileptogenic area. Postoperative improvement has been reported in some cases (Leblanc et al., 1991; Landy et al., 1992; Maehara et al., 1997; Cascino et al., 2004).

Acknowledgments The authors wish to thank Dr Adele Finardi for her contribution in preparing the manuscript.

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Disorders of neural crest induction of non-neural tissues Chapter 16

Neural tube programming and the pathogenesis of craniofacial clefts, part I: the neuromeric organization of the head and neck MICHAEL H. CARSTENS* Cardinal Glennon Children’s Hospital, Saint Louis University, Saint Louis, MO, USA

16.1. Introduction Developmental anatomy is the bedrock upon which all treatment methods for cleft and craniofacial anomalies must be based. Traditionally facial development has been considered to be independent of brain development but recent advances in molecular genetics demonstrate a more intimate neuroembryological relation than was previously appreciated. The well-known dictum of DeMeyer in 1964, ‘the face predicts the brain’, can be inverted to ‘the brain predicts the face’, as mechanisms of induction are now better understood. Many important insights into neuroembryology can be deduced from craniofacial anomalies and the results obtained by surgical intervention for their correction. A closer collaboration between craniofacial surgeons and neurologists should be of mutual benefit. Most facial anomalies represent defects in specific developmental fields. The success or failure of surgical manipulations permits a more accurate understanding of just exactly where these fields exist and how they behave. When a deficient developmental field is released from normal surrounding fields subsequent facial growth can be anticipated to be more normal. By the same token, persistent patterns of relapse after surgery are a strong indication of the site of the pathology. Nowhere is this more apparent than in the treatment of routine clefts of the lip and palate. Despite advances in presurgical orthodontics and operative techniques we continue to be faced with results that deteriorate over

time, most of our patients requiring multiple secondary interventions. Such a model cannot be correct. As an anatomist, I have always been struck by the inability of descriptive embryology to answer very fundamental questions about clefts, such as why the surgical approaches to unilateral and bilateral cleft formation are so different if the mechanisms are the same, what explains the spectrum of severity of clefts, what is the relationship between cleft palate and cleft lip, why isolated palatal clefts are more likely to be associated with additional birth defects and why, if isolated genes are to blame for clefts, there is no evidence of such misexpression all over the body. The purpose of this chapter is to propose a new and clinically relevant model of cleft pathogenesis based on concepts of developmental biology and neuroembryology that are as yet little known in medicine. Much of the relevant literature is less than 20 years old. Many key discoveries have come from technologies that are still evolving. All plastic surgeons dealing with congenital anomalies are witnesses to nature’s variations. Properly interpreted, such information can produce, for the first time, a new ‘gross anatomy of the embryo’. Craniofacial surgeons and neurologists have an indispensable role to play in the discovery of this knowledge. Applications of neuromeric anatomy provide a potential embryonic ‘map’ of all craniofacial structures with important implications for diagnosis and surgery. Exclusive of the cranial base (basisphenoid and posterior) and parietal bone, the craniofacial skeleton is

*Correspondence to: Michael H. Carstens MD, FACS, Associate Professor of Plastic Surgery, Director of Craniofacial Surgery, Cardinal Glennon Children’s Hospital, St Louis University, 3635 Vista, St Louis, MO 63110, USA. E-mail: [email protected], Tel: þ1-314-577-8793, Fax: þ1-323-268-5062.

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made exclusively from neural crest. Thus the cell populations producing the ethmoid, presphenoid, premaxilla and vomer all originate in anteroposterior order from the neural folds in genetic register with the first rhombomere (r1). The rostral aspect of the second rhombomere gives rise to the premaxilla and vomer while the inferior turbinate, palatine bone, alisphenoid, maxilla and zygoma arise from the more caudal neural crest of r2. The squamous temporal, mandible, malleus and incus are r3 neural crest bones. Disturbances at a particular neuromeric level can cause individual or multiple fields to be deficient or absent. Thus, isolated cleft palate (unassociated with cleft lip) represents a deficiency state of the vomer. This occurs as a spectrum. As the vomer is progressively smaller it lifts away from the plane of the palatal shelves and the cleft extends forward towards the incisive foramen. In Treacher–Collins syndrome all r2 developmental fields of the midface are affected: the maxillary, palatine and zygomatic bones are all small. The septum, vomer and premaxilla (being r1 structures) are unaffected. Developmental fields form in a specific spatio-temporal sequence. Each one builds upon its predecessors. Making a face is similar to assembling a house with magical pieces of LegoW, each one of which will grow over time. Imagine a house made from 20 pieces (four on the floor and five stories high). All pieces are growing independently. If a cornerstone piece is removed, the 19 remaining pieces undergo a deformation and the house tilts into the deficiency site. The missing piece in cleft lip is the premaxilla. Surgical reconstruction of this field is the key to repair of the common cleft.

The nature of this pathological sequence has been identified (Carstens, 1999a, 1999b, 1999c, 2000a, 2000b, 2000c, 2002a, 2004a, 2004b). First, a deficiency state exists in the functional matrix responsible for synthesizing the piriform margin. Within this abnormal developmental field, insufficient bone volume results. This causes a stereotypical displacement pattern of the soft tissue envelopment on both sides of the cleft. If the deficiency state is significant enough, it will affect the ability of adjacent developmental fields to perform soft tissue closure of the nostril floor and lip. The resulting division further aggravates tissue displacement. Over time, the effects of deficiency, displacement and division create a distortion of the soft tissue envelope. This results in an abnormal anatomy of the septum. Ongoing growth of the osteocartilaginous nasal vault, uncoupling of normal relationships between the skeletal elements and aberrant force vectors exerted by the perioral musculature result in the characteristic ‘opening-up’ of the cleft site so elegantly described by Delaire (Delaire 1975a, 1975b, 1978; Delaire et al., 1988; Markus et al., 1992; Precious and Delaire, 1992). The above concepts of cleft formation are known as ‘four-dimensional theory.’ These can be summarized by the pneumonic of ‘the 4 D’s’, each of which corresponds to a dimension. Deficiency is axial. Displacement is coronal. Division is sagittal. Deficiency is temporal. Interestingly enough, the order of these processes follows the order of axis specification in the embryo: anteroposterior, then mediolateral and finally right–left.

16.3. The biological significance of relapse 16.2. The 4 D theory of cleft formation: the common labiomaxillary cleft The pathological anatomy of the labiomaxillary cleft is a four-dimensional problem. The principles of its surgical management must be conceptualized in the same manner. How the cleft site appears in the newborn is very different from its anatomy in utero. Indeed, the initiation of the cleft problem may occur as early in embryogenesis as gastrulation (the process by which the germs layers of the embryo are established) at 15–18 days gestation (Carlson, 1999; Gilbert, 2003). At the time of initiation of the cleft site, four pathological processes are unleashed; these processes exert their effects in a strict sequential order (Carstens, 1999a). A spectrum of presentations is thereby produced, ranging from the ‘cleft-lip nose’ with an apparently normal lip, to a full-blown, wide separation of tissue elements of the lip, primary palate and secondary palate (Carstens, 2000a).

All surgeons involved with cleft care know full well the frustration of seeing well executed repairs in infancy degenerate into a predictable sequence of secondary deformities requiring further correction. Even in the best of hands, re-operation rates may reach as high as 85% (Mulliken, 2003). What exists here is not failure of technique but an inadequate biological model of the problem in the first place. If the pathological anatomy of the cleft site hinges on a deficiency state in a specific developmental field, and if the surgical correction of the cleft does not include reconstitution of that defective field such that it will grow normally over time and will cease to perturb the growth of its neighboring fields, then all forms of cleft surgery are condemned over time to varying degrees of relapse. However, in pediatric craniofacial surgery, all patterns of relapse unequivocally indicate the original pathology. Relapse is nothing more than the manifestation over time of a deficient developmental field.

NEURAL TUBE PROGRAMMING AND CRANIOFACIAL CLEFTS, PART I Knowledge of the embryology of the face conceived in terms of specific fields (zones of soft tissue and the bone that they produce) will enable surgeons to conceive of new surgical approaches based not on geometry but upon developmental anatomy. Cut-and-paste tissue manipulation will be supplanted by biological principles of field reconstitution and reassignment.

16.4. Beyond descriptive embryology: developmental fields and the functional matrix Surprisingly, the drawings of cleft lip pathogenesis depicted even in recent texts reflect an understanding of facial embryogenesis that is nearly a century and a half out of date. Descriptive embryology as a science began from pioneering observations by Wilhelm His in the 1870s using light microscopy and histological staining (His, 1901, 1902). The approach was morphological rather than cellular – to the end of his career His vigorously opposed the idea that genetic information could be contained in the nucleus. Terms introduced by His such as ‘lateral nasal process’ can be found in all textbooks, but just what the constituent parts of a ‘process’ are and where in the embryo they come from is less clear. Surgeons know that lefts, for example, occur in a comprehensible spectrum of presentations but, unfortunately, concepts such as ‘failure of fusion’ or ‘failure of mesoderm penetration’ do not explain the varying degrees of pathology. For most of us, embryology seems a mere jumble of terms with no clinical relevance. However, without a detailed understanding of the developmental anatomy of the face based on modern developmental biology, genetics, comparative anatomy and neuroembryology, pediatric plastic surgery is a collection of techniques without any scientific basis. Based upon clinical observations of secondary cleft patterns, I arrived at the following hypotheses: 1) unidentified developmental fields might constitute the ‘building blocks’ of the face; 2) a mesenchymal deficiency state in such a field would be characterized by an inadequate osteosynthetic capability; 3) an osseous deficiency state exists in the inferolateral piriform fossa/lateral nasal wall of cleft patients; 4) such a functional matrix deficiency state might account for the relapse pattern observed in cleft patients after primary repair. To test these ideas it seemed logical as a first approximation to study the relative contributions of the internal carotid artery (ICA) and external carotid artery (ECA) circulations to the skin and epithelium of the nasal fossa. Contrast injections were performed into isolated internal carotid arteries in a series of aborted fetuses (Carstens, 2002b). I found, to my surprise, that the upper border of the inferior turbinate combined with the skin/

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mucosa junction of the inferolateral piriform fossa just anterior to the inferior turbinate constituted a potential field interface zone. At this site three distinct biological systems (vascularization, innervation and genetic programming) functioned in precisely the same manner: 1) The internal carotid supplied the mucosa (but not skin) of the lateral nasal wall but only as far as the upper border of the inferior turbinate. The mucosa beneath the turbinate and the skin margin along the infracartilaginous nostril were unperfused. 2) The sensory innervation followed exactly the same distribution pattern. The epithelium supplied by the ICA corresponded to sensory supply from the first branch of the trigeminal nerve, while that supplied by the ECA was innervated by V2. 3) The inferolateral piriform fossa also represents an interface zone between three entirely different developmental zones of the embryonic neural crest. Neural crest cells refer to those cells that arise during embryogenesis from a border zone between the more lateral zone ectoderm responsible for forming skin and the more axial zone of ectoderm responsible for forming the brain and spinal cord. Neural crest cells migrate widely and form many structures, such as dermis, bone and cartilage, usually associated with mesoderm. These cells also form components of the nervous system such as Schwann cells and autonomic ganglia. For this reason neural crest is often referred to as an ectomesenchyme and the great extent of its derivatives has often resulted in it being called ‘the fourth germ layer’. Neural crest cells do not make their appearance until well after gastrulation is complete. The three traditional germ layers are all recognized at the time of gastrulation. The behavior of neural crest cells in terms of their migration pathways and derivatives depends largely on what part of the neural folds they originate from. The names of these neural crest zones correspond to the original three parts of the developing central nervous system: the prosencephalon (forebrain), mesencephalon (midbrain) and rhomboencephalon (hindbrain). The clinical relevance of this model can be seen in the developmental anatomy of the piriform fossa. The lateral wall of the nasal cavity has an upper zone populated by prosencephalic neural crest (PNC), innervated by V1 and irrigated by the ICA. The lower zone of the lateral nasal wall is populated by rhomboencephalic neural crest (RNC), innervated by V2 and irrigated by the ECA. The ‘breakpoint’ between these two zones is the inferior turbinate. This outer lamina of the piriform margin belongs to the ascending process of the maxilla. The medial wall of the nasal cavity has an upper zone populated by PNC, innervated by V1 and irrigated by the ICA. This zone consists of the perpendicular plate of the ethmoid and the septum. The lower zone of the medial nasal wall is populated by mesencephalic neural

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crest (MNC), innervated by V2 and irrigated by the ECA. This zone contains the vomer and the premaxilla. Details of the experimental data and their many implications were published in Carstens (2002a). The purpose was to describe a preliminary but clinically relevant ‘map’ of developmental fields as they applied to the facial midline. Details of the medial nasal wall fit a model quite analogous to that of the lateral nasal wall. In this model the septum and perpendicular plate of the ethmoid form a sharp developmental boundary with the vomer and premaxilla characterized by blood supply (ICA vs ECA), innervation (V1 vs V2) and neural crest origin (PNC vs MNC/RNC). This model permits us to assemble an accurate picture of how the premaxilla and maxilla develop and interface with one another. Isolated deficits in components of this system explain the pathological anatomy of all forms of cleft. The premaxillary developmental field has three subdivisions: central incisor, lateral incisor, and a vertical ascending process. Formation of an intact alveolar arch involves fusion between the premaxilla and the maxilla. This brings the two ascending processes (premaxilla and maxilla) into apposition and fusion. The piriform margin is therefore bicortical and serves as a buttress for reconstruction. Loss of the medial (premaxillary) ascending process leads to a scooped-out piriform fossa and is the cause of the cleft nasal deformity (Tessier type 2 cleft). Loss of the lateral (maxillary) ascending process creates a cleft extending to the medial inferior orbital rim but leaving the piriform fossa intact (Tessier type 4 cleft). More extensive neural crest deficiency in the premaxillary field leads to loss of alveolar bone in a labiolingual gradient. This creates a cleft of the primary palate and, frequently, loss of the lateral incisor.

16.5. Neuromeres: the clinical significance of the neuromeric map Certain cells born in the neural plates at the exact boundary between neural ectoderm and non-neural ectoderms are termed neural crest and have a very important role to play in development of the head and neck. Neural crest can be ‘mapped’ to very precise zones of origin. Each such zone corresponds to a developmental unit of the neural plate. These segmented units, called neuromeres, are distributed in a transverse fashion along the entire neural axis of the embryo (Berquist, 1952; Vaage, 1969; Graham et al., 1989; Landmesser, 1989; Butler and Hood, 1997). The nervous system is thus divided into transverse developmental units just like the body of an earthworm. Each such segment has a genetic definition. Certain genes or combinations of genes express

products only in a particular zone. The anatomical extent of these protein products constitutes the domain of the neuromere. Each neuromere has a certain neuroanatomical content characterized by nuclei and ascending or descending tracts. The neural crest sitting just outside the neural tube in the domain of a given neuromere will express the same defining set of proteins as those cells within the neural tube. Furthermore, neural crest cells from a given neuromeric level will supply certain zones of ectoderm and mesoderm. This model allows us to see the nervous system as the master integrative agent of development. In summary, the highlights of the neuromeric model are as follows. The CNS of all vertebrates is divided into three classes of neuromeres (Puelles and Rubenstein, 1993, 2003; Rubenstein and Puelles, 1994; Rubenstein et al., 1994). The forebrain is formed from six prosomeres (previously thought to be four in number). From caudal to cranial these are numbered p1 to p6. They are subdivided into two tiers, dorsal (alar) and ventral (basal). The telencephalon forms from the alar tiers of p6 and p5. The basal tier of p6 has much to do with the olfactory system while basal p5 is associated with the visual apparatus. Puelles and Rubenstein propose that the midbrain is constructed from two mesomeres, m1 and m2. These contain, respectively, the superior and inferior colliculi. (An anatomical boundary between the two has not been demonstrated as it is in the borders between the rhombomeres.) In this model, the hindbrain is described as being made up from 12 well defined rhombomeres, r0–r11. An alternative viewpoint held by Sarnat (personal communication, 2003) considers r0 to be the principal neuromere of the midbrain and r1 the neuromere of the isthmic region (metencephalon, from which develops the pons and cerebellar cortex. Neural crest from r0 and r1 (the two mesencephalic neuromeres) is involved in the construction of the orbit. The remainder of the hindbrain (myelencephalon) is made from rhombomeres r2–r11. These form the medulla. Neural crest originating from neural folds associated with rhombomeres r2–r11 supplies the pharyngeal arch system. When the neural crest cells migrate they swarm over the surface of the mesoderm lying just outside the neural tube. This mesoderm is called paraxial mesoderm (PAM) and is segmented in direct register with the neuromeric system. Each segment of PAM is called a somitomere (Sm) and is shaped like a ball. The first seven somitomeres (corresponding to r1–r7) are incompletely separated. All somitomeres from Sm8 caudally undergo anatomical rearrangement into somites. Thus, Sm8– Sm11 form the four occipital somites and Sm12 becomes the first cervical somite. Developmental biologists refer to mesoderm from Sm1–Sm7 as cephalic mesoderm.

NEURAL TUBE PROGRAMMING AND CRANIOFACIAL CLEFTS, PART I Each mesenchyme of pharyngeal arch consists of mesoderm from two units of PAM (somitomeres or somites) plus the neural crest. The original number of pharyngeal arches in primitive aquatic vertebrates was seven. With the tetrapod transition to a land-based existence this number was reduced to five. Thus, the mesenchyme of each pharyngeal arch (PA) is composed of PAM and neural crest associated with a pair of rhombomeres. Neural crest migration is a physical process in which the cells move along 1) pathways of least resistance (anatomic cleavage planes) or 2) molecular ‘guidewires’ such as vimentin. Neural crest cells swarm over the surface of PAM somitomeres, taking three routes: 1) they can move laterally outward in the plane between the non-neural ectoderm/endoderm and the somitomeres; 2) they may remain interposed between the neural tube and the somitomeres; and 3) they may migrate ventral to the neural tube and then travel caudally, stopping along their way to form the sympathetic chain. Fate mapping experiments show that the r1 neuromere is subdivided into a rostral zone that forms the cerebellar cortex and a caudal zone that gives rise to the deep cerebellar nuclei. Neural folds corresponding to the caudal region of r1 neural plate give rise to neural crest with a unique fate that has direct relevance to the formation of cleft lip and cleft palate. Rapid growth of the head causes the embryo to fold. This cephalic flexure forces Sm1 into a new spatial relationship with respect to Sm2. Sm1 now lies medial to and cranial to Sm2. This repositioning means that Sm1 and r1 neural crest are now in physical contact with the first pharyngeal arch. For this reason, cephalic mesoderm originating from Sm1 forms structures not directly associated with PA1 (e.g. the penetrating vasculature assigned to V1-innervated dura and the basisphenoid bone) while r1 neural crest is divided between frontoorbital structures and pharyngeal arch structures. The cephalic fate of r1 neural crest includes formation of extraocular muscle fascia, all dura innervated by V1, and the presphenoid bone. All these structures are supplied by the ICA and innervated by V1. In the first pharyngeal arch, r2 neural crest from the most rostral zone of can combine gives rise to the vomer and the premaxilla. These are supplied by the ECA and innervated by V2. This is important in simplifying discussion of the contributions made by mesencephalic neural crest and paraxial mesoderm belonging to the first somitomere associated with r1. An understanding of the developmental anatomy of the extraocular muscles and their motor nuclei hinges on a proper understanding of this terminology.

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The rhombomeres were discovered first, in the late 19th century, but their properties were not understood until the mid 1980s. Furthermore, the exact number of rhombomeres was uncertain and their anatomical role at the most caudal medulla was unclear. In 2000 Puelles published work in the avian model demonstrating the existence of ‘pseudorhombomeres.’ Previously it was thought that the rhombomeric series terminated at r8, which included the entire spinal cord. Puelles’s work suggested that the final number of rhombomeres was 12 (Wingate and Lumsden, 1996; Cambronero and Puelles 2000). The existence of neuromeres in the more rostral CNS required advancements in gene mapping. These were not reported until 1993. Over the ensuing decade further investigation of the neuromeric system has proceeded at a frenetic pace at neuroscience laboratories around the world. Here we shall be using the Puelles and Rubenstein model with Sarnat’s modification. At this juncture we must take note of a caveat that applies to concepts about prosomeres held widely within the scientific community. Many descriptions of brain anatomy extent in the literature assign four neuromeres to the forebrain, two for the telencephalon and two for the diencephalon. These are known respectively as T2/T1 and D2/D1. This nomenclature is elegantly presented in the brain development section of texts such as those of O’Rahilly and Muller (O’Rahilly and Gardner E, 1974; Muller and O’Rahilly, 1997; O’Rahilly and Muller, 2001). Based on sophisticated gene mapping techniques previously unavailable, the Rubenstein–Puelles model has by no means been universally incorporated into the thinking of neurologists, neuropathologists, and researchers (Figador and Stern, 1993; Larsen, 2001). However, from the standpoint of craniofacial surgery, the Rubenstein–Puelles model constitutes an extremely sensitive instrument with which to analyze patterns of deformity. The clinical significance of the neuromeric model is that it enables us to map out the anatomical site of origin for all zones of ectoderm and mesoderm supplied by a given zone of the nervous system. The role of neural crest populations in those zones, specifically what structures they make, can also be understood on the basis of their neuromere of origin. The premaxilla, for example, could develop from a precursor cell population in the ‘premaxillary zone’ of MNC along the neural fold corresponding to the second rhombomere. A deficiency state in the population (inadequate cell number, defective migration, abnormal post-migratory rates of mitosis or cell death) will lead to a small or absent premaxilla. Furthermore if the premaxillary MNC has several subsets, aligned in craniocaudal order along the neural fold (ascending process, lateral

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incisor and central incisor) then the spectrum of deficiency states seen in the premaxilla of cleft patients can be understood as progressively greater degrees of disturbance in the premaxillary MNC precursor population.

16.6. Segments, somitomeres and somites Segmentation as a concept is highly intuitive. Repetitive functional units are observed in the centipede, in the multiple ribs of the snake and in the dermatomes of the human thorax. All vertebrates possess the fundamental elements of a vertebral body, muscle units attached thereto, sensorimotor nerves arising at that same level to innervate those muscles and well defined geographical zones of skin. As the future spinal cord all vertebrate embryos have primitive organizational blocks of mesoderm called somites (Hall, 1988; Ordahl, 1993; Fan et al., 1994; Tam and Trainor, 1994; Brand-Saberi et al., 1995; Christ and Ordahl, 1995; Hall and Miyake, 1995; Pourquie et al., 1996; Tam and Behringer, 1997; Dietrich et al., 1998; Kato and Aoyama, 1998; Tajbakhsh and Spurle, 1998; Nowicki and Burke, 1999; Venters et al., 1999; Bonner-Fraser, 2000; Burke, 2000; Pourquie, 2000; Tam et al., 2000). Each somite is preceded by an earlier, incompletely segmented structure, called a somitomere (see below). These flank the neural tube from the cranial base to the tail. Each vertebra at a given neuromeric level is the combination of the caudal half-somite from the neuromeric level above immediately rostral to it and the cranial half-somite at that same neuromeric level. Human embryos possess 42–44 pairs of somites: four occipital somites contribute to the posterior brain case. These are followed by eight cervical, 12 thoracic, five lumbar, five sacral and eight to ten coccygeal somites. The rostral first cervical somite contributes to the foramen magnum and its caudal half to the atlas. For this reason there are eight cervical nerves but only seven visible vertebrae. Given the highly regular organization of the spinal cord it seemed logical to anatomists of the 19th century that a similar pattern might exist in the CNS as well. Darwin’s concepts of evolution were bolstered by tremendous progress in paleontology and comparative anatomy. As the anatomical logic of vertebrate structure became defined it was natural for scientists to look for commonalities in development as well. Neuroanatomists had noted the presence of small bulges on the surface of the embryo at the hindbrain region; these seemed to correlate in a regular way with cranial nerves. They also appeared to relate in some manner to the occipital somites and, rostrally, to the pharyngeal arches. These developmental zones of the rhomboencephalon were termed rhombomeres but their biological rationale was

uncertain. Furthermore, the physical presence of neuromeres appeared to die out at higher levels of the neuraxis. Elaborate attempts were made by comparative anatomists to understand the organization of the head based on somites. This culminated in Goodrich’s work (1930) based on fishes, emphasizing dorsoventral relationships between mesoderm and cranial nerves. (59) This comparative approach was applied to the bones of the skull by Sir Gavin de Beer (1937) and later by Jarvik (1980). Given the limitations of the data, the anatomy of the head and neck remained unexplained. As so often is the case in science, new technologies would be required before dramatic advances in knowledge could be made, and a new model, based on neuroembryology and genetics, would emerge. The advent of scanning electron microscopy opened a new window on the morphology of development. Exhaustive work by Hinrichsen (1985) described the external development of the face. Within the neural tube, scanning electron microscopy proved the existence of rhombomeres as segmental diverticula of the lateral wall. The boundaries of these neuromeres and their specific anatomical content were confirmed using contrast injections and immunofluorescence. At the same time Meier and Jacobson observed a somite-like organizational pattern of paraxial mesoderm (that mesoderm closest to the neural tube) in a wide variety of vertebrates (Meier, 1980, 1981a, 1982a, 1982b, 1984; Meier and Tam, 1982; Jacobson, 1986, 1993). In mammalian embryos, seven of these incompletely separated masses termed somitomeres were noted. Beginning with r1, a one-to-one correspondence between somitomeres and rhombomeres was observed. Note that r0 (mesencephalic neuromere) does not participate in the ‘match-up’. Neural crest from r0 may well interact with prechordal plate mesoderm instead. At the level of Sm8 a completely separate somite surrounded by an epithelial coat was noted. This is termed ‘the first occipital somite’: four such somites were documented rostral to the first cervical somite (Hunter, 1935; Huang et al., 1997, 2000). Recent cell labeling studies have conclusively demonstrated that somitogenesis begins at the eighth somitomere, but in a subtle manner. A typical somite is transformed from a cuboidal mass of mesoderm into bone, muscle and dermis. In development antecedents of these tissues within the somite occur as transient structures: the sclerotome, myotome and dermatome. The sclerotome produces the chondral bone of the cranial base (basioccipital, exoccipital and supraoccipital bones) and the vertebrae. The dorsal dermatome and dorsal myotome produce respectively the dermis and paraspinous muscles supplied by the posterior (dorsal)

NEURAL TUBE PROGRAMMING AND CRANIOFACIAL CLEFTS, PART I ramus of the spinal nerve. These are known as epaxial structures. The dermis of the remainder of the body wall and the extremities and their corresponding muscles are all hypaxial structures supplied by the anterior (ventral) ramus of the spinal nerve. These mesodermal structures arise from the ventral dermatome and the ventral myotome respectively. All somites arise from the transformation of a previously existing somitomere but the first seven somitomeres never become somites. A somitomere becomes a somite when it displays complete separation from its neighbors, when its outer layer of cells becomes an epithelium and when it displays the three primary subunits of sclerotome, myotome and dermatome. The first such transformation (that of Sm8 to the first occipital somite) is externally difficult to recognize. The rostral half of Sm8 is incompletely separated from Sm7. It behaves just like a somitomere. In contrast, the caudal half of Sm8 undergoes a complete somitic transformation. The back end of the resulting first somite is thus completely separate from the rostral aspect of the second somite (Sm9). In addition, the anatomical organization of occipital somites differs from that of all other somites. Although they have sclerotomes and ventral myotomes, they possess neither dorsal myotomes nor dermatomes. A dorsal myotome appears for the first time in the first cervical somite. This produces the muscles of the suboccipital triangle. A true dermatome does not appear until the second cervical somite. For this reason, there is no skin innervated by C1. Although C1 is well described in amphibians, in mammals it appears only transiently, providing neither sensory nor motor innervation in the mature state (Sarnat, personal communication). C1 is well defined in amphibians. The dorsal branch of nerve C2 supplies the skin over the retroauricular skin while the ventral branch of C2 supplies the mastoid and the rostral anterior triangle of the neck as part of the cervical plexus. For these reasons documentation of occipital somite anatomy requires a molecular, rather than a strictly morphological, approach. When these structures are considered in terms of their associated rhombomeres the anatomical pattern becomes quite clear. Somitomeres and occipital somites follow similar trends in muscle formation (Bock, 1974; Noden, 1979, 1983a, 1983b, 1986, 1989, 1991a). Pharyngeal arches are hypaxial structures, the muscles of which are organized into two layers, deep and superficial. In this model, internally placed myoblasts of Sm6 and Sm7 would produce the uvulus, levator veli palatini and palatopharyngeus while externally placed myoblasts form stylopharyngeus. All four occipital somites make the muscles of the tongue from the internal plane of their

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myotomes. Sm8 (the first occipital somite) would be expected to produce a tongue muscle associated with the Sm7 palate; this is palatoglossus. Labeling studies show that the sternocleidomastoid and trapezius muscles originate from the external plane of the occipital somites. These two muscles logically represent the superficial plane of the first occipital myotome. Both these muscles originate from the osseous product of the Sm7, the mastoid process. Neuromeric theory allows for an accurate assignment of muscle origins according to their somitomere/somite of origin (based on the location of the motor nerve within the neuraxis). This approach to origin and insertion is rational and consistent, although in many cases it proves to be the reverse of that described in traditional textbooks of anatomy. Somitomeres provided a revolutionary new construct for the embryogenesis of the head. For the first time, an organization of mesoderm was observed that correlated with the previously described model of pharyngeal arches. Somitomeres form in register with the rhombomeres of the hindbrain. The advent of genetic mapping to the neural plate provided a means of understanding the anatomical relationships existing among the neuromeres of the medulla, the neural crest cells corresponding to each neuromeric level and the zones of outlying mesoderm innervated by those neuromeres and populated by those neural crest populations. The mesoderm of the human head was conceived as having 11 somitomeres divided into two distinct zones. The first seven belong to what was termed head mesoderm. The next four somitomeres constitute the segmental plate. Segmental plate somitomeres go on to condense into somites while head somitomeres do not undergo a condensation. The transitional nature of the Sm8 is quite apparent. Just at the time when Sm19 makes its appearance the first somite transformation takes place at Sm8. Thereafter, the appearance of each new somitomere is accompanied by the formation of a new somite. Strict timing is associated with this process, pointing to the existence of a so-called ‘cellular clock’ that regulates somitogenesis. It is as if there is a difference between the first two neuromeres r0 and r1 associated with the mesencephalon, which produce at gastrulation a somitomere (Sm1) that is unassociated with the pharyngeal arches, and the more caudal 10 neuromeres of the rhomboencephalon. The latter ten developmental levels produce, at the time of gastrulation, the ten somitomeres (Sm2–Sm11) that pair up to form the five pharyngeal arches. Note that the numbering system used in this chapter for the transformation sequence of somitomeres to somites may differ from that used by textbooks of descriptive embryology. First, evidence that there are four occipital somites in mammals is at odds with the

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avian five-somite model. Second, only eight rhombomeres are described in most papers, the eighth being represented as quite large. Puelles described the eighth rhombomere as broken up into four individual segments matching the cranial nerve anatomy and foramina of the skull; these he called ‘pseudorhombomeres’. Because his description fits the rest of somitogenesis so well, I have dropped the term ‘pseudo’ in the interests of clarity. Thus diagrams of somitomere–somite transformation may depict the appearance of the first somite at Sm8 matching that of Sm12 because a four-occipital-somite model is being used. Comparative anatomy has much to contribute to our understanding of the significance of the r7/r8 transition zone. Fish brains are small. The position of the roots of cranial nerve XI and the hypoglossal lie outside the piscine skull. These nerves can reach their targets without traversing the skull. The expansion of the vertebrate skull associated with tetrapods in the posterior fossa incorporated these nerves into the jugular foramen and four occipital foramina (subsequently reorganized in mammals into a single occipital foramen). This points to the transformation of somites 1–4 from a truncal to a cranial fate.

16.7. How segmentation of the mesoderm matches that of the neural tube Scanning EM studies in chicks by Meier in 1980 demonstrated that the segmentation of the somitomeres was also in register with segmentation of the intermediate mesoderm and lateral plate mesoderm (the somatic lamina) (Meier, 1981b, 1982a). All forms of mesoderm come into being during gastrulation. The latter term refers to the process by which populations from a single layer of cells, the epiblast, rearrange themselves to form a three-layer ‘sandwich’ consisting of ectoderm, mesoderm and endoderm. A pear-shaped embryonic disc results; in the center of the disc an axially oriented neural plate develops. The ectoderm then becomes divided into two zones, neural ectoderm (the future nervous system) and surface ectoderm, the future epidermis of the body. The edges of the flattened neural plate roll up into cigar-like neural folds. These will ultimately contact each other in the dorsal midline and seal up, forming the neural tube. This process is called neurulation (Schoenwolf, 1991, 1997). Neural crest cells almost constitute a fourth germ layer of the embryo. These cells are found at the interface between the neural ectoderm and the surface ectoderm, i.e. atop the neural fold. In mammals these neural cells ‘migrate’ well before neural tube closure occurs but after completion of gastrulation. Note that the concept of cellular migration may actually be a misnomer. An

alternative viewpoint, advocated by Anderson, is that neural crest ‘movements’ represent local forms of cell proliferation that are physically directed by the microanatomy of their environment. In this model the distinct manner in which neural crest populations reach their targets is a passive consequence of environmental passageways and roadblocks (see below). At the time of formation of the trilaminar embryonic disc (and before neurulation) mesoderm spreads out laterally to form various zones (Larsen, 1997). Those mesodermal cells closest to the neural tube and notochord are called paraxial mesoderm (PAM), which starts at the rostral tip of the notochord and is organized into somitomeres (and somites) all the way down to the tail. Mesoderm migrating further laterally stays flat; it is known as the lateral plate mesoderm (LPM). LPM has a natural separation plane (the embryo will eventually take advantage of this to form the intraembryonic coelom). The outer (dorsal) lamina of the LPM is called the somatic lateral plate mesoderm (LPMs). LPMs is responsible for forming all the nonaxial bones of the body. From the clavicle on down, every bone (except the vertebrae) develops from LPMs. LPMs is overtly segmented in register with PAM. The inner (ventral) lamina of the LPM is called the visceral lateral plate mesoderm (LPMv). LPMv forms the mesoderm of the respiratory system and gut. It also forms the heart and blood vessels. After formation of PAM and LPM an intercalated zone of intermediate mesoderm (IM) is formed; this runs down the length of the embryo as a segmented cord. IM is responsible for the production of the genitourinary system. This brings in another important revolution in biological thinking. The familiar germ layer system is probably incorrect. It is certainly useful in terms of describing the organization of the vertebrate body via gastrulation and hence will probably persist in textbooks. But it is now known that a germ layer can produce cells associated with a different germ layer. For example, the hypoblast (the temporary second layer derived from the epiblast) lines the yolk sac, which in turn produces the extraembryonic mesoderm (EEM). The intraembryonic mesoderm (IEM) produced later in time by gastrulation is in physical continuity with the EEM but does not produce it. The endodermal lung bud emanating from the esophagus produces its own mesoderm. Thus the mesenchyme of the lung does not result from the endoderm interacting with another (unknown) source of mesoderm but is actually produced by the bud itself. Neural crest, of strictly ectodermal lineage, has the ability to produce structures such as fascia and bone that are normally considered outside the head and neck as strictly mesodermal derivatives.

NEURAL TUBE PROGRAMMING AND CRANIOFACIAL CLEFTS, PART I Embryonic tissues seem to be organized into morphologic units called pharyngeal arches. With these definitions in mind we can now explore how the somitomeric system fits this model. We shall then discuss the discovery of homeobox (Hox) genes provides the genetic basis for hindbrain segmentation. Thereafter we shall complete the neuromeric map of the central nervous system by discussing how non-Hox genes define a more complex, but still logical, system of neuromeres rostral to the hindbrain. A population of neural crest is associated with each rhombomere, located at its respective sector along the neural fold. In like manner each rhombomere is matched up with a corresponding somitomere. The exception to this is rh0. Neural crest from this neuromere interacts with prechordal plate mesoderm (PCM). In this way the r0 neural crest provides the inferomedial sclera and the fascia for the inferior oblique, inferior rectus and medial rectus while the PCM provides the myoblasts. In humans the first 11 somitomeres are organized into six head segments (De Robertis et al., 1990; Christ et al., 1998). The first segment consists solely of Sm1. This structure is much larger than the subsequent somitomeres; it is aligned with r1. PAM from Sm1 is considered to not interact with the pharyngeal arch system. It contributes to the orbit by forming the basipostsphenoid bone and the superolateral extraocular muscles. A new model (see below) posits SM1 to interact with neural crest from r0 and r1 to produce a new premandibular arch. The second head segment consists of Sm2 and Sm3, which together make up the first pharyngeal arch. The third head segment consists of Sm4 and Sm5. Together these make up the second pharyngeal arch. The fourth head segment consists of Sm6 and Sm7, which together make up the third pharyngeal arch. The fifth head segment consists of Sm8 and Sm9 (occipital somites 1 and 2). These form the fourth pharyngeal arch. The sixth head segment contains Sm10 and Sm11 (occipital somites 3 and 4). These form the fifth pharyngeal arch. The arch numbering system for the pharyngeal arches here differs from that of most texts. Each arch derives from two neuromeres. The ten rhombomeres of tetrapods (r2–r11) produce five pharyngeal arches. This chapter proposes the following principle: neuromeres are not ‘lost’ in evolution; their anatomic content is merely altered. Every line of evidence regarding the homeobox genes encoded in the hindbrain suggests that this system is strongly conserved throughout evolution. Thus, a terminology based on a mysterious ‘loss’ of the fifth arch while the sixth is preserved is not logical. The neuromeric approach simplifies discussion of the comparative anatomy of pharyngeal arches (Kardong, 2002).

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Note that the first two pharyngeal arches are larger and exist as a dimer (pair). The mesenchyme (neural crest þ paraxial mesoderm) of the second arch migrates beneath the ectoderm and endoderm of the first arch and engulfs the first arch mesenchyme like two slices of bread surround a slice of cheese. In this process the ectoderm of PA2 is reduced to a small sector of skin covering the anterior external auditory canal. The endoderm of PA2 within the oral cavity is found only over the dorsum of the tongue. The last three pharyngeal arches are considerably smaller. The reason that they (PA3–5) are reduced in size is that some of their somitomeric mass must be shared to form somites whereas the paraxial mesoderm of somitomeres 2–6 is completely dedicated to the formation of arch structures. The anatomy of a somitomere consists of a whorl of PAM cells surrounding a central lumen. Though lacking the defined structures of a somite (dermatome, myotome and sclerotome) the mesoderm of a somitomere has an intrinsic spatial orientation. Distal somitomeric PAM probably represents cells ingressing early at the neuromeric level during gastrulation while PAM nearer the midline represents later-arriving cells. This spatial organization means that different PAM derivatives may come from distinct ‘compartments’ of the somitomere. Each compartment may be characterized by the expression of different genes or by varying degrees of expression of the same gene. These expression patterns may determine if the derivative is to be muscle or bone. Alternatively, if the mesenchyme streaming from a somitomere differentiates in response to signals found in its target epithelium the physical disposition of cells within the somitomere may determine what they will become. In Sm1 for example, extraocular muscles may come from the distal margin of the somitomere while the mesenchyme of the basipostsphenoid originates from the internal aspect of the somitomere. Recall that most bone from mesenchyme originating from r0–r7 is not from PAM but from neural crest. Neural crest cells from each rhombomeric zone migrate downward from the neural fold and spill over the outer surface of their corresponding somitomere much like caramel poured over an apple. Subsequent folding of the embryo places pairs of these neural crest/somitomere units into special configurations known as pharyngeal arches. In each arch the more cranial member of the somitomere pair is located in the proximal/dorsal half of the arch while the more caudal member is located in the distal/ventral half of the arch. An aortic arch artery supplies each pharyngeal arch transiently. The arterial axis enters each arch ventrally and exits dorsally to join with the ipsilateral dorsal aorta. Thus the ventral aspect of each

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pharyngeal arch is metabolically more ‘mature’. For this reason, products of the distal arch such as bones and muscles appear before their proximal counterparts. The mandible (r3) is formed prior to the maxilla (r2). The facial muscles of the lower face (r5) appear earlier in time than those supplying the upper face (r4). Although most craniofacial bones are formed from neural crest, PAM from the somitomeres has important derivatives as well. Sm1 does not participate in pharyngeal arch formation. Neural crest from r1 forms the presphenoid and orbitosphenoid (lesser wing) while corresponding PAM from Sm1 produces the basisphenoid. Sm1 also provides myoblasts for the superolateral extraocular muscles (superior rectus, levator palpebrae superioris). In mammals PAM from Sm2 and Sm3 forms the parietal bone. Sm6 PAM produces the petrous temporal bone while the PAM of the nonsomitic part of Sm7 produces the mastoid process. Despite their lack of formal myotomes, the first six somitomeres provide the myoblasts for many important craniofacial muscles. These include the extraocular muscles, the muscles of mastication, the infrahyoid muscles, the facial muscles and selected muscles of the palate. Beginning with Sm7 all somitomeres change their configuration into fully separate somites having characteristic dermatomes, myotomes and sclerotomes. The myotome of the first occipital somite produces two large external and ventral muscles, the sternocleidomastoid and the trapezius. The tongue musculature comes from the exclusively internal and ventral myotomes of the first to fourth occipital somites. The fascial envelopes of all muscles of the head and neck are derived from neural crest corresponding the neuromeric level of the myoblasts.

16.8. Molecular basis of segmentation: introduction to homeobox genes Vertebrates have a segmental body plan. Molecular biology has, since 1990, literally revolutionized our comprehension of the mechanisms by which segment formation and segment specialization are accomplished. The genes that control this development have an incredible degree of phylogenetic conservatism. Nucleotide sequencing studies show that genes found in primitive organisms such as Drosophila and worms exist in mammals as well and that these genes share common functions. A surprisingly limited number of genes are required. A given gene may have multiple roles depending upon the period of development in which it is expressed and the organ in which it is expressed. The molecular products of these genes can be broken down into two main types: 1) intracellular

transcription factors and 2) extracellular signaling molecules. Transcription factors stay within the cell. They can bind to genes to initiate patterns of expression at key steps in development (Lobe, 1992). One class of transcription factor, the basic helix–loop–helix protein, has a short sequence of amino acids in which two alpha helices are separated by a ‘loop’ of amino acids. Immediately next door to this sequence is a basic region that binds to DNA. The helix–loop–helix causes dimerization. This is typical in muscle development. Another class having an unusual geometry is the zinc finger protein. Symmetrically spaced units of cystine and histidine along the polypeptide chain bind to zinc ions via four ligands. When this happens the four residues are drawn together and the polypeptide chain puckers up like a finger. This finger can subsequently insinuate itself into specific binding sites of DNA; activation of DNA sequences results. In understanding embryonic segmentation and, ultimately, formation of the head the most important class of transcription factor is the homeodomain proteins (Graham et al., 1989; Keynes and Lumsden, 1990; Hunt and Krumlauf, 1991, 1992; McGinnis and Krumlauf, 1992; Scott, 1992; Krumlauf, 1993; Mavilio, 1993; Duboule, 1994; Lumsden and Krumlauf, 1996; Stein et al., 1996; Puelles and Rubenstein, 2002). These all have a helix–loop–helix configuration consisting of the same 61 amino acids. The DNA coding for this region, the homeodomain, is a unique sequence of 183 nucleotides known as the homeobox; every single gene producing this type of protein has the same sequence. Because of this molecular anatomy these genes are called homeobox genes. In scientific terminology genes are written in italics, whereas the protein product is not. Thus the Sonic hedgehog protein, Shh, is produced by the gene Sonic hedgehog (shh for short). When the gene is human, the abbreviation is capitalized, i.e. SHH. As originally described in Drosophila, eight homeobox-containing genes exist on a single chromosome. These are divided into two sections, the anterior antennapedia complex and the posterior bithorax complex. Mammals possess 38 Hox genes analogous to those of the fruit fly. These are located on four different chromosomes and are arranged into 13 paralogous groups. What is amazing is that the genes are distributed anatomically along the chromosome. Hox genes for anterior mammalian segments are located at the 30 end while more caudal segments are at the 50 end. Because the genes are activated and expressed in the 30 to 50 direction, segment formation in all organisms is a craniocaudal developmental sequence.

NEURAL TUBE PROGRAMMING AND CRANIOFACIAL CLEFTS, PART I This order has major experimental and evolutionary significance. Hox genes create characteristic morphology in each segment. Hox specification of neuromeric levels c7 and t1 creates the 12th and 13th somites; the seventh cervical vertebra results. At levels c8 and t1 a different Hox ‘barcode’ results in a morphologically different structure, the first thoracic vertebra. When mutations in Hox genes occur morphological variations are seen in the segmental structures that would normally have been expressed. A posterior-to-anterior transformation occurs when the contents of a given segment resemble those of the next most anterior structure. This is called a loss of function mutation. An anterior-to-posterior transformation occurs when the contents of a given segment resemble those of the next most posterior structure. This is called a gain of function mutation (Kessel et al., 1990; Kessel and Gruss, 1991). Retinoic acid (vitamin A) is a potent posteriorizing agent (Morris-Kay, 1992). Exposure of mouse embryos to retinoic acid at a specific time in development causes an extra cervical vertebra to appear beneath the skull base. This structure, known as the proatlas, articulates with the skull with a single peg (Jenkins, 1969; Kemp, 1969). The two condyles disappear. The cervical system, now consisting of eight vertebrae, is also different. The morphology of the former atlas and axis reverts to that of standard murine cervical vertebrae. By the rules of vertebral formation, as the first of eight cervical vertebrae, the proatlas must be produced from the posterior half of the fourth occipital somite and the anterior half of the first cervical somite. The respective neuromeric levels are r11 and c1. Thus the evolution of the foramen magnum and the double condylar occipito-atlantal joint was quite possibly due to a ‘frameshift’ mutation of the Hox code causing a shared ‘loss of function’ between the fifth and sixth cervical somite. The paraxial mesoderm of these somites was ‘expropriated’ from the proatlas to: 1) expand the posterior braincase, 2) enable a double condyle system formerly between the proatlas and the second cervical vertebra to be ‘reassigned’ to the skull base and 3) reconfigure the geometry of the foramen magnum. The potential evolutionary consequences of these changes are: 1) expansion of the posterior skull, which permitted better accommodation for an expanded visual cortex; 2) an increase in rotation, flexion and extension of the skull, which was immediately useful for better predation and an augmented repertoire of feeding behaviors; and 3) better biomechanics for the occipitocervical junction adapted to tetrapod behavior on land – in primates this may even have facilitated the transition to an erect posture.

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Hox genes are so-named because the homeoboxbearing genes are analogous to the antennapedia/ bithorax complex. However recent work has disclosed other gene families with no relation to Drosophila but with the homeobox and additional conserved sequences. These include groups such as the Engrailed or Lim genes. The nine paired (Pax) genes all contain a paired domain of 128 amino acids; this is a DNA binding site (Wehr and Gruss, 1996). POU genes (pit-1, oct-1 and unc-86) all have a common 75 amino acid loop (for DNA binding) as well as a homeobox. As we shall see many of these non-Hox homeobox genes has been used in the mapping of the vertebrate forebrain. Some molecules produced by cells serve as extracellular ‘signal carriers’. Most inductions in embryology (interactions between tissues such as epithelium and mesenchyme) occur using peptide growth factors. The first molecule, nerve growth factor, was reported with great fanfare in the 1950s. Two large families of these molecules exist and their members are making their way into many scientific papers in the plastic surgery literature. The transforming growth factor-beta (TFGb) family is made up of more than 30 genes that are very active throughout embryogenesis and beyond. Induction of mesoderm, proliferation of myoblasts, and the invasive properties of angiogenic endothelium are all attributable to TGFb products (Kingsley, 1994). Nine genes make up the fibroblast growth factor family. FGF products perform a plethora of tasks form inducing growth of the limb bud to ensuring neuronal survival (and in their absence, neuronal death) (Wilkie et al., 1995). These molecules are now widely used and many applications have been made to cranial suture closure. However, the widespread and protean properties of the TGFbs, coupled with their ability to activate other cascades, makes interpretation of laboratory results difficult. This is because, once again, the action of the same gene can vary widely, for it depends heavily on the period of development and the location in which the gene is expressed. In craniofacial development one of the most potent signaling families, the hedgehog proteins, has only recently been isolated. In mammals three forms of this protein (called Sonic hedgehog, Indian hedgehog and Desert hedgehog) come from three different genes with the same name. Sites of action of shh genes include the primitive node and notochord, the neural floorplate, the ectoderm of facial ‘processes’. Shared shh activity in the apical ectoderm of the second pharyngeal arch and the epithelial buds of the lungs may explain seemingly unrelated pathological states (Roberts et al., 1995; Bellon, 1996; Helms et al., 1997; Ahlgren et al., 1999; Hu and Helms, 1999; Ramalho-Santos et al., 2000; Sukegawa et al., 2000).

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16.9. Craniocaudal pattern formation Gastrulation involves the creation of a three-layer ‘sandwich’ of ectoderm on top of the future organism. This comes from a single sheet of cells, the epiblast, sitting atop a nutrient source, the yolk sac (in the chick) or the gel-filled blastocele cavity (in mammals). A two-layer sandwich comes first. In the avian model epiblast cells are observed to loosen their intercellular bonds. Cells ‘drop out’ from the epiblast in random fashion, falling down into the blastocele cavity, where they reconnect to form a second, subjacent sheet (Azar and Eyal-Giladi, 1979; Eyal-Giladi, 1997). In mammals the proliferation of epiblast leads to direct formation of an underlying second layer (Gilbert, 2003). In either model the deeper cells are known as the hypoblast, or so-called primitive endoderm. A cellular cleavage plane now exists between the layers; this pathway will now permit gastrulation to happen. A groove up the midline axis of the epiblast forms called the primitive streak. At the rostral end of the streak sits a small, elevated pit called Hensen’s node. Like water draining from the bathtub down the drain, epiblast cells migrate toward the streak and enter the space below the epiblast. They are guided in their migration by the basal lamina of the epiblast above. In so doing, these migrating cells are like arctic divers plunging through a hole in the ice and then navigating beneath the ice by following its undersurface. It can be questioned whether cell ‘migration’ exists and, if it does, how the cells ‘know’ where they should go. If we conceive of cells following a route to a destination, how was the route set up in the first place? This evokes the dilemma of attributing to cells a form of ‘knowledge’ they cannot possess. Fortunately, we are rescued from biochemical theism by the pioneering work of German embryologist Erich Blechschmidt (1880–1955). Blechschmidt (1964, 1977) conceived of embryological events in very simple terms. Populations of cells in specific anatomical locations expand their numbers in proportion to their relative access to nutrition. As the populations increase, identifiable anatomical structures within the embryo (ligaments and flexures) channel the growth and ultimately affect the final three-dimensional form of these populations. Thus the final shape of a structure is determined by physical factors, not a genetic program. In the case of gastrulation, the Blechschmidt model postulates that the single-layer epiblast is attached to an unyielding superstructure, the amnion. Around the periphery of the epiblast/amnion junction run two vascular structures, the vitelline veins. These run from the connecting stalk (or the yolk sac) forward to the primitive heart. This implies that the highest availability of

nutrients to the cells of the epiblast occurs at the periphery. Multiplication of this peripheral population creates a physical force directed away from the constraining amnion and toward the midline. This force acts on the cells of the midline, causing them to ingress into the primitive streak. The midline epiblast cells are squeezed like toothpaste and forced out between the epiblast and hypoblast until they reach the periphery again. The situation is not unlike people lined up outside the door of a small theater. Once the doors open the pressure from the back of the crowd will propel those people directly in front of the doors right into the theater and down to the very first row. Thus the theater fills up from distal to proximal. We shall see that this same pattern recurs as the embryo ‘fills up’ with mesoderm. The most peripheral mesoderm (extraembryonic or cardiogenic) comes from epiblast cells nearest the primitive streak. These are followed by lateral plate mesoderm cells and finally by the future paraxial mesoderm cells. Cells ingressing from the epiblasts do so at four general spatial points: 1) Hensen’s node, 2) the remainder of the primitive pit, 3) the rostral streak and finally 4) the caudal streak. The fate of a migrating cell (what layer it will belong to and how far out it shall migrate) is determined by its anatomical site of ingression and the timing of ingression. Another analogy might be paratroopers jumping out of a plane from four different doors. Standing in line before their respective doors, each receives instructions on his mission just prior to exiting. So it is that, from the apex of Hensen’s node, the very first cells to make their exit will become endoderm and, in the exact midline, the notochord. These are followed by the medial somitic mesoderm, the lateral somitic mesoderm and, finally, the lateral plate mesoderm. When gastrulation is complete at a given level of the embryonic axis, a caudal regression of Hensen’s node and of the streak takes place (Schoenwolf, 1991; Tam and Beddington, 1987, 1992; Schoenwolf et al., 1992; Tam et al., 1993, 1997; Tam and Zhou, 1996; Lemire and Kessel, 1997). The very first cells to ingress produce the foregut endoderm. Next follows mesoderm tip of the notochord and most anterior mesoderm called the prechordal plate mesoderm (PCM). Gastrulation occurs over time as a craniocaudal process of cellular ingression and reassignment. Sonic hedgehog produced at the PCM and at the notochord does two very important things. First Shh from the PCM begins to specify the hindbrain while Shh from the notochord begins to organize the somites, i.e. a decision is made between ‘brain’ and ‘nonbrain’. Furthermore, the ingressing cells at a given neuromeric level receive a Hox code ‘identity’ that is identical with those epiblast cells that

NEURAL TUBE PROGRAMMING AND CRANIOFACIAL CLEFTS, PART I did not ingress. A unique pattern of Hox gene expression is thus a ‘barcode’ that persists in all the cells that originated at the region, regardless of where they eventually migrate. This is readily seen in form of the axial skeleton. Formation of a normal craniocaudal pattern of segment is reflected in the unique combination of Hox genes that specifies all vertebrae (Kessel et al., 1990). Noden applied these concepts higher in the neuraxis to the rhomboencephalon by correlating patterns of gene product expression with cranial nerves and neural crest, and rhombomeres (Tam and Beddington, 1987). A Hox ‘barcode’ could be found for rhombomeric levels r3 and caudal; similar gene definitions using krox-20 (the human form of this murine gene is EGR2). follistatin, Engrailed and wnt-1 permitted ‘mapping out’ rhombomeres r0, r1 and r2 (Noden, 1991b). At r0 (some texts describe this as the anterior border of r1), FGF8 and wnt-1 stimulate expression of Engrailed genes en-1 and en-2. The farther one moves away from the ‘signaling center’ the lower the concentrations of En-1/En-2 proteins becomes. Indeed, r0 as the principle rhombomere of the mesencephalon expresses many important genes that determine the spatial organization of the brain. These molecules stimulate the rostral region to become mesencephalon (midbrain) and the caudal region at r1 to become metencephalon (cerebellum). The midbrain subsequently forms two developmental units in which are located the superior and inferior follicular for visual and auditory connections. Puelles and Rubenstein refer to these as m1 and m2 but for our purposes here they both are derived from r0. The early forebrain is divided into six prosomeres. Just like the rest of the spinal cord, hindbrain and midbrain the prosencephalon possess a ventral (basal) zone and a dorsal (alar) zone. It should be noted that the use of the terms basal and alar is a descriptive analogy based upon the hindbrain. No sulcus limitans exists in the forebrain; therefore true ‘alar’ or ‘basal’ zones do not exist. We make use of this crude analogy simply to better understand the eventual differentiation of the prosencephalon into two major up of both subdivisions. The caudal diencephalon is made up of both alar and basal units of p1–p4 and contains the epithalamus, thalamus and hypothalamus. The rostral diencephalon is formed from the basal units of p5–p6. These contain (respectively) the apparatus of vision and olfaction. The telencephalon is made from the two remaining dorsal zones of p5 and p6. It contains the cerebral cortex. Much progress in mapping the derivatives of the prosomeric zones is due to elegant work by Rubenstein and Puelles (2003).

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16.10. Neuromeric basis of neural crest ‘migration’ and fate Now that we have a description of the neuromeric system firmly in mind we shall need a schematic way to visualize it. Using a mouse embryo in sagittal section, Rubenstein et al. have outlined the boundaries of the neuromeric zones. Key neuroanatomical landmarks are presented in a linear, schematic manner. Basal and alar regions of the entire neuraxis, including the prosencephalon, can be readily appreciated. These relationships are then projected on to an anatomical representation of the embryo. I have modified these drawings by applying a color code in which each neuromeric zone along the neuraxis has its own specific color. When tracing out all the derivatives of r3, all muscles and bones of the mandibular portion of the first pharyngeal arch will be depicted in green. This permits structures such as dermis and dura to be ‘mapped’ backward to their origins at a specific neuromeric level. The neural folds can be considered in terms of five zones. 16.10.1. Anterior prosencephalic neural fold: nonneural ectoderm Pioneering work by Couly and LeDourain established a detailed fate map of the avian neural plate (Couly and LeDourain, 1985, 1987, 1990; Couly et al., 1992, 1996; Hall, 1997; Le Douarin and Kalcheim, 1999; Cobos et al., 2001; Fernandez-Garre et al., 2002). In particular, they showed that neural crest cells do not occupy the entire length of the neural fold. The neural folds of the anterior prosencephalon (from p6 back to p4–p3) do not have neural crest cells. The neural folds of the posterior prosencephalon (from p3–p2 on backward) contain neural crest. All ectoderm lateral to the neural folds is destined to form epidermis. This ectoderm, plus that of the anterior prosencephalic neural folds is termed non-neural ectoderm. I shall describe the anatomical content of the non-neural ectoderm in cranial-to-caudal order, i.e. from p6 to p4. Folding of the embryonic head coupled with growth of the forebrain causes radical changes in the spatial relationships of these three zones to occur, especially between p6 and p5. These changes will be described below. The most anterior zones of non-neural ectoderm are termed p6. The first important structures of p6 are the adenohypophyseal placodes. Described as singular, these fused structures occupy either side of the midline. Pathology in either adenohypophyseal placode explains the occurrence of epithelial tumor localized to either the right or left pituitary. Posterior to each adenohypophyseal placodes the non-neural ectoderm

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becomes the nasal epithelium (NE). This forms the epithelial lining of the nasopharynx all the way forward from Rathke’s pouch and Waldeyer’s ring to the true skin of the nasal vestibule. The NE makes a boundary with true skin of the nose and upper lip (in the chick known as upper beak epithelium, UBE). Within the NE lies the nasal placode (NP). This specialized epithelium gives rise to three classes of neuron, all of which migrate into the brain. The lateral NP contains olfactory neurons for conventional odor detection. The medial NP contains accessory olfactory neurons, those involved with the detection of complex chemical signals, pheromones being the prime example. Finally, the medial NP contains neurons associated with gonadotropin-releasing hormone (GnRH). Animal behaviors related to detection of sexual cues from urine and sniffing of genitalia relate to this system. GnRH is related to development of secondary sex characteristics. Kallman’s syndrome (anosmia and/or hypogonadotropic hypogonadism) results from anatomical defects in the NP (Gauthier, 1960; Lieblich et al., 1982; Rugarli and Ballabio, 1993; LeDourain et al., 1997; Molsted et al., 1997). Note that the Spanish pathologist Maestre de San Juan first described this syndrome (Maestre de San Juan, 1856). The p5 zone of non-neural ectoderm comprises the skin of the nose and upper lip. This UBE (the term is derived from the avian model), constitutes the epidermis of the nose and philtrum of the upper lip but not the forehead. The lateral boundaries of the UBE are marked by sensory innervation of V1 and lie medial to the arcade formed by the facial artery–angular artery. Just caudal (posterior) to the UBE lies the optic placode. This forms the lens and is crucial for development of the globe (and ultimately for that of the orbit). The p4 zone of non-neural ectoderm forms the calvarial ectoderm. The epidermis of the forehead and the frontal bone sweep back over the cerebrum within this zone. In birds, the frontal bone is huge while the parietal bone is diminutive. Formation of the avian calvaria is exclusively from neural crest and is extremely rapid. Sutures are not observed; pathological craniosynostosis does not occur. In mammals the parietal bone forms from PAM of r2 and r3 derivation. The varying forms of synostosis all involve boundary between bones of dissimilar developmental derivation. These observations may have important implications for the relative incidence of craniosynostosis observed in humans. Without a dermis for vascularization and support, epidermis becomes rather worthless. Indeed all epithelia require a subjacent supporting layer. The production of all dermis and bone in the face is exclusively the responsibility of neural crest. So where does the neural crest come from and how does it ‘know’ what

to do? The answer to the first question lies in a clear conception of the three functional types of neural crest and of the manner in which they migrate (Serbedzija et al., 1990, 1992). The answer to the second question relates to the ‘programming’ function exerted upon the neural crest cells by the epithelial environments they inhabit (Tan and Morriss-Kay, 1985, 1986; Gui et al., 1993). We shall deal with the first question in the section below and discuss the second at the conclusion of this essay. 16.10.2. Prosencephalic neural crest The first zone of neural crest extends over the posterior prosencephalon from p3 back to p1. This PNC moves forward in the subectodermal plane as a large vertical sheet of cells in the midline. The PNC migrates from the dorsal part of the lamina terminalis, from which also is formed the colossal plate. For this reason, p6 deficiency associated with holoprosencephaly can result in hypoplasia or absence of the intercanthal ligament; hypertelorism results. This is contrasted with the r1 deficiency state seen in anencephaly. The r1 component of the frontal bone is absent, the absence of r1 dura and neural crest pericytes profoundly affects forebrain development and the sphenoid bone (an r1 structure) is small and misshapen. (For further details see Ch. 7.) The most anterior cells of the sheet will reach the p6 ectoderm, where they will be ‘instructed’ in situ by the epithelium above and/or by the neural ectoderm to make dermis and the upper lateral cartilages. Into this envelope MNC neural crest from r1 migrates to form nasal septum, the alar and lateral cartilages, the perpendicular plate of the ethmoid, the ethmoid labyrinth and crista galli and, finally, the upper and middle turbinates. The sixth prosomere neural crest also ‘activates’ the adenohypophyseal and nasal placodes. Without interaction with neural crest these placodes are nonfunctional. When neural crest arrives in the p5 zone it will form the nasal bones and the frontal bone, the nasal bones and the lower lateral cartilages. The nasal process of the frontal bone descends deep to the nasal bones to articulate with the facial process of the premaxilla. To digress briefly, the piriform rim is really a bilaminar structure. Its internal aspect is made from the nasal process of the frontal bone (p5), which extends downward just beneath the nasal bone. It abuts the frontal process of the premaxilla (a product of r20 neural crest). Just lateral is the frontal process of the maxilla (an r2 derivative). This abuts against the nasal bone itself. The bicortical structure of the piriform rim makes it stronger: it is capable of holding screws. Surgeons refer to the piriform as a ‘buttress’ and use it for placement of fixation plates. Additional bones of p5

NEURAL TUBE PROGRAMMING AND CRANIOFACIAL CLEFTS, PART I derivation are the orbital lamina of the ethmoid bone and the lacrimal bone. The boundary between the neural crest derivatives of zone p5 and those of zone p4 is uncertain at this time. The supraorbital margin of mammals and the roof of the orbit are p5 while the remainder of the forehead would logically be p4. Indeed the ossification centers of the frontal bone appear in caudal to cranial order. While the parietal bone of birds and mammals is probably of r2/r3 origin it differs in the two orders by being of neural crest origin in the former and of paraxial mesoderm derivation in the latter. In a subsequent part of this series this difference will be explored as a possible explanation for an exclusively mammalian innovation: the muscles of facial muscle expression. The fascial organization of these muscles constitutes a distinct layer enveloping virtually the entire head and neck. This fascial layer is referred to in the surgical literature as the superficial musculoaponeurotic system. The invention of this system may well be related to an organization of the spatial and functional relationships between the first and second pharyngeal arches, an evolutionary development of profound import and quite possibly at the heart of the ‘mammalian revolution’. 16.10.3. Mesencephalic neural crest The MNC lies over the mesencephalon. Unlike PNC, which moves as a large sheet, MNC proliferates as three distinct streams, described as r0, r1 and r20. As mentioned earlier, the development of the mesencephalon is stimulated by FGF8/Wnt-1 produced at the isthmus (levels r0 and r1). Neural crest associated with each of these neuromeres undergoes a remarkable expansion atop the rapidly growing mesencephalon. Although the midbrain is quite small in the mature state, in the embryo it is enormous. This premigratory MNC contributes to large surface areas of dura associated with the sensory distribution of V1. Mesencephalic neural crest migrates in three successive streams in craniocaudal order. That from r0 and r1 is dedicated to development of extraocular connective tissues and the posterior orbital wall. Neural crest from r0 migrates first and its final destination is the most medial and cranial of all MNC. It makes no bone. It lies internal and caudal to the optic vesicle. It therefore produces the inferomedial sclera and the fascia of the corresponding extraocular muscles innervated from the cranial oculomotor nucleus (inferior oblique, inferior rectus and medial rectus). A limited amount of dura over the base of the forebrain, the rhinencephalon, would logically come from r0 neural crest. The innervation pattern of the terminal nerve

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(cranial nerve 0) may well map out the contribution of r0 neural crest. Next to migrate is MNC from r1. The presence of the optic vesicle forces this stream to assume a superolateral position within the future orbit. It also fills in the space behind the globe and lateral to the future optic nerve. The presphenoid bone is formed from r1 MNC via a chondral intermediate. So too is the lesser wing of the sphenoid, being derived from pre-existing orbitosphenoid cartilage. As the superolateral half of the optic vesicle is enveloped by r1 MNC its scleral coat is acquired as well as the fasciae for the remaining extraocular muscles innervated by the caudal oculomotor nucleus (superior rectus and levator palpebrae superioris). A great deal of r1 neural crest is involved with dura synthesis. It covers the entire cerebral cortex innervated by V1. The most posterior (caudal) of the MNC streams is termed r20. It occupies the exact boundary between the mesencephalon and rhomboencephalon, known as the isthmus. It is last to migrate and its pathway must contend with an orbit that is now filled to capacity by the globe and its r0 and r1 MNC predecessors. Accordingly, r20 MNC is forced to descend behind and below the orbit to reach a more medial position into the future nasal cavity. It forms a groove in the midline of the ventral presphenoid bone. From this position it descends along a boundary formed from pre-existing p6 perpendicular ethmoid plate and septum and forms the vomerine and premaxillary bones. The pathway taken by r20 MNC can be traced by following its neurovascular axis, the sphenopalatine nerve and artery. This neural crest sits just above the second rhombomere. It is important to distinguish between r20 neural crest and r20 neural crest proper because of the migration pattern and function of these cells. The anteriormost part of r20 moves in a stream toward the orbit. In so doing, it has to pass forward through adjacent rhombomeric zones. Most importantly, r20 neural crest does not participate in the development of the first pharyngeal arch. Thus, although these neural crest cells originate over the rhomboencephalon proper, their biological behavior is that of MNC. Therefore the third and most caudal zone of MNC is the cranial neural crest of r2; it bears the name r20 MNC. Vascular supply of structures produced from MNC and RNC offers an additional and important means of distinguishing r1 from r20 from r2. All derivatives of PNC and MNC (r0 and r1) are supplied by the internal carotid artery. All derivatives of RNC (r2–r11) are supplied by the external carotid artery. Although r20 MNC does not participate in the pharyngeal arch system its blood supply comes from the terminal (anteriormost) branch of the internal maxillary artery, the sphenopalatine.

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16.10.4. Rhomboencephalic neural crest Rhomboencephalic neural crest does not travel in streams: at each neuromeric level it proliferates or ‘migrates’ as a segment into and around its corresponding somitomere. Furthermore, cells from one neuromere do not mingle with those of a neighboring neuromere. (Mixing between even or odd numbered neuromeres is permissible experimentally but not adjacent ones.) The posterior (caudal) neural crest from r2 does enter and interact with the PAM of the second somitomere. It is required for the proper formation of the first pharyngeal arch. Neural crest from r2 does not mix with that of r3. Because it obeys the ‘rules’, this neural crest from the most rostral rhombomere is named r2 RNC. RNC can be subdivided into two zones. The rostral zone (RNCr) contains neural crest interacting with those somitomeres that do not go on to become fullfledged somites. These include the head mesoderm of Sm1 and the first three pharyngeal arches. As r2–r7 reorganize themselves into arches the somitomeres seem to ‘pair up’. These dimers reorganize themselves into a single structure. Perhaps because the cellular volume of the first three pharyngeal arches is greater neural crest migration into them takes longer to complete than that of more caudal zones. RNCr migration is not completed until somite stage 14. The caudal zone of rhomboencephalic neural crest (RNCc) involves neuromeres responsible for populating the second group of pharyngeal arches. PA4–5 are smaller in size. It is therefore reasonable to expect that the neural crest population will take less time to migrate into these arches. Indeed RNCc migration is complete by somite stage 11. The manner in which the anatomy of a pharyngeal arch comes about and in which this affects the order in which different fields within a pharyngeal arch are expressed (as reflected in the ossification sequence of craniofacial bones) is discussed below.

16.11. Spatial reassignment of non-neural ectoderm Head folding coupled with forebrain growth causes changes in the spatial relationships of non-neural ectoderm p6–p4. Prior to closure of the anterior neural folds the p6 zone starts from the midline just rostral to the stomodeum, just in front of Rathke’s pouch, and projects backward (caudally) along the folds. The apical surface of this future nasal vestibular epithelium (NVE) faces dorsally. With closure of the neural folds both p6 zones come together in the midline. They acquire a population of PNC at this time. Head folding forces this zone to roll forward and ventrally

much like the tracks of a tank. The NE that once faced anteriorly is now drawn into the future nasal cavity. At the leading edge of the NVE lies the adenohypophyseal placode and, just behind it, the NP. The apical surface of the nasal vestibular epithelium now faces ventrally. As the p6 NE and NP are pulled into the future nasal cavity, similar changes occur with the p5 zone of non-neural ectoderm. This zone, the UBE, is slated to become the future epidermis of the nose and upper lip. Being in continuity with the p6 NE, the p5 UBE is pulled forward. The topology of the NE resembled the letter U turned 90 on to its side. The upper limb of the U is the keratinized p5 epithelium of the nasal dorsum. Neural crest that has migrated immediately below the p5 epithelium will be programmed by it to form dermis and the nasal bones. The lower limb of the U is the non-keratinized p6 epithelium of the nasal vestibule. The vomeronasal organ, located in the membranous septum, probably represents the remnant of the NP. Neural crest that has migrated immediately above the p6 epithelium will be programmed by it to form the alar and triangular cartilages. The future nose consists of two such p6/p5 systems. The medial walls contain p6 neural crest. As these approximate, the perpendicular plate of the ethmoid and the nasal septum result. Another way to visualize the topology of the nasal chamber is to imagine a condom placed on a flat surface. The tip of the reservoir represents the p6 NP while the remaining latex of the reservoir is the p6 NE. All the rest of the condom is p5 nasal skin. If the tip of the reservoir is glued to the table and the condom is inflated an invagination will occur. This will place the nasal skin on the outside and the NE on the inside.

16.12. Timing of neural crest migration Age in embryos is calculated by the presence of anatomical landmarks. Because somites appear at absolutely regular intervals and are readily counted, somite stage is a reliable means to measure time in a developing embryo. Neural crest migrates from different anatomical sites at different times. To study this pattern, Osumi-Yamashita labeled neural crest populations from four zones: rostral rhomoboencephalon RNCr, caudal rhomboencephalon RNCc, mesencephalon MNC and caudal prosencephalon PNC. Neural crest migration from each zone was studied and the timing of its completion (as measured by somite stage) was determined. At the 11-somite stage, RNCr (neural crest from r2–r7) migration was complete. This zone populates the first three pharyngeal arches in craniocaudal order.

NEURAL TUBE PROGRAMMING AND CRANIOFACIAL CLEFTS, PART I Thus first arch bones such as the mandible appear before third arch derivatives such as the greater cornu of the hyoid. At the 14-somite stage, two distinct zones complete their migrations. RNCc (neural crest from r7– r11) completes its migration, as does MNC from r0, r1 and r20. Both these migrations are also craniocaudal. At the 16-somite stage, PNC migration is complete. Unlike MNC and RNC, PNC proliferation appears to proceed in caudal-to-cranial order, like toothpaste being squeezed from a tube. Thus the p5 zone is populated by PNC prior to p6. Placodes are activated by the presence of underlying neural crest. Experimentally, it is known that the p5 optic placode certainly appears before the p6 olfactory placode. Pathologies tend to be more severe the earlier in development they strike. A very late ‘hit’ on neural crest migration to the midline could cause a mild holoprosencephaly due to absence of the ethmoid complex without affecting the orbit. The more severe is this type of neurocristopathy, the greater the degree of hypotelorism that will result.

16.13. Fate of the neural crest: the role of epithelial ‘programming’ In the preceding discussion we have seen how neural crest derivatives receive instructions from the nonneural ectoderm as to what structures they should form. Many important surgical implications flow from these considerations. NVE from p6 determines the size and shape of the upper lateral and septal cartilages of the nose whereas the UBE (nasal skin) from p5 will affect the formation of lower lateral nasal cartilages and the nasal bones. A similar role might perhaps be exerted by the foregut endoderm. Recent experimental work from the laboratories of Couly and LeDouarin is of enormous clinical significance in this regard for it supports the hypothesis that foregut endoderm (FGE) plays the decisive role in the formation of neural crest bones and cartilages of the pharyngeal arches. Neural crest cells, if left to themselves in the Petri dish, will form cartilage. Certain membranous bones of neural crest form via cartilaginous intermediates while others form directly within membrane. By applying techniques of surgical extirpation and transplantation in the previously described quail chick chimera model, Couly and LeDouarin mapped out distinct territories of FGE and found that specific zones were responsible for the production of specific bones and cartilages. This yielded a ‘map’ of FGE in which endoderm destined to form Sessel’s pouch was found to underlie the frontonasal bud while the remainder of FGE resulted from the summation of individual outgrowths of endoderm corresponding to the pharyngeal pouches.

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Following our previous discussion of gastrulation it is possible to conceive of FGE as having a neuromeric code as well. The zone corresponding to the mucosa of the vomer and premaxilla would come from r1 (or r20, to be consistent). With closure of the palate this mucosa becomes hidden from view but it is histologically distinct from that of the p6 NE. This is readily apparent on lateral dissection of the septum, in which the mucoperiosteum stops at the border of the vomer, the innervation changes from V1 (the medial nasal nerves) to V2 (the sphenopalatine nerve) and the blood supply switches from internal carotid along the upper septum to the external carotid sphenopalatine coursing along the septo-vomerine border. All the remaining endoderm of the pharynx and larynx would be coded from r2 to r11. Couly and Le Douarin made an important distinction between neural crest cells emanating from differing parts of the neural fold in terms of the expression of Hox genes. They found that rostral neural crest corresponding to PNC and MNC constituted a domain in which Hox genes were not expressed (Hox-negative). These neural crest cells gave rise to the membranous bones of the neurocranium, the nasal capsule and the first pharyngeal arch maxilla and mandible. Thus the Hox-negative domain extended to r3. In contrast, the domain corresponding to the pharyngeal arches 2–5 was considered to be Hox-positive and yielded the hyoid bone and cartilages of the visceral skeleton. Specific mapping of the hyoid-forming region of FGE revealed that extirpation of specific zones caused failure of formation of corresponding components of the hyoid. When the entire hyoid region was reversed 180 , the orientation of the hyoid bone was reversed as well. Thus pharyngeal endoderm is required from neural crest cells to become cartilage or membranous bones based on cartilage while ectoderm is required for neural crest to become membranous bone via the classic mechanism (no cartilaginous intermediate). Hox-negative neural crest is exclusively responsible for the generation of the facial skeleton but does not possess the information required to pattern the skeleton. For this to occur FGE is required. Defined areas of FGE induce the formation of specific bones and cartilages from cephalic neural crest. Information required to determine the axes of facial bones depends upon the spatial orientation of specific zones of FGE. The ability to respond to patterning cues from FGE is exclusive to non-Hox-expressing cephalic neural crest. Cells from different zones of the Hox-negative domain behave in an equivalent manner. Because of its enormous regenerative capability up to three-quarters of the neural fold responsible for cephalic morphogenesis can be

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removed with no consequences. This implies that the pathways taken by PNC and MNC are not inherent to the neural cells per se but are determined by the microenvironment through which the neural crest cells migrate/proliferate. Although the nature of the signaling employed by FGE to instruct neural crest is uncertain it has been shown that the spatial orientation of branchial pouch endoderm is established prior to the migration of neural crest into the pharyngeal arches. Protein markers such as BMP7, FGF8 and Pax-1 are found in different regions of the FGE irrespective of whether neural crest cells are present or not. Indeed, these polarities may well be established prior to formation of the pharyngeal arches, i.e. they may be determined by the spatiotemporal sequence of cell movements at gastrulation itself. These considerations allow us to think of facial bones as the products of specific biosynthetic units or fields corresponding to the concept of functional matrix first elaborated by Moss years ago. Individual deficits of the facial bones as manifested in craniofacial clefts would thus result from very early insults or aberrations of either epithelial programming units, in the neural crest cells that come to populate them or in the biochemistry of epithelial-mesenchyme interaction.

16.14. Conclusion We have now completed a brief synopsis of neuromeric organization. Neuromeres are developmental units of the nervous system with specific neurological content. Outlying each neuromere are tissues of ectoderm, mesoderm and endoderm that bear an anatomical relationship to the neuromere in three basic ways. This relationship is physical in that motor and sensory connections exist between a given neuromeric level and its target tissues. The relationship is also developmental because the target cells exit during gastrulation precisely at that same level. Finally, the relationship is chemical because the genetic definition of a neuromere is shared with those tissues with which it interacts. The model developed by Puelles and Rubenstein is used to describe the neuroanatomy of the neuromeres. Although important details of the model are currently being refined it has immediate clinical relevance for practicing clinicians. The physical size and shape of each neuromere are defined by the protein products of genes expressed within the confines of the neuromere. Many of the crucial genes in this system contain a unique sequence of DNA bases leading to a stereotypical amino acid sequence known as the homeobox. Homeobox genes are master regulators of other genes because the

homeobox unit unlocks other DNA sequences. Homeobox genes are divided into two classes: Hox genes are homeobox genes analogous to those originally described in Drosophila. Non-Hox homeobox genes possess the homeobox sequence of bases but bear no relationship to the Drosophila system. Neural crest developing in the neural fold above a given neuromere bears a similar relationship with that neuromere. Neural crest cell populations are organized into three main groups depending upon their location along the neuraxis. The physical behavior of neural crest migration is determined by the microanatomy of each of these three environments. Neural crest from the caudal prosencephalon moves forward as a cohesive sheet and populates the non-neural ectoderm of the rostral prosencephalon. PNC is responsible for the mesenchyme producing the bones, cartilages and connective tissues of the fronto-orbital-nasal mass. The nature, shape and size of these derivatives is not inherent in the cells of the PNC but results from the instructions given to PNC by specific ‘target’ zones of ectoderm (termed p6, p5 and p4) and foregut endoderm (r0, r1, r20) with which the neural crest cells interact. Mesencephalic neural crest is associated with neuromeres r0, r1 and r20 and travels as individual cells in anatomically distinct streams. This MNC is responsible for the bulk of the orbit, the sclera and the sphenoid. Its most caudal portion (r20) is of great clinical importance because it interacts with r20 endoderm to produce the vomer and premaxilla. Deficits in this epithelial-mesenchymal system are responsible for the most common forms of clefting involving the primary palate, the lip and the secondary palate (in association with cleft lip). Rhomboencephalic neural crest begins at neuromeric level r2 and continues to r11. Neural crest migration is segmentally segregated into pairs of outlying somitomeres and somites forming the five pharyngeal arches. This process occurs in cranio-caudal order and is completed over two time periods. Population of the first three arches (the rostral RNC or RNCr) coincides with that of MNC. A second wave (the caudal RNC or RNCc) populates pharyngeal arches 4 and 5. Thus we are able to think about the head and neck in terms of neuromeric terminology. Relationships between the processes of neurulation and gastrulation have been presented in this chapter to demonstrate the manner in which neuromeric anatomy is established in the embryo. We are now in a position to describe in detail the static anatomical structures that result from this system. The neuromeric ‘map’ of craniofacial bones, dermis, dura, muscles and fascia will be the subject of Chapter 17.

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Schoenwolf G (1997). Neurulation: coming to closure. Trends Neurosci 20: 510–517. Schoenwolf G, Garcia-Martinez V, Dias MS (1992). Mesoderm movement and fate during avian gastrulation and neurulation. Dev Dyn 193: 235–248. Scott MP (1992). Vertebrate homeobox nomenclature. Cell 71: 551–553. Serbedzija GN, Fraser SE, Bronner-Fraser M (1990). Pathways of neural crest migration in the mouse embryo as revealed by vital dye labeling. Development 108: 605–612. Serbedzija GN, Bonner-Fraser M, Fraser SE (1992). Vital dye analysis of cranial neural crest migration in the mouse embryo. Development 116: 297–307. Stein S, Fritsch R, Lenmire L, Kessel M (1996). Checklist: vertebrate homeobox genes. Mech Dev 9: 1–108. Sukegawa A, Narita T, Kameda T, et al. (2000). The concentric structure of the developing gut is regulated by Sonic hedgehog derived from endodermal epithelium. Development 127: 1971–1980. Tajbakhsh S, Spurle R (1998). Somite development: constructing the vertebrate body. Cell 92: 127–138. Tam PPL, Beddington RSP (1987). The formation of mesodermal tissues in the mouse embryo during gastrulation and early organogenesis. Development 99: 109–126. Tam PPL, Beddington RSP (1992). Establishment and organization of germ layers in the gastrulating mouse embryo. In: Postimplantation Development in the Mouse, Ciba Foundation Symposia 27–49165. Tam PPL, Behringer RR (1997). Mouse gastrulation: the formation of a mammalian body plan. Mech Dev 68: 3–25. Tam PPL, Trainor PA (1994). Specification and segmentation of the paraxial mesoderm. Anat Embryol 189: 379–390. Tam PPL, Zhou SX (1996). The allocation of epiblast cells to ectodermal and germ-line lineage is influenced by the

position of the cells in the gastrulating mouse embryo. Dev Biol 178: 124–132. Tam PPL, Williams EA, Chan WY (1993). Gastrulation in the mouse embryo: ultrastructural and molecular aspects of germ layer morphogenesis. Microsc Res Tech 26: 301–328. Tam PPL, Parameswaran M, Kinder SJ, Weinberger RP (1997). The allocation of epiblast cells to the embryonic heart and other mesodermal lineages: the role of ingression and tissue movement during gastrulation. Development 124: 1631–1642. Tam PPL, Goldman D, Camus A, Schoenwolf GC (2000). Early events of somitogenesis in higher vertebrates: allocatioon of precursor cells during gastrulation and the organization of a meristic pattern in the paraxial mesoderm. In: CP Ordahl (Ed.), Somitogenesis, Part 1. Academic Press, San Diego, pp. 1–32. Tan SS, Morriss-Kay GM (1985). The development and distribution of the cranial neural crest in the rat embryo. Cell Tiss Res 240: 403–416. Tan SS, Morriss-Kay GM (1986). Analysis of cranial neural crest cell migration and early fates in post-implantation rat chimeras. J Embryol Exp Morph 98: 21–58. Vaage S (1969). The segmentation of the primitive neural tube in chick embryos (Gallus domesticus). Adv Anat Embryol Cell Biol 41: 1–88. Venters SI, Thornsteindottir S, Duxton MI (1999). Early development of the myotome in the mouse. Dev Dyn 216: 219–232. Wehr R, Gruss P (1996). Pax and vertebrate development. Int J Dev Biol 40: 369–377. Wilkie AO, Morriss-Kay GM, Jones EY, Heath JK (1995). Function of FGFs and their receptors. Curr Biol 5: 500–507. Wingate RJT, Lumsden A (1996). Persistence of rhombomeric organization in the postsegmental hindbrain. Development 112: 2143–2152.


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Fig. 16.1. Four-dimensional model of cleft. A functional matrix deficiency at the lateral piriform fossa prevents lateral nasal mass A2 from uniting with medial nasal mass A1. A1 remains in false contact with the philtrum A0. It cannot participate in forming the nasal floor. The piriform deficit creates a mismatch between the p5 nasal skin (light blue) and the p6 vestibular lining (red). This explains the appearance of the cleft nose.

Fig 16.2. (A) Relapse pattern of left unilateral cleft after apparently successful surgical repair in infancy reveals how unbalanced growth forces act over time to maintain and exacerbate asymmetry of soft tissue and bone. (B) Untreated cleft maxilla grows normally but remains in a retrocessed position compared to the normal right side. The functional matrix is produced by the embryo long before bone production occurs. In cleft situations both sides are disconnected and grow asymmetrically. The cleft side functional matrix is thus displaced by the time of osteogenesis. Both soft tissue and bone are therefore normal but stuck in the wrong position. Without correcting force vectors surgically in infancy the cleft maxilla will function normally but remain malpositioned.

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Fig 16.3. Nasal walls showing ‘breakpoint’ between neuromeric coding, blood supply and innervation. The commonest cleft lip/nose deformity occurs precisely at this intersection.


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Fig 16.4. Neuromeric organization of the embryo. Each neuromere has a specific color. All derivatives of that neuromeric level will have the same color. Note the role of occipital somite in forming the posterior cranium and skull base. All bones formed by the somitomeres and occipital somites (up to O4) are segmental, i.e. individual sutures reveal field boundaries. The fourth occipital somite and first cervical somite mark the transition of a parasegmental system.

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Fig 16.5. Gastrulation (Blechschmidt hypothesis): The two-layer embryonic disc is attached at its posterior pole (left). The dorsal epiblast will eventually form the entire embryo. The ventral hypoblast is derived from ‘dropout’ of epiblast cells into the yolk sac. These former epiblast cells then coalesce to form a temporary layer (also called the primitive endoderm) designed to permit gastrulation. Blood supply is greatest at the periphery via the vitelline (umbilical) veins. High peripheral Po2 leads to a high local mitotic rate. This drives epiblast cells toward the midline, just like toothpaste squeezed from a tube. The primitive groove allows epiblast cells at its margin to be pushed into it. The first cells to enter push the hypoblast aside to form the definitive endoderm (upper inset). Cells entering subsequently intercalate themselves between the epiblast and the endoderm. This intermediate layer is the mesoderm (lower inset). Cells of the epiblast that do not participate in gastrulation form the ectoderm. All three primary germ layers are now present. Gastrulation proceeds from cranial to caudal. Homeobox genes expressed in varying combinations along the axis of the embryo define a segmental (neuromeric) organization at the time of gastrulation. Cells ingressing at a particular level will be assigned a hox code specific for that level. Segmentation of cells at gastrulation into genetically distinct anatomical levels provides the basis for the future organization of the embryo.

Fig 16.6. Gastrulation proceeds in a cranial to caudal fashion (here depicted as left to right). Cells are squeezed like toothpaste into the primitive streak. Cells entering first form the endoderm, here shown pushing the hypoblast out of the way. Cells migrating between the epiblast and the endoderm form the mesoderm. Those cells closest to the streak (1) will be first to enter and, once inside the bilaminar structure will be pushed most laterally (10). Cells further away from the streak (2) will enter later in time and, once inside, will remain more medially (20). When gastrulation is complete the epiblast becomes the definitive ectoderm. Once in their final position, mesodermal cells are patterned by chemical signals from either the ectoderm or endoderm with which they are in contact. These signals are region-specific: i.e. unique combinations of genes are expressed in specific anatomical sectors of the trilaminar embryo. Gene products causing mesoderm 10 to become lateral plate mesoderm originate at a finite distance from the midline. The organization of these anatomical sectors is neuromeric and organized around the homeobox genes. The hox code at level t1 is different from those of levels t2 and t3.


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Fig 16.7. (A) Invertebrate segmentation. Homeobox genes originally described in the fruit fly are called Hox genes. Similar Hox coding is found in mammals caudal to the third rhombomere. (B) Mammalian segmental organization is based on the Hox code. Note the four occipital somites and the four coccygeal somites. Human embryos have 38þ somites. Neuromeric units cranial to r3 are defined by non-Hox homeotic genes (Hox-negative).

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Fig 16.8. Hox coding of the mode and frameshift mutations (from Larsen). In mouse (D), application of retinoic acid causes an additional cervical vertebra to appear (Kessel, 1992). This is the primitive proatlas. Vertebrae 1 and 2 undergo transformation from atlas and axis to typical cervical vertebrae. The proatlas thus articulates directly with the cervical spine; atlas and axis are eliminated.

Fig 16.9. The primordial first cervical vertebra (the proatlas) accounts for the discrepancy between the number of cervical spinal nerves and vertebral bodies. The proatlas forms by parasegmentation between the fifth occipital somite and the first cervical somite. Incorporation of the proatlas into the skull base as condyles, dens and basioccipital–dental ligament provides a dual condyle system for craniovertebral flexion–extension.


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Fig 16.10. Migration of neural crest (Gui, Osama-Yamashita). PNC (p4, p5, p6) migrates as a sheet to populate the non-neural 0 ectoderm and create frontonasal structures. MNC (r0, r1, r2 ) migrates as individual streams to form orbitosphenoid structures. RNC (r2–r11) migrates as individual segments into the pharyngeal arches.

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Fig 16.11. An avian neural plate fate map demonstrates how neural crest cells above the caudal prosencephalon migrate forward as a sheet to populate the non-neural ectoderm (NNE) above rostral prosencephalon. NNE is divided into three zones. Zone A (red) ¼ p6, zone B (dark green) ¼ rostral p5, zone C (light green) ¼ caudal p5 (possibly p4).


Chapter 17

Neural tube programming and the pathogenesis of craniofacial clefts, part II: mesenchyme, pharyngeal arches, developmental fields; and the assembly of the human face MICHAEL H. CARSTENS* Cardinal Glennon Children’s Hospital, Saint Louis University, Saint Louis, MO, USA

17.1. Introduction Chapter 16 centered on the anatomical components of the neuromeric system and the identification of their derivatives. The emphasis was on mesenchyme (neural crest and mesoderm) because so many structures (bone, muscles, fascia and dura) arise from it. As discussed, the mesenchyme of any particular structure can be traced back (‘assigned’) to one or more neuromeric zones in the developing embryo. Excess, deficiency or outright absence of a developmental field can be attributed to pathological processes occurring at a neuromeric level very early in development. Craniofacial developmental fields function much like LegoW pieces built one upon another in a tightly regulated sequence. When a field ‘goes wrong’, neighboring fields cannot form correctly. The result is a facial cleft. I shall now focus on the actual process by which such clefts occur, beginning with a detailed analysis of individual fields, where they come from, in what order they form and what anatomical consequences are seen in the event of field failure. Contributions of various types of mesenchyme will be discussed. I shall then describe the physical manner by which fields are assembled over time. The clinical observations made by Paul Tessier (1969, 1976, 1981) regarding patterns of craniofacial cleft formation (derived on a strictly empiric basis) match closely patterns of neural crest migration. Three caveats: 1. Take time to study the definitions section at the beginning of Chapter 16 and the illustrations

section at the end of this chapter. This will give you a visual orientation to the terminology and concepts we are about to discuss. The legends make each illustration self-explanatory. Because these figures are referenced time and again, they are in a fixed intellectual order that is not in synch with the text. Start with Figs. 17.1–17.5. 2. My purpose is to explore an entirely new framework for understanding head and neck anatomy; works by Carlson (2004), O’Rahilly and Muller (2001, 2004), Gilbert (2006), Liem et al. (2001), Kjaer and Fischer-Hansen (1999) and others (Huang et al., 2000; Helms et al., 2005) will greatly enhance your understanding of development. 3. This work is interpretive but testable – concepts from diverse specialties are woven together to paint a coherent picture of how development might work. The model is my own best guess as to how this system works. You are encouraged to take it further.

17.2. Anatomy of craniofacial mesoderm 17.2.1. Paraxial mesoderm: somitomeres and somites Craniofacial clefts result from hypoplasia or absence of recognizable anatomical structures, such as bone, cartilage, muscle or dermis. These all originate from mesenchyme; this is to be distinguished from epithelium. Epithelial cells are polar. One surface faces an external, extracellular environment (such gut lumen or air) while the other surface is joined with a supporting cellular

*Correspondence to: Michael H. Carstens MD, FACS, Associate Professor of Plastic Surgery, Director of Craniofacial Surgery, Cardinal Glennon Children’s Hospital, St Louis University, 3635 Vista, St Louis, MO 63110, USA. E-mail: [email protected], Tel: þ1-314-577-8793, Fax: þ1-323-268-5062.

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network. Epithelial cells are interconnected with tight junctions, gap junctions and the like. They display internal polarity. Organelles such as mitochondria are localized to certain regions within each cell. Mesenchymal cells are nonpolar: they do not have ‘sides’. Mesenchymal cells are not attached to each other: they can migrate within an extracellular environment. Mesenchymal cells are internally homogeneous: organelles are not concentrated in specific zones within the cell. Mesenchyme of the head and neck come from two main sources: paraxial mesoderm (PAM) and neural crest. Neural crest mesenchyme will be discussed later. Paraxial mesoderm results from the physical act of gastrulation, the creation of a trilaminar embryo. At the time of gastrulation the embryo is a simple bilaminar structure consisting of two epithelial layers, a dorsal epiblast and a ventral hypoblast (also known as the primitive endoderm). The hypoblast floats on top of the yolk sac. A midline primitive streak forms in the epiblast appears first at its caudal end, the connecting stalk; it extends forward about two-thirds of the distance of the epiblast. It provides a means by which cells living near its border lose their epithelial characteristics and become individual mesenchymal cells capable of independent migration. These pass into the primitive streak. At any given neuromeric level, they form specific new structures depending upon the timing of their passage into the primitive streak (see Fig. 17.8). The first cells to ingress cluster beneath the midline to form the notochord; these multiply rapidly in a lateral direction. This causes the hypoblast cells to be pushed out laterally (O’Rahilly and Muller, 2001). Eventually the new layer completely covers the undersurface of the epiblast and lines the yolk sac. It is now called the definitive endoderm. Endoderm does not demonstrate overt signs of segmentation. Nevertheless, it is organized into developmental zones (endomeres) in perfect register with the neuromeres of the neural plate. Like the notochord, each endomere bears a unique pattern of homeobox gene expression. The next population of epiblast cells to enter the primitive streak can now migrate between the epiblast above and the endoderm below. These cells form the mesoderm. This consists of two zones. The first wave of migrating mesodermal cells enters the primitive streak early. These cells aggregate close to the midline to form the PAM. Genes elaborated in the midline notochord and in the midline neural plate induce the PAM to round up into discrete structures called somitomeres (Sm). Each neuromeric level, beginning at the first rhombomere (r1) produces a pair of somitomeres, one on either side of the notochord. As gastrulation proceeds in an orderly cranio-to-caudal sequence,

somitomeres make their appearance at regular time intervals. The first seven somitomeres are incompletely segmented. The PAM cells are oriented around a central cavity, the somitocele. When 11 somitomeres are produced (i.e. at the end of the medulla) the end of the central nervous system (i.e. r11) is reached (Tessier, 1969) (see Fig. 17.9). A later wave of mesodermal cells originates from more peripheral regions of the epiblast. These cells fan out toward the periphery of the embryo and forms a flat layer called the lateral plate mesoderm (LPM). Gene products in the lateral aspect of the epiblast will induce the LPM to form a dorsal, somatic layer (LPMs) and a ventral, visceral layer (LPMv). It makes sense that the LPMs is in register with its overlying ectomere and the LPMv is in register with the underlying endomere (see Fig. 17.10). The posterior aspect of the vertebrate braincase is produced not from somitomeres but from occipital somites. Like all somites, these possess sclerotomes that fuse together to form the basioccipital, exoccipital and supraoccipital bones. The number of occipital somites varies with the organism. Avian embryos possess five while mammalian embryos possess four (abbreviated O1–O4). In the avian model, at the appearance of the 19th somitomere an anatomical transformation of the eighth somitomere takes place. The caudal end of Sm7 becomes completely separate from that of Sm8. The cranial end of Sm7 remains incompletely separated from Sm6. This raises the question as to whether this is an anatomical basis for the parasegmentation pattern seen in all subsequent somite derivatives (see Fig. 17.23). In birds, Sm7 also develops a sclerotome and a myotome (but no dermatome); it becomes the first occipital somite (O1). Production of Sm13 coincides with the transformation of Sm8 into O1. This is the first completely epithelialized somite; both its cranial and caudal borders are distinct. Elegant studies by Huang (2000) have mapped the avian mastoid process, sternocleidomastoid and trapezius muscles to the level of Sm7/O1. The spatial pattern in which the five avian sclerotomes amalgamate to produce the skull base is also well demonstrated as well (see Fig. 17.20). In mammals, the pattern is slightly different. Sm8 is transformed into O1. This structure is completely epithelialized. All remaining somite formation proceeds in exactly the same manner to produce the vertebral column and peripheral mesodermal structures, all of which are unified in a segmental fashion by the peripheral nervous system. Despite these differences the topology used by mammalian sclerotomes to produce the cranial base can be considered to follow an analogous pattern. Mueller and O’Rahilly (1997,

NEURAL TUBE PROGRAMMING AND CRANIOFACIAL CLEFTS: PART II 2003) document the contributions of each occipital somite in human materia (see Figs. 17.21, 17.22 and 17.24). The notochord and neural tube serve as an axis dividing the embryo into dorsal and ventral sectors (Liem et al., 2001). All structures relating to the notochord and nervous system are considered epaxial; these are innervated by dorsal motor and sensory nerves. All other structures of the embryo are considered hypaxial; these are innervated by ventral motor and sensory nerves. Craniofacial PAM thus has two primary fates. Epaxial PAM will be involved in the production of the protection of the brain, i.e. it forms part of the neurocranium. Epaxial PAM structures include the entire skull base posterior to the pituitary, the parietal bone and the petro-mastoid temporal complex. Hypaxial PAM becomes reorganized into the muscles assigned to the pharyngeal arches, i.e. it is associated with of the splanchnocranium. The cartilages and bones of the pharyngeal arches are of neural crest origin. Thus the skeletal components of PA4 and PA5 come from neural crest (see Fig. 17.19). Careful mapping of derivatives in the chick embryo by Noden (1985, 1991a; Noden and Trainor, 2005) demonstrates great homology with mammals (see Fig. 17.25). The transformation of PAM into pharyngeal arches (in mammals numbering four) occurs concomitantly with the formation of the neural tube. Beginning with r1 each neuromeric level of the hindbrain is associated with flanking somitomeres. The first somitomere is large and is situated at the level of the pituitary. It was formerly considered not to be involved in pharyngeal arch formation. Recent evidence supports the existence of a previously unappreciated premandibular arch (Kuratani et al., 1997; Kuratani, 2005; Tapadeia et al., 2005). This structure is attached to the cranial base corresponding to the anterior cranial fossa. It is responsible for synthesis of ethmoid, sphenoid, vomer, premaxilla and maxilla. The premandibular pharyngeal arch is referred to here as PA0. The next three arches are formed by pairs of somitomeres. Sm2 þ Sm3 ¼ PA1, Sm4 þ Sm5 ¼ PA2, Sm6 þ Sm7 ¼ PA3. The same pairing probably applies to Sm8–Sm11: Sm 8 þ Sm9 ¼ PA4 and Sm10 þ Sm11 ¼ PA5. In each case, the formation of a pharyngeal arch involves a physical amalgamation of mesoderm. Derivatives of each arch come from spatially distinct sectors. These are distinguished by the expression patterns of genes unique to each sector of the arch (Lumsden and Keynes, 1989; Lumsden et al., 1991; Hunt and Krumlauf, 1992; Krumlauf, 1993; Lumsden and Krumlauf, 1996) (see Fig. 17.17). The spatial organization of each pharyngeal arch faithfully reflects the contributions of its predecessor

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somitomeres. For example, Sm2 forms the cranial half of PA1 while the caudal half develops from Sm3. The zygoma (formed from r2 neural crest) has no associated muscles from Sm2. The squamous temporal bone and the mandible both arise from r3 neural crest. These structures are connected by muscles of mastication originating from Sm3 and uniting both bones. The clinical model of macrostomia suggests that an embryological ‘fault line’ exists along the axis of the first and secondary pharyngeal arches. Embryonic folding turns these arches 90 into contact with the ventral surface of the prosencephalon. The future mesenchyme of the maxilla (Sm2 and r2 neural crest) is now dorsal to that of the mandible (Sm3 and r3 neural crest). The lateral facial cleft (#7) running from the oral commissure toward the ear reproduces this genetic boundary zone (Carstens et al., 2005). In this model, PAM behaves quite differently depending on whether it is epaxial or hypaxial. The epaxial component of each somitomere (or somite) retains a physical relationship to the nervous system. This is most easily recognizable in the vertebral column. Each vertebral body is formed from the sclerotome of two adjacent somites. This pattern, in which the cranial half of somite n combines with the caudal half of the somite above it, n-1, is known as parasegmentation (Lawrence, 1988; Larsen, 1997). For example, the third thoracic vertebra forms from the cranial half of somite T3 and the caudal half of somite T2. This pattern becomes less obvious at the skull base. The atlas is formed from cervical somites 1 and 2. The ancient proatlas (now incorporated into the foramen magnum) is formed from occipital somite 4 and cervical somite 1 (Kessel and Gruss, 1991; Larsen, 1997) (see Figs. 17.11 and 17.15). The sutures of the skull represent distinct segmental field boundaries. These bones (arising at rhombomeric levels 1–7) do not manifest parasegmentation. The mammalian parietal bone is produced by epaxial PAM from Sm2 and Sm3 (as is the temporalis muscle) while the temporal bone is synthesized from Sm6 and Sm7. In mammals, epaxial PAM from Sm4 and Sm5 is not involved in bone formation; its raison d’eˆtre is to provide source material for the muscles of facial expression assigned to the skull. Frontalis is probably an Sm4 derivative while occipitalis comes from Sm5. Parasegmentation first makes its appearance with the occipital somites (arising at rhombomeric levels 8–11). No obvious ‘cranial vertebrae’ exist. Instead, the sclerotomes of O1 through the cranial half of O4 fuse together to form all elements of the skull base posterior to the pituitary. The basioccipital, exoccipital and supraoccipital bones each receive contributions

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from all four occipital somites. These bones are composite fields. Parasegmentation is an epaxial phenomenon. Hypaxial PAM of the somitomeres and somites behaves in a very different manner. In the head and neck it is strictly segmented, being ‘assigned’ to an individual pharyngeal arch. All muscles of mastication arise from Sm3. Facial muscles assigned to the maxilla and mandible come from hypaxial Sm4–Sm5. Sm6 produces levator veli palatini while the superior constrictor comes from Sm7. Theoretical origins of middle and inferior constrictors would involve Sm9 and Sm11 respectively. The anatomical gaps between the constrictors could represent field boundaries of PA3, PA4 and PA5. Fig. 17.13 illustrates the assembly of vertebrae. The fate of hypaxial PAM in the trunk differs from that assigned to the extremities. We have seen that in somites the lateral dermatome and myotome form the skin and muscles of the body wall and extremities. Because trunk musculature develops in a very straightforward segmental manner the ventral (hypaxial) motor and sensory nerves that supply it are arranged in a logical, linear, spatial pattern. Muscles such as the external oblique arise from multiple myotomes. Their motor nerves receive contributions from several neuromeres. Sensory nerves such as the iliohypogastric reflect multiple dermatomes. Muscles assigned to the extremities have a different neuroanatomy. They arise from a unique portion of the somitic myotome and undergo complex migratory patterns to seek out their levels of insertion. For this reason the ventral motor and sensory nerves supplying the limbs are organized into complex ‘switchyards’ called plexuses. The topological arrangement of the roots, trunks, divisions, cords and branches of each plexus is a faithful replica of the migratory patterns of the target muscles. Shortly after gastrulation PAM becomes organized transversely into sequential blocks (somitomeres) in a strict cranio-caudal manner. Each somitomere is spatially divided into an epaxial zone and a hypaxial zone. The epaxial zone is sessile and relates to the nervous system. The hypaxial zone of the somitomeres proliferates into the pharyngeal arches. This occurs as soon as the somitomere is fully formed. This early proliferation explains why Sm8–Sm11 can form both pharyngeal arches and occipital somites. The transformation of mesenchyme into an arch takes place first. The remaining mesenchyme is subsequently converted into a somite. For this reason, pharyngeal arches are visible before occipital somites make their appearance. To consider the biological rationale for the timing of this arch/somite sequence it is necessary to return

to gastrulation. Cells exiting the primitive streak at any given neuromeric level do so in a rigid spatiotemporal sequence (Rossant and Tam, 2002). This is seen clearly in the formation of the various zones of mesoderm. The space into which mesodermal cells migrate can be thought of as a ‘pocket’. Two such pockets (left and right) exist at each neuromeric level. The earliest cells to enter the pocket travel most distally. They fall to the bottom of the pocket. Subsequent migrations occupy progressively more superficial levels of the pocket. When each pocket is full of cells, a finite amount of time has elapsed and a new pocket begins to form at the next most caudal neuromere. Maturation of cells also follows a timing sequence. The most distal cells of the pocket are the ‘oldest’; these are more likely to undergo population expansion than more recently arrived cells. If the distal population of each somitomere is fated to become pharyngeal arch this transformation will precede that of the proximal population. Thus, arch formation will always take place before somite formation. In development, the ‘decision’ as to what zone of mesoderm is fated to become lateral plate and what zone will be transformed into paraxial is a chemical one determined by gene products expressed in the overlying ectoderm and neural tube. These chemical signals can be thought of as radio waves emitted from broadcasting towers located at specific anatomical zones of the embryo. Signals from the ectoderm induce LPM and signals from the notochord and neural tube induce PAM. Each chemical signal diffuses outward, its concentration decreasing at a greater distance from its source. Mesodermal cells closer to the neuraxis will ‘listen’ more attentively to the stronger signals from the midline than to those from the periphery. They will organize into somitomeres. In a similar fashion each somitomere and somite can be though of as made up of distinctive zones, each reflecting the presence of specific combinations of gene products unique to that zone. This ‘genetic map’ specifies all future structures (dermis, muscle, cartilage and bone) that will develop from that somitomere. When Sm2 and Sm3 amalgamate into the first arch the caudal sector of PA1 contains (from distal to proximal) mandibular fields Mn1, Mn2, Mn3 and MnR. Because embryonic folding places Sm1 and Sm2 into direct contact, these amalgamate to form the theoretical premandibular arch (PA0). The maxillary fields of PA0/Mx1, Mx2, Mx3 and P are set directly opposite those of PA1. Similar genes in each arch are involved with the definition of each toothbearing field. This provides a genetic basis for occlusion. More posterior to the maxillary zone of Sm2 lies a separate genetic environment corresponding to the

NEURAL TUBE PROGRAMMING AND CRANIOFACIAL CLEFTS: PART II palatine bone. This mesenchyme has the capability of ‘programming’ neural crest cells that subsequently arrive to populate the zone such that they produce the actual palatine bone. All cells of a particular somitomere share a common Hox gene code with their corresponding neuromeric zone in the central nervous system. However, somitomeres subsequently display a complex array of genetic environments based on a very simple system of cellular age and distance from important sources of gene products produced in surrounding structures (see Figs. 17.14 and 17.15). This system is readily seen in the relative amounts of cellular material ‘assigned’ to form pharyngeal arches versus occipital somites. Proceeding caudally, the relative size of each pharyngeal arch becomes smaller and smaller compared to its corresponding somite. There is also a change in the type of product produced by the pharyngeal arch mesenchyme. Beginning with PA4, neural crest mesenchyme ceases to be transformed into bone. Instead it forms cartilage. By Sm12 no further pharyngeal arches are produced; all PAM cells are organized exclusively into the somite form. 17.2.2. Anatomy of pharyngeal arches All pharyngeal arches result from the fusion of proliferating cell populations from two adjacent somitomeres. These combine in a stereotypical manner. The proximal zone of each arch (i.e. that closest to the skull) is formed from the more dorsal/epaxial aspect of the somitomeres. The distal zone of each arch is formed from the more caudal/hypaxial somitomere. In knockout experiments conducted on the first arch, genetic expression in the distal zone affects the mandible while those causing disturbances in the proximal zone affect the maxilla. However, the so-called first arch may in fact be an amalgam of Sm1–Sm3 with Sm1–Sm2 making the premandibular arch while Sm2–Sm3 make up the traditional mandibular arch. Another set of observations pertains to the timing with which various anatomical structures make their appearance. The mandible (r3) forms before the maxilla (r2). The malleus is a homolog of the articular bone (a dermal bone of neural crest origin ensheathing the proximal end of Meckel’s cartilage). Thus the malleus is an r3 derivative. The primitive palatoquadrate cartilage of the upper jaw is analogous to Meckel’s cartilage. It has three components seen in the acanthodian skull. Of all jawed fishes (gnathostomes) the acanthodians have the earliest fossil record. The anterior palatoquadrate is known as the autopalatine cartilage. This provides the basis for the maxilla and palate. Immedi-

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ately behind is the metapterygoid cartilage, the future template for the alisphenoid bone. Most posterior is the quadrate cartilage. This is the origin of the incus. Not surprisingly, it is anchored to the prootic temporal bone. Thus the incus (an r2 derivative) lies internal to the malleus (an r3 derivative). Malleus forms before incus. In summation, all connections between the skull and PA1 are mediated through r2 structures. These represent the proximal portion of PA1. However, r3 structures always form before corresponding r2 structures. The same pattern obtains for derivatives of PA2 and PA3. Facial muscles develop in an absolutely stereotypical manner: from ventral to dorsal, deep (internal) to superficial (external), and lateral (caudal) to medial (cranial) (Gasser, 1966). It is logical to ‘assign’ the muscles of the upper division of the facial nerve to epaxial Sm4–Sm5 and those of the lower division to hypaxial Sm4–Sm5. The upper portion and lesser cornu of the hyoid come from PA2, as does the styloid process. The hyoid appears well before the styloid but the latter is attached to the temporal bone. As previously stated, pharyngeal arches result from cellular proliferation of the hypaxial aspect of somitomeres Sm2–Sm11 (r2–r11). The first somitomere (r1) is exclusively dedicated to the head. The epaxial portions of Sm2–Sm7 are anchored to the cranial base. The epaxial portions of Sm8–Sm11 are organized into O1–O4, from which form the basioccipital bone and the posterior cranial fossa (exoccipital, supraoccipital bones). In mammals the level of Sm11 marks the end of the pharyngeal arch system. All somitomeres from Sm12 caudally are transformed completely into somites with identifiable anatomical components (dermatome, myotome and sclerotome). When pharyngeal arches develop, the zone of adhesion between the caudal somitomere of the first pair and the cranial somitomere of the next pair must break down. This could simply result from forces produced by cellular proliferation and embryonic folding. When distal Sm3 migrates downward it must disrupt its connection with Sm4. Evidence of somitomeric pairing shows up in neural crest migration routes. Although neural crest cells are produced at each neuromeric level they appear to migrate out from only evennumbered rhombomeres. Thus all neural crest migration into PA1 proceeds via r2, migration into PA2 proceeds via r4 and migration into PA3 proceeds via r6. Some investigators postulate that something ‘goes wrong’ with neural crest cells at odd neuromeric levels. But this flies in the face of evidence regarding programming of neural crest that is neuromere-specific. For example, neural crest cells with the Hox gene ‘tattoo’ of r3 are never found in Sm2. If r3 neural crest

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cells are transplanted into the proximal (Sm2) portion of PA1, two mandibles are produced. The stacking model provides a more likely explanation from neural crest behavior. Rhomboencephalic neural crest migration takes place after the physical formation of the arches. For example, as soon as PA3 is assembled, neural crest cells enter it via r6. In the meantime, PA4 is developing. At this juncture we have a clear picture of how a pharyngeal arch is constructed. Furthermore, individual bones and muscles can be assigned to neural crest and PAM from specific neuromeric levels. Kjaer and co-investigators painstakingly catalogued the order of appearance of craniofacial bones. When neuromeric mapping is applied to these structures the initial results often appear contradictory and confusing. If the maxilla comes from level r2 and the mandible from r3, it is difficult to see why mandibular structures should appear first. Why does dental development follow a mesial to distal plan? Since gastrulation is a cranialcaudal process the cells of Sm2 are undoubtedly ‘older’ than those of Sm3. When it comes to assessing the ‘activation’ of PA1 it appears that it occurs in a distal to proximal manner, i.e. cells from Sm3 form products well before those from Sm2. Why biologically ‘younger’ cells produce derivatives before their elders is explained by the mechanism with which blood supply is distributed to the pharyngeal arches. 17.2.3. Vascularization of the pharyngeal arches: the nutritional basis of derivative formation Successive sprouting of aortic arches from the heart occurs in a strict spatio-temporal sequence (Noden, 1991b; Graham and Smith, 2001). Recall that, at the time of pharyngeal arch formation, the heart has migrated from the anterior aspect of the embryo. It comes to lie in a ventral position just beneath the future pharynx. At the time of aortic arch development the heart is in the process of descending into the body cavity. As it does so, it leaves behind it a series of paired aortic arch arteries, each one of which is ‘assigned’ to supply the pharyngeal arch immediately above it. The very first such artery connects with the cephalic margin of the ipsilateral dorsal aorta. This creates the so-called primitive maxillary artery. This vessel is eventually reconfigured to become the anterior cerebral artery supplying all areas of prosencephalon covered by dura innervated by cranial nerve V1. The exact anatomical manner in which the outflow tract from the heart supplies the pharyngeal arches is remarkably simple and elegant. Diagrams in books based on the gill arches of fishes are quite misleading. A typical illustration depicts a core artery running up the center of each gill arch in ventral–dorsal fashion

to anastomose with the dorsal aorta. How might this happen? One never sees a central vascular core running through the axis of a muscle. Instead, arteries and motor nerves travel in the interstices between muscles, i.e. they follow fascial planes. Each muscle unit is penetrated from without by a nerve. In development this occurs when individual neuromeric units of PAM (somitomeres) lying just outside the neural tube induce neural structures to grow outward and supply them. The arterial supply is derived by induction from the nerve via vascular endothelial growth factor (VEGF) (Ruberte et al., 2000; Mukouyama et al., 2002). As somitomeres are formed strictly from PAM and the pharyngeal arches arise by fusion of paired somitomeres, supplied by a single sensory nerve with nuclei in each rhombomere, the artery, in order to gain access to each pharyngeal arch, must follow an embryological plane between its constituent somitomeres. This results from an induction of surrounding PAM by the cranial nerve into an arterial axis. In the head and neck all muscles are surrounded by fascia derived exclusively from neural crest cells. As neural crest cells spread over each pharyngeal arch they penetrate the PAM via natural cleavage planes separating genetically myoblast populations. Chapter 16 described in detail the spatial relationships among the fascial planes of the body and stressed that relative positions of motor neurons in the neural plate (and neural tube) faithfully replicate the spatial location of their muscle ‘targets’. Muscles close to the vertebral axis such as the paraspinous group are classified as epaxial. Their motor neurons lie close to the midline in the medial lamina of the medial motor column (MMCm). Muscle groups hypaxial to the midline but not assigned to the extremities have neurons in the lateral lamina of the medial motor column (MMCl). Muscle groups of the extremities have motor neurons still more laterally located in the neural plate. These form the lateral motor column. Ventral muscles of the limbs are supplied by neurons from the LMCm. Dorsal muscles of the limbs are supplied by neurons from the LMCl. All motor nerves to striated muscles use Schwann cells for axonal insulation. These Schwann cells are of neural crest derivation. Because neural crest defines the fascial planes between muscles it is logical that all neurovascular structures make use of these planes in order to access their target muscles (see Fig. 17.18). The spatiotemporal order of appearance of muscles within the pharyngeal arches has a great deal to do with blood supply. The overall pattern is ventral to dorsal, caudal to crania, and medial to lateral (in that relative order). Muscle development requires metabolic activity; this in turn requires blood supply. Thus

NEURAL TUBE PROGRAMMING AND CRANIOFACIAL CLEFTS: PART II the order in which muscles develop within a pharyngeal arch reflects the penetration pattern of the arterial supply to that arch. Each aortic arch vessel begins as lateral plate mesoderm and ascends from the ventrally positioned heart to reach the caudal aspect of the corresponding pharyngeal arch. It connects with the PAM-induced vascular axis between the somitomeres and ascends upward to anastomose with the ipsilateral dorsal aorta. Thus, when blood supply is established, it will reach caudal myoblasts before cranial ones, deep myoblasts before superficial ones and posterior myoblasts before anterior ones. The spatial relationships between somitomeres and aortic arches with relation to the rhombomeres are shown in Fig. 17.16. In this model, the anatomy of the aortic arches can be interpreted as follows: 1. The first aortic arch (AA1) supplies Sm1 along its deep and caudal aspects. AA1 forms the primitive maxillary artery; it is eventually incorporated into the internal carotid axis as the anterior cerebral artery. The anterior cerebral artery supplies all fronto-orbital-nasal structures innervated by V1. It irrigates the sphenoid and orbit as well as the supraorbital and trochlear vessels. Thus AA1 provides blood supply to all derivatives of Sm1 and all neural crest derivatives from r1 forward. This is the arterial axis of all bone and soft tissue structures originating from prosencephalic and mesencephalic neural crest. The nucleus of V1 resides in r1. All dura innervated by V1 is synthesized from r1 neural crest. Thus it is natural that the blood supply for this dura arises from AA1. 2. The second aortic arch (AA2) supplies Sm2 and Sm3, i.e. PA1. The principal derivatives of AA2 are: a) hypaxial ¼ internal maxillary artery; b) epaxial ¼ middle meningeal artery. The remaining dura of the prosencephalon (temporal, parietal, and occipital lobes) is synthesized by neural crest from r2 and r3. Blood supply for dura comes from the middle meningeal artery. Quite logically, this arises as a separate branch of the internal maxillary artery. 3. The third aortic arch (AA3) supplies Sm4 and Sm5, i.e. PA2. The principal derivatives of AA3 are: a) hypaxial ¼ facial, posterior auricular (including the posterior tympanic); b) epaxial ¼ superficial temporal. 4. The fourth aortic arch (AA4) supplies Sm6 and Sm7, i.e. PA3. The principal derivatives of AA4 are: a) hypaxial ¼ ascending pharyngeal; b) epaxial ¼ posterior meningeal. 5. The fifth aortic arch (AA5) supplies Sm8 and Sm9, i.e. PA4, O1 and O2. The principal derivatives of AA5 are hypaxial ¼ superior thyroid.

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6. The sixth aortic arch (AA6) supplies Sm10 and Sm11, i.e. PA5, O3 and )4. The principal derivatives of AA6 are: hypaxial ¼ inferior thyroid. Important deductions about the neuromeric basis of the intracranial arterial system can be made from a careful analysis of dural innervation. Properly considered, dura covers only the prosencephalon. Dura is a PAM derivative. Below the tentorium cerebelli the midbrain and hindbrain are covered only by pia and arachnoid, both neural crest derivatives. The innervation of the prosencephalic dura comes from V1–V3 with small contributions from VII and IX (via X) to the basisphenoid/basioccipital. Prosencephalic dura lines the surface of the anterior and middle cranial fossa. The floor of the posterior cranial fossa below the tentorium is lined by periosteum. Posterior to the foramen magnum this is supplied by C2–C3. Anterior to the foramen magnum, posterior fossa dura has glossopharyngeal innervation via vagus twigs. The innervation to the remainder of the calvaria mimics that of the overlying scalp. Because the nuclei of the trigeminal, facial and glossopharyngeal nerves reside in r1–r7, all neural crest cells supplying sensory nerves to the dura arise from these levels. All mesoderm forming the dura and blood vessels to it arises from Sm1–Sm7. Thus, the origin of the internal carotid supplying the brain as well as the external carotid supplying the face arises from the aortic arches and aortic roots corresponding to Sm1–Sm7. These are, specifically, AA1–AA4.

17.2.4. Anatomy and fate of non-pharyngeal-arch paraxial mesoderm Not all PAM participates in the formation of pharyngeal arches (strictly hypaxial structures). Sm1 is exclusively dedicated to the orbitosphenoid and anterior cranial fossa. As discussed above, the ventral aspect of Sm2–Sm11 is dedicated to arch formation. The following model is proposed for the dorsal portion of a somitomere. 1) The epaxial regions of Sm2–Sm3 and Sm6–Sm11 maintain a direct anatomical relation to the brain. These somitomeres provide PAM mesenchyme for coverage of the middle and posterior cranial fossae. 2) In mammals, the epaxial regions of Sm4–Sm5 fuse, with along the external aspect of Sm2–Sm3. Myoblasts from Sm4–Sm5 give rise to the frontal and occipital muscles. These derivatives are all external to those of Sm2 and Sm3; therefore they are not involved in production of the skull. The formation of specific components of the skull from PAM is discussed by Mossi-Kay (2001) and Jiang

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et al. (2002). The parietal bone is made up of PAM from Sm2–Sm3. The squamous temporal bone is formed from Sm3. Sm6 forms the petrous temporal bone and Sm7 the mastoid temporal bone. Sm8–Sm11 are subsequently converted into occipital somites with sclerotomes. In the vertebral column proper the sclerotomes of each somite combine to form vertebral bodies. Signals involved in the induction of the medial somite arise from the notochord and neural tube. Hence it is not surprising that the vertebral bodies encase these structures. Remnants of the notochord persist as the nucleus pulposus. This situation exists in the cranial base as well. Sm11 and Sm12 combine to form the proatlas (the first true cervical vertebra, bringing the total to eight). In mammals a vestigial proatlas persists as three structures: the rostral tip of the dens, the dento-occipital ligament and the condyles of the exoccipital bone. The course of the rostral notochord is as follows. Via the dens and the dento-occipital ligament it gains access to the basioccipital bone. This is formed as the fusion of sclerotomes from O1–O4. The notochord then passes from the basioccipital bone to the basisphenoid bone. This is produced from the PAM of Sm1. The notochord terminates at the junction of the PAM basisphenoid and neural crest presphenoid. Thus the notochord occupies the center of PAM-derived basicranium from r11 forward to r1. Because PAM from somitomeres Sm2–Sm5 is not involved in cranial base synthesis, its derivatives remain lateralized and therefore peripheral to the notochord.

groups of neural crest, defined by their level of origin from the embryonic brain, mature in a fixed temporal order and migrate in unique patterns (Gui et al., 1993; Osumi-Yamashita et al., 1994; Creuzet et al., 2005). First to depart from the neural folds are those neural crest cells associated with the mesencephalon, i.e. r0, r1 and r20. These mesencephalic neural crest (MNC) cells do not participate in pharyngeal arch formation. Instead, they migrate forward in three distinct streams toward the orbit and interorbital midline. These pathways are long; therefore MNC migration is not complete until somite stage 14. Next to mature are cells from the rostral rhomboencephalon r2–r7. These rhomboencephalic neural crest (RNC) cells are assigned to pharyngeal arch formation. Rostral RNC (RNCr) cells migrate in a strictly segmental fashion. Although these cells begin traveling in time immediately after r20 they have a shorter distance to travel and thus arrive at their destination by somite stage 11. Cells from the caudal rhomboencephalon r7–r11 start later still. These cells also travel in segmental fashion a relatively short distance within their respective segments. It is therefore logical that these caudal RNC (RNCc) cells arrive at their destination concomitantly with those from the mesencephalon, i.e. at somite stage 14. Cells from the neural folds above the caudal prosencephalon (i.e. at neuromeric levels p1, p2, p3 and p4) migrate forward as a large sheet. They begin their journey after the departure of the MNC. These prosencephalic neural crest (PNC) cells travel a great distance forward; their migration is not complete until somite stage 16.

17.3. Anatomy of craniofacial neural crest 17.3.2. Prosencephalic derivatives (16-somite stage) All students of the nervous system are familiar with neural crest cells. The importance of these cells for development is so great that they are often referred to as ‘the fourth germ layer’. The biology of the neural crest is summarized in several authoritative reviews (Hall, 1999; Le Douarin and Kalcheim, 1999; Jiang et al., 2002). In this section I shall detail, neuromere by neuromere, the neuromeric organization of the neural crest and the manner in which distinct anatomical zone of neural crest migrate in distinctive ways. Because we must relay on nomenclature derived from neuroembryology, you should study Figs. 17.1–17.5. Figs. 16.4 and 16.11 (previous chapter) should be carefully studied because it shows the order in which the various neural crest populations migrate into their final position. 17.3.1. Three populations of neural crest Neural crest cells from the level of the midbrain posteriorly are produced in a cranio-caudal order. Three

The model as described below represents an amalgamation of the neural crest fate mapping studies of Couly and Le Douarin (1985, 1987) and the prosomeric system of Puelles and Rubenstein (Rubenstein et al., 1998; Cambronero and Puelles, 2000; Puelles and Rubenstein, 2002). However, the existence of six discrete prosomeres is not universally recognized by neuropathologists and neuroscientists, many of whom consider the forebrain to be made up of four prosomeres (Tessier, 1981). These well established studies, done with traditional anatomical methods such as Nissl stains, were carried out without the use of genetic markers. Furthermore, the prosomeric model has undergone recent iteration that clarifies the anatomy of p1–3 but make the boundaries of p4–6 more complex (Puelles and Rubenstein, 2003). What is certain is that the neural folds above the rostral prosencephalon are devoid of neural crest cells. Assignment of anatomical zones of these rostral neural

NEURAL TUBE PROGRAMMING AND CRANIOFACIAL CLEFTS: PART II folds to underlying prosomeric zones seems logical. The rostral folds contain specific structures such as the optic and olfactory placodes; these have been mapped out in avian embryos. Assignment of prosomeric zones as described here is strictly empiric in nature, based upon known craniofacial anomalies that seem to occur in each zone. Particularly difficult are the anatomical boundaries and contents of the p4 neural fold. Although the model I propose is hypothetical, current laboratory techniques should prove capable of refining the neural plate fate map in the mouse and correlating this map with the human prosomeric system (see Figs. 17.6 and 17.7). Migration from the prosencephalic neural crest is designed to provide the forebrain and upper facial midline with mesenchyme to synthesize dermis and bone. As mentioned above, the neural folds of prosomeres p6 and p5 are devoid of neural crest. These zones are made up of non-neural epithelium only. All epithelia require an underlying support network (usually in the form of mesenchyme) in order to survive. Neural crest cells appear over the posterior prosencephalon (at prosomeres p4, p3, p2 and p1). This prosencephalic neural crest population must advance forward and populate the subepithelial plane of p6– p5. In this way, PNC ensures the viability of the non-neural epithelial zones. It also provides the mesenchyme necessary for the synthesis of bone and cartilage structures specific to each zone. Cellular movement of PNC takes place more in the form of a sheet than as clearly identifiable streams. The order in which the target zones are populated would probably be caudal–rostral. This is supported by the known sequence of placodal development, which is: otic, optic and then olfactory (Webb and Noden, 1993; Streit, 2004). Failure of neural crest to populate the subplacodal zone leads to placodal dysfunction or outright absence. The role of neural crest in p4 is uncertain. It is likely to correspond to the frontal bone. Avian parietal bones are strictly neural crest but in mammals significant amounts of PAM (probably from Sm3) are involved in parietal osteogenesis. These mesodermal cells are associated with the temporalis muscle. Membranous ossification requires a ‘stimulating mesenchyme’ (neural crest dura or dermis) and a ‘responding mesenchyme’ (neural crest or PAM). In some parts of the skull, a responding mesenchyme may be sandwiched between two layers of stimulating ectoderm or endoderm. Bilaminar membranous bone formation occurs. In the calvaria the potential space between the laminae is manifested by the presence of a diploic space. Another example of bilaminar bone fields explains the anatomy of the sinuses of the skull. These occur at sites where mucosa from oropharyngeal

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cavity has anatomical access to that potential space. By exploiting field separation planes, the mucosa will expand into the frontal, ethmoid, sphenoid and mastoid bones to create their respective sinuses but it will never transgress a neuromeric boundary, i.e. a suture. All calvarial bones formed by neural crest and PAM cranial to the first motion segment (r11–c1 junction, the proatlas) are segmental, i.e. the bone boundaries correspond to neuromeres. At the craniocervical junction vertebrae become, for the first time, parasegmental, i.e. each vertebral body results from contributions of the caudal somite above and the rostral somite at that level. PNC migration into the frontonasal zones of p5 and p6 is a lateral to medial process that ‘recognizes’ and flows around pre-existing MNC populations that cover the forebrain and surround the optic vesicle. Like guided missiles, the cells of the PNC home in on three ‘target zones’ of non-neural ectoderm: the calvarial epithelium, the optic placode, the upper beak epithelium, the nasal epithelium and the nasal placode. In every case the migration of PNC into involves a succession of lateral to medial cell movements, each zone advancing further forward, building upon its predecessor. Thus the flow of PNC through prosomeric zone p5 is forward and medial: each zone of p5 is ‘newer’ than its predecessor. Neural crest entering the sixth prosomeric zone is the most recent of all. Pathological correlations follow from this anatomy. The adenophyseal placode is the most medial p6 structure. Thus, isolated failure of the adenophyphyseal placode, with consequent pituitary insufficiency, can occur in the presence of a normal face. The presence of p5 neural crest is a requirement for p6 PNC to reach its target. Heminose or arhinia can certainly occur in the presence of a normal eye. However, if an optic placode does not develop, the ipsilateral nasal structure will appear as a tubular appendage, often attached at the level of the canthus. This finding is characteristic in cyclopia (see Fig. 17.35). 17.3.2.1. Prosomere 5 The first zone of non-neural ectoderm zone to be populated with PNC mesenchyme is that of the calvarial epithelium. This migration is external to the future orbit and comes in above what will become the orbital rim. Formation of the prefrontal zones occurs from lateral (PFl) to medial (PFm). The frontal bone is considered by many comparative anatomists to represent the amalgamation of several distinct membranous bones in ancestral tetrapods. The orbital rim zones may thus represent the previously separate medial and lateral prefrontal bones, separated by the supraorbital

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neurovascular of V1. Neural crest then stacks up upon itself to form dermis beneath the calvarial epithelium. The skin of the forehead is built upon the skin of the orbital rims. So too is the synthesis of the frontal bone. For this reason ossification of the orbital rims takes place before that of the frontal tubers. After formation of the dermis PNC in the remaining p5 neural fold occupies a potential space between two osteoinductive substrates: the r1 forebrain dura previously laid down by MNC and the more recently produced p5 dermis. Each of these layers will induce membranous osteogenesis to take place in the residual mesenchyme. A bilaminar frontal bone results from a dermis-induced external lamina and a dura-induced internal lamina. This potential space communicates with the nasal cavity via the frontonasal duct. When nasal epithelium invades and exploits that potential space the frontal zone of the frontal sinus results. The second zone of non-neural ectoderm to be populated by PNC is that of p5, within which is located the optic placode. Neural crest flowing around the optic placode causes it to become ‘activated’. Interaction between the optic placode and the optic vesicle induces the latter to form the optic cup, into which the placode is incorporated as the lens. At the same time placodal neural crest mesenchyme flows inward to surround the globe. This neural crest is stimulated to form membranous bone by the dura of the basal forebrain laid down previously by r1 MNC. This can be called the cranial lamina of the fronto-orbital bone. At the same time r0/r1 dura/sclera associated with the globe serves as an inducing agent, causing placodal p5 neural crest to form a transient cartilaginous orbital capsule surrounding the globe. (In birds this forms a ring of chondral bones arranged rather like ball bearings around the orbital rim.) In mammals the orbital capsule is converted into the orbital lamina of the fronto-orbital bone. Exploitation of the potential space between these two layers by nasal epithelium results in the orbital zone of the frontal sinus. The p5 neural crest making up the two laminae of the fronto-orbital bone is divided into two zones by the supraorbital neurovascular pedicle. The lateral fronto-orbital zone (FOl) is the site of the Tessier #10 cleft. Frontal sinus pneumatization usually does not extend into this zone, hence FOl tends to be very thin. This may explain why clefts in this zone present as an encephalocele herniating into the lateral orbit. FOl is in continuity with PFl of the lateral orbital rim; therefore zone 10 clefts tend to affect the lateral eyebrow. The medial fronto-orbital zone (FOm) is the site of the Tessier #11 cleft. This zone is always pneumatized. Perhaps this is why encephaloceles are not as common here. FOm is in continuity with PFm

of the medial orbital rim; therefore zone 11 clefts tend to affect the medial eyebrow. Neural crest excess or tumors in either zone of the fronto-orbital bone can result in significant orbital dystopia. Extending caudally from FOm, the p5 neural crest forms a separate bone from the orbital capsule. This is the thin orbital lamina of the ethmoid, i.e. the lateral wall of the ethmoid sinuses. The ethmoid sinuses are in anatomical continuity with the frontal sinuses but these sinuses remain separate because they form within different fields. An excess accumulation of p5 neural crest within the orbital lamina of the ethmoid labyrinth will cause lateral expansion of the labyrinth; hypertelorism will result. The final p5 product within the orbit is the lacrimal bone. This bone sits anterior to the orbital lamina of the ethmoid. A medial extension of r2 called the frontal process of the maxilla (MxF) forms the most medial aspect of the inferior orbital rim and approaches the lacrimal bone but must straddle around the lacrimal sac to reach it. It accomplishes this goal by producing two tongues of mesenchyme, anterior and posterior. Where these contact the lacrimal bone the anterior and posterior lacrimal crests are produced. The lacrimal bone extends caudally downward from the orbit as a lamina that terminates just lateral to the inferior turbinate. The space between these two laminae (r2 medial and p5 lateral) is exploited by mucosa to form the lacrimal sac and duct. The proper formation of the p5 lacrimal bone is dependent on the physical integrity of the r2 inferior turbinate. If no inferior turbinate is present, the caudal portion of the lacrimal bone will be deformed or absent. On the other hand, excessive neural crest in the p5 lacrimal field or failure of this field to undergo appropriate apoptosis may result in lacrimal duct stenosis or frank obliteration of the lacrimal sac. Frontal bone anatomy is thus complex. The frontal bone proper is bilaminar and occurs between two inductive substrates of dermis and rostral forebrain dura. The fronto-orbital bone is also bilaminar; it occurs between the inductive substrates of basal forebrain dura and the periocular extension of dura (i.e. sclera). Anencephaly involves deficiency of r1 structures. The sphenoid is always deformed. The presence of normal orbital and nasal structures seen in anencephaly testifies to the interactions between p5 dermis and r1 dura, since in the face p5 is autonomous whereas in the forehead its development depends upon an underlying predecessor, r1 dura (see Fig. 17.32). A third zone of p5 non-neural ectoderm to be populated by PNC is that of the future skin covering the nose and philtrum. In birds this is known as the upper beak epithelium (UBE). In mammals the p5 external

NEURAL TUBE PROGRAMMING AND CRANIOFACIAL CLEFTS: PART II nasal skin is in continuity with that of the p6 internal nasal skin (nasal vestibular epithelium). Within each nostril the boundary between p5 skin and p6 vestibular epithelium is readily visible; it lies at the caudal margin of the upper lateral cartilage. The dermis of the UBE is capable of inducing p5 nasal bone formation via membranous ossification. Deficiency states of the p5 nasal fields would explain the clinical picture of short nasal bones described by Sheen and Sheen (1998). In summary: p5 neural crest reaches the facial midline via successive waves of lateral to medial migration, each more distal, anterior and midline than its predecessor. The order of formation of p5 components is as follows: orbital rim (prefrontal lateral, prefrontal medial, nasal process of frontal) with secondary upward growth of frontal bone, optic placode (frontoorbital medial, fronto-orbital lateral, orbital plate of ethmoid labyrinth, lacrimal. These are Tessier clefts #10 and #11. Additional pathology of frontal p5 would have to include developmental excess states as seen in frontonasal dysplasia (Balci et al., 1999; Guion-Almeida and Richieri-Costa, 2001; Gene et al., 2002; Richieri Costa and Guion-Almeida, 2004; Plock et al., 2007). These conditions demonstrate the p5 fields to be shieldshaped. Anencephaly can manifest as gross absence of the frontal bone in concert with loss of forebrain dura; at the same time the orbital rims and the remainder of the face are preserved (Dambska et al., 2003; Dias and Partington, 2004). The latter condition is fundamentally a problem with r1 but the areas of p5 so affected serve to distinguish what part of p5 develops in concert with r1 and what part is independent (see Fig. 17.34). 17.3.2.2. Prosomere 6 The final migration of PNC populates the non-neural ectoderm of the 6th prosomere. This has three subzones: the nasal epithelium, the nasal placode, and the adenohypophyseal (pituitary) placode. The nature of the placodes, coupled with the proliferation of surrounding neural crest mesenchyme, leads to the formation of bilateral nasal chambers. Also, the pattern of closure of the rostral neuropore has a great deal to do with how both sides of the facial approximate each other in the midline (Schoenwolf, 1997; Copp, 2005). Development of the face is dominated by the behavior of four important placodes: pituitary, nasal optic and otic. A placodal region is one in which the surface ectoderm is in direct contact with CNS neuroectoderm. Cellular migration of three types takes place from all placodes. 1) Specialized sensory cells

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are represented by structures such as the otoliths of the labyrinth and the vomeronasal organs flanking the septum. 2) Neuroblasts arising from the lateral olfactory placode provide the apparatus of smell, whereas those that form the medial olfactory placode transmit chemoreceptive data to the brain. 3) Mesenchymal cells are derived from neural crest and/or PAM; these differentiate into the cartilaginous capsules of the presphenoid, the nasal cavities, the orbits and the temporal bone. The natural behavior of placodes is to penetrate (more accurately to become incorporated) into the embryonic CNS. As these placodes ‘sink beneath the waves’ they interact with the brain to induce additional structures. The p6 nasal placodes contribute three sets of neurons that ultimately find their way to at least three different sites in the p6 telencephalon. Without this incorporation, areas of the basal and alar forebrain (the rhinencephalon) are hypoplastic. The p5 optic placode interacts with the p5 optic vesicle to form the lens of the eye. Without this incorporation no lens will form and the globe will be phthisic. Incorporation of the r5/r6 otic placode induces the cochlear and vestibular system. Abnormalities of these structures may represent forms of neurocristopathy. The adenohypophyseal placode is located directly in front of the anterior end of the notochord. It is directly connected to the diencephalon at the future neurohypophysis. These simple relationships are readily seen in the neural fold state prior to closure of the neuropore and head folding. Growth of the telencephalon at this point involves the formation of paired hemispheric vesicles that push laterally from the sidewalls of the neural tube. These cerebral vesicles push rostrally and caudally. They expand so greatly that they eventually envelop the neuraxis. Along with this proliferation of the brain mesodermal tissue (PAM) from Sm1 invades the zone around the adenohypophyseal placode to form the basisphenoid. At the same time r1 neural crest invades anteriorly to form the presphenoid. These mesenchymal tissues force the adenohypophyseal placode to assume a new position facing ventrally within the future pharynx. Work by Kjaer demonstrates that basisphenoid always forms before presphenoid. This is consistent with the idea that PAM from the medial part of a somitomere is laid down first in time before neural crest from surrounding zones of the same somitomere. A similar process explains how the p6 nasal epithelium gets drawn inside the nasal chamber. The traditional view holds that ingrowth of the nasal placodes is responsible for forming the inner nasal architecture. Instead, persistent placodal adherence to the rhinencephalon of the p6 basal forebrain is the key element

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here. Neural crest beneath the p5 nasal skin proliferates forcing the nose to grow outward from the face. The adhesion of the p6 nasal placode to the brain is so strong that the nasal epithelium is drawn inwards. At the same time, placodal traction may be responsible for neuroectodermal tissue being drawn out from the central nervous system as the olfactory bulbs. Alternatively, placodal structure may simply contain the cell adhesion molecules required for neuronal growth. The first zone of p6 non-neural ectoderm to be populated by PNC is that of the nasal vestibular epithelium. Because the PNC migrates in a subepithelial plane, initial location of the mesenchyme is ventral to the nasal epithelium. With head folding and p5/p6 proliferation this situation reverses itself 180 . The p6 nasal epithelium is within the nasal cavity and the neural crest now lies dorsal to it. The upper lateral cartilages are derived exclusively from p6 PNC. The actual ‘program’ that determines the size and geometry of the nasal cartilages resides in the p6 nasal vestibular epithelium. In a similar manner the lower lateral cartilages are programmed by the p5 epithelium. The nasal epithelium dictates to the neural crest exactly where to make the cartilages. For this reason, variations in vestibular lining may explain differences in size or angulation of the nasal cartilages. Prior to closure of the rostral neuropore (RNP) the two potential nasal cavities are widely separated. The remnant of the RNP is the foramen cecum (just above the nasal bones). Closure of the neural folds concludes neurulation and this process is initiated at O4, i.e. at r11. From that direction closure proceeds both rostrally and caudally. Closure of the rostral neuropore occurs before that of the caudal neuropore. However, closure of the RNP is a bidirectional process. Caudal to the RNP, the pattern of closure is directed forward (rostral). Rostral to the RNP, the pattern of closure is directed backward (caudal). Thus closure of the RNP brings together in the midline four zones that were previously separated p5 nasal skin/p6 nasal epithelium. Certainly no nasal chamber can be constructed without a placode. Unilateral or bilateral absence of placodes is the developmental basis for heminose or arhinia (Newman and Burdi, 1981; Govila, 1991; Meyer, 1997; Olden et al., 2001; Shino et al., 2005). The high position of the single nasal placode seen in cyclopia can be understood in terms of a gross deficiency of p5 mesenchyme (Toraynski and Jacobiec, 1982; Hendry et al., 2004; Nagasi et al., 2005; England et al., 2006). In this contracted state the nasal placode is drawn up to the position of the RNP and therefore appears above the common orbit (see Fig. 17.35). The second zone of p6 non-neural ectoderm to be populated by PNC is that of the nasal placode. Neural

crest initiates within the nasal placode three neuronal populations. The lateral zone contains neurons of the olfactory system. These process odors. The medial zone contains neurons of the accessory olfactory system. These process chemicals such as pheromones. Also contained in the medial zone are neurons associated with release of gonadotropin-releasing hormone (GnRH). Each set of neurons is relayed to separate areas of the frontal and temporal lobes (Carstens, 2002). Anterior to the perpendicular plates the r1 neural crest loses its ability to make bone. Instead two leaves of septal cartilage (which appear fused) develop, sandwiched between the two lateral walls of p6 nasal epithelium. The floor beneath the septum consists of two r20 vomerine bones. The septum develops temporally after the perpendicular plates. In holoprosencephaly the perpendicular plate and the septum can be completely absent. This is accompanied by a wide bilateral cleft lip and palate with absence of the vomer/premaxilla. In less severe states the septum and premaxilla may be attenuated. On the other hand, a widened bifid septum results if the p5/p6 nasal complex fails to approximate. The third zone of p6 non-neural ectoderm to be populated by PNC is that of the adenohypophyseal (pituitary) placode. With formation of the primary head fold, the most anterior non-neural ectoderm (zone p6) is tucked ventrally and caudally until it lies just beneath the presphenoid/basisphenoid (PS/BS) junction. This can be visualized by imagining the fingernail of one’s right index finger as the placode and the metacarpophalangeal joint of the same finger as the PS/BS ‘joint’. This is the topology of the neural plate. If one then makes a fist with the hands supine the tip of the index finger (containing the placode) will come to rest beneath the MP (PS/BS) joint. Each p6 zone contains an adenohypophyseal placode. At the anterior extreme of the embryo the p6 zones are continuous with each other, resembling a handlebar moustache. For this reason most texts describe the adenohypophyseal placode as if it were a single entity when, in point of fact, it is bilateral. For this reason the adenohypophysis can be smaller on the same side in the presence of additional forebrain pathology. Tumors of a given cell type can also be unilateral. The physical location of the frontal processes of the maxilla (MxF) and of the premaxilla (PMxF) beneath the PS/BS junction permits upward ‘penetration’ by these placodes to take place. Once within the ‘potential space of the sphenoids’, the adenohypophysis will ultimately be joined along its posterior aspect by extension of the most caudal diencephalons. This extension from basal p1, the neurohypophysis, will descend into the space. The marriage of the non-neural ectoderm adenohypophyseal placodes from p6 (now populated by

NEURAL TUBE PROGRAMMING AND CRANIOFACIAL CLEFTS: PART II neural crest) with the basal diencephalon from p1 creates the pituitary gland. At this site primitive tumors, such as craniopharyngiomas, can bulge beneath the mucosa into the pharynx. At times this can even disrupt palatal formation (DiRocco et al., 2006; Koral and Weprin, 2006; May et al., 2006). Sixth prosomere neural crest reaches the facial midline via successive waves of lateral to medial migration, each more distal (anterior and midline) than the other. The order of formation of p6 components is as follows: anterior cranial fossa (ethmoid labyrinth, crista galli, perpendicular ethmoid plates), nasal placodes, upper lateral cartilages, lower lateral cartilages, septum. These are basically Tessier zones #12 and #13. 17.3.3. Mesencephalic derivatives (14-somite stage) 17.3.3.1. Rhombomere 0: No bone derivatives The existence of r0 as a separate entity distinguished from r1 is a matter of conjecture. For the purposes of this discussion it is useful to think of the r0 population as located along the inferomedial aspect of Sm1. It is in direct contact with the midline prechordal mesoderm (the derivatives of which are controversial). Migration from the mesencephalic neural crest advances anteriorly and ventrally from its origin along the lateral aspect of the forebrain until it envelops Sm1 and occupies the space of the future orbit. The ‘proto-orbit’ is located external to the zone p5 of the basal forebrain. As the optic primordium emerges it encounters neural crest mesenchyme originating from r0 and r1. The optic cup pushes its way through this MNC like a fist through a sock. The future globe thus acquires a coating of neural crest that will provide the future sclera. Orbital MNC has two spatially distinct zones. The first MNC population comes from r0. This neural crest hugs the axis of the embryo. It moves directly in front of the notochord to interact with the prechordal mesoderm located between the rostral notochord and the buccopharyngeal membrane. When the primary head fold occurs these two clumps of r0 MNC and PCM come to lie anterolateral to the p5 diencephalon. The optic vesicle acquires a coating of r0 MNC along its inferomedial surface. The significant derivatives of r0 are dura covering the medial surface of the frontal lobe, the inferomedial sclera and the fascia of the three inferomedial extraocular muscles (inferior oblique, inferior rectus and medial rectus). 17.3.3.2. Rhombomere 1: presphenoid, orbitosphenoid, medial pterygoid plate, ethmoid lamina, ethmoid perpendicular plate, septum Migration from r1 occurs slightly later than that from r0. The pathway of r1 cannot violate that of r0; thus

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it must be external to that of r0. When the optic primordium emerges from the ventral diencephalon, it acquires an MNC ‘coating’ that becomes the sclera, which is divided into two hemispheres. The inferomedial sclera comes from r0; the superolateral sclera comes from r1. Many aspects of ophthalmic anatomy (e.g. the nasal and temporal vasculature) may perhaps be traced back to the binary source for its neural crest. Dura innervated by V1 comes largely from Sm1 and is continuous with the sclera of that side of the globe as well. For this reason, sensory nerves to the eyes are exclusively V1. Myoblasts from Sm1 associated with r1 form the superior rectus and levator palpebrae superioris. Fascia for these muscles logically comes from r1 neural crest. Bone formation occurs from r1 as well and is indicated on the chart as stream II. The first bone derivative is the basisphenoid, formed from paraxial mesoderm. Neural crest cells deposited forward from the basisphenoid bone become the presphenoid. The pituitary gland occupies the potential space between the basisphenoid bone and the presphenoid. Neural crest located lateral to the presphenoid condenses to form the orbitosphenoid cartilage (later the lesser wing of sphenoid). An inferior extension forms the medial pterygoid plate. Forward from the presphenoid the ethmoid capsule forms around the attachment of the p6 olfactory placodes to the basal forebrain (the rhinencephalon). For this reason, each nasal chamber has a medial ethmoid structure, the perpendicular plate, and a lateral ethmoid structure, the medial lamina of the ethmoid sinus. (The lateral lamina of the ethmoid sinus is a p5 derivative.) The timing between formation of bones made from PAM and those made from neural crest should be defined here. Formation of a somitomere precedes the migration of neural crest assigned to it. PAM of Sm1 is organized into a potential osteogenic zone located close to the neuraxis long before r1 neural crest invades the somitomere. For this reason formation of the PAM basisphenoid surrounding the most anterior part of the notochord occurs before the neural crest presphenoid is formed. As a matter of fact, neural crest never does have access to the notochord. Throughout its length, the notochord is flanked by PAM. Neural crest can’t migrate in between the two. The physical presence of the pre-existent basisphenoid is the reason that presphenoid is ‘added on’ in front of it. We arrive therefore at three extremely important generalizations. 1. At every neuromeric level, bone derived from somitomeric PAM will be laid down prior to that derived from neural crest. Basisphenoid precedes

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presphenoid. Temporal bone precedes squamous occipital bone. 2. PAM bones are found exclusively in the neurocranium, not the viscerocranium/pharyngeal arches. 3. All PAM bones of the skull are chondral, the sole exception being the parietal bone, in which r2–3 PAM is potentially mixed with p4 neural crest. 17.3.3.3. Rhombomere 20: premaxilla and vomer Now that we have accounted for the derivatives of r0 and r1 we come the final zone of the mesencephalic neural crest, r20. The prime sign is used because this neural crest arises from level r2 but it appears not to participate in the formation of PA1. Instead it migrates as a distinct stream into the facial midline. Nevertheless the vascular axis of this mesenchyme is that of the sphenopalatine artery. All pharyngeal arch derivatives are supplied by branches of the external carotid artery. The internal maxillary artery supplies PA1, the most distal and internal branch of which is the sphenopalatine artery. Somatic sensory supply to proximal PA1 is V2, the most distal and internal branch of which is the sphenopalatine nerve. All neural crest and PAM derivatives rostral to and internal to r20 are supplied by the internal carotid artery and innervated by V1. These correspond to the putative premandibular arch. Sharp boundaries of blood supply, innervation and genetic programming distinguish structures emanating from neuromeric levels r1 and r2. The most common clefts of the lip and palate occur at this interface zone and may well result from pathology in the cephalic zone of r2 at the earliest stages of embryogenesis. The anatomical pathway taken by r20 neural crest is dictated by the presence of three pre-existing fields of neural crest mesenchyme in the future orbit and nose. The presence of these fields forces r20 MNC to either follow along their boundaries or to make detours around them. Detours require a pathway for the MNC to follow. The mechanism of r20 MNC migration is to take advantage of previously established r1 MNC structures. Just as lateral plate mesoderm has a ‘leading edge’, the pioneering cells of which leave a trail for later mesoderm to follow, so too do the initial cells of a neural crest pathway leave signals behind for their successors. Furthermore succeeding waves of neural crest migration will follow each other based upon their ordinal anatomical site of origin along the neural fold. Unfortunately, playing ‘follow the leader’ may lead to obstacles. The r1 MNC blastema responsible for synthesis of the lesser sphenoid wing of the sphenoid and the medial pterygoid plate ‘pushes’ r20 MNC laterally and prevents it making any contribution to the orbit. In so doing r20 MNC is placed on a direct

collision course with the back wall of the greater wing of sphenoid (formed by r2 RNC). By the time r20 MNC arrives at the orbit the blastema of the r2 RNC alisphenoid mesenchyme is already in place. Accordingly, the r20 MNC pathway is forced to pass beneath the greater wing. By this ‘underhanded move’ it gains entrance to the pterygopalatine fossa. In its desire to constantly seek out the contours of r1 MNC, the r20 MNC turns medially from the pterygopalatine fossa and heads toward the presphenoid bone in the midline. In so doing it will constitute a physical obstacle to the future development of the upwardly growing palatine bone. The palatine bone is derived from r2 RNC. It is the very last field to be formed from the most external lamina of r2 neural crest (vide infra). The palatine bone consists of a horizontal plate that forms the posterior hard palate and a perpendicular plate projecting up into the orbit. At the posterior margin of these two plates a pyramidal process inserts itself just between the two pterygoid plates. From the superior border of the perpendicular plate are projected upward a posteriorly directed sphenoidal process and an anteriorly directed orbital process. Formation of these two processes is a consequence of the physical presence of the neural crest pathway of r20! Ossification of the palatine bone begins in membrane at the junction of the future horizontal and perpendicular plates. It then spreads medially into the horizontal plate and posteriorly into the pyramidal process. Last to form is the perpendicular plate, the ossification spreading superiorly. At the time that r20 MNC enters the pterygopalatine fossa, the perpendicular plate is growing. The sphenoid and orbital processes have not yet grown out from the superior border of the perpendicular palatine plate. The r20 MNC runs medially to ‘seek out’ the midline. In so doing, it passes above the perpendicular plate. Subsequently, superior growth of the perpendicular plate encounters the r20 MNC mesenchymal column. The sagittally oriented perpendicular plate is forced to split into two masses, posterior and anterior. These become the sphenoid and orbital processes. Thus, the presence of the sphenopalatine notch represents a ‘footprint’ of the migratory pathway used by r20 MNC on its way to the midline. All notches and foramina of the skull are tacit recognitions of the prior existence of neurovascular structures. We have said before that the derivatives of r20 are the premaxilla and the vomer and that these are bilateral field complexes. Beneath the presphenoid is a midline axial structure (probably bilateral in origin) the sphenoidal rostrum. Inserted on either side of the rostrum are two flanking laminae from the posterior margins of the vomerine bones. These are known as

NEURAL TUBE PROGRAMMING AND CRANIOFACIAL CLEFTS: PART II the vaginal processes. As the vomers extend anteriorly from the vaginal processes they descend beneath the perpendicular plates of the ethmoid (PPE). The ability of r20 MNC to follow along the fused midline laminae of r1, (i.e. the perpendicular ethmoid plates) determines whether or not one or both premaxillary bones will be produced. The r1 PPE is a bilateral structure. Holoprosencephaly is a p6 condition of the ventral and anterior forebrain and the r1 ethmoid plate upon which the brain rests (Lemire et al., 1981; Siebert et al., 1981, 1987; Cohen and Sulik, 1992). Because r1 neural crest and Sm1 PAM are necessary for forebrain dura and blood vessels, a possible cause of p6 hypoplasia is an r1 deficiency state. Holoprosencephaly can exist in a unilateral form that includes ipsilateral attenuation or absence of the perpendicular plate. In these cases hypoplasia or frank absence of the ipsilateral premaxilla can be present. If the palatal shelves come from r2 RNC and must fuse to the r20 MNC in order to achieve union at the midline, then unilateral holoprosencephaly, by destroying the pathway for r20 MNC migration, will create an ipsilateral palatal cleft (see Fig. 17.33). We shall now look at the derivatives of r20, the order in which they form and their relationship to their r1 ‘guideline’. Neural crest of r20 behaves in a mesencephalic fashion. All MNC neural crest derivatives are arranged in cranio-caudal order along the neural folds; r0 lies cranial to r1. The r1 neural crest contains (in order) the sclera, presphenoid, then the orbitosphenoid. All derivatives of r0 and r1 are produced by streams that flow forward toward the orbito-nasal midline in concentric arcs, adding from the midline outward. The lamination sequence of r20 MNC is completely different from that of r0 and r1. Building upon the PAM derivative basisphenoid, r1 neural crest flows forward and lateral beneath the forebrain to form the central anterior cranial fossa. There is no absence or deformation of the sphenoid in the presence of a normal ethmoid. The ethmoid capsule forms a lattice for r20 migration. Earliest in time to migrate is the premaxillary neural crest. At about the time PMx neural crest arrives at the anterior base of the presphenoid, the r1 perpendicular plates of the ethmoid are forming. These make up the ‘perpendicular plate pathway’ by which PMx passes along the future roof of the mouth and arrives at its final position in the facial midline. As PMx migrates it leaves behind it a molecular ‘slime trail’ for vomerine neural crest to follow. Once in the midline PMx forms a common alveolar process that subsequently splits into two independent zones. Neural crest ‘flow’ into zone 1 creates the central incisor and alveolus. This field is a pie-shaped wedge with the apex pointing toward the incisive

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foramen. A ‘split-second’ later, a second neural crest population containing the future lateral incisor and ascending process of the premaxilla arrives on the scene. It too has the form of a pie-shaped wedge with its apex at the incisive foramen. Thus zone 2 does not ‘spill over’ from zone 1 but arises just posterolateral to it. Thus zones 1 and 2 are in continuity, as are zones 3, 4 and 5. From its most distal aspect zone 2 PMx produces a cellular outgrowth of vital importance to plastic surgeons. This frontal process of the premaxilla (FPmedial) is responsible for the internal piriform rim. Deficiency states in PMxF are the cause of the cleft lip nasal deformity (see below) (Carstens, 2004). The vomerine neural crest migrates into position later than that of PMx. The premigratory position of vomer field cells is thus caudal to PMx along the neural fold. Isolated deficiency states of the vomer population can exist. It is therefore possible to have hypoplastic vomer in the presence of a perfectly normal lip and an intact premaxilla. This can lead to a spectrum of problems. Thus, even though primary contact between the premaxilla maxilla has resulted in fusion of the alveolar arch, small vomer bone may be physically unavailable for fusion with the ipsilateral r2 palatal shelf, leading to secondary contact failure and a midline cleft palate. Earlier it was stated that the migration pattern of r20 was different from that of r0 and r1. The former MNC zones produced streams of neural crest with an additive lamination pattern. PMx and vomer, on the other hand, follow each other ‘Indian file’ down a common pathway. This behavior is thus similar to that of RNC, save that r20 MNC does not participate in formation of PA1. The PMx neural crest is the ‘pathfinder’ for subsequent vomerine migration. For this reason, an absent premaxilla will always be accompanied by an absent vomer. On the other hand, total absence of the ipsilateral premaxilla (and vomer) can exist because of a failure of the r1 ‘perpendicular plate pathway’ to form in the first place (thereby making migration impossible). 17.3.4. Rostral rhomboencephalic derivatives (11-somite stage) The earliest neural crest migrations to be completed are those originating from the rostral rhomboencephalon, i.e. from r2–r7. This type of neural crest (RNCr) is intimately involved with the formation of the first three pharyngeal arches. RNCr pathways are segmentally restricted between a rhombomere and its corresponding somitomere. RNCr cannot participate in craniofacial development independent of the pharyngeal

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arch to which it is assigned. Rather, it is physically transported to the face (frontonasal mesenchyme) by 1) growth of the pharyngeal arch and 2) embryonic folding. The very first fold in the CNS, called the mesencephalic flexure, is hinged at the midbrain and tucks the CNS almost 180 on itself. The most rostral zones of the prosencephalon (p6 and p5) are brought ventrally until they are in almost direct contact with the rapidly growing PA1 and PA2. Epithelial contact between the frontonasal and lateral masses results in fusion. Neural crest and PAM mesenchymal derivatives then move into their appropriate locations. This type of RNCr migration is very different from that observed in neural crest of prosencephalic or mesencephalic origin. The biological behavior of PNC and MNC is not constrained by a secondary reorganization of PAM into a pharyngeal arch. These neural crest cells move right under the epithelium to their destinations, set up shop and do their thing. Timing is everything, especially in craniofacial development. We know that RNCr migration into the first three arches is complete by the 11-somite stage. On the other hand, the mesenchyme to which the RNCr will directly attach to form the facial skeleton comes from MNC. However MNC migration is not complete until the 14-somite stage. CNS folding occurs in this interval. At this point in time the PA1 and PA2 ‘sandwich’ has swung into position. All potential RNCr fields are ready to make contact with their MNC counterparts. It is for this reason that r20 MNC must ‘negotiate’ its way between the more internal fields of r1 and the more external outlying fields of r2. Note further that, although MNC interacts with PNC via the r1 perpendicular ethmoid plates, no direct anatomical contact exists fields from RNCr and those from PNC. When the pharyngeal arches arrive on the scene they are composite entities. As discussed above, the first seven somitomeres are not completely separate. The external surface of the somitomeres show deep grooves in the epithelium; these give the somitomeres the appearance of popcorn balls. These grooves are not full-thickness; it would be theoretically possible for PAM and neural crest from one somitomere to spread over to the next one. In reality, this does not happen. Curiously enough, although adjacent (even and odd) somitomeres do not exchange mesenchyme, even– even pairs and odd–odd pairs can do so with impunity. This has been accomplished in the laboratory. It also is the reason that PA1 and PA2, once formed, can promptly meld into each other to create a ‘sandwich’. In this case somitomeric PAM and neural crest cells from r2 interact with those of r4, whereas those from r3 interact and meld with those of r5. For this reason,

myoblasts from r4 populate the maxillary fields and those from r5 populate the mandibular fields. Turning our attention to the osseous derivatives of the first and second pharyngeal arches, by the time the PA1/PA2 sandwich arrives on the scene it has two visible components. An upper maxillary mass contains derivatives of r2 þ Sm2 and r4 þ Sm4. The maxillary mass comes into direct contact with the frontonasal ‘process’. A lower mandibular mass contains derivatives of r3 þ Sm3 and r5 þ Sm5. The mandibular mass bears no physical relationship to the orbitosphenoid complex. It is suspended from the cranial base r6 petrous temporal bone at the temporomandibular joint. 17.3.4.1. Rhombomere 2: inferior turbinate, palatine, maxilla, zygoma, alisphenoid and lateral pterygoid plate – the infraorbital tier of Tessier clefts explained Paraxial mesoderm corresponding to Sm2 is not organized into a definite sclerotome. Nonetheless, the superomedial portion of Sm2 remains sessile alongside the neural tube. It does not form part of PA1. Instead it contributes mesenchyme to the blood vessels penetrating all dura innervated by V2. It also forms the mesenchyme of the anterior third of the parietal bone. Some superomedial r2 neural crest remains sessile alongside the developing brain. It helps form the dura covering the prosencephalon. This r2 zone is defined by the sensory distribution of V2 to the dura. The neural crest responsible for synthesizing the maxilla and lateral orbit can be organized into three distinct osteogenic segments. The segments arrive at the scene via spatially distinct migratory pathways. These pathways do not conflict with each other. Neural crest cells within each segment produce bone derivatives in a strict time sequence. The first segment contains the various components of the maxilla. The overall shape of these fields is that of a five-sided box, the medial wall of which is partially open. The second segment contains the two fields of the zygoma, which forms secondarily in cartilage over the malar eminence of the maxilla. The third segment contains the lateral pterygoid wing and greater wing of sphenoid. These fields make their appearance in the following order. Clefts occurring below the axis of the orbit are classified by Tessier as #3–#7. All involve neural crest bones originating from r2. The best way to understand the anatomical distribution of these clefts is to consider the neurovascular supply of the maxillary dentition. The anterior superior alveolar artery (ASAA) and middle superior alveolar artery (MSAA) are both branches of the infraorbital artery. The posterior superior alveolar artery (PSAA) arises in the pterygopalatine fossa from

NEURAL TUBE PROGRAMMING AND CRANIOFACIAL CLEFTS: PART II the third part of the internal maxillary artery, proximal to the infraorbital artery. Thus, a developmental relationship exists between the developmental fields of the ASAA and the MSAA. As we shall see, the neurovascular distribution of the superior alveolar nerves and arteries divides the anterior wall of the maxilla into three developmental field zones. The most medial, Mx1, is defined by the anterior superior alveolar nerve (ASAN). The ASAN departs from the infraorbital nerve midway along its course through the canal. It traverses the canalis sinosus in the anterior wall of the maxillary sinus. The canalis sinosus swerves laterally away from the infraorbital canal and then hooks downward and medially to pass beneath the infraorbital foramen. It then runs downward to the canine and the incisors. The presence of the canalis is a tacit recognition of the prior existence of the anterior superior neurovascular bundle supplying these structures. The anterior superior alveolar artery arises from the infraorbital artery just prior to its exit from the infraorbital foramen. Just like its companion nerve, its course defines the canalis sinosus. Tessier clefts #3 and #4 occur here. Mx2 is defined by the middle superior alveolar nerve (MSAN). This arises from the inferior alveolar nerve in its course along the infraorbital groove. The nerve then tracks laterally and forward in the lateral maxillary wall. It supplies the premolar teeth. It is accompanied by the MSAA. The Tessier #5 cleft occurs here. Mx3 is defined by the posterior superior alveolar nerve (PSAN). The PSAN departs from V2 in the pterygopalatine fossa. It pierces the maxilla just along its infratemporal surface and descends beneath the sinus mucosa to supply the molars. It is accompanied by the PSAA. This will be the site of Tessier cleft #6. 17.3.4.1.1. Segment 1: IT, Mx1, Mx2, Mx3, and P Mx1 is the first major field set of the first segment of r2 neural crest. Comprises those structures located medial to a vertical line dropped from the inferior orbital foramen. Mx1 is composed of: 1) the mesial alveolus containing the canine (supplied by the anterior superior alveolar nerve and artery) and 2) the anterior maxillary wall medial to the infraorbital foramen. The orbital rim medial and the orbital floor medial to the infraorbital fissure are formed by Mx1. From the medial edge and from the superomedial edge of Mx1 arise two extremely important structures for plastic surgeons; these are crucial for understanding Tessier clefts #3 and #4. These structures are the inferior turbinate bone (IT) and MxF. This thin lamina extends upward from the canine region, past the lateral margin of the nasal bone, and terminates

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by abutting with the medial prefrontal bone (PFm), i.e. the medial orbital rim. Defects of IT cause the #3 cleft while defects of MxF cause the #4 cleft. The inferior turbinate is a discrete bone forming one-third of the lateral nasal wall. This structure is of critical importance in explaining the pathological anatomy of the Tessier clefts #2, #3 and #4. To understand the clinical presentations of these clefts we must explore the role that IT plays in the separation of the nasal cavity from the maxillary sinus. Anterior to IT lies the ascending process of the premaxilla. Posterior to IT is the vertical plate of the palatine bone. IT is situated above the two premolars, hence it probably belongs to the Mx1 field complex. As IT projects into the nasal cavity it forms a caudally directed scroll. Beneath this scroll is the terminus of the lacrimal duct. Thus the lower half of the lateral nasal wall is formed by three r2 neural crest derivatives (in anteroposterior order): MxF, IT and the palatine bone. The upper half of the lateral nasal wall can likewise be divided into three discrete zones. The posterior zone is made from the vertical plate of the r2 palatine bone. The central zone contains the middle turbinate. This bone represents the caudal border of the r1 ethmoid complex. MT sits above IT and has the form of a Roman arch. An aperture between MT and IT results. This is hidden by a scimitar-like projection of MT into the nasal cavity, the infundibulum. It is via this field boundary that the maxillary sinus drains. The anterior zone of the lateral nasal wall is made from the ascending process of the maxilla. The cranial margin of MxF abuts against the p5 nasal bone medially, the p5 nasal process of the frontal bone posteriorly and the p5 lacrimal bone laterally. Thus the maxillary sinus is a six-sided box, five sides of which are exclusively of r2 derivation. The sixth side, the medial wall (the lateral nasal wall), is the combination of an upper tier of r2 and p5 fields with a lower tier of exclusively r2 fields. As a mucus-producing structure the maxillary sinus must have an obligatory escape route for its secretions. Fortunately, our six-sided box is not watertight. The field boundary between the anterior and posterior zones of the p6 lower ethmoid provides just the exit point for the maxillary sinus. It thus drains into the inferior aspect of the infundibulum, dripping out from beneath the middle turbinate. This messy situation neatly reinforces our previously described model of sinus development. All sinuses result from the expansion of oral mucosa into a potential space between fields. Thus the mucosa seizes the opportunity to insert itself into the opening between the anterior and posterior ethmoid zones and insert itself into the potential cavity between p6 and r2.

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The structural integrity of IT is the prerequisite for the proper formation of two other structures that appear later. First, the r20 frontal process of the premaxilla (PMxF) is constructed upon the scaffolding of IT. Second, p5 neural crest cells descend along the lateral surface of IT to create the lacrimal bone. Because IT is such a crucial component of the lateral nasal wall, IT deficiency states will create a severe cleft condition that begins at the lateral piriform margin, eliminates the medial wall of the maxillary sinus and ascends into the lacrimal system. This is the pathology of the Tessier #3 cleft. The second important element to be produced from Mx1 is the frontal process of the maxilla. As stated before, MxF in all but humans and higher primates is physically distinct from PMxF. MxF also depends on IT for its construction. The physical presence or absence of PMxF is not required for MxF synthesis. When MxF is deficient, additional forms of clefting can occur. Recall that the inner aspect of the piriform margin is composed of two laminae; a small upper one coming down from p5 and the lower one ascending from r20. The superior lamina, the descending nasal process of the frontal bone (Fn) buttresses the undersurface of the nasal bones. The inferior lamina, the frontal process of the premaxilla (PMxF) arises from the distal (versus mesial) margin of the premaxilla just above region of the lateral incisor region. The lateral piriform margin and the medial piriform margin are initially separated by the nasolacrimal groove. This closes over during fetal development, causing the lateral piriform rims to approach the midline from either side. They eventually overlap the medial piriform margins. Because of this overlap no suture is observed in the term fetus. This has led some physical anthropologists to question the existence of the premaxilla in humans (Ashley-Montague, 1936). Definitive proof regarding the premaxilla has been provided by Barteczko and Jacob (2004). In the Tessier #4 cleft, the persistence of this groove spares the medial piriform margin. All the incisors ipsilateral to the cleft are intact. Tessier #4 and #5 clefts occur as gradations of severity within the Mx1 field. Therefore the #5 cleft is simply a more severe form extending all the way to the infraorbital foramen. Loss of dental units within Mx1 can also occur. The developmental significance of the bilaminar piriform margin is that deficits of the r20 PMxF will allow the r2 MxF to sit more laterally and the vertical height of the lateral piriform margin will be lower. This describes the pathological osseous anatomy of the isolated cleft lip nose. In those r20 deficiency states in which the PMxF is hypoplastic or missing, the piriform margin is unilaminar and extremely thin. This

means that the actions of the nasalis and paranasalis muscle complexes acting over time will exert a more pronounced distracting force to displace the piriform margin out laterally. Mx2 constitutes the next field set of first segment r2 neural crest. Mx2 is composed of the alveolus housing the premolar teeth (supplied by the middle superior alveolar nerve and artery) and the maxillary wall lateral to the infraorbital foramen. The orbital rim lateral to the intraorbital foramen and the orbital floor lateral to the infraorbital fissure are formed by Mx2. It is thus in contact with the greater wing of the sphenoid. In zoologic terms this is called the alisphenoid. As we shall see, neural crest forming the alisphenoid arrives after Mx2 and thus potentially must be constructed upon it. An isolated defect of Mx2 causes a Tessier #5 cleft. When the alisphenoid is also affected one sees a Tessier #5, #9 cleft. Mx3 constitutes the final field set of first segment r2 neural crest. Mx3 is composed of the alveolus housing the molars (supplied by the posterior superior alveolar nerve) and the maxillary wall behind the buttress. This includes the zygomatic process of the maxilla. The Tessier #6 cleft is associated with hypoplastic states in this zone. The postorbital field of the zygoma sits directly above Mx3 buttress. The Tessier #8 cleft localizes to the postorbital field. Dual affectation leads to the Tessier #6, #8 cleft. Mx1, Mx2 and Mx3 contribute to an important medial structure, the palate. The palatal shelves extend from each of these fields. Just above the palatal shelves is located the medial maxillary wall. This is made up of three coplanar structures all lined up in a row. The fused frontal processes (PMxF and MxF) form the anterior third of the wall. IT forms the middle third of the wall. It is a derivative of Mx1m. The ‘sprouting’ of IT from Mx1 occurs prior to that of MxF. The lateral ascending process is constructed upon an intact IT. Thus a cleft in zone 3 will always destroy zone 4 but not vice versa. An isolated defect of MxF causes a Tessier #4 cleft. Immediately behind Mx3 lies the palatine bone. This structure ossifies after the maxilla. It can be considered the most proximal field of the first segment. The Tessier #7 cleft is associated with the palatine bone. Deficiency states have been described with severe hypoplasia or absence of this structure. In such cases the soft palate musculature will have nothing to insert upon. These muscles will remain as unfulfilled mesenchymal blobs over their sites of origin, i.e. the eustachian tube (tensor veli palatini) and petrous apex (levator veli palatini). In the literature this condition is described as an ipsilateral absence of the soft palate. This has occurred in the context of severe Goldenhar syndrome.

NEURAL TUBE PROGRAMMING AND CRANIOFACIAL CLEFTS: PART II 17.3.4.1.2. Second r2 segment: jugal and postorbital The second segment of r2 neural crest belongs to the zygoma and is ossified in cartilage after the maxilla is formed. The zygoma is not a single bone. It is the composite of two previous bones seen in lower vertebrates. The jugal bone forms the temporal process of the zygoma and the inferior half of the malar eminence. It is the most distal and is synthesized first. It begins along the axis of the zygomaticofacial neurovascular bundle and spreads downward to contact the Mx buttress. It also spreads forward to contact the Mx2 lateral orbital rim. The postorbital bone forms the frontal process of the zygoma and the superior half of the malar eminence. It also begins at the zygomaticofacial axis and spreads upward to contact the p5 zygomatic process of the frontal bone. It also spreads backward to contact the r3 zygomatic process of the squamous temporal bone. The anatomical split between the postorbital and jugal is indicated by the zygomaticofacial neurovascular axis. Tessier clefts #8 and 7 correspond respectively to these two fields. These are commonly affected in Treacher– Collins syndrome where the zygomatic arch is absent and the malar eminence is reduced or absent. 17.3.4.1.3. Third r2 segment: lateral pterygoid and alisphenoid The third segment of r2 neural crest contains first the lateral pterygoid lamina (LPt) and second the alisphenoid. Both fields are confluent with each other at the pterygopalatine fossa. From their juncture each field is joined to the presphenoid bone by an osseous process. Where these processes straddle the pre-existing sensory nerve V2, the foramen rotundum is created. Within the orbit isolated deficiencies of the alisphenoid have been described that communicate to the external postorbital region. Perturbations of neural crest within the membranous alisphenoid are the basis of the Tessier #9 cleft. The alisphenoid would appear to be an extension of the cranial base. It represents, however, an attachment of PA2 to the skull. This is a very ancient arrangement. The primitive palatoquadrate cartilage (from which the maxilla is derived) had three components: autopalatine, metapterygoid, and quadrate. The most anterior is the precursor of the maxilla and palate, the middle one forms the alisphenoid, and the latter forms the incus. 17.3.4.2. Rhombomere 3: The mandible and derivatives of the ear The original lower jaw of tetrapods consisted of nine membranous bones organized around Meckel’s cartilage. Over time some of these fields disappeared (or

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were amalgamated). The most proximal bone fields became incorporated as components of the ear. Only the dentary bone remains to form the mammalian mandible. The mandible can be divided into four developmental fields. Three of these belong to the toothbearing alveolar bone. The ramus field contains also the coronoid process and the condyle. It develops after the alveolus. For this reason, in cases of craniofacial microsomia or Treacher–Collins syndrome, partial or total absence of the ramus can occur with preservation of the dental mandible. Details of the formation of these fields are well described by Kjaer. No clearer account of mandibular development can be found in the literature. Our purpose here is simply to identify the r3 fields and discuss their temporal order of formation. These concepts serve to rationalize the Kjaer’s observations and permit additional clinical correlations (such as the anatomical rationale of the muscles of mastication). PAM corresponding to Sm3 is not organized into a definite sclerotome. Nonetheless the superomedial portion of Sm3 remains sessile alongside the neural tube. It does not participate in the formation of PA1. Instead it contributes mesenchyme to the blood vessels penetrating all dura innervated by V3. It also forms the mesenchyme of the posterior two-thirds of the parietal bone. Some superomedial r3 neural crest remains sessile alongside the developing brain. It helps form the dura covering the prosencephalon. This r3 zone is defined by the sensory distribution of V3 to the dura. 17.3.4.2.1. First r3 segment: Mn1, Mn2 and Mn3 The alveolar zones of the mandible are similar to those of the maxilla. Three distinct sensory nerves supply the dental units of each zone. Mn1 contains both the incisors and the canine. Mn2 contains the premolars. Mn3 contains the molars. Each zone is supplied by a distinct sensory nerve. Early in development each nerve has its own separate canal. These canals are organized in a strict time sequence. Derivatives in zone Mn1 represent the ‘oldest’ mesenchyme; the incisors are the first teeth to erupt. Accordingly the nerve to Mn1 occupies the most caudal canal. Just cranial to it lies the canal for nerve Mn2. Mn3 follows the same pattern. The three canals eventually become roofed over by the medial lamina of the mandible growing upward from its lower border. Thus the inferior alveolar nerve is ‘three nerves in one’. 17.3.4.2.2. Second r3 segment: ramus, condyle and coronoid The ramus forms via membranous ossification later in time than the alveolar bone. A portion of this

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periosteum converts to a cartilage cap that eventually forms the condyle. Pathologies affecting this zone may result in hypoplasia or absence but tend to spare neighboring fields. For example, craniofacial microsomia tends not to affect the tooth-bearing region of the alveolus. This coronoid forms at about the same time as the condyle. The cartilage goes on to form chondral bone. From this same PAM the temporalis muscle is formed. For this reason in Treacher–Collins syndrome absence of the coronoid is associated with an absent temporalis muscle.

17.3.4.6. Rhombomere 7

17.3.4.2.2. Third r3 segment: derivatives ‘assigned’ to the ear

The formation of PA4 and PA5 occurs in a serial fashion caudal to PA3. The muscles and cartilages are all coplanar. They form the true pharynx and, via a ventral extension, the larynx. Because rhombomeres r8– r11 form later than r2–r7, the final migration of neural crest from these more caudal levels occurs later as well, being completed by somite stage 14 (versus 11).

The original angular bone becomes the tympanic bone. The reflected lamina of the angular held the original tympanic membrane; this is incorporated into the modern tympanic bone. The prearticular forms the anterior process of the malleus, the articular forms the malleus proper and the quadrate forms the incus. In nonmammalian vertebrates the jaw joint is represented by the quadrate and articular bones. 17.3.4.3. Rhombomere 4

Sm7 forms the mastoid temporal bone. PAM myoblasts from Sm7 produce stylopharyngeus and superior constrictor. In concert with myotomes of somites 1– 8 (Sm8–Sm15) forms sternocleidomastoid and trapezius. This r7 neural crest (possibly in combination with lateral plate mesoderm) forms the caudal half of the hyoid bone and the greater cornu. 17.3.5. Caudal rhomboencephalic derivatives (somite stage 14)

17.3.5.1. Rhombomere 8 The middle constructor (produced from Sm8) is covered with neural crest fascia. Intrinsic muscles of larynx are probably PAM (vs lateral plate).

This neural crest may represent the ancient hyoid suspension of the mandible. It probably forms the styloid process of the temporal bone. The limited bone produced by r4 is offset by the very extensive production of a fascia enveloping the entire anterior head. All muscles innervated by upper division of facial nerve are enveloped by r4 neural crest fascia. This is the superficial investing fascia (SIF) or the superficial musculo-aponeurotic fascia (SMAS).




This neural crest forms the upper half of the hyoid bone. It participates with r4 in forming the stylohyoid ligament. The SIF/SMAS fascia produced by r5 neural crest is extensive, enveloping the posterior head and the neck. Because the posterior digastric (a Sm5 PAM derivative) attaches to the hyoid, r5 contributes to the fascia layer enveloping this muscle. The deep investing fascia is in-continuity with that enclosing all other first arch muscles. Quite logically, the deep investing fascia lies deep to the SIF.

The cricoid cartilage is formed by neural crest in combination with lateral plate mesoderm. r 11 also contributes to inferior constrictor.

17.3.4.5. Rhombomere 6 Sm6 PAM forms the petrous temporal bone. PAM myoblasts from Sm6 produce levator veli palatini and superior constrictor Fascia surrounding these muscles is neural crest.

The thyroid cartilage is formed by neural crest in combination with lateral plate mesoderm. r9 also contributes to middle constrictor. 17.3.5.3. Rhombomere 10 The inferior constrictor (produced from Sm10) is covered with neural crest fascia.

17.4. Formation of the larynx and trachea The exact contributions of neural crest and lateral plate mesoderm to the laryngeal, arytenoid and cricoid cartilages are unclear but these structures arise from levels r8–r11. The nuclei of the vagus nerves supplying these structures are distributed along the length of this segment of medulla. The tracheal cartilages display a unique structure that is anticipated by the cricoid. These cartilage units are U-shaped, being incomplete posteriorly. Because endoderm provides the pattern for so many other cartilages in the oropharynx it is tempting to think that lateral plate mesoderm responds

NEURAL TUBE PROGRAMMING AND CRANIOFACIAL CLEFTS: PART II in the same way. The endodermal bud grows downward into an unorganized central mass of LPM and forces it to form rings of cartilage. The origins of LPM in the neck that could contribute to the trachea are the same as those of the cervical esophagus. The cervical esophagus displays a curious vascular pattern of axial segmental vessels, four or five in number. At the level of the thorax, the esophageal vasculature comes from the celiac axis. In mammals, this situation suggests that eight to ten neuromeric units are involved in producing the esophagus. At the same time, the musculature of the cervical esophagus gradually changes from striated to smooth, the transition being complete at the thoracic inlet. It would appear that there is something segmental about the cervical esophagus. The reason for this might be that LPMs and LPMv are unsplit in the neck. The formation of the bony body wall requires that these two layers separate, with LPMv forming the internal mesoderm of the body cavity while LPMs encircles the visceral organs. Indeed the anatomy of ribs is nothing more than segmental extensions of PAM from each vertebra that bud outward into the mass of LPMs. We know that somatic LPM follows the same Hox code as the vertebrae. Thus, within the neck, lateral plate mesoderm maintains an occult segmental pattern. Although cervical LPM is an unsplit fusion of visceral and somatic laminae, it is the somatic component that imposes a segmental order to the esophagus and trachea. As soon as LPMs and LPMv part company (at the thoracic inlet) all segmentation of visceral structures disappears. The endoderm may therefore confer cartilage-forming signals on the unsplit LPM from neuromeric levels c1 to t1–2. The spaces between the tracheal rings are occult manifestations of individual neuromeric units of LPM. As soon as LPM splits apart at the inlet, gene expression within LPMv would then respond by formation of a carina and the appearance of lung buds. The lungs are appropriately enclosed within the ensheathing visceral pleura. Sensory innervation of the visceral pleura is nonsegmental. It is organized around the neuromeric units of the sympathetic autonomic nervous system (SANS). The parietal pleura, on the other hand, is formed from LPMs, innervated segmentally and thus is capable of exquisite, localizing pain.

17.5. Spatial relationships of the pharyngeal arches Mammalian facial anatomy represents a significant departure from the initial arrangement of pharyngeal arch muscles in other tetrapods. These muscles were designed to be coplanar. Deep layer muscles fulfill a

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sphincteric function while superficial muscles act as dilators. The original branchial arch skeleton has both dorsal and ventral components. I advance the hypothesis here that the neuromeric arrangement of the pharyngeal arches is a holdover of this anatomical plan. The cranial neuromere produces dorsal structures while the caudal neuromere produces ventral structures. In evolution the neural crest and paraxial mesoderm changed roles. What had formerly been ‘assigned’ to produce cartilaginous gills became fascia. Bones originally designed to open or close the gills became transformed into the muscular sling of the mammalian pharynx, i.e. they begat the constrictors. Only a few PA2 derivatives remain coplanar with those of the first arch. Most PA2 mesenchyme moves forward to envelop PA1 with the facial muscles positioned superficially with a entirely separate fascia, the SMAS. PA3 (r6–r7) remains coplanar with the first two, however. It abuts up against the r8/O1 muscle, the sternocleidomastoid. Perhaps the amalgamation of PA2 with PA1 occurs concomitant with or shortly after the formation of PA3. PA4 and PA5 are formed in continuity with the caudal aspect of PA3 and thus are ‘tucked in’ deep to the plane of the first three arches. Thus at the level of PA3 the pharynx becomes a tube within a tube.

17.6. Formation of the cranial base Analysis of the order of ossification of the facial skeleton from a neuromeric perspective reveals several important observations. The development of the neurocranium is in direct proportion to the need for brain coverage. Bone made from somitomeric PAM will at all times form before that formed by neural crest. For example, basipostsphenoid (PAM from Sm1) forms before the presphenoid (r1 neural crest). We can imagine a spatial relation between the neuraxis and the somitomeres versus the eventual position of the pharyngeal arches. Although the somitomere may not possess a sclerotome per se, the physical position of the PAM next to the neural tube makes it likely to be converted into paraxial bone. PAM represents the mesodermal ‘first response’ to brain formation and the need for structural support. The timing of neural crest migration occurs well after somite formation. Neural crest thus represents a ‘second response’ to brain formation. As in the building of a house, the foundation of the cranial base is first laid down by PAM, because this mesenchyme is physically situated at the ground floor. Subsequently neural crest takes over, making the ensheathing bones of the calvarial roof. This happens as the sidewalls of the neural tube grow upward and approximate. Neural crest from the neural folds

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follows this process right along. It is thus no accident that the ossification sequence of the frontal and parietal bones is ventral to dorsal. Cranial base anatomy displays both segmentation and parasegmentation. PAM forming the basisphenoid is strictly an r1 derivative. So, too, the temporal bone comes from Sm6–Sm7 plus corresponding r6–r7 neural crest. Parasegmentation begins at the spheno-occipital suture. Each of the four occipital somites contribute to the basioccipital, exoccipital and supraoccipital bones in a laminar manner. If the sclerotomes of these somites produced vertebrae, it would be as if the four bodies were fused in succession, forming the basioccipital bone. The lateral elements around the foramina would likewise be laminated, with r8 on the outside and r11 closest to the ring. These laminae would produce the exoccipital bone. Finally, fusion of the neural arches would yield the supraoccipital bone. Experimental work by Huang and by the O’Rahilly/ Mueller team provides solid evidence that this is so. The avian model described by Huang et al. (2000) has five occipital somites. Using tracing-maker methodology the contributions of each somite to the skull base are depicted in color. Muller and O’Rahilly (2003) demonstrated that the mammalian model has only four somites. The pattern of these is similar to that of birds so it is reasonable to suppose that the same topology of assembly holds true as well. 17.6.1. Clinical example of sequential field assembly: the fronto-orbitosphenoid model We can now understand facial bone formation in terms of neuromeric derivatives. This is a visual exercise involving migratory pathways by which neural crest and PAM arrive at their final destinations in the face and skull. A description of this entire process is beyond the scope of this chapter. Nonetheless, we can use the fronto-orbito-sphenoid complex as a convenient model to study how these migrations can be understood on a neuromere-by-neuromere basis (see Fig. 17.28). This science has direct relevance for the surgical correction of congenital deformities. Tessier (1969, 1976, 1981) documented the fact that craniofacial clefts seemed to follow certain occult anatomical patterns, which he classified numerically. The Tessier system, derived purely on an empiric basis, has a remarkable fit with neuromeric fields. Through understanding the face as an assembly of fields with a neuromeric basis, clefting takes on an entirely new relevance for neuroscientists and surgeons alike. The analysis of craniofacial clefts provides the means by which we may understand the developmental anatomy of the fields themselves. A theoretical diagram of the

orbitosphenoid complex might color-code each field to match its neuromeric level of origin. The pathways by which neural crest cells arrive at the orbit and the structures created by each neural crest would be indicated by color-coded arrows. The physical location of all neural crest cells is understood to be from sites above and behind the orbitosphenoid complex. Derivatives listed most distally along each pathway migrate earlier and develop earlier. Fronto-orbital derivatives of p5 neural crest appear in light blue. The ossification center for each frontal bone centers around the axis of the supraorbital artery. This is the penultimate branch of the internal carotid/ anterior cerebral artery, the most distal being the anterior ethmoid arteries. The supraorbital artery divides the frontal bone into four developmental zones. Below the horizontal axis of the artery the orbital rim is divided into a medial prefrontal field (PFm) and a lateral prefrontal field (PFl). Above the axis of the artery are the medial fronto-orbital field (FOm) and the lateral fronto-orbital field (FOl). FOm and FOl form by a wave of ossification ascending from the orbital rim. These fields are exquisitely sensitive to perturbations in the underlying r1 zone; PFm and PFl prove to be resistant. This difference is demonstrated by the presence of orbits and face seen in anencephaly. The mediolateral axis of the frontal fields displays a clear cut time line. The more medial fields form last. Thus, global deficiency states first manifest themselves medially and, with greater severity, progress laterally. Hypotelorism occurs well before cyclopia. Orbitosphenoid derivatives of r1 neural crest and PAM appear in lilac. The anterior surface of the neural crest presphenoid (presphenoid) is facing us. Hidden behind it is the PAM basisphenoid (BS). Although these two bones have differing mesenchymal sources both presphenoid and the basisphenoid bone belong to the axial cranial base. Both are formed by chondral ossification. All components of the cranial base form in cartilage. Arriving later in time, two neural crest orbital cartilages flank the presphenoid. These ossify to make the orbitosphenoid, better known as the lesser wing. The orbitosphenoid is joined to the presphenoid via two bony pedicles. These form an arch around the pre-existent optic nerve, the optic foramen. Projecting like a beak from the anterior inferior (rostral-ventral) surface of the pharyngeal arch is the pharyngeal tubercle. Hanging down caudally from the underbelly of the presphenoid are two neural crest laminae, the medial pterygoid plates (MPt). From each membranous neural crest MPt a secondary chondral extension later develops: the pterygoid hamulus. Most neural crest bones of the face form via membranous ossification. However, when neural crest cells

NEURAL TUBE PROGRAMMING AND CRANIOFACIAL CLEFTS: PART II are grown in isolation in vitro they form cartilage. Craniofacial neural crest, in order to form a membranous bone, requires signaling from epithelium. In the absence of such interaction cartilage will form. But this does not determine that the cartilage will automatically become chondral bone. Subsequent signals can convert a neural crest cartilage to a membranous bone. Another example of this process is the zygoma, attached first to the maxilla as a neural crest cartilage derivative and subsequently converted into membranous bone. Orbitosphenoid derivatives of r2 neural crest and PAM appear in purple. Attached only to the ventrolateral aspect of the presphenoid is the alisphenoid bone, forming the greater wing of the sphenoid. Ossification of alisphenoid occurs in two ways. The lateral margin of the superior orbital fissure forms in cartilage. The remainder of the alisphenoid is a membranous bone. Hugging the external face of the r1 MPt is a second laminar bone, the lateral pterygoid plate (LPt), which is of r2 RNC derivation. Just like MPt, LPt is formed in membrane. Sitting directly in front of the medial pterygoid plates just like two bookends are the r2 palatine bones. These strictly membranous bones have perpendicular and horizontal laminae joined at a 90 angle. At the anterior and superior corner of the perpendicular lamina each palatine bone projects a quadrangular, superiorly directed orbital process. These orbital processes are not obvious within the eyesocket. Just posterior to the orbital processes smaller prominences project upward to make contact with the anterolateral corners of the presphenoid. Between these anterior orbital processes and the posterior sphenoid processes lies the sphenopalatine foramen. This is an absolutely critical anatomical landmark with enormous significance for the formation of cleft lip and palate. Orbitosphenoid derivatives of r20 neural crest for the vomer and premaxilla appear in blue-grey. Through the sphenopalatine foramen passes the most distal branch of the external carotid artery, the sphenopalatine artery. The most midline sensory branch of V2, the sphenopalatine nerve, passes through this foramen as well. Neurovascular pedicles are like paleontological footprints: they serve as evidence of the earliest cellular migrations in the embryo. In this case, the sphenopalatine axis shows us the pathway by which r20 mesencephalic neural crest passes forward toward the orbit, is forced by pre-existent r1 mesenchyme to take a posterior and inferior route, and gains access to the midline of the future nasal cavity. This r20 population contains within it two neural crest osteosynthetic ‘packets’. The most anterior is that of the premaxilla; this is followed up by the vomer. The presence of r1 neural crest in the roof of the nasal midline guides the r20 blastema into the midline of the future

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face. Without this r1 ‘superhighway’ r20 cells will be unable to arrive at their destination. Pathologies of the p6/r1 zones, such those found in holoprosencephaly, will accordingly be accompanied by varying degrees of deficiency or outright absence of the r20 premaxilla. We shall now consider the construction of the facial midline in a neuromeric manner, on a zone-by-zone basis. The timeline here is very tight and precise. The average production time of a somite in human embryos is about 3 hours. If RNCr migration is complete by the 11-somite stage and PNC migration is complete at the 16-somite stage, the entire time for facial assembly is 15–18 hours. Initially the frontoorbital part of the head is physically disconnected from the pharyngeal arches, which are forming further back along the embryonic axis. Like an origami puzzle, the face ‘comes together’ when two lateral components (the pharyngeal arch complexes on either side) sprout out from the rhomboencephalic zone of the embryo, extend towards each other in the midline like pincers and make contact with a central pair of units organized around the forebrain. This occurs in two steps: 1) the contributions of PNC and MNC to the creation of the fronto-nasal–orbital unit; and 2) the contributions of RNCr to the creation of the lateral orbito-zygomatico-maxillary unit. Neural crest migration in mammals occurs after organization of the mesoderm, after somitogenesis begins, immediately before closure of the neural folds is initiated and before primary head folding has taken place. Although experimental work has defined the end point of neural crest migration, the start point on a zone-by-zone basis is less clear. My working hypotheses are: 1) Neural crest populations migrate in a strict time sequence. 2) A developmental zone contains several populations, each producing a specific anatomical structure or field. 3) Within a developmental zone, successive neural crest populations build upon preexisting cell populations; therefore fields appear in a strict time sequence. 4) Within a developmental zone, if a neural crest field N is deficient or absent, any subsequent population dependent upon N for cellular orientation and migration will likewise be deficient or absent. 5) Developmental zones are autonomous; if a neural crest field N in zone X is deficient, it will not affect the ability of fields to develop appropriate cell mass in adjacent zone Y. 6) The presence of a deficiency in zone X will affect the physical shape of fields in adjacent zone Y. These fields will have normal volume and surface area but, with growth, they can undergo secondary deformation as a compensation for the deficiency site. This is known as deficiencyinduced field mismatch.

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17.6.2. Neuromeric origin of facial soft tissues In subsequent sections we shall be discussing the mechanisms by which a spectrum of clefts are produced using a neuromeric field model in which deficits in specific fields lead to specific types of clefts. To do so, we shall need to expand our vocabulary one step further. Neuromeric concepts must be applied to soft tissue structures as well. To this point, the anatomy of facial fields has been presented in terms of the mesenchymal components that make them up, emphasizing neural crest and paraxial mesoderm. Our discussion of neural crest has focused on: 1) where the neural crest components arise in the embryo prior to their migration, 2) the sequence in which these neural crest fields migrate to the face, 3) the pathways they use to get there, 4) the spatial arrangement of the fields once in final position and 5) the developmental relationships that exist among fields that enables them to fit together in a precise time sequence to build the face. Our discussion of mesoderm has focused on: 1) distinguishing its various anatomical types (paraxial PAM, lateral plate LPM), 2) its mechanism of formation during gastrulation, 3) the neuromeric basis of segmentation as it applies to the gastrulation process, 4) somitomeres and somites as segmental units, 5) neural crest contributions with these mesodermal units, 6) the reorganization of facial mesenchyme into head mesoderm, pharyngeal arches and occipital somites and 7) the spatial origami of head folding by which these units are positioned for final integration into the face. Because the facial bones are so readily distinguished and because they form in such a strict time sequence, much of our discussion of developmental fields has centered on these bony building blocks. But we must now change our emphasis completely to that of the soft tissues. The bones of the craniofacial skeleton, like all bones in the body, are merely the products of soft tissue developmental fields (functional matrices, if you will). Any defect in a bony is merely the manifestation of a problem in the functional matrix that produced it. A field/functional matrix disturbance can therefore manifest itself in bone, in soft tissue structures or in both. The skin of the forehead, nose and philtrum is composed of p5 non-neural ectoderm epidermis and p5 neural crest dermis. The upper eyelid epidermis comes from p5 NNE and the dermis is made from r1 MNC. The epidermis of the lower eyelid could also be made of p5 NNE or r2 ectoderm while the dermis is likely produced by r2 RNC. For this reason the sensory supply to the upper lid is from V1 while that of the lower

lid is from V2. The remainder of the facial skin is produced in accordance with its innervation patterns. Ectoderm corresponding to r2–r11 is found throughout the five pharyngeal arches but actual epidermis and dermis probably is produced exclusively from ectoderm and RNC corresponding to r2 and r3. The dermatomes of the occiput come from cervical neuromeres c2–c4. Neural crest probably produces the hair of the face and scalp from r1–r3 and from c2–c4. It is intriguing that the hair pattern corresponds to the dermis innervated by the dorsal (epaxial) branches of C2–C4 while the dermis supplied by ventral (hypaxial) branches of C2–C4 is hairless. In the same manner, a dorsal/ventral pattern might be seen in V2 and V3 derived dermis. The epaxial dermis would produce hair while the hypaxial dermis would produce beard. Most patterns of human hair formation fit this model. Eyebrows, for example, would occur at the interface between r1 RNC dermis and p5 PNC dermis. Facial muscles supplied by the upper division of the facial nerve are likely to originate from the proximal second arch (i.e. from Sm4 PAM) while those supplied by the lower division of the facial nerve would originate from the distal second arch (i.e. from Sm5 PAM). Oral mucosa is probably formed from the first three pharyngeal arches like a series of car tires stacked one aside the other. The even neuromeres would lie cranial to the odd neuromeres along a line from the commissure to the midpoint of Waldeyer’s ring. Salivary gland formation would result from PAM mesenchyme invaded by oral epithelium. The connective tissue within the salivary glands is neural crest. This has implications for understanding the derivation of the parotid gland. Because this structure is penetrated by the facial nerve it may be reasonable to assign it to r4 and r5 (being distributed along the upper and lower divisions respectively). Facial fat is a neural crest derivative. 17.6.3. The biological basis of the developmental fields The question of whether the functional matrix concept has any provable experimental basis is best answered by a plethora of papers stemming from the quail-chick chimera system popularized in developmental biology by Couly and LeDourain (Le Douarin and Kalcheim, 1999). These investigators showed that visible differences in neural crest cells existed in quail and chick embryos such that microsurgical extirpation experiments could be carried out. When neural crest cells from one type of embryo are transplanted into the other their derivatives can be distinguished under the light microscope. Using the neuromeric map these

NEURAL TUBE PROGRAMMING AND CRANIOFACIAL CLEFTS: PART II authors were able to demonstrate what derivatives came from what levels. For example the mandible, malleus and incus are all neural crest products from level r3. The Couly–LeDourain model also allowed for assessment of programming function. Mesenchyme responds to an occult program in the surrounding epithelium. Ectodermal zone patterns influence the development of dermal bone (Hu et al., 2003). More recently, foregut endoderm has been shown to instruct neural crest what to do (Ruthin et al., 2003). By taking out a certain zone of foregut endoderm, specific parts of the hyoid bone fail to appear. The spatial layout of the hyoid fields is faithfully reflected in the organization of the foregut endoderm. Finally, if a given zone of foregut endoderm is reversed 180 then the corresponding part of the hyoid bone is reversed as well. Thus, all neural crest bone and cartilage derivatives associated with of the foregut arise as the result of programming embedded in the endoderm. Because the endoderm in each region arose from a spatially dedicated zone of epiblast cells it can truly be said that the overall organizational plan of the organism is set up prior to gastrulation. Thus a developmental field is a form of genetically programmed epithelial–mesenchymal signaling that results in the creation of specific anatomical structures. Future research will undoubtedly result in the understanding of the fate of foregut endoderm. Specific zones will correspond to specific structures. Each zone will ultimately be categorized by a unique pattern of gene expression, alterations of which will lead to predictable abnormalities in the neural crest products associated with that zone. We shall return precisely to this concept at a later point when discussing the pathogenesis of the labiomaxillary cleft.

17.7. Assembly of craniofacial developmental fields Developmental fields do not occur in isolation. Interaction with other fields is often required. This is particularly well demonstrated in the formation of facial bones. Many of these structures result when specific populations of neural crest migrate from their nascent position in the neural fold to distant locations. Here they interact with local epithelial cells from which they receive signals that determine cellular mitotic rate (volume) and the physical confines in which such population expansion may take place (shape). Moreover, the presence of one field may be required in order for another field to correctly develop. The footplate of the lacrimal bone is positioned just lateral to the inferior turbinate. The inferior turbinate forms earlier

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in time than does the lacrimal bone. A disturbance in the formation of the inferior turbinate may lead to defective or absent lacrimal system. By the same token problems within the lacrimal system (stenosis of the duct) can occur without affecting the inferior turbinate. It should be thus apparent that developmental fields are the results of tightly regulated sequence of field creation, field positioning and field assembly. Processes such as flexion of the embryonic neuraxis and programmed cell death (apoptosis) are required in order for field assembly to take place correctly. Brain growth is critical to the development of the face. It should be understood, up front, that the migration of neural crest cells refers to a point in time before embryonic folding takes place. All the populations have arrived and are positioned with respect to epithelial developmental zones and pharyngeal arches. Positional genes, such as the DLX system, map out the arches into spatial regions, each with its own developmental fate. Thus, the spatial position of a neural crest population on the neural fold does not confer specificity. Once the cells arrive at their destination, they are ‘instructed’ as to their final anatomical form. Thus, r1 neural crest does not ‘know’ to become the sphenethmoid complex. Those cells that are physically positioned in front of (or inside) the anterior zone of somitomeres 1 will become first the presphenoid and later the ethmoid. The purpose of this chapter is to use neuromeric concepts to understand how the face forms. From this an integrative theory of cleft formation will be presented. I shall begin with facial bones and then proceed to soft tissue structures. This distinction is completely artificial. Facial bones are mesenchymal responses to an epithelial (soft tissue) environment. Bone does not ‘grow itself’: it is the product of a developmental field. Each developmental field is neuromeric in nature and includes all soft tissues associated with a given bone. For example, the zone of bone from which a muscle ‘originates’ corresponds to the same neuromere(s) as the motor innervation of the muscle. The supraspinatus (innervated from neuromere c5) arises from the medial scapula formed from c5 LPM. The larger infraspinatus (innervated from c5 and c6) arises from a larger portion of medial scapula formed from contributions of both c5 and c6 LPM. Facial bone synthesis occurs in a rigid spatiotemporal order. These bones arise almost entirely from neural crest mesenchyme. Understanding the pathways by which neural crest cells migrate into position is a crucial first step. There are three general populations of neural crest involved in constructing the face; the behavior of each depends on its anatomical zone of

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origin. PNC arises from the neural folds above the caudal forebrain. PNC migrates forward like a glacier-like sheet to populate the neural folds of the rostral forebrain (which are lacking in neural crest). PNC is responsible for the synthesis of the anterior cranial fossa, the primary nasal cavity and the superomedial orbit. MNC arises from the neural folds above the midbrain (an enormous structure in the embryo). MNC departs in cranio-caudal order as individual streams and migrates forward beneath the non-neural ectoderm outside the brain to form the remainder of the medial orbit and sphenoid and the caudal components of the medial nasal chamber (the vomer and premaxilla). RNC arises from the neural folds above the hindbrain. RNC migrates in strictly defined segments into the pharyngeal arches. It completes the caudal components of the lateral secondary nasal chamber, the floor and posterolateral wall of the orbit, and forms the oropharynx and larynx. A detailed description of the anatomy of these populations, how they relate to the neuromeric system and how isolated defects in a component population lead to a craniofacial cleft, is the subject of this chapter, concentrating on the fronto-naso-orbito-maxillary complex, as facial clefts occur within this region and examining first how the bone fields form and then the soft tissue anatomy of the nose and mouth. The interactions between underlying bone fields and soft tissue field that characterize the common labiomaxillary cleft will be detailed as these provide a common biological model for craniofacial cleft formation in general (see Figs. 17.26, 17.27 and 17.29–17.32). 17.7.1. The assembly of the face: a method of study Understanding of how component fields of paraxial mesoderm and neural are assembled to form the face and skull involves visualizing structures arising from one anatomical site and then moving into new positions via migration or folding. This is a four-dimensional process. The physical development of certain fields may be dependent upon the correct development of precursor fields. Errors in this developmental sequence lead to varying forms of craniofacial clefts. Craniofacial development is thus like a complex play in several acts. 17.7.1.1. Step 1: Summary of ideas Major ideas introduced by this chapter and Chapter 16 include: 1) the segmental organization of the early embryo based upon the neuromeric system, (2) the mechanism by which gastrulation forms a trilaminar embryo, (3) the anatomy of the resulting germ layers,

including the neural crest, (4) the segmental reorganization of mesenchyme into somitomeres and somites, (5) formation of pharyngeal arches and neural crest migration patterns and (6) derivative analysis of final anatomical structures. 17.7.1.2. Step 2: Key definitions and point of clarification Craniofacial development is an interdisciplinary study. Several contemporary texts are worth consulting. Review the definitions and anatomical abbreviations presented at the beginning of Chapter 16. 17.7.1.3. Step 3: Staging of embryos (general principles) Embryology is the analysis of relationships between order and form. The timing with which anatomical structures make their appearance is all-important to understanding the overall process. Several parameters traditionally used to describe development in the first 8 weeks of life make the literature confusing. These parameters are: 1) time/age, 2) crown–rump length, 3) Carnegie stage and 4) the observable number of somite pairs. Each system has its own degree of (im) precision and overlap. Lumping the events of embryogenesis into weeks is quite imprecise. Critical events may take place within the space of a single day. Fusion of lateral nasal prominence and the medial nasal prominence may occur in as little as 6 hours. Embryo size (measured as crown–rump length in millimeters) is a more accurate measurement of maturation. The Carnegie staging system, originally described by Streeter and refined by O’Rahilly, categorizes development into a series of stages defined by the formation and maturation of key structures. The system derives its name from the leadership role played by the Carnegie Institution as a sponsor of embryological research in the early 20th century. Each stage is tightly linked to crown–rump length, intervals of which are as small as 2 mm through stage 15 (3–36 days). Morphological studies correlating developmental stage with the number of observable somites are useful but are limited to a certain window of embryogenesis. Carnegie stage 9 (days 20–21) is defined by the appearance of somite pairs 1–3. O1, previously described by von Baer and others and confirmed by Huang, may indeed be present at Carnegie stage 8. It turns out that the O1 is incompletely segmented at its rostral end (i.e. from Sm7) but fully epithelialized at its caudal end. Thus the boundary between Sm6 and Sm7 is indistinct but that between somite 1 and somite 2 is complete. Thus Carnegie stage 9 would include up to four somite pairs. Using this numbering system, formation of the

NEURAL TUBE PROGRAMMING AND CRANIOFACIAL CLEFTS: PART II 31st pair (observed between days 29 and 31) would define Carnegie stage 13. Although the number of somite pairs ceases to be of importance in defining subsequent embryonic stages these entities play a vital role in the segmentation of the spine and the organization of the trunk. In addition to four occipital somites, human spines are constructed of eight cervical, 12 thoracic, five lumbar and five sacral somites. Fusion of these latter to form the sacrum is evidenced by the presence of four individual foramina. The coccyx is constructed with an additional two to five somites, bringing the total count in humans to approximately 37–40 pairs. The final somite bearing a myotome is the first coccygeal; therefore the total number of spinal nerves sums to 31 pairs. 17.7.1.4. Step 4: Carnegie staging system: (with special reference to the head and neck) Major events of craniofacial formation can be summarized by Carnegie stage. Key aspects of these will be discussed in greater detail later as they apply to specific anatomical sites (e.g. cranial base synthesis, the formation of the nose). Here, an overview of the developmental timeline is given that may be referred to in the course of the chapter (O’Rahilly, 1987). Measurements of crown– rump length are in millimeters. The beautiful SEM studies of human embryos by Hinrichsen (1985) and Jirasek (1983) constitute an excellent way to visualize these processes. Carnegie stage 1 (0.1 mm, 1 day): fertilization of oocyte. Carnegie stage 2 (0.1 mm, 2–3 days): 16-cell morula (solid sphere of cells). Carnegie stage 3 (0.1 mm, 4 days): blastocyst with central fluid-filled cavity, unattached. Carnegie stage 4 (0.1 mm, 5–6 days): blastocyst attaches to endometrium. Carnegie stage 5 (0.1–0.2 mm, 7–12 days): bilaminar embryo implants into uterine wall. Carnegie stage 6 (0.2–0.3 mm, 13–15 days): primitive streak. Carnegie stage 7 (0.4 mm, 16–17 days): gastrulation, notochordal process. Carnegie stage 8 (1.0–1.5 mm, 18–19 days): Hensen’s node and the primitive pit present. Notochord is now formed and ready to give signals (at next stage) that will regulate somite formation. Neurulation of embryonic nervous system ¼ neural plate þ neural folds (origin of neural crest cells). Carnegie stage 9 (1.5–2.5 mm, 20–21 days): 11 somitomeres present (neuromeric levels r0–r11). In 48 hours the entire CNS is laid out. First appearance of somites (body segmentations). This includes the

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transformation of the eighth somitomere into the first occipital somite. Traditional Carnegie staging does not take this somite into account; one somite must be added mentally to each stage described. Four (in actuality five) somites present. All occipital somites synthesized. Occipito-cervical junction established. Peripheral nervous system development begins at first myelomere (Sm12). Neural folds elevate. Neural crest cells present. Carnegie stage 10 (2.0–3.5 mm, 22–23 days). General: 4–12 somites. Scanning electron microscopy discloses the formation of optic and nasal fields. These are seen as thickening on thin-section micrographs. Neural crest migration from PNC and MNC is under way into the face. 11-somite stage: RNCr migration from r1–r7 is completed, invading Sm1–Sm7 (future PA1–PA3). Neural fold closure begins. Carnegie stage 11 (2.5–4.5 mm, 24–25 days). General: 13–20 somites present. (In reality there are 14–21 somites, so this extends as far caudally as neuromeric level thoracic 8.) The cranial neuropore closes. All neural crest migration is completed during this stage. MNC and RNCc migration terminates at the 14-somite stage. PNC migration is complete at the 16-somite stage. All future neural crest bone fields of the head and neck are now in place. Embryonic flexion takes place. Eyes: optic vesicle. Mouth: PA1 and PA2 are formed. Carnegie stage 12 (3–5 mm, 26–27 days). General: 21–29 somites. (At 30 somites this extends to neuromeric level lumbar 5; only the sacral and coccygeal levels are lacking.) The caudal neuropore closes. Upper limb buds and tail bud. Nose: nasal fields can be discerned. Ears: otic vesicle. Mouth: the primitive oral opening is bounded above by the (apparently) singular prosencephalon and below by the fused first arch swellings of the primitive mandible (mandible precedes maxilla in development). PA3 and PA4 are formed. Carnegie stage 13 (4–6 mm CRL, 28–31 days). General: 30þ somites (At no. 31 the sacrum is beginning to form.) Lower limb buds (l3–s2). Brain: the axis of the nasal placodes to the brain at the end of stage 13 is 110 . The rostral corpus changes rapidly. The future forehead is initially coplanar with the pharyngeal arches but, with rapid brain growth, it projects forward and a prominent vertical midline furrow appears. This marks the beginning of prosencephalic segmentation. It is of the utmost significance that development of the nasal placodes and future fronto-orbital-nasal neural crest fields is associated with forebrain development. Holoprosencephaly always manifests itself as abnormalities

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involving first p6 fields, and then (if more severe) p5 fields. Nose: at 5 mm the epithelium of the nasal placode thickens. The epithelium surrounding the p6 nasal placode becomes convex as p5 neural crest surrounds it and proliferates. Eyes: at 6 mm the optic vesicle becomes cup-like and a rounded optic placode is present (the future lens). Ears: otic vesicle separates from epithelium to ‘sink in’ and form the cochlear–vestibular apparatus. Mouth: PA5 is formed. No true mouth is present in stage 13: PA1 and PA2 (destined to form the future sidewalls of the oral cavity) have not apparently moved forward into position. They are still positioned well caudal to the plane of the fronto-orbito-nasal prominences. When seen from below, the future roof of the nasopharynx shows two thickened ridges of tissue running backward from each nasal placode to converge at Rathke’s pouch. This structure (Rathke’s pouch) is an extremely important anatomical landmark marking the position of the future sphenoid and the posterior border of the anterior cranial fossa. This is the ‘hinge point’ of the frontal lobes. The ridges contain p6 neural crest and serve as conduits for neurons connecting the nasal placodes with the basal forebrain. The covering epithelium of the primitive stomodeum is endoderm (not neuromerically coded). With embryonic folding the combined first and second pharyngeal arches (formed at stage 10) have now moved into position and are merging with the FONPs. The maxillary swelling is made from levels r2 and r4. The mandibular swelling is made from levels r3 and r5. Carnegie stage 14 (5–7 mm, 32 days). General: Mesenchyme (already present) rapidly multiplies and appears to ‘fill out’ the furrow of the epithelium the brain. These epithelial zones are p5 (frontal) and zone p5 (nasal); they flank the optic and nasal placodes. The mesenchyme beneath these zones probably originates from prosencephalic neural crest. The maxillary swelling is now in direct contact with inferolateral aspect of nasal placode and forms LNP (see below). Nose: significant transformations in the nasal placodes. At 6.5 mm epithelial convexity surrounding the placode is maximal; the center begins to ‘sink in’ to the embryonic mesenchyme. This creates the nasal pits. The plane of each pit is much more frontal. This stage is remarkable for the appearance of primary choanae. On either side of the nasal pits a semicircular proliferation of neural crest occurs, creating two opposing ‘parentheses’ of tissue. These masses are referred to by the literature as the medial nasal process (MNP) and

the lateral nasal process (LNP). Although these terms are very imprecise I shall use them for simplicity. Each nasal placode has specific medial and lateral neuroanatomical content. The medial placode contains neurons of the olfactory system. The lateral placode contains neurons of the accessory (pheromonal) olfactory system and neurons associated with GnRH. Neural crest flanking the medial aspect of the placode is of p5 origin and forms the MNP. Neural crest flanking the lateral aspect of the placode is of r2 origin and forms the LNP. Blood supply to the MNP is from the internal carotid while that to the LNP comes from the external carotid. External clefts along the nasal rim noted by Newman and Burdi (1981) occur at this field interface. When the neuroectodermal ‘tunnels’ are complete between the placodes and Rathke’s pouch, cellular instability occurs in the floor of each tunnel. Probably because of apoptosis, each floor opens up in anteroposterior fashion. The interior of the future nasal cavities can now be seen. For the first time, p6 ectoderm of the nasal pits is in contact with the endoderm of the future roof of the oral cavity. Eyes: lens vesicle and optic cup. Mouth: along the oral margin of the medial nasal wall is a mesenchymal swelling representing an invasion of r20 neural crest. This mesenchyme comes from above the mesencephalon, tracks beneath the level of the sphenoid body and then proliferates forward and forward along the inferior margin of the p6 ethmoid neural crest. This r20 MNC forms the vomer and premaxilla. It terminates anteriorly against the p5 mesenchyme in continuity with the MNP, i.e. the future prolabium. Carnegie stage 15 (7–9 mm, 33–36 days). General: Hand plates. Fusion of 1st and 2nd pharyngeal arch creates lamination of fields. Brain: cerebral hemispheres present. Nose: cellulous strands present between nasal pit and olfactory fields in the brain. Nasal pits depressed and well-sculpted due to the proliferation of neural crest mesenchyme surrounding each pit. Brain growth causes movement of the nasal masses toward the midline. Outwardly they appear to diverge at a 120 angle but on histological section the axes of the nasal pits are nearly parallel. When the primarer nasenboden break down to create the primary choanae, the p5 neural crest mesenchyme of the lateral nasal cavity comes into direct contact with r2 neural crest mesenchyme from the maxillary swelling. Eyes: furrow now separates maxillary swelling from LN. This is the future nasolacrimal duct.

NEURAL TUBE PROGRAMMING AND CRANIOFACIAL CLEFTS: PART II Mouth: the sidewalls of the primitive oral cavity are created by the fusion of first arch mesenchyme with the lateral nasal wall. Downward projection of this mass represents the future maxilla. The most internal field of the maxillary complex is the palatine bone, followed by the inferior turbinate and thence the tooth-bearing fields. Carnegie stage 16 (8–11 mm CRL, 37–40 days). General: Foot plates. Nose: nasal pits are now deep holes into the facial mesenchyme. These are completely separated from the oral cavity by tissue termed by Hinrichsen the primarer nasenboden, the neuromeric anatomy of which will be described later. Eyes: retinal pigment. Ears: auricular hillocks begin. Mouth: when seen from below by looking into the primitive oral cavity the primarer nasenboden appear as two swellings running posteriorly toward Rathke’s pouch like the letter V. These swellings represent downward proliferation of p6 neural from the future ethmoid complex underlying the midline of the anterior forebrain. Upper lip formation begins. Carnegie stage 17 (11–14 mm, 41–43 days). General: Finger rays. Nose: olfactory nerve has two separate plexuses; terminal nerve present. The external surface of the nose is significantly different. Furrows in the primary nasal floor of the nose prior to separation. Eyes: nasolacrimal groove represents future duct. Ears: six auricular hillocks complete. Mouth: the nasal floor remains undivided, but proliferation of neural crest on either side causes the r2 and r20 swellings to project downward into the future oral cavity. These represent the maxilla laterally and the premaxilla/vomer medially. No distinct formation of an alveolar ridge at this stage. No labiobuccal sulcus present. Carnegie stage 18 (13–17 mm, 44–47 days). General: Toe rays. Nose: olfactory bulbs present. Nasal tip first appears. Eyes: eyelids begin to develop. Medialization of nasal chambers and frontalization of eyes makes face look ‘human’. Furrows separating anterior facial swellings are smoothed out (e.g. nasolacrimal groove internalizes to complete lacrimal system). Furrows persist behind zygoma and developing ear; these are later developing structures. Mouth: sulcus separates lip from alveolus and prolabium from premaxilla. Premaxillae completely separate from maxillary alveolus. Faint groove marks labial–prolabial junction. Carnegie stage 19 (16–18 mm, 48–50 days). General: Midgut herniation. Nose: olfactory, vomeronasal and terminal neurons present. Olfactory bulbs and nuclei differentiating. No nasolabial angle. Mouth:

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intraoral fusion of premaxillae begins. Maxillary alveolar ridges present. Closure of primary palate begins as fusion between premaxilla and maxillary alveolar ridges. This process follows a strict lingual to buccal, cranial to caudal sequence. Carnegie stage 20 (18–22 mm, 51 days). General: Fingers distinct but webbed. Scalp vascular plexus. Nose: olfactory bulbs complete. Mouth: MxP shelves fully developed and elevating in preparation for closure of hard palate. Carnegie stage 21 (22–24 mm, 52–53 days). General: Fingers free, toes distinct but webbed. Carnegie stage 22 (23–28 mm, 54–56 days). General: Toes free, eyelids open. Mouth: alveolar fusion (primary hard palate) complete. (Secondary) hard palate begins front-to-back closure Carnegie stage 23 (27–31 mm, 57þ days). General: Eyelids fused. Mouth: (secondary) hard palate fusion complete. Soft palate fused half from palatine shelf to free margin of uvulae. 17.7.2. Major themes of craniofacial development 1. The formation of mesenchyme is a more primordial event than the development of the brain. Gastrulation sets up the trilaminar embryo in 48 hours during stage 7. Neural plate and folds appear in stage 8. From a teleological standpoint, proper protection of the nervous system must be ensured. Brain development must take place in the presence of a pre-existent mesenchyme into which it can expand. The covering layers outside the CNS come from either neural crest or PAM. 2. Formation of mesoderm via gastrulation is a cranio-caudal process beginning at the level of the tip of the notochord. The anterior extent of the notochord lies at the junction of the presphenoid and basisphenoid bones. This corresponds to neuromeric level r0. Mesodermal segmentation results as a response to gene signals emanating from different levels of the notochord. No definitive endoderm and intraembryonic mesoderm can be produced anterior to r0. 3. The forebrain sitting in front of the notochord is an evolutionary ‘afterthought’. It arises from induction signals from the tip of the notochord, the anterior visceral endoderm (just in front of the notochord) and a zone of ectoderm located at the extreme anterior aspect of the trilaminar embryo. No mesoderm is associated with forebrain development. All mesenchyme associated with forebrain coverage comes via two mechanisms: a) migration of neural crest from the caudal prosencephalic neural folds and b) migration of PAM from somitomeres and somites arising at level r1 and back.

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4. The forebrain arises as a vesicular structure that, under normal conditions, becomes divided into two hemispheres. Only the forebrain has dura and it is of exclusively neural crest origin. Growth of the forebrain occurs in all directions such that the two hemispheres eventually surround the midbrain and hindbrain. Intracranial dural anatomy can be mapped on the basis of sensory supply to neural crest from r1, r2 and r3. 5. Epithelium surrounding the brain is not stable without a supporting layer of mesenchyme. Neural crest creates the dermis underlying ectoderm above the skull (the future skin of the face and scalp). Neural crest creates the submucosa underlying the endoderm of the oropharynx beneath the skull base. 6. The brain case (neurocranium) has two primary units. The membranous neurocranium consists of dermal bones formed by signals (and some PAM) differentiated as a result of signals from the dermis above and meninges below to an intermediate layer of mesenchyme. The chondral neurocranium forward from the sella turcica is of neural crest origin. Backward from the sella turcica it all comes from PAM. 7. By Carnegie stage 10 all sources of PAM for the growing brain are synthesized. Eleven somitomeres have formed. Transformation of the last four (Sm8– Sm11) creates the occipital somites. 8. By Carnegie stage 11 the physical provision of mesenchyme to the brain and face is complete. This requires four elements: a) PAM, b) neural crest, c) pharyngeal arches and d) folding. Organization of paraxial mesoderm into somitomeres begins with notochord development in stage 8. In stage 9 all somitomeres contributing to the head are present. This includes the transformation of Sm8–Sm11 into the four occipital somites. All neural crest migration to the head (begun at stage 9 in mammals) is complete by stage 11. Pharyngeal arches first appear as Sm2–Sm5 and are transformed into PA1 and PA2. Embryonic flexion brings all these mesenchymal sources into contact with the forebrain. Melding of individual fields to form the face is not a slow process. Although the four types of neural crest that migrate into the head and neck (PNC, MNC, RNCc and RNCr) complete their migrations at different times of embryonic development, all neural crest are in place within about 15 hours. These components of the future face are initially located in parts of the embryo that are widely separated. The PNC and MNC are positioned together initially around the brain while the rostral RNC is found in the pharyngeal arches. Furthermore, by the time PNC has populated the future frontonasal zone of the embryo (16-somite stage), reorganization of the first 11 somitomeres into pharyngeal arches and occipital somites is complete. These quite

disparate components must be physically approximated in order for facial assembly to occur. Cephalic folding accomplishes this goal in less than 24 hours. Human embryos at day 22 have cephalic neural folds that are broad and thick. These stick up in the air like the fins of some ancient Cadillac limousine. The reason for this neural fold projection is the tremendous proliferation of head mesenchyme lying just beneath them. This mesenchyme is, of course, a product of the explosive growth of MNC and RNC populations combined with the contribution of the first somitomeres. These somitomeres lie astride the future cranial base like saddlebags. Sm1 does not participate in pharyngeal arch formation. Sm1 PAM will form the basisphenoid and part of the extraocular musculature. The rapidly growing embryo lies atop a yolk sac that is not growing much at all. As the cephalic part of the embryonic axis expands, its yolk sac ‘tether’ forces the neural plate to bend at specific sites. The first of these flexures occurs at the site of the future mesencephalon. At day 22 the cranial (mesencephalic) flexure bends the prosencephalon ventrally toward the pharyngeal arches. In less than 24 hours the angle between the forebrain and the rest of the neuraxis decreases from >150 to lateral incisor B) is a manifestation of the relative biological ‘maturity’ of the mesenchymal field within which each tooth develops. The frontal process field (PMxF) is a vertical offshoot of PMxB; this subfield is the biologically ‘newest’ tissue. Pathology affecting the premaxilla occurs as a spectrum based on this original developmental pattern. A deficiency state of the premaxilla will first occur in the most distal aspect of the frontal process (i.e. at its most cranial extent). As the mesenchymal deficit worsens, the frontal process will be reduced in a cranialcaudal gradient. ‘Scooping out’ of the piriform rim results; the nasal lining is pulled down as well. This causes depression of the alar base and a downward– lateral displacement of the lateral crus. Biological signals from PMxF do not affect lip formation. Therefore the forme fruste manifestation of premaxillary deficiency is a cleft lip nose with a perfectly normal lip. Once the frontal process is eliminated, the deficiency state shows up in the lateral incisor field. Progressive degrees of premaxillary deficiency in the lateral incisor field cause progressive loss of alveolar bone substance. Alveolar bone development follows a gradient from the incisive foramen forward. Mild deficiency causes notching on the labial surface. As the deficiency worsens the notch deepens backward toward the incisive foramen. A critical lack of alveolar bone mass results in outright failure of lateral incisor development. The mechanisms by which bone is affected in a cleft have already been covered, but a little more detail is required on how a deep osseous field can affect the overlying soft tissue. Tessier’s series includes colobomas, eyebrow absence, as well as skin deficits. I propose that the soft tissue deficits seen in craniofacial clefts represent either failures of fusion or failures of formation. The notch in the alar rim is a boundary zone fusion failure, whereas the absence of eyelashes on the lateral lower eyelid represents the failure of that field to produce a product. Neural crest mesenchymal cells that participated in lash formation in an adjacent field fail to behave correctly in the target field. Failure of formation is a more difficult topic to discuss (and beyond the scope of this chapter) but the mechanism

of fusion may well be universal throughout the head and neck. The role of the LNP–MNP nasal bridge can be examined as a case in point. Migration of myoblast containing mesenchyme along the Simonart’s band cannot occur without a generalized breakdown of the epithelium covering the lateral lip element, the r2–r20 skin bridge, and the p5 prolabial skin. Stability of the epithelia in facial processes is maintained by repression activity of Sonic hedgehog (SHH) within the skin. BMP4 causes de-repression of SHH; epithelial breakdown results. Thus absence or deficiency of an appropriate BMP4 signal will lead to restricted expression of SHH, abnormal persistence of epithelium and failure of mesenchymal fusion. Since the signal emanates from the PMx is diffuses down from the piriform fossa to the lip. Therefore, as the BMP4 signal is progressively weaker, lip cleft severity worsens: i.e. from a vermilion notch to an incomplete form involving half of the lip and finally to a complete cleft lip. In summary, the volume of the premaxilla determines whether or not lip closure can occur. First, small premaxillae make small amounts of BMP4. The amount of BMP4 produced is critical to produce the epithelial breakdown necessary to permit mesenchymal merger. Second, when the premaxilla is too small, the physical distance between it and maxilla will exceed a critical dimension. Epithelial bridge formation between the alar base and the prolabium cannot occur. Third, if this critical distance exists at the level of the incisive foramen, a cleft of the secondary palate will form. This is because the horizontal repositioning of the palatal shelf from the maxilla must make contact with the vomer just posterior to the incisive foramen. The process is just like a zipper. If initial contact is not made, fusion of the palatal shelf to the vomer cannot take place. Even if initial contact is made, a secondary palatal cleft can still result due to displacement of the vomer away from the midline. The vomer can become warped by the inequality of growth forces on either side of the cleft. Thus, the zipper may get started anteriorly but as its process proceeds posteriorly, when is encounters the deviated vomer, a palatal cleft will ensue.

17.8. Conclusion The idea of common neuromeric definition provides us with a new understanding of the anatomical rationale behind the bones, muscles and fascia of the craniofacial skeleton. These structures, whether derived from neural crest, PAM or both, can be traced back to the level of the embryo from which its cells originated. The neuromeric system offers a unique perspective

NEURAL TUBE PROGRAMMING AND CRANIOFACIAL CLEFTS: PART II on craniofacial deformities. Pathological states involving a particular neuromeric level can affect one or all of its derivatives. Deformities involving seemingly unrelated bones or muscles can be understood in terms of common neuromeric levels of origin. Genes found at multiple levels of the embryo may be misexpressed within a single neuromeric level or on only one side of the body. The purpose of this chapter was to outline the principles of the neuromeric system and to describe the manner in which the craniofacial skeleton originates. Each bone was assigned to a neuromere(s) of origin using a color code. The time course of assembly of the craniofacial bones was discussed. This information was then related to the clinical pattern of the common labiomaxillary cleft. The application of these principles to the rare craniofacial clefts described by Tessier enables us to understand that these pathologies are variations in the craniofacial field system.

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Schoenwolf G (1997). Neurulation: coming to closure. Trends Neurosci 20: 510–517. Serbedzija GN, Bonner-Fraser M, Fraser SE (1992). Vital dye analysis of cranial neural crest migration in the mouse embryo. Development 116: 297–307. Serbedzija GN, Fraser SE, Bronner-Fraser M (1990). Pathways of neural crest migration in the mouse embryo as revealed by vital dye labeling. Development 108: 605–612. Sheen J, Sheen AP (1998). Aesthetic Rhinoplasty, 2nd edn. Quality Medical Publishing, St Louis. Shino M, Chikamatsu K, Yasuoka Y, et al. (2005). Congenital arhinia: a case report and functional evaluation. Laryngoscope 115: 1118–1123. Siebert JR, Kokich VG, Cohen MM, Lemire RJ (1981). The facial features of holoprosencephaly in anencephalic human fetuses. II. Craniofacial anatomy. Teratology 23: 305–315. Siebert JR, Kokich VG, Warkany J, Lemire RJ (1987). Atelencephalic microcephaly: craniofacial anatomy and morphologic comparisons with holosprosencephaly and anencephaly. Teratology 36: 279–285. Streit A (2004). Early development of the cranial sensory nervous system: from a common field to individual placodes. Dev Biol 276: 1–15. Tapadeia MD, Cordero D, Helms JA (2005). It’s all in your head: new insights into craniofacial development and deformation. J Anat 207: 461–477. Tessier P (1969). Fentes orbito-faciales verticales et obliques (colobomas) completes et frustes. Ann Chir Plast 19: 301–311. Tessier P (1976). Anatomical classifications of facial, cranio-facial, and laterofacial clefts. J Maxillofac Surg 4: 69–92. Tessier P (1981). Plastic Surgery of the Orbit and Eyelids (trans. SA Wolfe). Mosby Inc. (Masson), Philadelphia. Toraynski E, Jacobiec FA (1982). Cyclopia and synophthalmia: a model of embryologic interactions. In: FA Jacobiec (Ed.). Ocular Anatomy, Embryology, and Teratology. Vol I of TD Duane, EA Jaeger (Eds.), Biomedical Foundations of Ophthalmology. JB Lippincott, Philadelphiach. 6. Webb JF, Noden DM (1993). Ectodermal placodes: contributions to the development of the vertebrate head. Am Zool 33: 434–447. Zhang Z, Song Y, Zhao X, et al. (2002). Rescue of cleft palate in Msx-1 deficient mice by transgenic bmp4 reveals a network of BMP and Shh signaling in the regulation of mammalian palatogenesis. Development 129: 4135–4140.

Fig. 17.1. Structure of a homeodomain protein. This highly conserved region contains 60 amino acids that form a DNA-binding helix–loop–helix structure. These are encoded by a180 nucleotide region called a homeobox. Humans contain 39þ Hox genes found in four clusters of four different chromosomes. (Reproduced from Carlson, 2004.)

Fig. 17.2. Homeobox-containing regions of Drosophila and the mouse and their segmental expression. This follows a craniocaudal sequence. Spatiotemporal expression follows a strict 30–50 direction, exactly following the physical location on the chromosomes. (Reproduced from Carlson, 2004.)

Fig. 17.3. Anatomy of the three-part (A) and five-part (B) human brain. The nomenclature of neuromeres is derived from these terms. (Reproduced from Carlson, 2004.)

Fig. 17.4. Schematic representation of signaling centers acting on and within the early embryonic brain. (A) In response to signals (greens arrows) from the anterior visceral endoderm, the prechordal plate and the notochord, the neural tube expresses oyx-2 in the future forebrain/midbrain and gbx-2 in the hindbrain/spinal cord. (B) Later in development, signals fFGF-8 (green) and wnt-1 (yellow) from the isthmic organizer induce decreasing gradients of en-1 and en-2 (blue) on either side. Sonic hedgehog (shh, red) is secreted from the other two organizers, the anterior neural ridge and the zona limitans, as well as the ventral part (floor plate) of the neural tube. D, diencephalon; Mes, mesencephalon; r, rhombomere; T, telencephalon. (B adapted from Lumsden and Krumlauf, 1996; figure as a whole reproduced from Carlson, 2004.)

Fig. 17.5. Generalized vertebrate embryo showing neuromeres and distribution of major signaling molecules. Midbrain/hindbrain signaling regions ¼ arrows just rostral to r1. Arrows between r2 and r3 represent a hypothetical signaling region in the forebrain (Bally-Cuif and Wassel, 1995). (Reproduced from Carlson, 2004.)

Fig. 17.7. The rostral prosencephalic folds (overlying prosomeres p6 to p5/p4) have no neural crest cells. They contains placodes. When neural crest arising from the caudal prosencephalic neural folds (overlying prosomeres p4/p3) flows forward in sheet-like fashion, the mesenchymal substrate of the frontonasal process is complete. The submucosa of the nasal vestibular lining, the dermis of the philtrum, columella, nose and forehead result. Neural crest mesenchyme forms the nasal and lacrimal bones; it cooperates with r1 neural crest to form the lateral lamina of the medial orbital wall and the frontal bone. (Modified with permission from Schoenwolf, 1997.)

Fig. 17.6. Early stages in the formation of cranial ectodermal placodes (blue) in the chick embryo, as viewed from the dorsal aspect. These specialized zones of the neural folds interact with neural crest cells to produce the neurosensory–endocrine apparatus of the embryo (the pituitary, olfactory systems, lens, inner ear and specialized ganglia). (Reproduced from Carlson, 2004.)

Fig. 17.8. Human mesoderm development, both extra- and intraembryonic, in staged cross-sections. Note: (1) Vesicle formation causing splitting of the lateral plate. (2) Paired dorsal aortae are seen caudal to the heart. In the head, aortic arches ascending through the pharyngeal arches connect to the dorsal aortae (these latter will fuse). (3) Somatic mesoderm LPMs makes up body wall and the limb bones, and ‘programs’ the muscles of the extremities. (4) Visceral mesoderm LPMv remains closely applied to the endoderm, forming the muscle layers surrounding the gut. (Reproduced from Carlson, 2004.)

Fig. 17.9. Relationship between somitomeres and somites. Cranial somitomeres (open circles) take shape along Hensen’s node until seven pairs appear. All somitomeres caudal to Sm7 have a different fate (ovals): they become transformed into somites (rectangles). As the most anterior of the caudal somitomeres transform into somites, additional somitomeres take shape posteriorly. For a while, the equilibrium between somitogenesis anteriorly and somitomeres synthesis posteriorly keeps the number of caudal somitomeres at 11. (Reproduced from Carlson, 2004.)

Fig. 17.10. The sclerotome differentiates before the dermomyotome as it surrounds the spinal cord to form the vertebral arches. The medial sclerotome from dorsal to ventral forms the vertebral body, intervertebral disk, proximal rib and connective tissue. The lateral sclerotome forms the vertebral arch, the pedicle, the distal rib and connective tissue surrounding the ganglion. (Reproduced from Larsen, 1997, with permission from Elsevier.)

Fig. 17.11. Hox gene expression in relationship to vertebral development in the mouse (note the additional thoracic and lumbar vertebrae compared to humans). Green asterisk, definite expression of Hox gene; purple asterisk, caudal limit where hox expression fades. (Reproduced from Carlson, 2004.)

Fig. 17.12. The dermomyotome (cervical neuromere c2 and below) differentiates into a dermatome and a myotome. These split into epaxial and hypaxial regions. The epimere forms the deep muscles of the back. The hypomere in the thorax forms three layers of the chest wall. In the abdomen, a fourth division ventral segment splits off to form the rectus. This multilevel muscle arises from a vertical fusion from neuromeric levels t12–l5. (Reproduced from Larsen, 1997, with permission from Elsevier.)

Fig. 17.13. Parasegmentation. Sclerotomes recombine to form vertebrae. As segmental spinal nerves grow out to innervate the myotomes, the cranial segment of each sclerotome recombines with the caudal segment of the next superior sclerotome to form the vertebral rudiment. (Reproduced from Larsen, 1997, with permission from Elsevier.)

Fig. 17.14. Patterns of gene expression in relation to neuroanatomical landmarks. Cranial sensory nerves derived from neural crest and placodal precursor are laid out in proper register. CRABP, cytoplasmic retinoic acid-binding protein; RAR, retinoic acid receptor. (Reproduced from Carlson, 2004.)

Fig. 17.15. Origin of cranial nerves in relation to rhombomeres (r) in the chick brain. Note that pharyngeal arches 1–3 occupy pairs of rhombomeres. Pharyngeal arches 4–5 are in register with pseudorhombomere 8–11. (Reproduced from Carlson, 2004.)

Fig. 17.16. Lateral view of organization of head and pharynx of a 30-day-old human embryo with individual tissue components in register. The identity of germ layer derivatives with respect to the neuromeric level or origin is maintained throughout ontogeny. (Reproduced from Carlson, 2004.)

Fig. 17.17. Pharyngeal arch developments seen in scanning electron micrograph of a 4 mm human embryo 30 days old. 1–3, pharyngeal arches; H, heart. Note physical continuity between mesenchyme of Sn1 (putative premandibular arch PA0) with PA1 posteriorly and the developing brain anteriorly. (Originally from Jirasek 1983; modified from Carlson, 2004.)

Fig. 17.18. Pharyngeal arches contain mesoderm derived from paired somitomeres and an arterial axis. Prior to actual assembly of the arch neural crest cells in neuromeric register with the arch pour over each somitomeres like taffy over an apple. The cranial somitomere of each pair contains myoblasts dedicated to forming axial muscles, specifically those assigned to the eye and the tongue. These are supplied by general somatic efferent neurons the nuclei of which reside in the medial motor columns. The caudal somitomere contains myoblasts dedicated to forming pharyngeal arch muscles. These are supplied by general visceral efferent neurons the nuclei of which reside it the lateral motor columns. Because all the muscles serving the head and neck are striated, the word ‘visceral’ in ‘general visceral efferent’ is anatomically outdated. (Reproduced from Sadler, 1990.)

Fig. 17.19. Organization of major components of the vertebrate skull. (A) Primitive aquatic vertebrate showing chondrocranium (green), viscerocranium (orange) and dermocranium (brown). (B) Human infant. (Reproduced from Carlson, 2004.)

Fig. 17.20. Vertebral embryology and its relationship to formation of the cranial base. The dens of the axis (tan color) is analogous to a vertebral body. It is a fusion of components from somites 4–6 (r11–c2). The basioccipital bone forms the cranial base anterior to the foramen magnum. It, like the dens, is a fusion of the ventral neural arches. The lateral aspect of each occipital somite is analogous to the vertebral pedicle. These sum up to form the exoccipital bone, the caudal aspect of which articulates with the tubercles of the atlas. (Reproduced from Carlson, 2004.)

Fig. 17.21. Embryogenesis of the posterior cranial base involves amalgamation of the first four occipital somites. These can be considered as primitive vertebrae. The ventral hemal arches combine to make the basioccipital bone(s), the lateral pedicles produce the exoccipital bones and the neural arches form the supraoccipital bone(s). Purple, r1 presphenoid and basisphenoid; turquoise, r8 first occipital somite; red, r9 second occipital somite; pink, r10 third occipital somite; blue-green, r11 fourth occipital somite.

Fig. 17.22. Although somites 1–4 initially appear as somitelike masses, they undergo a topological transformation in which they become inclined and stack up sequentially inside each other like Russian dolls. Under the influence of occipital lobe development the fused neural arches undergo expansion posteriorly and superiorly. Each of the occipital somite myotomes contributes myoblasts to the tongue. The low hairline and large tongue seen in Down syndrome reflect misallocation of paraxial mesoderm away from the occipital braincase and toward the tongue muscles.

Fig. 17.23. Five occipital somites form the avian occipital skull. This model has been subsequently applied to the human skull (vide infra). (Reproduced from Huang et al., 2000 with permission.)

Fig. 17.24. Skull base, demonstrating the spatial territories on a neuromeric basis. Bone derivatives listed – Blue, r2 alisphenoid (greater wing), lateral pterygoid plate and anterior parietal. Green, r3 andible, posterior parietal, squamous temporal, tympanic, malleus and incus plus muscles of mastication. Derivatives involving the second and third pharyngeal arches (r4–r7) and somitomeres 4–7 are not shown. Orange, r4 styloid process and stapes plus superior oblique. Yellow, r5 muscles of facial expression. Hot pink, r6 petrous temporal plus lateral rectus. Lilac, r7 mastoid temporal plus levator veli palatini. (Modified from Agur and Dalley, 2004.)

Fig. 17.25. Map of cephalic mesoderm in the chick embryo. Note transformation of Sm8 into O1. (Reproduced from Noden, 2005.)

Fig. 17.26. Paul Tessier’s numeric classification of craniofacial clefts recognized two tiers, above and below the orbit. Maxillary clefts are numbered 0–7 while orbitofrontal clefts are numbered 8–14. Empirically the two zones display pairing in which the sum of the lower and upper clefts is 14. The system has several minor flaws. It does not distinguish between states of field deficiency and field excess. Lumping together of developmental fields occurs thus: the common cleft involving PMx belongs to zone 2 rather than the inferior turbinate zone 3. Clefts in zone 3 are much more devastating because they involve an entirely different mechanism. (Reproduced from Tessier, 1981 with permission from Elsevier.)

Fig. 17.27. The Tessier clefting system can be understood as a series of individual developmental field knockouts. These involve successive zones of prosencephalic, mesencephalic and rhomboencephalic neural crest. All elements of the ethmoid complex (including the septum) are r1 derivatives and should bear the same color code as the sphenoid. Notice the spatial correspondence between the four mandibular fields (Mn0, incisors; Mn1, canine and bicuspids; Mn2, molars; MnR, ramus) and the premaxillary/maxillary fields (PMx, incisors; Mx1, canine and bicuspids; Mx2, molars). Mn0 is analogous to Mx0/PMx. The inferior alveolar nerve and the superior alveolar nerve have three branches each. PMx (Mx0) contains Tessier clefts #0, #1 and #2; Mx1 contains Tessier clefts #3, #4 and #5; Mx2 contains Tessier cleft #6; MxP contains Tessier cleft #7.

Fig. 17.28. Orbitosphenoid model showing the timing of neural crest migrations. Note the uncanny spatial manner in which the Tessier clefts line up as an maxillary tier (clefts 0–7) and a fronto-orbital tier (clefts 8–14. Neuromeric theory explains certain inconsistencies of the classification such as clefts occupying common zones. Both #3 and #4 belong to Mx1. #0 and #14 are not clefts occupying common zones. Both #3 and #4 belong to Mx1. #0 and #14 are not clefts per se (no fields are deficient or absent); they present failure of midline approximation.

Fig. 17.29. The frontal processes of the premaxilla (PMxF) and maxilla (MxF) are shown color-coded for r20 and r2 respectively. PMxF arises as a byproduct of the lateral incisor field of the premaxilla (PMxB). Tessier #1 clefts manifest between the central and lateral incisors or as abnormalities of PmxA per se.

Fig. 17.30. In situ anatomy of the premaxilla is seen here in the fetal skull. Growth vectors of both PMxF and MxF are depicted by arrows. (Reproduced from Barteczko and Jacob, 2004 with permission.)

Fig. 17.31. Neuromeric coding of the medial nasal wall. The relation of PMxA central incisor field to the nasal bone field (N) is reflected by the combination of Tessier clefts #1 and #13.

Fig. 17.32. Neuromeric coding of the lateral nasal wall. The key position of the inferior turbinate field (IT) is shown. This is the earliest field of the maxillary complex to develop. IT serves as the scaffold for the frontal process of the maxilla (MxF) and, subsequently, the frontal process of the premaxillary (PMxF). In Tessier cleft #3 IT failure blocks normal development of MxF and, PMxF and subsequent formation of the lacrimal field (L). Tessier cleft #11 can cause isolated deficits in L alone (i.e. lacrimal duct stenosis).

Fig. 17.33. A holoprosencephalic fetus demonstrating complete absence of the premaxilla and philtrum. Without an adequate r1 perpendicular ethmoid plate, r20 mesenchyme destined to become the premaxilla and vomer cannot migrate into position. (Reproduced from Larsen, 1997, with permission from Elsevier.)

Fig. 17.34. Frontal bone embryogenesis occurs via interaction between p5 prosencephalic neural crest forming the forehead dermis and r1 mesencephalic neural crest forming the dura. This anencephalic fetus demonstrates outright failure of forehead skin innervated by V1 and V1-innervated dura. This localizes the defect to the first rhombomere. Anencephaly is also characterized by a misshapen sphenoid (an r1 product). Residual r1 derivatives of orbital roof, upper eyelids and ethmoid complex are unaffected. Skin coverage over the nose and philtrum (p5 dermis) is likewise normal. (Reproduced from Larsen, 1997, with permission from Elsevier.)

Fig. 17.35. Cyclopia, demonstrating deficiency states of p6 (near total) and p5 (subtotal). Only a single nasal placode is present. The stalk of the nasal proboscis lies above the plane of the unitary orbit. This reflects the gross absence of p5 nasal mesenchyme with contraction directed toward the foramen cecum. (Reproduced from Carlson, 2004.)


Chapter 18

The oral–facial–digital syndromes JOSEPH R. SIEBERT* Children’s Hospital and Regional Medical Center and University of Washington, Seattle, WA, USA

18.1. Introduction The first description of oral–facial–digital syndrome (OFDS) in the modern literature was by PapillonLeáge and Psaume (1954). Given the wide variety of findings, it is conceivable, however, that patients were described earlier, and in fact a report of apparent Mohr syndrome can be found as Case 460 (‘Monstrorum humanum hexadactylum’) in Otto’s 1841 volume Monstrorum sexcentorum descriptio anatomica (J.B. Beckwith, personal communication). The first English description was by Gorlin and colleagues (1961). Several hundred patients have been described since. Malformations commonly involve the oral (hypertrophic frenula, lingual hamartomas, cleft palate), facial (ocular hypertelorism, cleft lip) and digital (brachydactyly, polydactyly, syndactyly) regions (Fig. 18.1 and Table 18.1). The association of specific anomalies and inheritance patterns forms the basis for recognizing at least 12 variants (Table 18.2). A variety of terms has been used to describe the cluster of syndromes known as OFDS, including dysplasia linguofacialis, Mohr syndrome, Mohr–Majewski syndrome, orodigitofacial dysostosis, orofaciodigital syndrome, oro-facio-digital syndrome, Thurston syndrome and Varadi syndrome. The first reported cases were female and suggestive of X-linked dominance with prenatal lethality in males. Rimoin and Edgerton (1967) reported additional kindreds in which males and females were affected. The presence of consanguinity in some families was taken as evidence of autosomal recessive inheritance, and indeed the majority of OFDS variants are autosomal recessive. Although most cases are X-linked dominant or autosomal recessive, another X-linked subtype, which is not lethal in males, has been described and may represent an allelic variant of OFDS type

I (Edwards et al., 1988). The inheritance of variants known only as single cases is not understood. The incidence of OFDS is estimated at 1:50 000– 1:250 000 live births. Among individuals with cleft lip or palate, the frequency is much higher, at 8–16 per 1000 (Jacquemart et al., 1980). Because of the lethality to males in some cases, the overall incidence of females is greater than males, approaching 2:1. It is unclear if OFDS exhibits any predilection for particular ethnic groups. OFDS type VI (Varadi syndrome) was initially described in a large cohort of European gypsies (Varadi et al., 1980), but cases have since been noted in other ethnic groups as well (Mu¨nke et al., 1990). OFDS type V (Thurston syndrome) has been ascribed only to persons of Indian background, although a child showing phenotypic overlap with types V and VI has been reported from China (Chung and Chung, 1999). Classification systems continue to develop and will probably be refined as additional phenotypic and genetic information is gained (Fenton and Watt-Smith, 1985; Neri et al., 1995). At present, extensive phenotypic overlap results in jumbled classification schemes and complicates attempts to distinguish new types of OFDS (Gorlin et al., 1990; Gurrieri et al., 1992; Camera et al., 1994; Toriello et al., 1997; Moran-Barroso et al., 1998). Intermediate forms or new variants of OFDS have been reported, largely on the basis of novel characteristics (Toriello, 1993; Moran-Barroso et al., 1998; Toriello and Lemire, 2002; Yildirim et al., 2002). However, listings such as those laid out in Table 18.2 remain somewhat arbitrary and do not necessarily reflect complex interrelationships. For example, type I exists in mild and severe forms (Driva et al., 2004). Some have suggested abandoning type VII entirely. It has been described in a mother and daughter, both of whom have an OFD1 mutation. Therefore the type is either allelic to

*Correspondence to: Joseph R. Siebert PhD, Department of Laboratories (A-6901), Children’s Hospital and Regional Medical Center, 4800 Sand Point Way NE, PO Box 5371, Seattle, WA 98105, USA. E-mail: [email protected], Tel: þ1-206-987-2581, Fax: þ1-206-987-3840.

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Fig. 18.1. (A) General appearance. This hydropic male fetus with OFDS was delivered at 18 weeks and exhibited disproportionately shortened limbs, short tubular bones, small or absent ossification centers in multiple vertebrae and dysplastic epiphyses. Skeletal dysplasia is a hallmark of OFDS type IV. Additional autopsy findings included hypothalamic hamartoma, bilateral cystic renal dysplasia, pulmonary hypoplasia and shortening with malrotation of the small bowel. (B) Facial features. Note nuchal thickening, small mandible, notched upper lip and ocular hypertelorism. Ears were low-set and posteriorly rotated. Multiple oral frenula and lingual hamartomas were apparent on further examination. (C) Polysyndactyly of hands. Eight digits were apparent on each hand (left shown). Patterns of syndactyly were not symmetrical. (D) Polysyndactyly of feet. Seven digits were apparent on the right foot (shown) and six on the left. Patterns of syndactyly were not symmetrical.

OFD1 or exhibits variable expression of the gene (Nowaczyk et al., 2003; Toriello et al., 2004). The classification of types VIII and IX has been questioned by some workers (Camera et al., 1994), whereas the Gabrielli variant may come to be recognized as a legitimate type (Ferrero et al., 2002; Obregon and Barreiro, 2003). Universal agreement does not yet exist for types XI and XII.

The distinction between autosomal recessive OFDS and other syndromes has also been challenged (Hingorani et al., 1991; Lin et al., 1991; Mu¨nke et al., 1991; Verloes et al., 1992; Franceschini et al., 1995; Neri et al., 1995). In particular, some patients with Beemer–Langer syndrome, Pallister–Hall syndrome and Majewski short-rib polydactyly syndrome have

THE ORAL–FACIAL–DIGITAL SYNDROMES Table 18.1 General findings in the oral–facial–digital syndromes Oral features Hypertrophic or duplicated frenula Lobulated or nodular tongue (lingula hamartoma) Cleft (bifid) tongue Absent or supernumerary teeth, especially incisors (primary and secondary) Widely spaced teeth Odontogenic keratocyst High arched palate Cleft palate (primary or secondary; often irregular or asymmetric) Cleft alveolar ridge Cleft uvula Facial features Frontal bossing Ocular hypertelorism / telecanthus Nasal changes: Broad, flat nasal bridge Broad nasal tip Short columella Notched or hypoplastic alae Thin nares Choanal atresia Lowset or posteriorly rotated ears Hypoplastic maxilla Cleft lip (median, pseudocleft) Micrognathia Digital features Polydactyly (central, pre- or postaxial) Syndactyly Brachydactyly Clinodactyly Duplicated hallux (generally involving great toe) Other anatomical changes Central nervous system (see Table 18.3 for details) Polycystic or ectopic kidney; hydronephrosis Cardiac malformation (endocardial cushion defect, tetralogy of Fallot, aortic stenosis) Skeletal dysplasia, especially mesomelic Short, variably bowed tibiae Shortened arms Narrow thorax; short sternum; abnormal ribs Wide metaphyses Talipes deformities Genital defects (agenesis or hypoplasia of penis, hypospadias, shawl scrotum, cryptorchidism, testicular hypoplasia, ambiguous female genitalia) Defects of upper airway (paresis of hypopharynx; hypoplasia of epiglottis, larynx, or trachea; tracheomalacia) Cutaneous findings (milia, alopecia) Source: from Mu¨nke et al., 1990; Toriello, 1993; Ade`s et al., 1994; Toriello et al., 1997

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phenotypic features that are virtually indistinguishable from variants of OFDS.

18.2. Phenotypic manifestations Phenotypic changes in OFDS are widely variable and account for many of the diagnostic and taxonomic uncertainties over this group of disorders. For example, significant phenotypic differences have been observed within families and genetic forms are not always distinguishable on the basis of phenotypic appearance. 18.2.1. Oral anomalies A variety of anomalies are recognized for the oral region. Frenula are broad and short and may involve maxillary, mandibular or lingual tissues; accessory frenula may be present, although these may be more suggestive of Pallister–Hall syndrome (Mintz et al., 2005). Lingual hamartomas appear as single or multiple nodules and consist of skeletal muscle, adipose tissue or salivary glands. Some lesions have a more lipomatous than hamartomatous appearance (Ghossaini et al., 2002) and may also involve the palate (Velepic et al., 2004). Abnormalities in the frenula restrict movement of the tongue. Teeth, especially the incisors, are commonly absent, although supernumerary incisors have also been identified in both primary and permanent dentition (Leonardi et al., 2004). 18.2.2. Facial anomalies Among facial changes, cleft lip and palate are common findings in OFDS. Clefts may be ‘true’ clefts, but more often a midline notch or ‘pseudocleft’ involves the vermilion border of the upper lip and gives a feline appearance. Clefts in the primary or secondary palate may be midline and complete, or submucous; they occur frequently, as do defects in the alveolar ridges. The bridge of the nose is often broad and flat, and the eyes widely separated. The latter change may be a manifestation of classic ocular hypertelorism or dystopia canthorum. 18.2.3. Digital anomalies Digital anomalies vary in severity and include brachydactyly, polydactyly and syndactyly, which can involve any of the digits of one or more extremity. Polydactyly may be preaxial, postaxial, central or develop from a combination of these types.

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Table 18.2 Variants of oral-facial-digital syndromes Type

Major features

Inheritance

I

Hyperplastic frenula Lobulated tongue Hypoplasia of nasal cartilage Cleft lip or palate Digital malformations Cutaneous milia Porencephaly Agenesis of corpus callosum Sparse, brittle hair Ocular hypertelorism Micrognathia Hydrocephalus Bifid or duplicated hallux ‘See-saw’ winking

X-linked dominant; prenatally lethal in males

Autosomal recessive

Skeletal dysplasia

Autosomal recessive

Cleft lip Postaxial polydactyly Early dental loss Central polydactyly Lingual, sublingual swellings Cerebellar dysgenesis Facial asymmetry Hydronephrosis Short tibiae or radii Preaxial and postaxial polydactyly Retinochoroidal coloboma Dandy-Walker malformation Retrobulbar cysts

Autosomal recessive (increased prevalence in individuals of Indian ethnicity) Autosomal recessive

Fibular aplasia

Unknown

Central polydactyly ?? No cerebellar anomalies ?? Myelomeningocele Stenosis of aqueduct of Sylvius Dysplasia of atrioventricular valves Natal teeth Fragmented alveolar ridges Absent central incisors Duplicated vomer Cleft ethmoid bone Cleft vertebral bodies Usual oral and digital changes Micrognathia Optic nerve colobomata Dandy-Walker malformation

Unknown

(Papillon-Leáge-Psaume syndrome)

II (Mohr syndrome)

III (Sugarman syndrome) IV (Mohr-Majewski syndrome) V (Thurston syndrome) VI (Varadi or Varadi-Papp syndrome) VII (Whelan syndrome) VIII (Edwards et al., 1988) IX (Gurrieri et al., 1992; Jamieson and Collins, 1993; Nagai et al., 1998) X (Figuera et al., 1993) XI (Camera et al., 1994) XII (Moran-Barroso et al., 1998) Unclassified type (Lubinsky and Denny, 1997) Unclassified type (Gabrielli et al., 1994) Unclassified type (Toriello and Lemire, 2002)

Source: Reproduced from Siebert 2005 with permission from MedLink Corporation

Autosomal recessive

Autosomal dominant or X-linked dominant X-linked, without prenatal lethality in either sex Autosomal recessive

Unknown

Unknown, but possibly recessive (consanguinity in one case) Unknown

Unknown

THE ORAL–FACIAL–DIGITAL SYNDROMES 18.2.4. Other anomalies Other anomalies are encountered as well, and helpful in diagnosing specific variants of OFDS (Table 18.2). This approach does not necessarily simplify understanding, however. In one patient, for example, penile agenesis and flattened clavicles were interpreted as type II, VI or a new form of OFDS (Yildirim et al., 2002). Polycystic kidneys may manifest tubular or glomerular cysts and are associated occasionally with hepatic cysts. Cardiac malformations are most often endocardial cushion defects, tetralogy of Fallot or aortic stenosis. Skeletal dysplasia (particularly mesomelic) and airway anomalies (hypoplasia, tracheomalacia) are recognized. Skin and hair exhibit several changes. Milia tend to present in infancy but may resolve, leaving scars. Hair may be dry and brittle, or absent (alopecia). Malformations of the central nervous system are described in a following section.

18.3. Genetic mechanisms Despite the high number of phenotypic variants, only one gene has been identified to date for OFDS. Mutations in OFD1 are associated with OFDS type I and have been identified in both familial and sporadic cases (Ferrante et al., 2001). A few patients with OFDS type VII and VIII have mutations in OFD1, suggesting that these types may be allelic to OFD1 or a manifestation of variable expressivity (Nowaczyk et al., 2003). OFD1 maps to Xp22.2–p22.3, and spans a 12 Mb interval (Ferrante et al., 2001). At least 18 mutations have been recognized, including single base pair changes, deletions, splices or frameshifts. One double deletion was probably the result of unequal recombinations of homologous sequences (Morisawa et al., 2004). The finding of OFDS type I in one of monozygotic twins suggests that the condition may have resulted from a postzygotic mutation (Shotelersuk et al., 1999). The OFD1 protein occurs in two forms, CXORF5–1 and CXORF5–2. The function is unknown but the protein is a core component of the centrosome and may be important to a number of developmental steps. Its expression in the kidney and contribution to mesenchymal-epithelial transition, microtubule formation, and mechanical sensation to urine flow may help explain the renal anomalies found in some individuals (Romio et al., 2004). Testing of patient DNA is currently available, but only on a research basis (Toriello et al., 2004). Relationships to other genes remain unclear. One gene, STK9, has been localized to the same Xp22 region where the genes for OFDS type I, Nance–Horan

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syndrome (X-linked, with dental abnormalities, cataracts, other dysmorphic features, mental retardation) and nonsyndromic sensorineural deafness are mapped (Montini et al., 1998; Toutain et al., 2002). Mutations in the GLI3 gene have been identified in families manifesting autosomal dominant Pallister–Hall syndrome, a condition with striking phenotypic similarities to OFDS (Kang et al., 1997a, 1997b, 1997c). Much remains to be learned about the developmental and genetic relationships between these two conditions. Although the etiology for OFDS is clearly genetic, little information exists concerning the mechanism(s) by which genetic changes translate into pathogenesis. Most mutations appear to cause premature truncation of protein synthesis (Stoll and Sauvage, 2002; Romio et al., 2003). It is also possible that truncated proteins act with a dominant-negative effect (Ferrante et al., 2001). X-inactivation of OFD1 occurs in the mouse model but not the human (Ferrante et al., 2003). Haploinsufficiency seems unlikely, since normal males have only one intact allele. The increase in prenatal lethality suggests that gene products are important to organogenesis and hence to survival (Feather et al., 1997). The presence of alternative splice forms of mRNA (OFD1a and OFD1b) in oral and nasal tissues, limb, brain and metanephros of first-trimester human embryos may result in nonfunctional proteins or unstable transcripts; OFD1 may be involved in metanephric precursor cell differentiation (Romio et al., 2003). OFD1 is conserved in vertebrates and absent in invertebrates (Ferrante et al., 2003). A mouse model (X-linked dominant Xpl mutant), manifesting polydactyly and renal cystic disease, maps to the homologous region on the X chromosome (Feather et al., 1997). In situ RNA studies of the mouse homolog ofd-1 have demonstrated expression of the gene in all of the tissues affected in OFDS type I (Ferrante et al., 2001). Specifically, expression is moderate to high in tissues of the craniofacial complex and central nervous system, and somewhat lower in skin, lung, thymus and kidney; expression is also observed in multiple structures of the oral and nasal cavity.

18.4. Neuropathology A host of neuropathological findings are recognized in OFDS (Table 18.3). Like the anomalies of oral, facial or digital tissues, changes in the CNS do not differentiate X-linked from autosomal recessive forms of OFDS (Leao and Ribeiro-Silva, 1995). Most often, these findings have been reported as part of case series and based upon clinical imaging rather than dedicated anatomical study of the CNS (Townes et al., 1976; Towfighi et al., 1985; Anneren et al., 1990; Leao and

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Table 18.3 Malformations of the central nervous system in the oral–facial–digital syndromes Malformation

Additional findings/comment

Reference

Hydrocephalus Holoprosencephaly/arhinencephaly Cerebellar dysgenesis; Dandy–Walker malformation; variable hypoplasia or aplasia of vermis Occipital encephalocele Occipital meningocele Cerebral atrophy Agenesis of the corpus callosum Cystic change: hydranencephaly; porencephaly; intrahemispheric (intracerebral), ependymal or arachnoid cysts Gyral abnormalities: lissencephaly; polymicrogyria; pachygyria Berry aneurysm Neuroglial heterotopia

Communicating or obstructive Rare finding in OFDS Enlarged fourth ventricle

Whelan et al., 1975 Varadi et al., 1980 Mu¨nke et al., 1990; Stephan et al., 1994

Dandy–Walker malformation Posterior fossa cyst Other CNS anomalies Other CNS anomalies Cysts are usually multiple, asymmetrical

Suresh et al., 1995 Reardon et al., 1989 Burn et al., 1984; Toriello et al., 1997 Salinas et al., 1991 Reuss et al., 1962; Salinas et al., 1991; Odent et al., 1998; Thauvin-Robinet et al., 2001

Holoprosencephaly (in type IV)

Townes et al., 1976; Towfighi et al., 1985 Salinas et al., 1991 Hingorani et al., 1991

Hypothalamic hamartomas Dysplastic or variably absent pituitary gland Asymmetrical, hypoplastic or dysplastic brainstem Chorioretinal or optic nerve coloboma

Retinal hamartoma

Rare May be found in meninges, cerebral cortex, basal ganglia, hypothalamus, brainstem May extend into thalamus, brainstem, anterior pituitary gland Smooth sella turcica by MRI Vascular malformation of brainstem; asymmetry of posterior cerebrum Microcephaly

May resemble retinoblastoma by imaging studies

Ribeiro-Silva, 1995). Thus, a substantial amount of information concerning the involvement of the CNS remains unknown for the variants of OFDS, especially at histochemical and molecular levels. Likewise, much remains to be learned regarding pathogenesis. In one review of the neuropathological findings in OFDS type I, the prevalence of abnormal gyral patterning (14.3% of reported cases) and gray matter heterotopia (21.4%) implicated defects in neuronal migration (Holub et al., 2005). Other mechanisms are surely involved, however, given the diversity of CNS findings. Anomalies of the hindbrain are considerably more common in patients with OFDS than are those of the forebrain. Cerebellar dysgenesis, including complete Dandy–Walker malformation and variable absence of the cerebellar vermis, are noted in several forms. Holoprosencephaly is, by contrast, very rare and is

Somer et al., 1986; Hingorani et al., 1991; Verloes et al., 1992; Boyko et al., 1991 Shashi et al., 1995; Al-Gazali et al., 1999; Buno et al., 2000 Co-Te et al., 1970; Townes et al., 1976; Towfighi et al., 1985; Ade`s et al., 1994 Gurrieri et al., 1992; Jamieson and Collins, 1993; Nevin et al., 1994; Stevens and Marsh, 1994; Sigaudy et al., 1996 Tsai and O’Brien, 1999

recognized infrequently in only two variants, OFDS type IV and type VI (Varadi et al., 1980; Ade`s et al., 1994). Interestingly, holoprosencephaly is found in two closely related conditions, hydrolethalus and Pallister–Hall syndrome. Some workers have suggested that these two syndromes and OFDS type IV may represent a single genetic entity (Hingorani et al., 1991; Mu¨nke et al., 1991). Recently, it has been shown that a mutation in a novel gene HYLS1 is responsible for hydrolethalus (Mee et al., 2005), so it may be possible to differentiate the condition from OFDS type IV on a genetic basis. 18.4.1. Neurological deficits In addition to malformations, neurological deficits are recognized in affected individuals. Psychomotor

THE ORAL–FACIAL–DIGITAL SYNDROMES retardation, developmental delay, seizures, hypotonia, ataxia, deafness, precocious puberty and failure to thrive have all been reported. The severity of mental retardation varies greatly, but may affect 30–50% of patients (Reuss et al., 1962; Doege et al., 1968; Rimoin and Edgerton, 1967; Fenton and Watt-Smith, 1985). It is usually mild and associated, albeit inconsistently, with CNS anomalies. Seizures occur in a small number of patients with OFDS but are not well characterized; generalized seizures are rare (Mohr, 1941; Papillon-Leáge and Psaume, 1954; Co-Te et al., 1970). Blepharospasm, nystagmus, oculomotor apraxia, esotropia and ‘see-saw’ eye winking occur in some patients (Sugarman et al., 1971; Toriello, 1988; Mu¨nke et al., 1990). Hearing loss has been associated with recurrent otitis media and cleft palate, and little is known regarding anatomical defects. Not all neurological symptoms are CNS-mediated per se. Anomalies of the oral region may impair speech, and digital malformations interfere with fine motor movements. Intermittent apnea, which may be central or obstructive, and hyperpnea have both been reported in numerous patients (Mu¨nke et al., 1990). Werdnig– Hoffman disease has been diagnosed in one patient with OFDS type I but is not well understood and may even be coincidental (Hashimoto et al., 1998). However, the association warrants consideration, for Werdnig– Hoffman disease arises from a deletion in the survival motor neuron (SMN) gene on 5q (Melki et al., 1990, 1994), and the 5q deletion syndrome shares some phenotypic resemblance with OFDS (Kleczkowska et al., 1993).

18.5. Diagnosis and clinical course The phenotypic changes associated with OFDS overlap with a number of other syndromes and in this way hinder diagnosis. The differential diagnosis of OFDS is quite wide and includes Majewski short-rib polydactyly, Beemer–Langer syndrome, Pallister–Hall syndrome, Joubert syndrome and Smith–Lemli–Opitz syndrome type II. The phenotypic similarities between some autosomal recessive variants of OFDS and several of these conditions, particularly the autosomal recessive skeletal dysplasias, Majewski short-rib polydactyly and Beemer–Langer syndrome, are so great that their very existence as distinct entities has been questioned (Fenton and Watt-Smith, 1985; Silengo et al., 1987; Hingorani et al., 1991; Neri et al., 1995). In each condition, short mesomelic limb segments, bent tibiae and polydactyly are recognized features. The presence of cerebellar anomalies also complicates diagnosis. Partial or complete cerebellar dysgenesis is a central finding in Joubert syndrome, an

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autosomal recessive disorder characterized by partial or complete absence of the cerebellar vermis, hyperpnea and abnormal eye movements (Joubert et al., 1969; Gustavson et al., 1971; Egger et al., 1982). Many patients with Joubert syndrome have polydactyly and one infant had ‘fleshy tongue nodules’ suggestive of lingual hamartomas (Egger et al., 1982). Cerebellar changes have also been reported in OFDS type VI (Varadi syndrome), as has abnormal superior cerebellar peduncles and deep interpeduncular fossa, the socalled ‘molar tooth sign’ (Gleeson et al., 2004). The presence of these findings in a number of other syndromes (e.g., Dekaban–Arima, Senior–Loken and COACH) suggests similarities in development, although details remain unknown. Hypothalamic hamartomas present a special challenge to diagnosis as well as classification (Hingorani et al., 1991; Verloes et al., 1992). The tumors are observed in some cases of OFDS but also resemble the masses found in Pallister–Hall and hydrolethalus syndromes (Verloes et al., 1992). The former condition resembles OFDS and is characterized by oral frenula, palatal defects, median cleft lip, polydactyly and cerebral malformations (Clarren et al., 1980). Anal atresia, found in Pallister–Hall syndrome, is rare in OFDS. Hypothalamic hamartomas can also present as sporadic lesions or as a familial trait with autosomal dominant transmission (Grebe and Clericuzio, 1996). A subset of families with Pallister–Hall syndrome manifest autosomal dominant transmission, which correlates with mutations in the GLI3 gene (Kang et al., 1997c) but differs from the autosomal recessive and X-linked patterns of inheritance ascribed to OFDS. Prenatal diagnosis of OFDS relies on ultrasound findings, which can be confirmed or expanded by autopsy in those fetuses or infants who do not survive (Shipp et al., 2000; Thauvin-Robinet et al., 2001). However, ultrasound examination may be complicated by the range of phenotypic change and severity (Atahan et al., 2004). In order to ascertain patterns of inheritance, a detailed family history, including information about recurrent pregnancy losses and examination of parents and siblings, is necessary. Physical examination requires care with appropriate measurements, of the entire body. Special attention must be given to oral and ocular tissues, and the digits must be carefully inspected for subtle changes such as clinodactyly or partial syndactyly. Radiographs of hands and feet should be obtained, even in the absence of external changes, to exclude bifid or abnormally ossified bones. Radiographs of the long bones should be obtained, especially from short or disproportionately short individuals. At appropriate ages, dental X-rays should be obtained to screen for missing teeth. Renal ultrasound examination,

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cranial computed tomography (CT) or magnetic resonance imaging (MRI), echocardiography, and olfactory and endocrine evaluation should be pursued as indicated. Clinical laboratory evaluation of renal function should be pursued with urinalysis, serum creatinine and other serum chemistries. Management must be customized to individual patients. The presence of lethal anomalies (e.g. severe pulmonary hypoplasia, congenital heart defects or devastating brain anomalies) may well dictate the patient course. Surgery is often required to remove extra digits or correct oral clefts, frenula, odontogenic keratocysts, hypoplastic nasal cartilages or tumors (Gunbay et al.,1996; Sakai et al., 2002; Lindeboom et al., 2003; Velepic et al., 2004). Speech therapy may be necessary. Hypothalamic hamartomas may produce morbidity because of their location (Squires et al., 1995). Precocious puberty has been reported in one child with a hypothalamic hamartoma, and pituitary dysfunction is a potential complication, although patients can respond satisfactorily to hormonal replacement therapy (Al-Gazali et al., 1999). Conductive hearing loss has been reported in many patients. Patients are at risk for otitis media. Renal insufficiency in one female with OFDS type I required transplantation at 19 years (Stoll and Sauvage, 2002). Gastrostomy may be necessary for feeding difficulties or failure to thrive. Comprehensive auditory and visual examinations and genetic counseling for patients and families are part of standard care. The prognosis for patients with OFDS depends upon the extent and severity of malformations and is thus highly variable (Toriello et al., 1997). Patients with congenital heart malformations or pulmonary hypoplasia often die in the perinatal period. Complications such as apnea, aspiration or pneumonia may lead to death in later infancy or childhood. The latter are often associated with moderate or severe mental retardation. Patients without life-threatening malformations or neurological impairment appear to have a normal life expectancy.

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Shashi V, Clark P, Rogol AD, et al. (1995). Absent pituitary gland in two brothers with an oral–facial–digital syndrome resembling OFDS II and VI: a new type of OFDS? Am J Med Genet 57: 22–26. Shipp TD, Chu GC, Benacerraf B (2000). Prenatal diagnosis of oral–facial–digital syndrome, type I. J Ultrasound Med 19: 491–494. Shotelersuk V, Tifft CJ, Vacha S, et al. (1999). Discordance of oral–facial–digital syndrome type I in monozygotic twin girls. Am J Med Genet 86: 269–273. Siebert JR (2005). Oral–facial–digital syndromes. In: S Gilman (Ed.). MedLink Neurology. MedLink Corp, San Diego. Available on line at www.medlink.com. Accessed 14 June 2005. Sigaudy S, Philip N, Gire C, et al. (1996). Oral–facial–digital syndrome with retinal abnormalities. Am J Med Genet 61: 193–194. Silengo MC, Bell GL, Biagioli M, et al. (1987). Oro-facialdigital syndrome II. Transitional type between the Mohr and the Majewski syndromes: report of two new cases. Clin Genet 31: 331–336. Somer M, Lindahl E, Perheentupa J (1986). Precocious puberty associated with oral–facial–digital syndrome type I. Acta Pediatr Scand 75: 672–675. Squires LA, Constantini S, Miller DC, et al. (1995). Hypothalamic hamartoma and the Pallister–Hall syndrome. Pediatr Neurosurg 22: 303–308. Stephan MJ, Brooks KL, Moore DC, et al. (1994). Hypothalamic hamartoma in oral-facial-digial syndrome type VI (Varadi syndrome). Am J Med Genet 51: 131–136. Stevens JL, Marsh JL (1994). Ocular anomalies in the oral– facial–digital syndrome. J Pediatr Ophthalmol Strabismus 31: 397–398. Stoll C, Sauvage P (2002). Long-term follow-up of a girl with oro-facio-digital syndrome type I due to a mutation in the OFD1 gene. Ann Genet 45: 59–62. Sugarman GI, Katakia M, Menkes J (1971). See-saw winking in familial oral-facial digital syndrome. Clin Genet 2: 248–254. Suresh S, Krishnamurthy R, Suresh I, et al. (1995). Prenatal diagnosis of orofaciodigital syndrome: Mohr type. J Ultrasound Med 14: 863–866. Thauvin-Robinet C, Rousseau T, Durand C, et al. (2001). Familial orofaciodigital syndrome type I revealed by ultrasound prenatal diagnosis of porencephaly. Prenat Diagn 21: 466–470. Toriello HV (1988). Heterogeneity and variability in the oral–facial–digital syndromes. Am J Med Genet Suppl 4: 149–159. Toriello HV (1993). Review: oral–facial–digital syndromes, 1992. Clin Dysmorphol 2: 95–105. Toriello HV, Lemire EG (2002). Optic nerve coloboma, Dandy–Walker malformation, microglossia, tongue hamartomata, cleft palate and apneic spells: an existing oral– facial–digital syndrome or a new variant? Clin Dysmorphol 11: 19–23.

THE ORAL–FACIAL–DIGITAL SYNDROMES Toriello HV, Carey JC, Suslak E, et al. (1997). Six patients with oral–facial–digital syndrome IV: the case for heterogeneity. Am J Med Genet 69: 250–260. Toriello HV, Guimaraes IN, Moretti-Ferreira D (2004). Oral– facial–digital syndrome type I. In: Gene Reviews at GeneTests: Medical Genetics Information Resource (database online), University of Washington, Seattle. Available on line at http://www.genetests.org. Accessed 17 June 2005. Toutain A, Dessay B, Ronce N, et al. (2002). Refinement of the NHS locus on chromosome Xp22.13 and analysis of five candidate genes. Eur J Hum Genet 10: 516–520. Towfighi J, Berlin CM, Ladda RL, et al. (1985). Neuropathology of oral–facial–digital syndrome. Arch Pathol Lab Med 109: 642–646. Townes PL, Wood BP, McDonald JV (1976). Further heterogeneity of the oral–facial–digital syndromes. Am J Dis Child 130: 548–554. Tsai PS, O’Brien JM (1999). Retinal hamartoma in oral– facial–digital syndrome. Arch Ophthalmol 117: 963–965.

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Varadi V, Szabo L, Papp Z (1980). Syndrome of polydactyly, cleft lip/palate or lingual lump, and psychomotor retardation in endogamic gypsies. J Med Genet 17: 119–122. Velepic MS, Sasso A, Velepic MM, et al. (2004). Combined anomalies of the palate in Mohr syndrome: is preoperative electromyography of the palate useful? J Pediatr Surg 39: 220–222. Verloes A, Gillerot Y, Langhendries JP, et al. (1992). Variability versus heterogeneity in syndromal hypothalamic hamartoblastoma and related disorders: review and delineation of the cerebro-acro-visceral early lethality (CAVE) multiplex syndrome. Am J Med Genet 43: 669–677. Whelan DT (1975). The oro-facial-digital syndrome. Clin Genet 8: 205–212. Yildirim S, Akan M, Deviren A, et al. (2002). Penile agenesis and clavicular anomaly in a child with an oral facial digital syndrome. Clin Dysmorphol 11: 29–32.


Other dysgeneses Chapter 19

Congenital vascular malformations in childhood IGNACIO PASCUAL-CASTROVIEJO* Ex-Chairman of the Pediatric Neurology Service, University Hospital La Paz, Madrid, Spain

Congenital vascular malformations are defined as 1) the absence of vessels that are normally present, 2) the presence of vessels that usually do not appear postnatally, 3) the presence of vessels of abnormal morphology and/or size or 4) the presence of abnormalities of the arterial, capillary or venous walls, occurring in isolation or in combination, with some or all structures. The different identity of arterial and venous blood vessels was thought to arise in response to hemodynamic forces such as blood pressure and the direction of the blood flow. However, recent data indicate that acquisition of artery or vein identity during vascular development is governed, in part, by genetic mechanisms. Molecular differences between arterial and venous endothelial cells are apparent well before the onset of the circulation. The transmembrane ligand ephrin-B2 is expressed in a specific fashion in arteries within both extraembryonic and embryonic blood vessels before circulation (Wang et al., 1998), and experimental studies in mouse and zebrafish suggest that Notch signaling is required for the proper development of arterial and venous blood vessels (Lawson et al., 2001).

19.1. Embryological development To better understand the various anomalies of the extraand intracranial vessels, it is necessary to make a chronological summary of the embryonic development of the cerebral arteries, including both their origin at the aortic arch and their intracranial trajectories, i.e. the ontogeny of their cerebral vasculature (Mall, 1912; Streeter, 1918; Congdon, 1922; Padget, 1948). All the arteries that vascularize the brain begin their development in the aortic arch. Two stages of this development are recognized: the primary or brachial stage,

which appears at about 22 days and shows the appearance of a vascular apparatus destined to become the precursor of the posterior arteries, and the second or postbrachial stage, in which the vascular apparatus mentioned is replaced by the adult arterial system during a period lasting about 28 days. The brachial stage begins with the formation of the first aortic arch and terminates somewhat arbitrarily with interruption of the sixth arch. The sequence in the appearance of the different structures is developed between the appearance of the first aortic arch in the human embryo of 1.5 mm (3 weeks) and that of the sixth aortic arch in the 5 mm (4.5 weeks) embryo. The formative process is sequential and the appearance of one aortic arch follows the disappearance of the previous one. The third arch of each side contributes to the development of the common and internal carotid arteries in the 3 mm embryo during the third and last phase of the brachial stage. At a length of approximately 7–12 mm (32–35 days) the internal carotid artery is already filling with blood in its intracerebral course; when the embryo is 12–14 mm (35–38 days), the common carotid artery may be identified and for the first time the cerebral ramification of the internal carotid artery and of its collateral vessels may be described as representing the earliest adult configuration. When the embryo is 3 mm (3.5 weeks), the trigeminal artery begins to sprout from the first aortic arch. It constitutes the first source of blood supply to the posterior part of the primordial brain. The formation of the basilar artery is accompanied by involution of the trigeminal or its annexation by the internal carotid. When the trigeminal artery persists after birth, it constitutes an important vascular malformation anastomosing the internal carotid system with the basilar artery, a function normally substituted by the posterior communicating arteries. The

*Correspondence to: Ignacio Pascual-Castroviejo MD, PhD, Orense str 14, 10 E, 28020 Madrid, Spain. E-mail: [email protected].

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vertebral arteries begin to form when the embryo is 9 mm (32 days) by longitudinal anastomosis between superior longitudinal segmental arms of the dorsal aorta with subsequent obliteration of all aortic connections, except for those of the seventh cervical segmental arches, which remain as the subclavian trunk. The vertebral arteries conclude their maturation when the embryo reaches 14–16 mm (between 36 and 40 days), with their origin having been displaced from the wall of the aorta to the level of the ductus arteriosus.

19.2. Congenital arterial anomalies The existence of intracranial vascular anomalies has been known for more than a century, the first descriptions corresponding to anatomical findings (Batujeff, 1889). However, knowledge of the vascular pathology began with Egas Moniz (1927) who described the conventional arteriography by direct injection of a contrast material into the arteries which permitted visualization of the normal and abnormal intracranial vessels in vivo. The main congenital vascular anomalies are seen in Table 19.1. 19.2.1. Nonpathological abnormalities of arteries originating at the circle of Willis Most often this refers to nonpathological anatomical variations, without any influence in the blood supply of the brain. The main variations are: 1) origin of one or both posterior cerebral arteries from the internal carotids; 2) origin of both anterior cerebral arteries from the same internal carotid; and 3) important differences in size of both internal carotids, both anterior cerebral arteries, both posterior cerebral arteries or both posterior communicating arteries. In these cases the vascular deficit caused by the narrow artery of one side is compensated by an increase in the size of the other (Fig. 19.1) Some 28 different normal anatomical variations of the circle of Willis are described (Lazorthes and Gronaze´, 1968). 19.2.2. Nonpathologic morphology abnormalities of extra- and intracranial arteries Apart from the generalized elongation of the arteries seen in Menkes disease (Fig. 19.2), most anomalies of this group are incidental angiographic findings. The most common varieties are: 1) Carotid or vertebral coiling or kinking at the neck (Fig. 19.3). This anomaly is seen in 25% of children below 8 years and in 50% of cases of Sturge–Weber syndrome (Pascual-Castroviejo, 1983). 2) Fenestration of arteries: also known as arterial duplication, this very rare anom-

Table 19.1 Congenital vascular malformations in childhood A. Congenital arterial anomalies 1. Nonpathologic abnormalities of the arteries originating at the circle of Willis 2. Nonpathologic morphology abnormalities of extraand intracranial arteries a. Coiling or kinking at the neck b. Fenestration of arteries 3. Morphologic abnormalities that may or may not be pathological a. Carotid or vertebral hypoplasia b. Megadolicoarteries 4. Persistence of embryonic arteries a. Trigeminal b. Hypoglossal c. Stapedial or otic d. Proatlantal intersegmental 5. Absence of cerebral arteries 6. Congenital malformations of arterial walls a. Moyamoya disease b. Fibromuscular dysplasia c. Intracranial aneurysms d. Capillary hemangiomas B. Cerebrovascular anomalies 1. Arteriovenous malformations 2. Venous angiomas 3. Capillary telangiectases 4. Vein of Galen malformations 5. Intracranial cavernous malformations 6. Cortical venous anomalies a. Vascular malformations of the leptomeninges b. Dural arteriovenous malformations c. Aneurysmatic dilatation of the torcula d. Sinus pericranii

aly only involves a short portion of the arterial course. It presents more frequently in the vertebrobasilar territory but also may be occasionally seen in the carotid system (Ito et al., 1977). 19.2.3. Morphological abnormalities that may or may not be pathological The two main types are carotid or vertebral hypoplasia and megadolicoarteries. 1. Carotid or vertebral hypoplasia is an anomaly that is often associated with neurocutaneous diseases, especially with neurofibromatosis type1 (Greene et al., 1974) and cutaneous facial or neck hemangioma or vascular malformations as a part of the syndrome described by Pascual-Castroviejo in 1978. However, this anomaly is usually detected incidentally during angiographic study of any

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Fig. 19.1. MRA revealing important differences in size of the internal carotid arteries, demonstrating these to be very narrow on the right side (arrow) and very wide on the left side (arrowhead).

disease because of the lack of neurological symptomatology as a result of good collateral vascular compensation through branches of other intra- and extracranial arteries. This collateral circulation may be occasionally supplied by an intercavernous anastomotic vessel connecting the internal carotids (Chen et al., 1998). Even rarer is the location of the carotid bifurcation at an asymmetrical level of the neck (Fig. 19.4), as may be seen associated with cutaneous hemangioma in the Pascual-Castroviejo type II syndrome (Pascual-Castroviejo et al., 2003). 2. Megadolicoarteries consists of a pathological congenital dilatation of the arterial lumen and an elongated course that the artery has to follow, giving the impression that it is too long for the trajectory it has followed in the brain. We have found this anomaly in cases of hypomelanosis of Ito ipsilateral to the hemifacial hypertrophy, and also associated with intracranial or facial hemangioma (Pascual-Castroviejo et al., 1996) or with arteriovenous malformation. However, isolated megaartery, especially of the basilar artery, can compress cranial nerves or the brainstem (Frasson et al., 1977) along its course in the posterior fossa.

19.3. Persistence of embryonic arteries Fig. 19.2. Conventional arteriography of the vertebrobasilar system showing many incurvations and generalized elongation of the extra- and intracranial arterial courses in a patient with Menkes disease.

Persistence of fetal anastomoses between carotid and verebrobasilar arteries after birth is a well known anomaly. These vessels are the primitive trigeminal, otic or stapedial, hypoglossal and proatlantal intersegmental arteries.

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Fig. 19.3. Conventional arteriography showing double kinking in the extracranial part of the internal carotid artery (asterisks).

third of the basilar artery. It may reach the basilar artery in one of two ways. In approximately 50% of cases, the trigeminal artery penetrates the sella turcica, perforates the dura near the clivus to then join the basilar artery between the origins of the anterior inferior cerebellar arteries and the superior cerebellar arteries (Fig. 19.5). In the other half of cases, the trigeminal artery leaves the cavernous sinus, goes with the trigeminal root or runs between the sensory trigeminal root and the lateral side of the sella in the groove of the posterior petrosal process, and then joins the basilar artery at the same level as in the other 50%. Occasionally, the ipsilateral or both vertebral arteries are hypoplastic. Formation of the basilar artery is accompanied by involution of the trigeminal artery or its annexation to the internal carotid, becoming complete in the 14 mm (5–5.5 weeks) embryo. The incidence of trigeminal artery probably approaches 1% (Yilmaz et al., 1995). Persistent trigeminal artery is seen in 30% of cases of facial and neck hemangioma (Chen et al., 1998). The prevalence of aneurysm associated with persistent trigeminal artery is approximately 3%, which is similar to the prevalence of aneurysms in the general population (Cloft et al., 1999). 19.3.2. Persistence of the hypoglossal artery

Fig. 19.4. MRA of the neck in a case with left facial and neck hemangioma, disclosing markedly asymmetrical carotid bifurcation (stars).

This is a rare anomaly, although it is the second most common persistent carotid–vertebral anastomosis. It has been seen in 0.25% of important angiographic series (Debaene et al., 1972). It may be associated with severe encephalopathy and cerebellar malformations (Pascual-Castroviejo et al., 1975). The hypoglossal artery is an anomalous artery which originates in the internal carotid artery between C1 and C3 levels and traverses the hypoglossal canal to join the basilar artery but does not traverse the foramen magnum. The

Persistent trigeminal artery is the most frequently seen, whereas the others are most uncommon. The trigeminal artery represents about 85% of the carotid– basilar anastomoses (Yilmaz et al., 1995). These embryonic arteries supply the brain during the first weeks of fetal life. The presence of these arteries may appear to be incidental, but they are often associated with some type of pathology. 19.3.1. Persistence of the trigeminal artery The trigeminal artery connects the proximal intracavernous portion of the internal carotid artery with the distal

Fig. 19.5. MRA showing persistence of the trigeminal artery (arrow) in a case with ipsilateral facial hemangioma.

CONGENITAL VASCULAR MALFORMATIONS IN CHILDHOOD posterior inferior cerebellar artery (PICA) may be absent bilaterally (Pascual-Castroviejo et al., 1975) and the ipsilateral vertebral artery is frequently hypoplastic or absent (Fig. 19.6). The hypoglossal artery has been reported to cause glossopharyngeal neuralgia and hypoglossal nerve paralysis (Yilmaz et al., 1995). 19.3.3. Persistence of the otic or stapedial artery This rare anomaly has been documented by means of angiography in a few cases (Patel et al., 2004). Persistence of the otic artery is one of four transient carotid– basilar anastomoses and usually the first to disappear during embryogenesis. This anomaly originates from the hyoid at about the 4th week of embryogenesis. When the embryo is 12–15 mm, the otic artery is divided into a dorsal branch that will form the middle meningeal artery and a ventral branch that will form the maxillary and mandibular arteries. Both hyoid and otic arteries disappear at about the third fetal week. The primitive otic artery arises from the carotid artery within the carotid canal, emerging from the

Fig. 19.6. Conventional carotid arteriography showing a persistent hypoglossal artery that originates in the internal carotid at the C2 level (asterisk).

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internal acoustic meatus, and joins the basilar artery at a caudal point. Its anomalous presence may disturb the normal structures of the anatomical area and may cause neurological symptomatology. Persistence of the otic artery has been described in association with a variety of anomalies in the normal cerebral vasculature, such as absence of the internal carotid artery on one side and of the external carotid artery on the other (Teal et al., 1973) and the presence of multiple intracranial aneurysms and fetal posterior cerebral artery (Patel et al., 2004). However, some authors are skeptical about the presence of the otic artery and its association with aneurysms distant from the persistent vessel because they have not seen any anatomical or angiographic evidence of a persistent otic artery and even suggest that it might not exist (Croft, 2004). 19.3.4. Persistence of the proatlantal artery Persistence of the proatlantal artery is a rare anomalous communication between the carotid and vertebrobasilar system (Fig. 19.7). Since it was first anatomically

Fig. 19.7. Conventional carotid arteriography showing persistence of the proatlantal artery, which arises from a hypoplastic external artery (arrowhead) at the C4 level. A, artefact.

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described in the 19th century, more than 40 cases have been reported in the literature (Luh et al., 1999; Basekim et al., 2004; Gumus et al., 2004). This congenital anomaly is the most dorsally placed of the four persistent embryonic arteries, trigeminal, otic, hypoglossal and proatlantal, from cephalic to caudal direction. Diagnostic criteria are based on the origin from the common carotid artery bifurcation, external carotid artery or internal carotid artery at C2–C4 levels; it joins the vertebral artery in the suboccipital region and enters the skull via the foramen magnum. Two types of proatlantal artery have been described (Lasjaunias et al., 1978): type I corresponds to the first segmental artery and arises from the internal carotid artery; type II corresponds to the second segmental artery and arises from the external carotid artery. Type II is more frequent than type I. Most cases of persistent types 1 or 2 proatlantal arteries in the literature are unilateral, but this arterial malformation has been reported with bilateral persistence in at least four patients (Woodcock et al., 2001; Gumus et al., 2004). Approximately 50% of cases of proatlantal artery may have aplasia or hypoplasia of one or both vertebral arteries proximal to the anastomosis. Association with unilateral or, more rarely, bilateral absence of the external carotid arteries has been reported in some cases (Lasjaunias et al., 1978; Gumus et al., 2004). The proatlantal arteries persist until the vertebral arteries develop, after regressing otic, hypoglossal and trigeminal arteries. During the 7–12 mm embryonic stage, the vertebral arteries are formed with important participation of the proatlantal artery. The persistent proatlantal artery and the hypoglossal artery arise from the carotid system outside the cranium and join the vascular system intracranially. Differentiation between the type 1 proatlantal artery and the hypoglossal artery requires a differential diagnosis. There are two important differentiating features (Gumus et al., 2004): 1) the suboccipital horizontal course is characteristic of the vertebral and proatlantal arteries. The hypoglossal artery lacks this horizontal course. 2) The proatlantal artery enters the skull through the foramen magnum, whereas the hypoglossal artery enters the skull through the hypoglossal canal. Therefore, the proatlantal artery extends much more posteriorly and horizontally, whereas a small dorsal curve will be enough for the hypoglossal artery.

19.4. Absence of cerebral arteries Congenital absence of the internal carotid arteries has been known since the 17th century. This anomaly was considered a rare finding before being described in association with ‘cutaneous hemangioma or with

vascular malformation’ (Pascual-Castroviejo, 1978; Pascual-Castroviejo et al., 1996). Absence of one carotid (Fig. 19.8) or one vertebral (Fig. 19.9) artery is rarely associated with neurological

Fig. 19.8. Three-dimensional MRA showing absence of the right carotid artery. Left carotid and basilar arteries appear enlarged.

Fig. 19.9. MRA showing absence of the left vertebral artery and pronounced differences of both carotid arteries, size being very enlarged in the left side (asterisks). The right primitive carotid artery is rather narrow and ends in the external carotid artery (arrow) without giving the internal carotid artery, which is absent. The right vertebral artery appears very enlarged (arrowheads).

CONGENITAL VASCULAR MALFORMATIONS IN CHILDHOOD disease, probably because of the abundant supply through the collateral circulation (Pascual-Castroviejo et al., 1996; Given et al., 2001; Torres-Mohedas et al., 2001). The prevalence of absence or hypoplasia of an internal carotid artery in cases of facial hemangioma is over 30% and of a vertebral artery is 35% (Pascual-Castroviejo et al., 1996; Torres-Mohedas et al., 2001). Absence of both carotids without severe encephalopathy has been reported (Dilange, 1975). We studied a case with absence of both vertebrals associated with almost complete cerebellar aplasia that showed severe motor abnormalities. The external carotid artery also can be absent in cases of facial hemangioma (Pascual-Castroviejo et al., 1996). Many cases with absence or hypoplasia of one carotid or one vertebral artery have shown cutaneous facial, neck or scalp hemangioma or vascular malformation during the first years of life. Cutaneous hemangiomas disappear after a few years but vascular malformations persist (Pascual-Castroviejo, 2004). Absence of a cerebral artery – carotid or vertebral – associated with the presence of an embryonic artery, mainly the trigeminal, usually corresponds to cases that show or have shown hemangioma or vascular malformation on the neck, scalp or facial region, usually located on the same side of the vascular malformations (Pascual-Castroviejo, 1978; Pascual-Castroviejo et al., 1996), although it can be located on the contralateral side as well.

19.5. Congenital malformations of the arterial walls

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and 24% occur in a parent and offspring, and occurrence in parents and siblings of patients with moyamoya disease is 30–40-fold higher than in the general population (Kanai and Fukuyama, 1992). Although moyamoya disease was long thought to be caused mainly by environmental factors, molecular genetic studies performed during recent years demonstrate a linkage between the disease and markers located in chromosome 3 p24.2–p26 (Ikeda et al., 1999), 17q25 (Yamauchi et al., 2000) and 6 (Inoue et al., 2000) in families affected with moyamoya disease. The diagnosis of moyamoya disease is based on angiographic features that consist of stenotic or occlusive changes in the supraclinoid portion of the internal carotid artery (Fig. 19.10) on both sides, frequently extending to the proximal portions of the anterior and middle cerebral arteries, abnormal netlike vessel formations at the base of the brain (moyamoya images) and development of extra- and intracranial transdural leptomeningeal collaterals between the pial vessels and those arising from the branches of external carotid artery or ophthalmic artery (Fig. 19.11). The pathogenesis of moyamoya disease is enigmatic. There are two major hypotheses: genetic and acquired. Epidemiological data suggest that moyamoya disease is caused by genetic rather than environmental factors. Both medical and surgical therapies have been used. Vasodilators, corticosteroids, antiplatelet agents, calcium antagonists, and low-molecular-weight dextran have been used, but their efficacy is difficult to assess. Various surgical treatments have been used in pediatric patients, including direct bypass surgery.

19.5.1. Moyamoya disease This is an occlusive disease of the circle of Willis characterized by bilateral stenosis or occlusion of the terminal portion of the internal carotid artery and by the development of abnormal net-like vessels at the base of the brain (moyamoya phenomenon). At the same time, collateral circulation gradually develops as a result of occlusion of the carotid fork at a younger age. Moyamoya disease occurs more frequently in females. It is found at any age but is most prevalent among children. Children and juvenile patients with moyamoya disease initially present with ischemic symptoms such as transient ischemic attacks, completed strokes and subsequent motor and/or intellectual impairment. Adults, on the other hand, present with intracranial hemorrhage. About 10% of patients with moyamoya disease have a family history of the disease (Ikezaki et al., 1997; Wakai et al., 1997). In Japan, approximately 70% of cases among family members occur in siblings

Fig. 19.10. Conventional carotid arteriography showing occlusive changes of the supraclinoid portion of the internal carotid artery and abnormal net-like vessels at the base of the brain. Presence of Bernasconi–Cassinari artery (arrowhead) as collateral vascularization.

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Fig. 19.11. MRA in axial view showing abnormal net-like vessels at the base of the brain (arrowheads), collateral and transdural circulation through branches of the external carotid arteries (arrows), and hypertrophy of the ophthalmic arteries (asterisks).

Long-term evolution of patients with moyamoya disease is poorly documented (Ezura et al., 1995). We have one patient who showed symptoms of psychomotor retardation during the first year of life and has been followed up for 38 years. She had a normal pregnancy and delivery at 26 years without any complication (Alonso-Martinez and Pascual-Castroviejo, 1999). She was treated only with nicardipine, the effectiveness of which is uncertain. 19.5.2. Fibromuscular dysplasia Fibromuscular dysplasia (FMD), also known as fibromuscular hyperplasia, is a nonatherosclerotic and noninflammatory segmental arteriopathy of unknown origin. The disease was first described by Leadbetter and Burkland in 1938 (43), although McCormack et al. (1958) reported the first accurate description of this entity. It is encountered most often in adult women and is rare in children. Although the disease can involve any artery of the body, it usually affects the renal arteries, followed by the mesenteric, carotid and vertebral arteries. Generalized FMD has occasionally been reported but is uncommon, although the exact frequency is unknown (McCormack et al., 1958). FMD is frequently reported in adults because of stroke, rarely in children, also related to stroke, and during intercourse, almost always in women. Caucasians apparently show a predisposition, as do people with hereditary disorders of connective tissue or with Ehlers–Danlos syndrome.

Affected girls usually show very feminine phenotype, which is similar to their mothers. These women and girls exhibit a thin and tall body with hyperextension of the joints. Symptoms of cervicocephalic FMD may include syncope, stroke, seizures, tinnitus, vertigo, lightheadedness and motor disturbances. Ischemic infarction in the cerebral region supplied by the affected artery is the most common sequela. Internal carotid, middle and anterior cerebral arteries are the most commonly affected vessels. The clinical presentation and severity may vary from an asymptomatic condition to a multisystem disease that mimics necrotizing vasculitis, depending on the arterial segment involved, the degree of stenosis and the type of fibromuscular dysplasia. The diagnosis is based on the characteristic radiographic changes. Duplex ultrasonography of the carotid arteries may demonstrate irregular patterns of stenosis and aneurysm. Conventional angiography and MRA disclose narrowing or occlusion of the affected arteries, which exhibit the appearance of a ‘string of beads’ that corresponds to zones of stenosis alternating with abnormally wide poststenotic arterial areas. Magnetic resonance angiography (MRA) has several advantages over conventional angiography (Fig. 19.12), the most important being the possibility of good visualization of all extra- and intracranial arteries in the same study without using contrast enhancement; also it is less invasive and dangerous and allows the presence of intracranial aneurysms to be ruled out in patients with FMD (Slovut and Olin, 2004). Pathological angiographic manifestations include four subtypes: 1. Medial fibroplasia: this is the most common finding and shows an accumulation of dysplastic fibrous tissue in the media, whereas the intima, internal elastic lamina and adventitia are preserved, although it can involve the external lamina as well. 2. Intimal fibroplasia: subendothelial mesenchymal cells accumulate within the fibrous tissue. These fibroplasias occur in less than 10% of patients with arterial fibrodysplasia. Angiographically, the lesion may appear as a focal, concentric stenosis. 3. Perimedial dysplasia: elastic tissue underlying the adventitial layer increases pathologically. This dysplasia accounts for less than 1% of arterial stenosis and may be indistinguishable angiographically from intimal fibroplasia. 4. Medial muscular hyperplasia: smooth muscle hyperplasia occurs without fibrosis within the media.


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Fig. 19.12. MRA in coronal view showing a narrow right internal carotid artery and its intracranial branches, which also exhibit a ‘string of beads’ appearance (arrowheads).

The cause of intracranial FMD is not known. Increased familial incidence points to a genetic predisposition but other possibilities, such as trauma, hormonal defects, infection and toxic injury to the endothelium, have been suggested as well. During recent years, the association of intracranial aneurysms in 20–50% of patients with FMD, the possible link between FMD and a1-antitrypsin deficiency in children with this disorder and the possible role of a1antytrypsin deficiency in the rupture of intracranial or abdominal vessels and arterial dissection (Maeda et al., 1997; Mawad et al., 2002; Proust et al., 2003; Ciceri et al., 2005; Ellegala and Day, 2005), suggest that the cause of FMD could be an underlying congenital connective tissue disorder (So¨lder et al., 1997). This disorder shows a variable evolution. Most of our cases have a chronic course but we also have a few cases with spontaneous resolution of internal carotid FMD. Treatment of FDM of the intracranial carotid artery or its intracranial branches consists of the use of aspirin, antiplatelet therapy or calcium antagonists, especially nicardipine or nimodipine. Surgical treatment is not common for carotids or its intracranial branches but it has been used for renal and other arteries with FMD. Percutaneous angioplasty has become the preferred treatment for symptomatic cerebrovascular FMD, especially in adults. The use of cerebral protection devices may reduce the frequency

of ischemic neurological events during stenting of the carotid artery in patients with atherosclerotic carotid artery disease (Al-Mubarak et al., 2001; Ohki et al., 2002).

19.6. Intracranial aneurysms Intracranial arterial aneurysms are uncommon in the pediatric population. They rarely occur in the neonatal period and only a few cases, distributed with similar frequency among males and females, have been identified to date (Gallia et al., 2005). However, in support of the congenital origin of intracranial aneurysms is the fact that anomalies of the circle of Willis are commonly associated with intracranial aneurysms. Intracranial hemorrhages caused by aneurysms are extremely rare in infancy. Intracranial aneurysms in children account for 0.5–4.6% of all aneurysms (Kanaan et al., 1995). About 20% are giant aneurysms. Compared with adult intracranial aneurysms, there is a male predominance and a higher incidence of giant aneurysms in children, with a predilection for the internal carotid artery bifurcation and the posterior circulation (Vin˜uela et al., 1997). Aneurysms consist of a pathologic and localized dilatation of a small part of the arterial wall due to a congenital defect of the elastica or the media layers. The most frequent locations are the basilar tip, anterior communicating,

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posterior communicating, posterior cerebral, carotid periophthalmic, vertebrobasilar junction, superior cerebellar, and middle cerebral bifurcation (Vin˜uela et al., 1997). Subarachnoid hemorrhage is a major cause of morbidity and mortality caused by intracranial aneurysms. Conventional arteriography and especially MRA demonstrates the presence of the aneurysms, their precise location, size, type of aneurysm and size of its neck (Fig. 19.13). A favorable outcome is achieved in only 50% of cases and the mortality rate is a devastating 30% (Dilange, 1975). Intraoperative complications occur in 30% of procedures (Fridriksson et al., 2002). Outcomes after aneurysmal subarachnoid hemorrhage have improved substantially over the past 30 years, particularly in highly specialized neurosurgical centers with high-volume practices (Berman et al., 2003; Ellegala and Day, 2005). Intraoperative complications and mortality are elevated (Kassell et al., 1990; Vin˜uela et al., 1997; Fridriksson et al., 2002; Mawad et al., 2002) but the literature shows a favorable response to emergency surgical clipping of the aneurysms, because of the significant mortality and morbidity rates from aneurysms rebleeding and a delayed cerebral ischemia caused by vasospasm that occurs during the waiting period. Aneurysms located along the anterior communicating artery are the most frequently treated at the circle of Willis, representing about 40%. Microsurgical clip application should be the preferred option for those aneurysms in this location with anteriorly directed fundi and endovascular packing for lesions with posteriorly directed fundi, depending on morphological criteria (Proust et al., 2003). Spontaneous partial thrombosis of basilar artery giant aneurysms in children has been

Fig. 19.13. Coronal view of three-dimensional MRA showing an aneurysm with a wide neck in the proximal portion of the right middle cerebral artery.

described in a few patients (Kanaan et al. 1995; Maeda et al., 1997; Ciceri et al., 2005). Other treatments mostly consist of endovascular methods attempting to obliterate the aneurysms. These types of endovascular treatment are difficult even when performed by expert interventional neuroradiologists. The literature reports several methods that include the use of detachable balloons, pushable microcoils, liquid embolic agents, a Guglielmi detachable coil (Vin˜uela et al., 1997), a combination of metallic stent with a liquid polymer injection (Mawad et al., 2002), and others. Results with the different types of treatment are encouraging but not completely satisfactory. The long-term outcome of patients treated by Guglielmi detachable coils remains unknown. Imaging follow-up by several techniques, especially contrast-enhanced MRA, after selective embolization of intracranial aneurysm seems to properly predict early aneurysm recanalization (Gauvrit et al., 2005). The administration of a calcium antagonist, mostly nimodipine, with surgical or interventional neuroradiological procedures, is common. However, treatment of intracranial aneurysms still constitutes a challenge.

19.7. Capillary hemangiomas Hemangiomas of the brain and spinal cord are considered to be malformations or hamartomas. They are benign tumors or tumor-like lesions that originate from blood vessels. These lesions are usually seen in the skin and soft tissues. In the neurocutaneous syndrome known as ‘cutaneous hemangiomas: vascular anomaly complex’ or ‘Pascual-Castroviejo type II syndrome’ (Pascual-Castroviejo, 1978, 2004; Pascual-Castroviejo et al., 1996; 2002), cutaneous hemangiomas can be associated with intracranial hemangiomas. Capillary hemangiomas of infancy are considered to be immature forms of capillary hemangiomas. Histologically, capillary hemangiomas are composed of nodules of small capillary-sized vessels, with each lobule fed by an artery. These lesions are most frequently located in subcortical areas and usually are fed by pial arteries (Fig. 19.14). The behavior of capillary hemangiomas is different in children from in adults. In children, they are most frequently associated with cutaneous hemangiomas and both intracranial and cutaneous hemangiomas follow a parallel evolution. They grow in size over months and more rarely over years because of rapid endothelial cell proliferation, and then they spontaneously involute to disappear (Mulliken and Glowacki, 1982) without treatment. Capillary hemangiomas of the CNS can be surgically removed and cured in adults who show neurological symptoms (Abe et al., 2004).


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Vascular endothelial growth factors – the tyrosine kinases Flt–1 and Flk–1 and angiopoietins and their receptors – appear to be necessary for angiogenesis and for proliferation, migration, adhesion and tube formation (Sato et al., 1995; Shalaby et al., 1995, 1997; Hatva et al., 1996; Risau, 1997; Plate, 1999; Hashimoto et al., 2000). A newly discovered group of cytokines, and their receptors – Tie-1 and Tie-2 – play a major role at later stages of vascular development, mediating endothelial cell matrix interactions that are essential to vascular maturation and remodelling (Sato et al., 1995; Davis et al., 1996; Maisonpierre et al., 1997). AVMs and cavernous malformations show some differences in their expression of endothelial cell angiogenesis receptors and structural proteins (Kilic et al., 2000; Uranishi et al., 2001). 19.8.1. Arteriovenous malformations Fig. 19.14. Axial view of the MRA showing a capillary hemangioma in the subcortical area of the left temporal lobe, which is vascularized by pial arteries (arrowhead).

19.8. Cerebrovascular anomalies The existence of intracranial vascular malformations (VMs) has been known for almost a century (Maraire and Awad, 1995). The first histopathological classification of VMs was presented in 1966 by McCormick, who modified the classification in 1984. In this classification, McCormick divided VMs into four groups: 1) arteriovenous malformations (AVMs), 2) cavernous malformations, 3) venous angiomas (VAs), and 4) capillary telangiectasia. Other rarer types of VM are ‘mixed’ vascular malformations of the brain that show distinct clinical, radiological and pathological profiles (Garcia and Anderson, 1991; Awad et al., 1993; Chang et al., 1997). Other modified classifications of CNS VMs, based on a combination of location, morphology and etiology, have been presented (Challa et al., 1995). VMs are dynamic lesions in which angiogenesis appears to take place constantly. During recent years, different groups have reported interesting data that allow correlation of the findings of immunochemical investigations, especially growth factors and extracellular proteins, with traditional concepts related to the histology of intracranial VMs (Folkman and Klagsburn, 1987; Folkman and D’Amore, 1996; Rothbarth et al., 1996; Risau, 1997). Studies carried out in recent years have revealed the fact that endothelial cell specific protein tyrosine kinase receptors mediate various facets of blood vessel formation during vasculogenesis and vascular response to injury and disease states (Uranishi et al., 2001).

Arteriovenous malformations are defined as direct communications between one or more arteries and one or more draining veins, without the intervention of a capillary bed (McCormick, 1984; Challa et al., 1995). Direct shunting of blood is associated with increased blood flow, great distention of the involved arteries and duplication or destruction of the elastica, fibrosis of the media and focal thinning of the wall. The involved veins also show distension, tortuosity and secondary changes in their walls. The most morbid and frequent presentation of AVM is cerebral hemorrhage, which occurs in about 60% of cases (Arteriovenous Malformation Study Group, 1999). Deleterious effects of AVMs on brain function include several pathological symptoms, including headache, hydrocephalus, a loud bruit over the head, nausea or vomiting, neurological deficits, seizures, disturbances in consciousness, mass effects or even sudden death (Urgelle´s et al., 1996). The existence of intracranial AVMs is discovered more often in adults than in children, and they may be found anywhere in the intracranial spaces, intraparenchymatous or meningeal or combined, either supratentorially or infratentorially. AVM may appear as an isolated pathology or associated with other diseases (Urgelle´s et al., 1996). The molecular mechanisms involved in the genesis and maintenance of AVM have not been elucidated (Uranishi et al., 2001). The involved vessels in AVM may show calcification and spontaneous occlusion (Pascual-Castroviejo et al., 1977). AVM size can range from small or even microscopic to giant, involving a large part of an entire hemisphere. Most visible AVMs are pyramidal-shaped lesions with the apex toward the center of the brain and the base in the meninges (Challa et al., 1995).

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Fig. 19.15. (A) Coronal view of three-dimensional MRA disclosing an AVM in the territory of the left middle cerebral artery. The study shows a big ‘nidus’ and a wide draining vein (asterisk). (B) Axial view of the MRA in the same patient showing drainage of the AVM to the right lateral venous sinus (arrow).

Conventional arteriography or MRA shows the AVM with the arterial feeders and draining veins (Fig. 19.15). Most frequently, there are less than three or four sizable feeders, but multiple small feeders are occasionally seen. The dilated draining veins connect the AVM to a nearby dural venous sinus. Enhancing the study with gadolinium may be necessary in some cases, especially in small AVMs that may be missed by conventional angiography. The radiological features of parenchymal AVMs consist of a mass of tangled vessels in the center of the malformation that is often known as the ‘nidus’. Some AVMs show a simple fistulous connection without a nidus. Given that AVMs are amenable to surgical or embolization treatment, it is necessary to grade AVMs radiologically based on size, location, number of major intracranial arteries supplying the feeders, age of the patient and clinical state, to better define the most effective and least dangerous treatment. Many deep AVMs are considered inoperable. Embolization and proton-beam radiation have fewer risks than surgery. Gamma knife radiosurgery appears to hold some promise in the treatment of deep-seated AVMs but size greater than 3 cm is a contraindication to its application. The reported obliteration rates following this treatment vary significantly, perhaps reflecting the different methods and timings of the imaging studies used

(Shin et al., 2004). Radiosurgery significantly decreases the risks of hemorrhage in patients with cerebral AVM, although it is not completely eliminated, after obliteration (Maruyama et al., 2005). Stereotactic radiosurgery has definite advantages compared with microsurgical resection for brainstem lesions. It provides complete obliteration of AMVs in two-thirds of patients (Maruyama et al., 2004). Better three-dimensional imaging studies and conformal dose planning reduce the risk of adverse radiation effects. Embolization can reduce the size of the AVM and possibly make it more treatable by radiosurgery and reduce the possibility of radiation injury (Liu et al., 2003). Embolization, mostly used preoperatively, is the preferred method of treatment in pediatric patients with multiple AVMs. However, there are cases of AVM in which preoperative embolization does not achieve sufficient occlusion of some arterial feeders to ensure control of intraoperative bleeding and the resection of the AVM becomes necessary. In these cases, low-flow deep hypothermic cardiopulmonary bypass may be necessary to control intraoperative bleeding (Meyer et al., 1997; Bendok et al., 1999; Dufour et al., 2001). Pregnant women with bleeding AVM have a much greater risk of rebleeding during the same pregnancy, especially between the 15th and 20th weeks of gestation (Challa et al., 1995).

CONGENITAL VASCULAR MALFORMATIONS IN CHILDHOOD 19.8.2. Venous angiomas Venous angiomas consist of one or more dilated veins and their tributaries. No arteries appear in the lesion. VAs were thought to be rare before the introduction of MRA but actually they may be the most common incidental vascular malformation detected by neuroradiological studies (Fig. 19.16) (Truwit, 1992), especially if contrast enhancement is used. Their clinical significance seems very low. Microscopically, VAs show modified structure of the veins, with the walls tending to be thicker and the lumens larger than those of normal veins. Images of VAs show a system of convergent small veins (the medusa head) terminating in one large central vein that empties into a meningeal or subependymal vein directly or through still larger veins. The smaller veins of a VA may appear no different from a capillary telangiectasis, especially if the draining vein is not seen. Despite being congenital, VAs are rarely diagnosed in infancy (Truwit, 1992). They can be found located in any part of the brain. Given their non-neoplastic nature, they are considered to be a type of venous developmental anomaly (Lasjaunias et al., 1986) and it is believed that they may represent a primary dysplasia of capillary and small

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veins (Ostertun and Solymosi, 1993). Although VAs can be seen by MR in T2, MR in T1 with contrast enhancement may better demonstrate the malformation, and even better visualization if MRA is performed. However, two-dimensional time-of-flight MR venography (2D TOF MRV) is probably the technique most widely used to study the cerebral venous structures (Rollins et al., 2005). Very few patients with VAs need any type of treatment. 19.8.3. Capillary telangiectases Telangiectasis means a single lesion composed of multiple dilated capillaries. Capillary telangiectases are usually small (< 2 cm) and they can occur in any part of the brain or in the spinal cord. They are frequently incidental findings at autopsy, especially in the pons. Microscopically, they show enlarged thin-walled, capillary-like vessels with red cells in the lumen. The exact nature of the capillary telangiectases is uncertain. The question of whether the involved vessels are true capillaries or simply dilated venules is still not elucidated (Challa et al., 1995). Histopathological distinction between telangiectasia and hemangioma include not only the size and structure of the vascular channels but also the presence of normal neural tissue between the vessels of telangiectasia and gliotic brain and absence of neurons between the hemangiomatous vessels. 19.8.4. Vein of Galen malformations

Fig. 19.16. MRA in axial view showing a venous angioma in the cerebellum (arrowhead).

Vein of Galen malformations (VGMs) or vein of Galen aneurysmal malformations (VGAMs) are recognized as rare congenital abnormalities that can cause severe morbidity and mortality in neonates and, less commonly, in infants and older children. VGMs became generally well known after the paper of Jaeger et al. in 1937. By 1987 only 245 cases had been reported in the English literature (Johnston et al., 1987). Neonates usually present with high-output cardiac failure, which is often fatal despite medical management (Hoffman et al., 1982; Johnston et al., 1987; Lasjaunias et al., 1989, 1995). Children usually present with heart failure, developmental delay, hydrocephalus and seizures. Prenatal symptoms are common. Mortality in the neonatal period is high (Johnston et al., 1987). Ultrasound transcranial sonography and MR studies may be useful during prenatal or perinatal periods and at any age before the closure of the anterior fontanelle (Abbitt et al., 1990; Goelz et al., 1996). Computed tomography (CT), conventional arteriography, transcranial sonography with colour Doppler (which can

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quantify the blood flow velocity and size of the aneurysm and also provide a useful baseline for follow-up) and especially three-dimensional MRA allows a complete exposure of the major vessels of supply (Fig. 19.17), tortuosity of arterial access, venous system and parenchymal and ventricular status. These diagnostic procedures aid in early endovascular intervention (Mitchell et al., 2001) and have been

Fig. 19.17. Three-dimensional MRA in (A) sagittal and (B) axial views showing a choroidal VGM with abundant blood supply from the territories of both carotids (small asterisks) and the basilar (large asterisk) vascular systems.

associated with improved outcome among survivors (Rodesch et al., 1994). Several proposed classifications have been used to describe VGMs (Raybaud et al., 1989; Houdart et al., 1993; Campi et al., 1996). Lasjaunias et al. (1991, 1995) proposed that VGMs be classified into two distinct types: choroidal and mural. Choroidal VGMs are usually seen in the neonatal period because of cardiac failure, abundant and usually bilateral blood supply from choroidal arteries and pericallosal arteries; many cases also show additional supply from transdiencephalic or transmesencephalic perforating vessels (usually thalamoperforating arteries). Occasionally, the middle cerebral artery may supply VGMs, but this is more likely in neonates than in older children (Raybaud et al., 1989; Mitchell et al., 2001). Mural-type VGMs receive their arterial supply from the collicular and posterior choroidal arteries, which may be unilateral or bilateral and drain into the median prosencephalic vein and then to the dural sinuses. Clinically, mural-type VGMs present in infants with macrocephaly or failure to thrive and mild cardiac symptoms (Lasjaunias et al., 1991; Halbach et al., 1998). Several general theories on the possible cause of VGMs have been proposed. We can summarize some of them. The vein of Galen could be dilated because of an increase of the arterial pressure (O’Brien and Schecter, 1970), or the vein of Galen could be a varix associated with an AVM (Nicholson et al., 1989), or VGM may also represent an ectasia secondary to an increased flow associated with an obstruction of a dural sinuses distal to the VGM (Lasjaunias et al., 1987). The presence of a persistent falcine sinus and postulated development around the 10th intrauterine week, however, may indicate that the anomaly represents a persistent median prosencephalic vein of Markowski, with absent development of a normal vein of Galen (Raybaud et al., 1989; Truwit et al., 1994; Lasjaunias et al., 1991b). Causes of the neurological symptomatology, apart from the direct pressure of the VGMs – obstructive hydrocephalus and elevated intracranial pressure – are reduced cerebral perfusion secondary to venous hypertension and to absorption of a great part of the intracranial vascularization by the VGAM that may cause ischemic zones and brain herniation. The treatment of VGMs has evolved from surgical procedures to interventional neuroradiological management (Lasjaunias et al., 1987, 1991a, 1991b; Ciricillo et al., 1990; Campi et al., 1998; Halbach et al., 1998; Mitchell et al., 2001). Surgery offers little benefit, with fatal outcomes in 80–100% of cases (Hoffman et al., 1982; Lasjaunias et al., 1989; Ciricillo et al., 1990). Results have improved with endovascular

CONGENITAL VASCULAR MALFORMATIONS IN CHILDHOOD management in infants and children with any muraltype or choroidal-type of VGM (Johnston et al., 1987; Ciricillo et al., 1990; Lasjaunias et al., 1991a; Yamashita et al., 1992; Rodesch et al., 1994; Campi et al., 1998; Mitchell et al., 2001). Several endovascular techniques have been described. Treatment in later infancy by the transarterial approach may be ideal (Lasjaunias et al., 1991a; Halbach et al., 1998) but the transvenous route is useful in the first week of life when there is an urgent indication for treatment of the neonate (Mitchell et al., 2001). Mortality is increased in the neonatal period if no treatment is offered. In some patients with persistent cardiac failure, multisystem failure can be prevented by urgent endovascular treatment (Mitchell et al., 2001). Transfemoral and transtorcular embolizations of the vein of Galen have been described using various approaches, catheters and embolic agents. The circumstances of every patient with VGM must be carefully analyzed before initiating the technique in order to select the most convenient therapy. Spontaneous thrombosis of a VGM is occasionally reported (Six et al., 1980; Whitaker et al., 1987). 19.8.5. Intracranial cavernous malformations Cerebral cavernous malformations (CCMs) (MIM 116860) or cavernous angiomas are characterized by abnormally enlarged capillary cavities without intervening brain parenchyma (Russell and Rubinstein, 1987). The vascular lesion consists of endotheliumlined caverns filled with blood at various stages of thrombosis and organization, separated by a collagenous stroma devoid of mature vessel wall elements (Maraire and Awad, 1995). CCMs were initially described by Virchow in 1863, and occur in an estimated 0.45% to 0.90% of the population (Robinson et al., 1991; Maraire and Awad, 1995), with male and female patients equally affected. These lesions are identified relatively commonly, especially after the development of contrast-enhanced CT and MR studies, but symptomatic disease is considerably less frequent (Steiger et al., 1989). Presentation of this lesion in children may be between 20% and 25% (Herter et al., 1988). Some are described in newborns (Moritake et al., 1985; Hubert et al., 1989), but most patients are older than 10 years (Simard et al., 1986) and are most often young adults. The malformations can occur anywhere in the CNS but are most common in subcortical white matter, pons, cerebellum and the external capsule region (Robinson et al., 1991). Those located in the temporal lobe and elsewhere in the brain are causally related to intractable epilepsy that can be cured by surgery. Rare localization, such as in the

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cavernous sinus, intraventricular and pontocerebellar zones, has also been described (Voci et al., 1989). CCMs may be sporadic or hereditary. The proportion of patients developing symptoms is higher in the hereditary form than in the sporadic form. CCMs commonly manifest as seizures, gross intracranial hemorrhage and focal neurological deficits. Lesions are frequently multiple in the same patient and may behave aggressively with repetitive hemorrhages and cumulative disability, although they may remain quiescent for many years. Hemorrhagic risk and neurological disability may be related to several factors, such as lesion location, previous hemorrhage, age, gender and stage of the reproductive cycle. The proportion of patients developing clinical symptoms is higher in the hereditary form than in the sporadic form. CCMs have been divided into two types, cavernomas (malformations in the CNS) and cavernous hemangiomas (true tumors with proliferative potential) (Lasjaunias et al., 1999). Studies following the introduction of magnetic resonance imaging (MRI) have emphasized the predominance of CCMs among the larger of angiographically occult vascular malformations of the brain (Gomori et al., 1986; Del Curling et al., 1991). Angiographic results are normal in the majority of patients with CCMs (Gomori et al., 1986; Rigamonte et al., 1988; Rapacki et al., 1990; Del Curling et al., 1991; Lasjaunias et al., 1999). Computed tomography (CT) study shows a well-circumscribed, nodular lesion of uniform or variegated mixed density with juxtaposition of calcifications, hemorrhage, and cystic components (Maraire and Awad, 1995). MRI is the most sensitive diagnostic tool for the evaluation of CCMs (Gomori et al., 1986; Rigamonte et al., 1988; Rapacki et al., 1990; Del Curling et al., 1991). The lesion typically appears as a well-defined, lobulated lesion, with a central core of reticulated mixed signal surrounded by a rim of signal hypointensity (Robinson et al., 1991; Maraire and Awad, 1995) (Fig. 19.18). Areas of hyperintensity correspond to acute or subacute hemorrhages and different stages of thrombus organization. The presence of cysts most probably represents residua of previously expanded hemorrhagic caverns that have since involuted with thrombus organization and resolution. Despite the great variety of images seen on MRI and the possibility of differential diagnosis, other types of intracranial diseases include calcified neoplasms, thrombosed AVMs, inflammatory lesions, infectious and granulomatous disorders. MRA cannot usually demonstrate any lesion during arterial phases but it shows CCMs during the venous system study. It is common to find another lesion near

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Fig. 19.18. MR study in T1-weighted view showing a central core of reticulated mixed signal surrounded by a rim of signal hypointensity (arrow) in the left cerebral hemisphere.

the region where another CCM has been removed some time earlier in a patient who seeks consultation because of signs of a lesion in another part of the brain (Fig. 19.19). CCMs may be found as a sporadic and unique lesion or manifested as multiple lesions with familial presentation in an autosomal-dominant inheritance form (Rapacki et al., 1990; Labauge et al., 1998). Clinical penetrance is incomplete and it has been suggested that it may depend on the CCM locus involved (Craig et al., 1998; Denier et al., 2004).

Fig. 19.19. MR study in coronal view reveals a bleeding CCM in the brainstem and cerebellum (arrow) and another CCM in the left cerebral hemisphere (arrowhead) close to a region where another CCM was removed some time before.

The study of a large Hispanic-American family (Dubousky et al., 1995) and of other families showed that all Hispanic-American families were linked to chromosome 7 and shared a common haplotype, suggesting that they had a common ancestor (Gu¨nel et al., 1995; Polymeropoulos et al., 1997). Analysis of other North American and European (French, Spanish and Portuguese) families demonstrated that some of these families were not linked to chromosome 7, demonstrating genetic heterogeneity (Labauge et al., 1998; Gu¨nel et al., 1998; Yung et al., 1999; Lucas et al., 2000, 2001; Gamero et al., 2001). Mutations in the gene encoding KR1T1 have been recently found as the cause of this hereditary type of CCM (Serebriisku¨ et al., 1997; Laberge-Le Couteulx et al., 1999; Sahoo et al., 1999). Several CCM loci have been mapped and loss of function mutations were identified in the KR1T1 (CCM1) gene, located in chromosome 7q (Dubousky et al., 1995; Gu¨nel et al., 1995; Polymeropoulos et al., 1997); in the MCC 4607 (CCM2) gene, located in chromosome 7p (Craig et al., 1998; Denier et al., 2004) and in the PDCD10 (programmed cell death 10) gene as the CCM3 gene, located in chromosome 3q (150). About 40% of kindred with CCMs are linked to CCM1, 20% to CCM2 and 10% to CCM3 (Dubousky et al., 1995; Craig et al., 1998; Bergametti et al., 2005). Neuroimaging penetrance diagnosis of CCMs is much higher than clinical penetrance. A large number of sporadic cases with multiple lesions are, in fact, familial cases (Labauge et al., 1998). The dynamic nature of CCMs is well documented (Hayman et al., 1982; Simard et al., 1986; Lechevalier,1989; Zabramski et al., 1994). Growth of lesions and image changes have been reported in more than a third of patients (Hayman et al., 1982; Robinson et al., 1991; Zabramski et al., 1994). New lesions, with an average rate of 0.4 new lesions per patient per year, have been reported in 29% of patients with familial CCMs after a follow-up of a several years (Zabramski et al., 1994). The management strategies are mostly determined by the symptoms. Asymptomatic patients with single or multiple lesions may not require any urgent treatment but a watchful attitude with periodic reimaging may suffice. Epilepsy, mostly caused by CCMs in a temporal lobe, should be treated with anticonvulsant medication, at least during the time that the seizures respond to medication. However, surgical resection of the lesion is necessary for cases with intractable epilepsy and the results usually are satisfactory with seizure control in most cases if the resection of the CCM is complete (Del Curling et al., 1991; Robinson et al., 1991; Scott et al., 1992). Favorable results have

CONGENITAL VASCULAR MALFORMATIONS IN CHILDHOOD also been reported in the resection of accessible symptomatic supratentorial lesions, and in brainstem lesions that appear at the pial or ventricular surface (Del Curling et al., 1991; Robinson et al., 1991; Zimmerman et al., 1991; Scott et al., 1992). There is no consensus as to whether the excision of the these lesions should be performed after an initial bleed or should await recurrence of symptoms or progression (Maraire and Awad, 1995). There is evidence to suggest that ‘de novo’ lesions develop in certain patients (Ogilby et al., 1993). The morbidity and mortality risks from surgery for brain lesions are high. Radiosurgical treatment of CCMs is controversial. The use of standard doses gives a poor clinical response and a high rate of complications. Lower doses are used, preferably for deep and inoperable CCMs, and may provide more promising results (Lunsford et al., 1992). 19.8.6. Cortical venous anomalies Intracranial cortical venous anomalies usually consist of abnormal drainage, mostly associated with anomalies of neuronal migration. These usually correspond to persistence of a cortical vein that drains the blood into the superior sagittal sinus after following the primitive sylvian sulcus (Barkovich, 1988). The size of this vein is directly related to the extent of the cortical malformation and may denote its severity. This vein may correspond to the primitive sylvian vein, which follows the fetal sylvian fissure, similar to those observed in the normal fetus of 20 and 26 weeks gestation (Lemire et al., 1975). This implies that at this time there is an arrest in cortical development in the affected region, including the venous development. 19.8.7. Vascular malformations of the meninges

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the upper eyelid show the choroidal angiomas, mostly associated with glaucoma. Leptomeningeal angiomas can also be associated with facial hemangioma (Pascual-Castroviejo et al., 2005). This vascular anomaly can also be found in Pascual-Castroviejo type II syndrome (Fig. 19.20). 19.8.7.2. Dural arteriovenous malformations The clinical behavior and morphology of dural AVMs are similar to those seen with cerebral parenchymal examples. Common clinical findings include repeated headaches or subarachnoid or subdural hemorrhages. The enlarged, tortuous veins and the dural sinus are always more prominent than the arterial feeders. Multiple dural AVMs have been reported in children (GarcıáMońaco et al., 1991). The venous sinuses very rarely show lacunar enlargement and this occurs locally and most often in a lateral sinus. The local enlargement of the sinus usually is the final part of an AVM (Newton and Cronquist, 1969; Gordon et al., 1977). We studied a case with a giant dilatation of a lateral sinus and a medium-sized dilatation of the other lateral sinus associated with an ipsilateral facial lymphangioma (Scavone et al., 1980). The treatment and prognosis of these lesions depend on the clinical symptomatology and size. Small lesions usually are asymptomatic and most do not need treatment. Those of medium size may be treated either by surgery or ablated by intravascular injection of thrombotic agents. Large AVMs are mostly treated by surgery. 19.8.7.3. Aneurysmal dilatation of the torcula This is a very rare malformation when it is a primary dilatation (Gu¨rsoy et al., 1979). Usually this anomaly is a secondary dilatation associated with a vein of Galen

Vascular malformations can be found in the leptomeninges, in the dura and in the venous sinuses. 19.8.7.1. Vascular malformations of the leptomeninges The most frequent are associated with the Sturge– Weber syndrome. The lesions can be located in any region of the leptomeninges over the hemisphere ipsilateral to the facial nevus flammeus affecting the area innervated by the first sensory branch of the trigeminal nerve. Only 23% of patients with bilateral facial nevus flammeus show bilateral leptomeningeal angiomatosis (Pascual-Castroviejo et al., 1993). The enhancement of MRI with gadolinium has become an obligatory study in these patients because it demonstrates not only the leptomeningeal lesions but also the ocular choroidal angiomas. Only patients with nevus flammeus affecting

Fig. 19.20. MR enhanced with gadolinium showing a leptomeningeal hemangioma in the upper area of the left cerebellar hemisphere (asterisks).

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Fig. 19.21. Sagittal view of MRA study showing the sinus pericranii (arrowhead) in a 3-day-old neonate.

malformation. Clinical features consist of bilateral exophthalmus, facial edema and psychomotor regression. Angiographic studies show generalized dilatation of the venous system, including the torcula. 19.8.7.4. Sinus pericranii This is a rare congenital anomaly of the diploic veins that consists of enlargement of the extracranial non muscular venous vessels which communicate directly with an intracranial venous sinus (Fig. 19.21) (Stromeyer, 1850). When the head is in a low position, the blood passes from the sinus to the extracranial diploic veins and its stasis causes a swelling that disappears with direct pressure. The vascular malformation may appear in the neonatal period or during the first years of life. MRA in sagittal view best demonstrates sinus pericranii. Treatment of this anomaly consists of surgical resection of the lesion, usually followed by good results (Stromeyer, 1850; Villarejo and Pascual-Castroviejo, 1977).

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Chapter 20

Acquired, induced and secondary malformations of the developing central nervous system HARVEY B. SARNAT* University of Calgary Faculty of Medicine and Alberta Children’s Hospital, Calgary, Alberta, Canada

20.1. Introduction The terms cerebral dysgenesis or malformation of the brain generally conjure up the wide range of genetically programmed defects that affect developmental processes in embryonic and fetal life. Many nongenetic insults also can affect ontogenesis, however. Acquired malformations may be induced by parenchymal infarcts or hemorrhages during fetal life or postnatally, particularly in infants born prematurely, by exposure to teratogenic drugs and toxins, fetal radiation exposure, intrauterine infections, fetal hydrocephalus, trauma and maternal malnutrition. Well known specific examples of these events include excessive maternal vitamin A intake in the first trimester, which can result in neural tube defects, the fetal alcohol syndrome, congenital cytomegalovirus (CMV) infection and fetal exposure to maternal antiepileptic medications. This chapter considers these secondary malformations of the nervous system.

20.2. Ischemic/Hypoxic infarcts in the fetal brain Despite major advances in perinatal and neonatal medicine and in obstetrics, and the recognition that many causes of cerebral palsy and epilepsy cannot be attributed to ‘birth asphyxia’, hypoxic/ischemic encephalopathy remains a major cause of neonatal neurological morbidity in full-term infants as well as in prematures and is a major contributory factor in other complications such as germinal matrix hemorrhage. However, fetuses with primary malformations frequently present intrapartum complications not

anticipated in infants born with normal brains, so birth asphyxia may be overdiagnosed in some cases or may be a secondary additional insult (Montenegro et al., 2005). Neuroblast radial migration from the subventricular zone to the neocortical plate begins at about 8 weeks gestation. More than 90% of neuroblast migration is completed by 16 weeks but additional migratory cells continue to differentiate as neurons until near term, even though most of the postmitotic neuroepithelial cells of the germinal matrix are destined to become protoplasmic astrocytes of the cerebral cortex (Sarnat, 1987, 1992). If there is damage to the cortex, even postnatally or at maturity, resident stem cells of the subventricular zone may migrate to the cortex to become new neurons (Sundholm-Peters et al., 2005). If the fetus is subjected to a period of systemic hypotension, such as might occur in maternal shock, or abruptio placentae should occur, rendering the fetus relatively hypoxic because of the barrier to gas exchange, lesions may occur in the neonatal brain that interfere with developmental processes. The white matter is particularly vulnerable because of its immature microcirculation, with radial end-arterioles and little collateral circulation from adjacent vessels, resulting in a relatively large territory of parenchyma supplied by each such vessel. In the same region are the radial glial fibers. Fig. 20.1 illustrates three sites where injuries to the fetal or premature neonatal brain may interrupt radial glia or cause their retraction, so that migratory cells along each fiber cannot reach their intended destination and become heterotopic neurons (Sarnat, 1994). Ischemic injury also may interfere with other processes, such as synaptogenesis and myelination.

*Correspondence: H. B. Sarnat MD, FRCPC, Professor of Paediatrics, Pathology (Neuropathology) and Clinical Neurosciences, Alberta Children’s Hospital, 2888 Shaganappi Trail NW, Calgary, Alberta T3B 6A8, Canada. E-mail: [email protected], Tel: þ1-403-955-7131, Fax: þ1-403-955-2922.

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H. B. SARNAT physiological, neuroimaging and infectious disease aspects of congenital infections. Only a few common, representative infections are cited, but many others may also impede or distort cerebral development by similar mechanisms. 20.3.1. Cytomegalovirus

Fig. 20.1. Sites of lesions that may destroy radial glial cells or their radial processes, which guide migratory neuroblasts to the cerebral cortical plate. (1) Lesions at the ventricular wall (e.g. congenital infections; germinal matrix hemorrhage) may destroy both premigratory neuroblasts and also radial glial cells. (2) Lesions of the white matter (e.g. ischemic infarcts) damage radial glial fibers, even if their cell bodies are preserved, and also damage migratory neuroblasts. (3) Lesions at the cortical surface (e.g. subarachnoid hemorrhages; cerebral contusions during premature delivery) may damage the pial end-feet of radial glial fibers and cause them to retract into the white matter. (Reproduced from Sarnat, 1992 with permission of Oxford University Press.)

20.3. Congenital infections Intrauterine infections of the fetal nervous system are a frequent cause of nongenetic, acquired malformations of the brain. This section will focus on these dysgeneses and their pathogenesis and is not intended as a comprehensive review of the clinical, epidemiological, neuro-

The most frequent infection acquired transplacentally by the fetus is cytomegalovirus (CMV) (Stagano et al., 1986; Demmier, 1994; Boppana et al., 2001; Ross and Boppana, 2005). This infection may produce various systemic manifestations at birth, including many neurological symptoms and signs such as abnormal posturing, neonatal seizures including infantile spasms, apnea, dysphagia and lack of visual fixation and hearing loss (Riikonen, 1978; Boppana et al., 1992, 2005; Perlman and Argyle, 1992; Naessens et al., 1999; Ross and Boppana, 2005). These clinical features often are secondary to malformations of the brain that are not genetically programmed and lead to permanent neurological sequelae. The landmark study that elucidated the most important mechanism of pathogenesis in human fetal CMV was that of Marques Dias et al. (1984). They demonstrated that the viral invasion of endothelial cells of CNS capillaries and other vessels lead to perfusion failure, with cerebral tissue ischemia and infarction, especially microinfarcts in the periventricular region. Such lesions during fetal development may interfere with radial glial cells or their fibers, leading to impairment in neuroblast migration. Abnormal convolutions form, or pseudogyration appears earlier than normal gyration is expected (Fig. 20.2), and the architecture of the cortical plate and subsequent cerebral cortex is

Fig. 20.2. Cerebral mantle of a 21-week fetus with congenital CMV infection. (A) Section of frontal lobe, showing pseudogyri with gyral fusion in the cerebral cortex, at an age when gyri are not normally yet formed; this is not a ‘precocious’ development of convolutions. The ependyma lining the frontal horn of the lateral ventricle is discontinuous (arrowhead). The arrow at the surface of the cortex indicates the pseudogyrus shown in higher magnification in (B). (C) Cortical plate of the occipital lobe showing a similar pseudogyral formation due to disturbed neuroblast migration. (D) Infarction and deposits of dystrophic calcifications in the watershed zone of the tegmentum of the pons; these lesions were seen bilaterally and relatively symmetrically. (E) Thalamic degenerating probable neuron exhibiting intranuclear DNA viral inclusion (arrowhead), which stained red with eosin. Most of the lesions are vascular as a result of endothelial involvement.

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morphologically disturbed. Other abnormal patterns at birth resulting from the fetal migratory disturbance in CMV are lissencephaly/pachygyria and polymicrogyria (Norman et al., 1976; Hayward et al., 1991; Barkovich and Lindan, 1994), schizencephaly not associated with mutation of the EMX2 gene (Iannetti et al., 1998) and subcortical heterotopia (Barkovich and Lindan, 1994). Porencephaly and multicystic encephalomalacia may occur, especially late in gestation. Porencephaly may not be discovered for several months or years, even though due to congenital CMV infection (Tominaga et al., 1996). Abnormal convolutions in many cases may be demonstrated by imaging in the neonatal period and even sometimes prenatally (Barkovich and Lindan, 1994; Boppana et al., 1997). Periventricular leukomalacia is common and not necessarily related to premature delivery. More than 70% of newborns with congenital CMV have an abnormal neonatal computed tomography (CT) scan, intracerebral calcifications, particularly in the periventricular region but also in the convexities of gyri, being the most frequent finding (Perlman and Argyle, 1992; Barkovich and Lindan, 1994; Boppana et al., 1997). Magnetic resonance imaging (MRI) increases the likelihood of demonstrating abnormal gyration. Cerebellar hypoplasia, another common malformation in CMV, may also

be detected. Direct viral invasion of neurons and glial cells occurs as well in CMV (Fig. 20.2) but is probably less important to the mechanism of dysgenesis than are the vascular lesions. Reactive inflammatory cells include, in addition to T-lymphocytes and macrophages, large numbers of plasma cells and multinucleated giant cells, which are modified macrophages. Immunoreactivity to CMV antigen may be demonstrated in inflammatory cells, glial cells; particularly gemistocytes, neurons and endothelial cells of both parenchymal and meningeal blood vessels. The chorioretinitis and optic atrophy also are mainly due to vascular lesions of the retina and eye itself; corneal opacities also may be seen at birth. Experimental studies in the mouse provide evidence of two mechanisms of damage by CMV to the developing brain: migration of murine viral infected neuroblasts to the cortical plate and the strong affinity of the virus for endothelial cells of cerebral blood vessels; involvement of large arteries, such as the middle cerebral artery, leads to prenatal or postnatal infarcts including porencephaly (Tsutsui et al., 1989, 1991). CMV also alters function in the developing brain by other mechanisms, such as interfering with catecholamine and indoleamine metabolism (O’Kusky et al., 1991) and causing elevation of uric acid levels in

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CNS tissue (Boyes et al., 1989). CMV, a DNA virus, is a member of the Herpesviridae family, together with herpes simplex types 1 and 2, Epstein–Barr virus, varicella-zoster virus and others. 20.3.2. Toxoplasmosis Toxoplasma gondii is not a virus but a protozoan parasite that, in urban society, has its most frequent reservoir in household cats. Mothers harboring this organism may transmit it hematologically to their unborn fetus. Congenital toxoplasmosis is second only to CMV as the most frequent congenital infection in the fetus and neonate (Swisher et al., 1994; Naessens et al., 1999). Microcephaly, congenital ventriculomegaly and calcifications in the periventricular region and also in the cerebral cortex and white matter are frequent, largely by vascular mechanisms similar to those described above for CMV. Cerebral involvement is more severe in infants infected early than late in gestation. Serological testing and treatment are available during gestation or in the neonatal period. 20.3.3. Herpes simplex Herpetic encephalitis in the neonate is similar to the disease in older children or adults but multicystic encephalomalacia may result from the multiple infarcts, including many microinfarcts (Chang et al., 1990). Cerebral cortical gyral calcification associated with the encephalitis of herpes simplex in infants may resemble Sturge–Weber syndrome on CT images (Ketonen and Koskiniemi, 1983). 20.3.4. Varicella-zoster The varicella-zoster virus, a type of herpesvirus, may cause a congenital varicella embryopathy as well as a postnatally acquired disease (Alkalay et al., 1987). In the fetus, congenital cerebral malformations may occur and the mechanism seems to be related to fetal zoster more than varicella because it often is segmental and also involves peripheral nerves and dermatomes in a segmental distribution, as in zoster (Higa et al., 1987). Neonatal electromyelography (EMG) may show widespread denervation of muscle, associated with a rising titer of varicella-zoster-specific IgM and death in the neonatal period (Harding and Baumer, 1988). Destructive ischemic, microcystic and inflammatory lesions are demonstrated in the cerebral cortex, white matter, thalamus, brainstem and spinal cord, as well as periventricular leukomalacia and generalized ventriculomegaly (Harding and Baumer, 1988; Magliocco et al., 1992). Congenital malformations include polymicrogyria of

the insular cortex, cerebellar heterotopia and other disturbances of neuroblast migration and general hypoplasia of brainstem structures. In addition to the vascular lesions, trans-synaptic spread of the varicella/zoster virus from infected neurons in the visual system has been demonstrated (Rostad et al., 1989), but this type of spread in the fetus is not well documented. Even in older children with postnatally acquired varicella, acute or delayed onset of hemiparesis may occur secondary to narrowing and occlusion of the internal carotid or middle cerebral arteries (Liu and Holmes, 1990; Ichiyama et al., 1990), because of the endothelial vascular lesions induced by the virus, just as in the fetus. 20.3.5. Rubella Neurosensory hearing loss is the most frequent clinical manifestation of congenital rubella but a diffuse congenital meningoencephalitis, multicystic degeneration of white matter and dystrophic calcifications may occur, particularly if the time of the infection is early in gestation (Reef et al., 2000). These anatomical lesions can interfere with neuroblast migration and other developmental processes, resulting in acquired cerebral dysgenesis. 20.3.6. Mumps Mumps is the foremost among several viruses that, when infecting the fetal brain, cause a noninflammatory stenosis or atresia of the cerebral aqueduct, resulting in congenital obstructive hydrocephalus. Pathologically, the cerebral aqueduct is small in size but still patent, or totally atretic, forked and replaced by clusters and rosettes of ependymal cells that allow no egress of cerebrospinal fluid from the third to the fourth ventricles. Gliosis and inflammation are absent in most cases, although a periaqueductal proliferation of astrocytes may occur in some cases. Associated malformations of the brain are generally absent, even though the cerebral mantle and corpus callosum are thin because of the increased pressure within the voluminous lateral and third ventricles. In older children who acquire mumps, by contrast, ependymal changes in the cerebral aqueduct leading to partial or complete aqueductal obstruction are infrequent (Leheup et al., 1987). Mumps in childhood is associated with meningoencephalitis, often predominantly involving the brainstem and cerebellum, features not common in the congenital infection. In rare cases, acquired aqueductal atresia may occur postnatally, following mumps infections in infancy or early childhood, usually with a latency of 3 months to as long as 4 years (Spataro et al., 1976).

ACQUIRED, INDUCED AND SECONDARY MALFORMATIONS The pathogenesis in humans and experimental animals may be a propensity for the mumps virus to infect ependymal cells (Volpe, 1995). The symptoms in children are those of progressive intracranial hypertension. 20.3.7. Congenital acquired immune deficiency syndrome The human immunodeficiency virus (HIV) is transmitted transplacentally and fetuses of mothers who are infected may become infected as well and be born with congenital acquired immune deficiency syndrome (AIDS) (Hutto et al., 1989; Katz and Wilfert, 1989; Ehrnst et al., 1992). This sad reality is being reported with increasing frequency in Western countries but is even more prevalent in Africa and in impoverished developing countries throughout the world. Since the antiviral medications used to treat AIDS are teratogenic, infants of mothers treated during pregnancy may have secondary malformations of the nervous system due to both the virus itself and the effects of chemotherapy. The virus invades endothelial cells and causes a vasculitis and microinfarcts in the fetal brain. The teratogenic effects of the virus depend upon the stage of ontogenesis in which the fetus is first exposed to the virus, and even late gestational exposures may interfere with brain development (Ehrnst et al., 1991). In adult AIDS patients, secondary invasion of the central or peripheral nervous systems by other organisms is well demonstrated, and includes toxoplasmosis (Grant et al., 1990; Luft and Remington, 1992; Rousseau et al., 1997), CMV infection (Vinters et al., 1989; Cohen et al., 1993) and others. These secondary opportunistic infections are potentially a risk to fetuses if the immune-compromised mother acquires such a secondary infection, although this is rare.

20.4. Teratogenic drugs and toxins Numerous toxins and drugs affect the developing nervous system and hence are teratogenic. Many are employed in experimental animals to induce malformations. An example is methylazoxymethanol, which interferes with neuroepithelial mitotic proliferation and neuroblast migration (Fiore et al., 2004; de Groot et al., 2005; Dupret et al., 2005; McLaughlin and Juliano, 2005). Vitamin A, the alcohol of retinoic acid, given in a single dose to rodents at a precise time in early gestation, causes neural tube defects and a Chiari malformation (Marıń-Padilla and Marıń-Padilla, 1981); excess vitamin A intake in the first month of gestation in humans similarly results in lumbosacral meningomyelocele and Chiari II malformation. Many antiepileptic drugs taken by pregnant mothers, including phenytoin

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(fetal hydantoin syndrome) and valproic acid as the best documented examples, are associated with an increased incidence of CNS malformations in the fetus (Ng et al., 2005). The most frequent teratogenic drug used by humans is alcohol. Infants with fetal alcohol syndrome have not only intrauterine growth retardation and dysmorphic facies but also cerebral malformations, often expressed clinically as epilepsy, mental retardation, cognitive defects and motor disturbances and pathologically as disturbances in cortical lamination or architecture, hypoplasia or agenesis of the corpus callosum and micrencephaly (Wisniewski et al., 1983; West et al., 1995). Cocaine and other illicit ‘street drugs’ may induce malformations such as midline prosencephalic defects, neuronal heterotopia and ocular anomalies but more frequently cause slow growth, microcephaly and low brain weight at birth; neonatal seizures may occur (Domıńguez et al., 1991; King et al., 1995). Immunosuppressive and antimetabolic drugs used in chemotherapy affect the fetal brain, particularly mitotic proliferation of neuroepithelial cells and degeneration of incompletely differentiated neurons and glial cells, which are more vulnerable than mature cells. Many of the heavy metals, such as lead and mercury, may be teratogenic. Another category of toxins that induce malformations are insecticides of various types, with lethal effects in insects on their nervous systems. Defoliative chemicals used in war are similar. Agent orange was sprayed on the tropical forests of Cambodia by the USA during the Vietnam War in the early 1970s and resulted in congenital anomalies in infants born to mothers who were pregnant and exposed to this chemical. Effects are still seen even today because the chemically stable teratogenic toxin remains in the soil and may affect food grown by rural families or children who play in the soil of their villages or eat dirt (Richner, 1996a,b) (Fig. 20.3).

20.5. Fetal radiation exposure The teratogenic effects of ionizing radiation on the developing nervous system are well documented and modern obstetricians avoid X-ray exposure of their pregnant patients unless absolutely necessary. X-irradiation of the fetal brain is a traditional method of inducing malformations in experimental animals.

20.6. Fetal hydrocephalus Obstructive hydrocephalus in the fetus is usually a result of a primary malformation of the CNS, such as aqueductal stenosis, Dandy–Walker malformation or Chiari malformation. The two most frequent acquired

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Fig. 20.3. Child with large posterior encephalocele in Cambodia, who was exposed to Agent Orange sprayed on forests by the USA as a defoliant during the Vietnam War. The toxin remains in the soil and continues to be potentially harmful even years after its use. It is teratogenic in the brains of developing fetuses and is neurotoxic to mature brains. (Courtesy of Dr B. Richner, Hoˆpital Kantha Bopha, Phnom-Penh, Cambodia.)

causes of congenital hydrocephalus are fetal viral infections, particularly mumps, which can produce a noninflammatory atresia of the cerebral aqueduct, and nonencephalitic intraventricular hemorrhage due to a hypoxic–ischemic insult during mid- to late gestation.

It is remarkable that, despite severe ventricular dilatation and compression of the cerebral mantle (brain tissue) with stretching of radial glial fibers, actual malformations of the cerebral cortex are relatively uncommon unless the hydrocephalus is associated with a primary cortical dysplasia.

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20.7. Maternal malnutrition In many places in Africa, Asia and Latin America where pregnant mothers are severely malnourished for proteins and calories, as well as deficient in vitamins (e.g. folic acid), minerals and other important prenatal nutrients, the incidence of malformations in their fetuses is high. Which of several factors is most to blame is difficult to isolate, but malnutrition of the fetus causes more than just intrauterine growth retardation: it also can impair the development of multiple organs, including the brain.

20.8. Maternal trauma Abdominal trauma during pregnancy may cause direct contusion of the fetal head and brain and intracranial hemorrhage, or may cause spasm of placental and umbilical arteries with impairment of perfusion and fetal ischemia.

20.9. Conclusions Most malformations of the brain are genetic disorders but cerebral dysgeneses can also be acquired in utero as secondary impairments of developmental processes. The most frequent causes of these acquired malformations are fetal hypoxia, ischemia or hypoperfusion and congenital infections. The principal mechanism is congenital infection. Cytomegalovirus is the prototype and the most studied; CMV induces cerebral dysgenesis less by direct viral invasion of neurons and glia than by viral involvement of endothelial cells of cerebral blood vessels leading to impaired perfusion and multiple infarcts. In the fetal brain, infarcts may damage radial glial fibers that guide migratory neuroblasts and glioblasts; abnormal gyration of the cerebral cortex and heterotopic neurons in the white matter ensue. Other insults to the developing brain may be due to toxins, including alcohol and teratogenic drugs, maternal malnutrition, trauma to the fetus and fetal exposure to ionizing radiation. When fetal or infant brains are demonstrated by neuroimaging to exhibit pachygyria, lissencephaly, schizencephaly and subcortical heterotopia, one should not assume that the cause is necessarily a genetic mutation. Identifying nongenetic causes is of great importance in genetic counseling.

References Alkalay AL, Pomerance JJ, Rimoin DL (1987). Fetal varicella syndrome. J Pediatr 111: 320–323. Barkovitch AJ, Lindan CE (1994). Congenital cytomegalovirus infection of the brain: imaging analysis and embryologic considerations. Am J Neuroradiol 15: 703–715.

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Higa K, Dan K, Manabe H (1987). Varicella-zoster virus infections during pregnancy: hypothesis concerning the mechanisms of congenital malformations. Obstet Gynecol 69: 214–222. Hutto C, Parks WP, Lai S, et al. (1989). A hospital-based prospective study of perinatal infection with human immunodeficiency virus type 1. J Pediatr 118: 347–353. Iannetti P, Nigro G, Spalice A, et al. (1998). Cytomegalovirus infection and schizencephaly: case reports. Ann Neurol 43: 123–127. Ichiyama T, Houdou S, Kisa T, et al. (1990). Varicella with delayed hemiplegia. Pediatr Neurol 6: 279–281. Katz SL, Wilfert CM (1989). Human immunodeficiency virus infection of newborns. N Engl J Med 320: 1687–1689. Ketonen L, Koskiniemi M-L (1983). Gyriform calcification after Herpes simplex virus encephalitis. J Comput Assist Tomogr 7: 1070–1072. King TA, Perlman JM, Laptook AR, et al. (1995). Neurologic manifestations of in utero cocaine exposure in near-term and term infants. Pediatrics 96: 259–264. Leheup BP, Feillet F, Roland J, et al. (1987). Le´sions des noyeaux gris centraux au cours des oreillons. Evolution clinique et neuroradiologique d’un cas. Rev Neurol (Paris) 143: 301–303. Liu GT, Holmes GL (1990). Varicella with delayed contralateral hemiparesis detected by MRI. Pediatr Neurol 6: 131–134. Luft BJ, Remington JS (1992). Toxoplasmic encephalitis in AIDS. Clin Infect Dis 15: 211–222. McLaughlin DF, Juliano SL (2005). Disruption of layer 4 development alters laminar processing in ferret somatosensory cortex. Cerebr Cortex 15: 1791–1803. Magliocco A, Demetrick D, Sarnat HB, Hwang WS (1992). Varicella embryopathy. Arch Pathol Lab Med 116: 181–186. Marıń-Padilla M, Marıń-Padilla MT (1981). Morphogenesis of experimentally induced Arnold–Chiari malformation. J Neurol Sci 50: 29–55. Marques Dias MJ, Harmont-van Rijckevorsel G, Landrieu P, Lyon P, et al. (1984). Prenatal cytomegalovirus disease and cerebral microgyria: evidence for perfusion failure, not disturbance of histogenesis, as the major cause of fetal cytomegalovirus encephalopathy. Neuropediatrics 15: 18–24. Montenegro MA, Cendes F, Saito H, et al. (2005). Intrapartum complications associated with malformations of cortical development. J Child Neurol 20: 675–678. Naessens A, Jenum PA, Pollak A, et al. (1999). Diagnosis of congenital toxoplasmosis in the neonatal period: a multicenter evaluation. J Pediatr 135: 714–719. Ng Y-T, Sotero de Menezes M, Flores L (2005). Fetal anticonvulsants. In: MedLink (previously Neurobase). Arbor Publishing, San Diego. Norman MG, Roberts M, Sirios J, et al. (1976). Lissencephaly. Can J Neurol Sci 3: 39–46. O’Kusky JR, Boyes BE, Walker DG, McGeer EG (1991). Cytomegalovirus infection of the developing brain alters

catecholamine and indoleamine metabolism. Brain Res 559: 322–330. Perlman JM, Argyle C (1992). Lethal cytomegalovirus infection in preterm infants: clinical, radiological and neuropathological findings. Ann Neurol 31: 64–68. Reef SE, Plotkin S, Cordero JF, et al. (2000). Preparing for elimination of congenital rubella syndrome (CRS). Summary of a workshop on CRS elimination in the United States. Clin Infect Dis 31: 85–95. Richner B (1996a). Kantha Bopha. Combat d’un me´decin suisses au Cambodge. La Nouveau Quotidien ESL SA, Lausanne. Richner B (1996b). Kantha Bopha. Als Schweizer Arzt in Kambodscha. Verlag Neue Zuücher Zeitung, Zu¨rich. Riikonen R (1978). Cytomegalovirus infection and infantile spasms. Dev Med Child Neurol 20: 570–579. Ross SA, Boppana SB (2005). Congenital cytomegalovirus infection: outcome and diagnosis. Semin Pediatr Infect Dis 16: 44–49. Rostad SW, Olson K, McDougall J, et al. (1989). Transsynaptic spread of the varicella zoster virus through the visual system. Hum Pathol 20: 174–179; erratum 20: 820. Rousseau F, Pueyo S, Morlat P, et al. (1997). Increased risk of toxoplasmic encephalitis in human immunodeficiency virus-infected patients with pyrimethamine-related rash. Clin Infect Dis 24: 396–402. Sarnat HB (1987). Disturbances of late neuronal migrations in the perinatal period. Am J Dis Child 141: 969–980. Sarnat HB (1992). Cerebral Dysgenesis. Embryology and Clinical Expression. Oxford University Press, New York. Sarnat HB (1994). Impairment of late neuroblast migrations by ischemia. In: HC Lou, , G Greisen, JF Larsen (Eds.). Brain Lesions in the Newborn. Alfred Benzon Symposium 37. Munksgaard, Copenhagen, pp. 105–119. Spataro RF, Lin SR, Horner FA, et al. (1976). Aqueductal stenosis and hydrocephalus: rare sequelae of mumps virus infection. Neuroradiology 12: 11. Stagano S, Pass RF, Cloud G, et al. (1986). Primary cytomegalovirus infection in pregnancy: incidence, transmission to fetus, and clinical outcome. JAMA 256: 1904–1908. Sundholm-Peters NL, Yand HKC, Goings GE, et al. (2005). Subventricular zone neureoblasts emigrate toward cortical lesions. J Neuropathol Exp Neurol 64: 1089–1100. Swisher C, Boyer K, McLeod R (1994). Congenital toxoplasmosis. Semin Pediatr Neurol 1: 4–25. Tominaga I, Kaı¨hou M, Kimura T, et al. (1996). Infection foetale par le cytome´galovirus. Porence´phalie avec polymicrogyrie chez un garçon de 15 ans. Rev Neurol (Paris) 152: 479–482. Tsutsui Y, Kashiwai A, Kawamura N, et al. (1989). Susceptibility of brain cells to murine cytomegalovirus infection in the developing mouse brain. Acta Neuropathol 79: 262–270. Tsutsui Y, Kashiwai A, Kawamura N, et al. (1991). Postnatal porencephaly induced in mouse by murine cytomegalovirus. Acta Neuropathol 82: 435–441.

ACQUIRED, INDUCED AND SECONDARY MALFORMATIONS Vinters HV, Kwok MK, Ho HW, et al. (1989). Cytomegalovirus in the nervous system of patients with the acquired immune deficiency syndrome. Brain 112: 245–268. Volpe JJ (1995). Neurology of the Newborn, 3rd edn. WB Saunders, Philadelphia, pp. 31–34.

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West JR, Chen WJ, Pantazis NJ (1995). Fetal alcohol syndrome: the vulnerability of the developing brain and possible mechanisms of damage. Metab Brain Dis 9: 291–322. Wisniewski K, Dambska M, Sher JH, et al. (1983). A clinical neuropathological study of the fetal alcohol syndrome. Neuropediatrics 14: 197–201.

Section II Comparative manifestations of central nervous system malformations


Chapter 21

Epilepsy in patients with cerebral malformations LORIE D. HAMIWKA AND ELAINE C. WIRRELL* University of Calgary and Alberta Children’s Hospital, Calgary, Alberta, Canada

21.1. Introduction Epilepsy is amongst the most common neurological disorders in the pediatric age range affecting approximately 1% of children. Although most children with epilepsy have a favorable long-term outcome, approximately 10–20% experience seizures that prove intractable to antiepileptic medication. Intractable epilepsy places the child at risk of physical injury, learning and cognitive disability, social embarrassment, inability to achieve independence, unemployment and inability to drive, and will have a major impact on family life and the development of personal relationships. Epilepsy syndrome and etiology have major impacts on intractability. The term ‘symptomatic’ epilepsy implies that the seizures are secondary to a pre-existing brain abnormality that is either identified from the history, examination or investigation, or is assumed to exist because of the presence of a static encephalopathy, such as cerebral palsy or mental retardation, prior to the onset of the seizures. Several studies have demonstrated significantly higher rates of intractability amongst children with partial or generalized symptomatic epilepsies, than among those with idiopathic syndromes (Berg et al., 1996, 2001; Hauser et al., 1996; Sillanpaa et al., 1998).

21.2. Epidemiology and importance of the malformations of cortical development in intractable epilepsy Malformative neocortical lesions are the most common identified cause seen in children with intractable epilepsy, with other etiological entities including neoplastic, metabolic or genetic, infection or inflammatory, vascular, traumatic or hippocampal sclerosis.

Malformative lesions can be further categorized into 1) cortical dysplasias, in which there is disruption of development of the neocortex, 2) lesions associated with tuberous sclerosis complex, 3) Sturge–Weber syndrome (encephalofacial angiomatosis), 4) neurofibromatosis type II and 5) vascular malformations. Of these, cortical dysplasias account for the majority of malformations but exhibit a wide range of severity from focal microdysgenesis to lissencephaly or hemimegalencephaly. However, many epilepsies beginning in very early life are due to severe malformations of cortical development and commonly have associated focal neurological deficits or mental handicap. There is a statistically significant negative correlation between the severity of cortical dysplasia and age at clinical presentation, with more severe cortical dysplasias in neonates and infants (Mischel et al., 1995). Identification of an anatomical abnormality concordant with electrophysiological data correlates with improved seizure-free outcomes in patients both with temporal (Kuzniecky et al., 1993) and extratemporal epilepsy (Mosewich et al., 2000) who undergo surgery. Presurgical detection of cortical dysplasia relies on high-resolution MRI and new imaging techniques are evolving to enhance detection rates. MR image quality is improved by increasing signal to noise ratio, either by using higher strength magnets, e.g. 3 T or 7 T, or by using phased array surface coils, which sample smaller volumes of tissue, resulting in less noise in the image. Grant found that phased array surface coil MRI at 1.5 T resulted in increased lesion detection and improved lesion diagnosis in 64% of patients with medically refractory epilepsy that was not mesial temporal in origin (Grant et al., 1997). Using a 3 T magnet resulted in the detection of new lesions in 37.5% of patients with epilepsy who had previously undergone

*Correspondence to: Elaine C. Wirrell MD FRCPC, Associate Professor of Pediatrics and Clinical Neurosciences, Alberta Children’s Hospital, 2888 Shaganappi Trail NW, Calgary, Alberta AB T3B 6A8, Canada. E-mail: Elaine. [email protected], Tel: þ1-403-955-2296.

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routine 1.5 T imaging (Grant, 2004). Quantitative image analysis may lead to improved detection of smaller areas of cortical dysplasia. Accurate interpretation of an MR epilepsy study may be time-consuming, as cortical lesions can be very subtle. With quantitative image analysis, suspicious areas may be identified using an automated program and then reviewed by a neuroradiologist (Fischl et al., 2002). Diffusion tensor imaging provides information on the organization of white matter tracts, which may be focally disturbed in epileptogenic regions. The use of this technique may assist in lesion identification on MRI (Eriksson et al., 2001). Fiber tracts can be displayed in three-dimensional space, using a modification of diffusion tensor imaging called ‘tractology’ (Grant, 2004). Finally, combining high-resolution MRI with techniques such as magnetoencephalography, which provides spatially localized neuronal activity at high temporal resolution, further enhances lesion detection and definition (Grant, 2004).

of 30 consecutive intractable epilepsy patients (adults and children) with a temporal lobe developmental malformation diagnosed on MRI, Ho noted that the dual pathology is often bilateral, which may influence the success of surgical resection (Ho et al., 1998). In this series, 87% had hippocampal atrophy (nine unilateral, 17 bilateral) and 60% had atrophy of the amygdala (three unilateral, 15 bilateral). Two possible explanations for this dual pathology have been postulated. First, the presence of cortical dysplasia or a developmental tumor may predispose a child to suffer a prolonged seizure with fever in early life, a known risk factor for mesial temporal sclerosis (Liu et al., 1995). Second, it has also been proposed that cortical dysplasia may in fact be induced by the seizure rather than causing it. Early-onset seizures and closed head injury have been reported to lead to abnormal neurons with ectopic localization and abnormal connections (Parent et al., 1997; Lombroso, 2000; Scharfman et al., 2000).

21.2.1. Temporal lobe epilepsy

21.2.2. Extratemporal epilepsy

There is significant variation among neuropathological series of epilepsy surgery patients in the prevalence of various pathological lesions. This variation is probably explained by surgical selection criteria for that center, including age of patient. While mesial temporal sclerosis has been identified as the most important pathological entity of intractable temporal lobe epilepsy in adults, seen in approximately 65% of surgical cases (Babb and Brown, 1987), it is less commonly reported in children (Duchowny et al., 1992; Bocti et al., 2003). Rather, cortical dysplasia or low-grade tumors are the major pathological substrates identified in childhood temporal lobe epilepsy, with dysplasia being reported in 9–67% and low-grade tumors in 9–48% (Adelson et al., 1992; Duchowny et al., 1992; Jay et al., 1993; Wyllie et al., 1998; Bocti et al., 2003). Furthermore, when mesial temporal sclerosis is found, there is a high incidence of dual pathology, particularly in children (Jay et al., 1993; Cendes et al., 1995; Nishio et al., 2000; Lee et al., 2001; Pasquier et al., 2002). In Bocti’s series of pediatric temporal lobectomy specimens, dual pathology was found in eight (seven cortical dysplasia, one ganglioglioma) of 12 cases with proven mesial temporal sclerosis (Bocti et al., 2003). In a surgical series of 34 children and adolescents with MRI-documented mesial temporal sclerosis on the side of electroencephalographic (EEG) seizure onset undergoing temporal lobectomy, Mohamed reported a high incidence of dual pathology – 79% had mild to moderate cortical dysplasia in addition to hippocampal sclerosis (Mohamed et al., 2001). In a radiological series

Extratemporal resection, including multilobar resection or hemispherectomy, is the most common curative surgery in infants and children, in contrast to adults where mesial temporal resections predominate. In the Cleveland Clinic series, these procedures comprised 44% of surgeries in adolescents, 50% of surgeries in children and 90% of surgeries in infants (Wyllie et al., 1998). In the series from Miami Children’s Hospital, 84% of children under the age of 3 had extratemporal resection or hemispherectomy (Paolicchi et al., 2000). In intractable epilepsy outside the temporal lobe, cortical dysplasia plays an even more significant etiological role. Wyllie reported that 46% of 49 pediatric cases of extratemporal or multilobar resection were found to have cortical dysplasia on pathology (Wyllie et al., 1998). Similarly, 56% of 75 children undergoing epilepsy surgery (46 extratemporal/multilobar) at the Miami Children’s Hospital were found to have cortical dysplasia, although this study did not differentiate pathology seen in extratemporal versus temporal resections (Paolicchi et al., 2000). In a series of 135 extratemporal resections for chronic epilepsy in children and adults, Prayson and Frater (2003) found that 52 (39%) had pathologically confirmed cortical dysplasia, with the most common patterns being diffuse cortical disorganization, neuronal cytomegaly and increased molecular layer neurons. In a German series of 63 consecutive patients operated on for extratemporal epilepsy, malformations were found in 25% and low-grade neoplasms in an additional 19% (Wolf et al., 1993).

EPILEPSY IN PATIENTS WITH CEREBRAL MALFORMATIONS

21.3. Why do malformations of cortical development cause epilepsy? Studies on the epileptogenesis of cortical dysplasia have been performed both on resected human tissue and in animal models. Animal models of cortical dysplasia include in utero manipulation (methylazomethanol [MAM] injection, gamma irradiation), manipulations in the newborn (ibotenate cortical injection, cortical freeze lesions) and spontaneously epileptic animals with cortical malformations (Ihara’s genetically epileptic rat, telencephalic internal structural heterotopia [TISH] rat and knockouts [PAFAHb subunit knockouts, tuberous sclerosis complex 2 knockouts]). While these models may help in determining why dysplastic cortex is hyperexcitable, it is less clear how well they can be related to the human condition, as phenotypes in animal models are often much milder and many, although showing enhanced sensitivity to seizure-inducing agents, are not associated with spontaneous seizures. Epileptogenicity in cortical dysplasia may be explained by either abnormal cells or abnormal neuronal circuitry. In general, more focal malformations of cortical development often have abnormal cells, which may act as pacemakers of epileptiform discharge (Schwartzkroin et al., 2000). By contrast, more diffuse malformations of cortical development have normalappearing cells but abnormal connectivity of the cellular aggregate (Schwartzkroin et al., 2000). 21.3.1. Abnormal neurons Epileptiform discharge in a large group of neurons can be triggered by a small group of abnormal cells that generate bursting behavior. The most dramatic examples of abnormal neurons in cortical dysplasia are the ‘balloon cell’, characteristic of tuberous sclerosis, which stains positively for markers of both neurons and glia, and has multiple ‘dendritic trees’ that show little orientation specificity, and the ‘giant cell’, which is often found together with balloon cells in Taylortype focal dysplasia. These neurons have been known to display intrinsic hyperexcitability (Mathern et al., 2000), possibly due to modification of NMDA receptors predisposing to hyperexcitability. NR1 and NR2 are the two subunits of the NMDA receptor. In vitro studies have shown that NR2 alone is nonfunctional while NR1 alone produces only weak currents to glutamate. Heteromeric coassembly of NR1 and NR2 subunits however, leads to marked increases in the NMDA channel current (Monyer et al., 1992). In studies on human surgical specimens, Ying noted that dysplastic neurons have increased NMDA receptor subunits NR1 and NR2A/B (Ying et al.,

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1998). Furthermore, in human epileptic focal cortical dysplasia, NR1 coassembled with NR2 (Mikuni et al., 1999), and NR1–NR2A/B were coexpressed in single dysplastic neurons (Ying et al., 1999), leading to neurons that are hyperexcitable to glutamate. MAM-treated rats also show an increased number of bursting neurons in regions of heterotopia and dysplastic cortex (Baraban and Schwartzkroin, 1995), which supports the concept that epileptogenesis may result from a few abnormal cells. Evidence of decreased binding to GABA receptors using autoradiography (Zilles et al., 1998) and decreased sensitivity of GABAA receptors to zolpidem, a benzodiazepine agonist (Habiltz and DeFazio, 2000) in the freeze lesion cortical dysplasia model also suggest a role for decreased inhibition in dysplasia. 21.3.2. Altered synaptic connectivity Cortical dysplasias are associated with reorganization of cortical circuitry. Abnormal connectivity has been best studied in the freeze lesion model of cortical dysplasia. In this model, the epileptogenic zone is actually adjacent to the microgyrus of the lesion and the cells in this adjacent region appear normal. Jacobs has shown that the pyramidal neurons adjacent to the dysplastic microgyrus receive more excitatory input, perhaps due to hyperinnervation by excitatory cortical afferents that were originally destined for the microgyrus (Jacobs and Prince, 2005). Furthermore, axons that would normally project out of the epileptic zone may also be interrupted and instead make excitatory synapses locally. Additionally, in the MAM model of cortical dysplasia, aberrant connections have been shown between the heterotopic cell regions and other, more distant brain regions, facilitating spread of abnormal epileptiform activity (Colacitti et al., 1998). Decreased numbers of inhibitory neurons have also been noted in and around dysplastic lesions, which would contribute to the overall hyperexcitability (Spreafico et al., 1998; Roper et al., 1999).

21.4. Clinical syndromes associated with the various malformations of cortical development Certain malformations of cortical development may be more commonly associated with specific seizure types, with more severe malformations associated with profound developmental disabilities and early-onset seizures and milder malformations presenting at various ages in individuals with relatively normal cognitive function. For example, generalized and bilateral forms of malformation, including bilateral periventricular nodular heterotopia, lissencephaly, subcortical band

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heterotopia, Aicardi syndrome, bilateral schizencephaly and bilateral perisylvian polymicrogyria, may manifest with one of the epileptic encephalopathies early in life (Ross, 2002) such as West syndrome or Lennox–Gastaut syndrome (Palmini et al., 1991; Guerrini et al., 1992; Ricci et al., 1992). In more focal forms, including both non-neoplastic entities (such as focal cortical dysplasia, tuberous sclerosis and hemimegalencephaly) and neoplastic entities (such as gangliogliomas and dysembryoplastic neuroepithelial tumors), refractory partial seizures, often with secondary generalization, are the rule (Whiting and Duchowny, 1999; Bentivoglio et al., 2003). Drop attacks are frequently seen when central regions are involved, presumably due to spread of ictal discharge to the frontal cortex and then contralaterally through callosal connections (Palmini et al., 1991). Conversely, one malformation may cause a variety of seizure types, depending on the maturational stage of the brain and its ability to generate focal or generalized seizures. An example of this tendency would be an infant with a malformation of cortical development who initially presents with infantile spasms but evolves over time to refractory partial complex seizures. The type of cortical malformation and associated seizures is an important determinant in the prognosis for favorable neurological outcome. 21.4.1. Seizure types seen in specific malformations of cortical development In 1996, a classification system for malformations of cortical development was proposed based on the timing at which the developmental process was disturbed (Barkovich et al., 1996). In 2001, this classification was revised as a result of increased knowledge of biological mechanisms and the discovery of new malformations (Barkovich et al., 2001). A subsequent revision was undertaken in 2005, predominantly related to genetic and neuroimaging advances (Barkovich et al., 2005). A similar classification has been also been proposed by Sarnat and Flores-Sarnat (2004). Using these recent classification schemes, seizure patterns associated with malformations of cortical development will be reviewed. Generally, seizures associated with abnormalities in cortical development are usually partial; however, in cases where there are diffuse or bilateral abnormalities a generalized pattern prevails. 21.4.1.1. Abnormal neuronal and glial proliferation or apoptosis This group of malformations is characterized by an increase or decrease in neuronal (and often glial)

number and proliferation of abnormal cell types. The abnormality may be diffuse or localized. 21.4.1.1.1. Malformations associated with decreased neuronal number These include microencephaly with normal to thin cortex, microlissencephaly and microcephaly with polymicrogyria or other cortical dysplasias. These disorders are sometimes associated with neonatal onset seizures, typically multifocal clonic or myoclonic seizure with severe developmental and neurological disability (Donat and Lo, 1994). 21.4.1.1.2. Malformations associated with increased proliferation These include megalencephaly and megalencephaly– polymicrogyria–hydrocephalus syndrome. While a genetic model of generalized megalencephaly has been associated with epilepsy in chickens (George et al., 1910), no human cases have been reported. 21.4.1.1.3. Malformations associated with abnormal proliferation These include non-neoplastic abnormalities (those associated with tuberous sclerosis, cortical dysplasia with balloon cells or hemimegalencephaly) and neoplastic abnormalities (dysembryoplastic neuroepithelial tumor, ganglioglioma or gangliocytoma). Seizures occur in 90% of children with tuberous sclerosis complex and often begin in the first months of life, frequently as infantile spasms (Fig. 21.1). Partial seizures are also common and may evolve from or to, or coexist with, infantile spasms. A number of children who present with focal seizures or spasms develop multifocal intractable epilepsy and an EEG pattern that resembles that seen in Lennox–Gastaut syndrome. Although the prognosis is generally poor in these children, they may rarely achieve complete seizure remission. Severe cortical dysplasias are characterized by the presences of cytomegalic neurons and balloon cells, often referred to as focal cortical dysplasia – Taylor type. These malformations are typically associated with medically resistant partial epilepsy of mainly extratemporal origin. In hemimegalencephaly, seizures are the most common and significant neurological symptom, occurring in 93–100% of those affected (Vigevano et al., 1996; Flores-Sarnat, 2002). Seizures often begin in the neonatal period and result in catastrophic epilepsy (Rintahaka et al., 1993; Di Rocco and Iannelli, 2000). Hemimegalencephaly is frequently associated with one of the earliest epileptic encephalopathies, Ohtahara syndrome, characterized by tonic spasms


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Fig. 21.1. 2-month-old with tuberous sclerosis complex, presenting with infantile spasms and focal seizures. (A) Axial flair MRI showing multiple cortical tubers with subependymal nodules (B) Awake interictal EEG showing a discontinuous background with multifocal epileptiform discharges.

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and a suppression burst pattern on EEG (Fig. 21.2). This syndrome has been uniformly associated with poor neurological outcome. Infantile spasms, often asymmetrical, are present in half of patients and partial seizures often coexist (Dulac et al., 1994). Progression to Lennox–Gastaut syndrome and epilepsia partialis continua has been reported. Dysembryoplastic neuroepithelial tumors, gangliogliomas and gangliocytomas are benign, slow growing, supratentorial, glioneuronal neoplasms in children and young adults with a history of medically resistant epi-

lepsy. These have a predilection for the temporal lobe and have been associated with adjacent cortical dysplasia (Im et al., 2002; Sakuta et al., 2005) (Fig. 21.3). 21.4.1.2. Abnormal neuroblast migration 21.4.1.2.1. Lissencephaly/subcortical band heterotopia spectrum Several subtypes of lissencephaly are now recognized, with subcortical band heterotopia considered the mildest end of this group of malformations. Children

Fig. 21.2. 3-month-old who presented with focal seizures and infantile spasms. (A) Axial T1-weighted MRI showing right hemimegalencephaly. (B) Interictal EEG showing bursts of epileptiform discharge throughout the right hemisphere, separated by epochs of relative suppression.


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Fig. 21.3. 12-year-old boy with simple/complex partial seizures. (A) Axial flair MRI showing a dysembryoplastic neuroepithelial tumor of the left temporal lobe. (B) EEG showing slowing over the left temporal region with anterior temporal sharp waves.

affected with classical lissencephaly have profound mental retardation and spastic quadriparesis. Seizures affect over 90% and often begin in the first 6 months of life (Guerrini, 2005). About 80% of children develop infantile spasms, which may not present with a typ-

ical hypsarrhythmic pattern but rather a modified hypsarhythmia pattern. Spasms are frequently associated with focal motor and generalized tonic seizures and these children ultimately develop multiple seizure types that are medically resistant to therapy.

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Epilepsy and developmental delay are the common clinical manifestation associated with subcortical band heterotopia; with seizures occurring in about 90% of patients (Habiltz and DeFazio, 2000). Barkovich (1996) has correlated both band thickness and severity of pachygyria with level of cognitive functioning. Pachygyria is associated with earlier seizure onset and an increased likelihood of developing Lennox– Gastaut syndrome or other generalized symptomatic epilepsies. Thicker heterotopia is also associated with a higher chance of developing symptomatic generalized epilepsy. 21.4.1.2.2. Heterotopia This group of malformations includes subependymal heterotopia, subcortical heterotopia and marginal glioneuronal heterotopia. Barkovich reviewed 13 patients with subcortical heterotopia and found that the majority had developmental delay with mild hemiparesis, and partial epilepsy with predominantly motor seizures (Barkovich et al., 1994). 21.4.1.3. Malformations due to abnormal late neuroblast migration and cortical organization This group of malformations includes polymicrogyria, schizencephaly, cortical dysplasia without balloon cells and microdysplasia. These malformations are believed to represent a defect in the later stages of cortical development, primarily involving cortical organization. 21.4.1.3.1. Polymicrogyria and schizencephaly Polymicrogyria and schizencephaly often coexist. Polymicrogyria has been associated with several malformation syndromes and may have a regional distribution or involve the entire cortical mantle. The clinical symptoms depend on the extent of involvement of the malformation, the most common neurological symptoms including seizures, focal neurological deficits and developmental delay. The most frequent seizure types are atypical absence seizures, tonic seizures, atonic drop attacks and tonic–clonic seizures, often occurring as Lennox–Gastaut-like syndromes (Kuzniecky et al., 1993) (Fig. 21.4). A minority of patients (about 25%) have focal seizures only, predominantly involving the facial region, and only a small number of children have infantile spasms. Electrical status epilepticus during sleep with atypical absence, focal motor and atonic seizures have been described in both unilateral and bilateral disease (Guerrini et al., 1998). Schizencephaly has a wide spectrum of anatomical presentations and thus a variable array of clinical symptoms. Seizures are most commonly focal, with up to 80% of patients having partial events. In bilateral cases these typically begin before the age of 3 years.

Fig. 21.4. (A) Axial T1-weighted MRI of a 4-year-old girl with familial polymicrogyria with greater involvement anteriorly. (B) Her initial EEG, performed at 18 months of age prior to onset of clinical seizures, showed diffuse fast activity. (C) At age 3.5 years, she developed multiple types of generalized seizure including epileptic spasms, atonic and tonic–clonic seizures. Her EEG now shows multifocal discharges on a poorly organized, slow background.

Children with unilateral schizencephaly may only come to medical attention after their first seizure (Barkovich and Kjos, 1992). 21.4.1.3.2. Cortical dysplasia without balloon cells With the availability of high-resolution MRI, focal cortical dysplastic lesions have been recognized with increasing frequency as a cause of medically intractable partial epilepsy (Fig. 21.5). Several surgical case series have found that children with cortical dysplasia without balloon cells had earlier onset of seizures with progression to medically resistant partial seizures and were more likely to have developmental delay and hemiparesis (Otsubo et al., 2005). 21.4.1.3.3. Microdysgenesis This microscopic malformation is characterized by a combination of various degrees of cytoarchitectural


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abnormality in the cortex and an increased number of neurons in the molecular layer of the cortex and/or in the subcortical white matter. Although there has been some controversy as to whether microdysgenesis is a normal variant or related to epilepsy, studies comparing patients undergoing epilepsy surgery to controls have shown a significantly higher incidence in the former group (Lawson et al., 2005). Clinically these children have onset of seizures at an early age and frequently associated mental handicap. Despite normal neuroimaging, seizures are often medically resistant. This diagnosis is made by pathological examination of the

surgical specimen. The prevalence of this abnormality in surgical specimens ranges from 10% to 30% depending on age and the location of the resection, with the highest frequency in children with extratemporal excision. 21.4.1.4. Other malformations of cortical development 21.4.1.4.1. Holoprosencephaly This group of disorders is characterized by failure of differentiation and cleavage of the prosencephalon,

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Fig. 21.5. 6 year old girl with very frequent partial complex seizures. (A) Axial flair MRI illustrating bottom of sulcus focal cortical dysplasia in the right frontal region. (B) EEG showing focal slowing over the right frontal region with admixed sharp waves.

and has been divided into three categories: alobar, semilobar and lobar. A large, multicenter study of 68 children with various severities of holoprosencephaly found that 49% had seizures (Plawner et al., 2002). Of patients over 12 months of age, severity of holoprosencephaly correlated with difficult-to-control sei-

zures, with the latter being seen in four of five (80%) with alobar, four of five (13%) with semilobar and only one of 11 (9%) with lobar holoprosencephaly. Cortical malformations were present in eight of 12 patients with difficult-to-control seizures, but only in 11 of 44 without difficult-to-control seizures.


21.5. Electrocerebral maturation and electroencephalogram features The immature brain exhibits an increased susceptibility to seizures, as reflected by the peak incidence of seizures in the first year of life (Hauser et al., 1993). Multiple factors play a role in this heightened susceptibility. While some of the increased incidence is related to the various symptomatic etiologies that promote seizures, including malformations of cortical development, the immature brain also has a lower threshold for seizures independent of associated neurological causes due to baseline hyperexcitability. A number of factors, such as high NMDA and AMPA receptor levels, altered composition of these receptors to favor excitation, prolonged action potentials, delayed maturation of post-synaptic inhibition and high synaptic density, play a role in this hyperexcitability (Moshe et al., 1983; Albala et al., 1984; Cavalheiro et al., 1987). The complex process of synaptogenesis dominates the period of early brain development, with the number of synapses produced in the early postnatal period greatly exceeding that of adults. This is followed by a period of synaptic pruning, a competitive process that depends on relative electrical activity. This results in the stabilization of successful synapses and elimination of unsuccessful ones. It has been suggested that highly patterned electrical activity, such as that seen in long-term potentiation, maintains synapses. 21.5.1. Electroencephalogram features associated with malformations of cortical development Characteristic EEG patterns have been recognized in association with some specific malformations of cortical development. The EEG in unilateral subcortical heterotopia shows slowing of the background with spike and spike-and-wave complexes over the affected hemisphere. Seizures have been reported originating from periventricular nodules and adjacent cortex. In band heterotopia, multifocal and generalized epileptiform discharges and generalized slowing are seen interictally, while ictal patterns are consistent with those seen in the symptomatic generalized epilepsies (paroxysmal generalized fast activity, irregular generalized spikeand-wave and high-amplitude slow-wave activity). Fast, rhythmic activity is often seen with dysplastic lesions. Localized, abnormal, fast activity is suggestive of a regional dysplastic lesion whereas more diffuse activity suggests more diffuse lesions such as agyrial pachygyria (Fig. 21.6). Long trains of rhythmical epileptiform discharges (spikes and sharp waves) have also been described on interictal scalp EEG in cortical dysplasia (Palmini

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et al., 1995; Otsubo et al., 2005) (Fig. 21.7). Positive spikes have been correlated with early seizure onset. Interictal electrocorticography showed continuous epileptogenic discharges; repetitive electrographic seizures and bursting discharges or continuous or quasicontinuous rhythmic spiking. Ictal electrocorticography showed paroxysmal fast and/or repetitive spiking.

21.6. Do uncontrolled seizures worsen longterm outcome in patients with malformations of cortical development? Children with severe and diffuse malformations of cortical development are often profoundly delayed. Whether superimposed, uncontrolled seizures worsen this outcome can be a difficult question to sort out clinically. These more severe malformations of cortical development often present early in life with one of the epileptic encephalopathies, such as infantile spasms or West syndrome. If seizures and/or frequent epileptiform discharges continue uncontrolled, cognitive development may be slow or arrest, behavior disorders may evolve and motor development may also be delayed. The key point in an ‘epileptic encephalopathy’ is that the slowing or regression of development is due to seizures or abnormal EEG, or both, rather than the underlying etiology of the epilepsy. Single EEG discharges have been shown to cause transient cognitive impairment (Aarts et al., 1984; Binnie, 2003). If single spikes can cause cognitive impairment, frequent multifocal discharges might be expected to more profoundly affect cognition. Frequent epileptiform discharges during sleep may have a particularly disruptive effect on memory consolidation by interfering with storage of memory in the neocortex (Kali and Dayan, 2004). Although the majority of infants with West syndrome are left with mental retardation, considerable evidence suggests that successful initiation of early treatment improves outcome (Zupanc, 2001). Infants who were medically refractory and underwent resective surgery showed significant increases in developmental levels postoperatively, with better outcomes occurring in those receiving surgery earlier (Asarnow et al., 1997). Children with infantile spasms, particularly those caused by tuberous sclerosis, are at risk of developing autism (Curatolo and Cusmai, 1987) and this risk may be decreased by early therapy (Askalan et al., 2003). Therefore, in West syndrome, the literature suggests that uncontrolled seizures do worsen long-term outcome. In some children who present with seizures later in life, epilepsy may also be progressive, with resulting cognitive decline. Oki reported on two children with focal cortical dysplasia in their dominant hemispheres, who experienced significant decline in verbal IQ with

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Fig. 21.6. 13 month old with past history of infantile spasms and complex partial seizures. (A) Axial T1 MRI showing diffuse pachygyria. (B) EEG showing generalized fast activity in keeping with pachygyria.

recurrence of frequent seizures in mid to late childhood (Oki et al., 2000). Even in older children, frequent epileptiform discharge can interrupt normal function in a focal area of cortex, with resultant developmental costs. Finally, there is clinical evidence that in certain patients, particularly those with recurrent, generalized seizures and/or status epilepticus, epilepsy may be a progressive disease, with cognitive deterioration, progressive brain atrophy and potential development of

intractable seizures. In a study of temporal lobe epilepsy, Hermann found that patients with childhood onset (< 14 years) had significantly reduced intellectual status and memory function compared to those with onset later in life, and the longer the epilepsy duration the worse the cognitive problems (Hermann et al., 2002). He also found reduced total brain tissue on volumetric MRI in the childhood-onset group. Other investigators have also documented progressive hippocampal (Fuerst et al., 2003) and extrahippocampal gray and


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Fig. 21.7. (A) Coronal gradient echo MRI of a 7-year-old girl with left parietal polymicrogyria. (B) EEG showing nearly continuous epileptiform discharge from this region.

white matter pathology (Bernasconi et al., 2004). There is ongoing debate as to whether ‘seizures beget seizures’, with this argument being predominantly refuted by large epidemiological studies and supported by experimental animal models of epilepsy (Berg and Shinnar, 1997; Sutula, 2004). While this phenomenon appears unlikely in many cases of human epilepsy, prolonged, recurrent seizures may lead to further brain damage and ‘rewiring’, enhancing susceptibility to further seizures.

21.7. Treatment and prognosis of epilepsies due to malformations of cortical development 21.7.1. Medical management Epilepsies due to the various malformations of cortical development are felt to have a high rate of medical intractability, as supported by the high rates of dysplasia found in epilepsy surgery specimens. However, there are very few studies that follow cohorts of newly

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diagnosed epilepsy due to cortical dysplasia, and case series from epilepsy clinics or surgical centers may select for more resistant cases. There are a wide range of lesions with cortical dysplasia and not all may lead to intractable epilepsy. Stephen reported on 63 adolescents and adults with radiologically diagnosed cortical dysplasia and localization-related epilepsy referred to a single epilepsy center over a 14-year period (Stephen et al., 2001). After a mean follow-up of 5 years, 54% had been seizure-free for at least 1 year and most were on monotherapy. Furthermore, no specific dysplastic lesion was associated with a particularly poor outcome. However, another hospital-based study on 2200 adult outpatients with epilepsy found that only 24% of those with cerebral dysgenesis attained seizure freedom (Semah et al., 1998). Unfortunately, there are no studies reported that recruited children with cortical dysplasia at the time of epilepsy diagnosis to clearly outline prognosis of these lesions in the pediatric age range. While Berg found that both remote symptomatic etiology and abnormal neuroimaging predicted a higher rate of intractability, malformations of cortical development were not assessed separately from other symptomatic causes (Berg et al., 2001). Lortie reported on 28 infants with focal cortical dysplasia and onset of their seizures in the first year of life identified from two pediatric epilepsy centers (Lortie et al., 2002). The majority initially presented with partial seizures but nearly half ultimately developed infantile spasms. While spasms were strikingly easy to control, all but one patient developed persisting partial or generalized seizures, and none of these achieved seizure freedom on antiepileptic drugs. Cieuta retrospectively reviewed 79 patients with cortical dysplasia seen in two European centres (Cieuta et al., 1996). Overall, in patients with diffuse and bilateral localized malformations such as agyria-pachygyria, band heterotopia and bilateral perisylvian or parieto-occipital polymicrogyria, partial seizures and epileptic spasms were more likely to be controlled with antiepileptic drugs but generalized seizures were intractable. In contrast, in lateralized malformations such as hemimegalencephaly or focal dysplasia, spasms tended to respond well to antiepileptic drugs; however, partial seizures were intractable. Ambrosetto reported on a small case series of five children with partial seizures due to unilateral opercular neuronal migration disorder who responded promptly to medication and who did not relapse after antiepileptic drugs were discontinued (Ambrosetto, 1993). Finally, while some patients may attain a period of seizure freedom of at least 1 year’s duration, an early benign course may not guarantee a good long-term

outcome. In a study of 333 patients presenting to one of seven surgical centers in the USA, 26% reported a prior remission of at least 1 year’s duration and 8.5% had experienced a remission of at least 5 years (Berg et al., 2003). Prior remission was more likely if epilepsy had its onset early in life. 21.7.1.1. Are certain antiepileptic drugs more effective in malformations of cortical development? Using the MAM model of cortical dysplasia, Smyth found that valproate, ethosuximide, lamotrigine, phenobarbital and carbamazepine had no effect on interictal epileptiform bursting (Smyth et al., 2002). Furthermore, pretreatment with valproate did not prolong seizure latency after kainate administration in MAM-exposed rats. This work suggests a dramatically reduced sensitivity to commonly used antiepileptic drugs in one model of cortical dysplasia. In humans, few studies have specifically focused on antiepileptic drug efficacy in malformations of cortical development. There is convincing evidence in the literature, however, to support the use of vigabatrin as the drug of first choice in infantile spasms due to tuberous sclerosis. In a European retrospective study, vigabatrin suppressed spasms in 68% of 192 infants, with a remarkable 96% response in those with tuberous sclerosis (Aicardi et al., 1996). Two prospective, randomized trials were published the following year, comparing vigabatrin to steroids. In a study of 22 infants with tuberous sclerosis and infantile spasms, all 11 vigabatrin-treated (150 mg/kg/day) patients but only five of 11 hydrocortisone-treated (15 mg/kg/ day) patients became spasm free ( p < 0.01) (Chiron et al., 1997). In addition, the mean time to disappearance of spasms on vigabatrin was significantly shorter than with hydrocortisone (3.5 vs 13 days, p < 0.01) and vigabatrin was better tolerated. A second study compared adrenocorticotropic hormone (ACTH; 10 IU/day) to vigabatrin (100–150 mg/kg/day) in 42 infants with both cryptogenic and symptomatic, newly diagnosed spasms (Vigevano and Cilio, 1997). While the efficacy of both drugs was similar for cryptogenic cases, vigabatrin was superior for tuberous sclerosis and cerebral malformations, and ACTH for perinatal asphyxia. A meta-analysis confirmed the unique response seen with vigabatrin for infantile spasms due to tuberous sclerosis (Hancock and Osborne, 1999). A total of 73 of 77 (95%) patients with tuberous sclerosis versus only 170 of 313 (54%) with other conditions had cessation of spasms with vigabatrin. An evidence-based literature review by Mackay et al. (2002) reached a similar conclusion. Vigabatrin may also improve

EPILEPSY IN PATIENTS WITH CEREBRAL MALFORMATIONS developmental quotient and autistic behavior in these children (Jambaque et al., 2000); however, larger studies are needed to confirm these impressions. Concerns about visual field constriction may limit duration of therapy but no evidence-based guidelines exist. In partial or generalized seizures due to cortical dysplasia there are no studies comparing antiepileptic drug efficacy and therefore no ‘drugs of choice’ can be identified. Treatment is best guided by choosing an appropriate medication for the seizure type and epilepsy syndrome. A recent review of efficacy of the new antiepileptic drugs in refractory seizures suggests that gabapentin, lamotrigine, oxcarbazepine and topiramate are appropriate for adjunctive treatment of refractory partial seizures in children; however, the subgroup with cortical dysplasia was not analyzed separately (French et al., 2004). 21.7.1.2. The ketogenic diet The ketogenic diet is a high-fat, low-carbohydrate diet that has documented efficacy in intractable childhood epilepsy, with 7–16% of children achieving seizure freedom and 40–55% achieving a more than 50% reduction in seizure frequency (Freeman et al., 1998; Vining et al., 1998; Lefevre and Aronson, 2000). It is most indicated in young children with refractory seizures who are not candidates for definitive surgical resection, and may have a particular benefit in children with malformations of cortical development or tuberous sclerosis. A recent review of 12 cases of tuberous sclerosis treated with the ketogenic diet in two centers noted that 92% had a reduction in seizure frequency of more than 50% and 67% experienced a reduction of more than 90% at 6 months on the diet (Kossoff et al., 2005). An Italian multicenter study reported that 64% of children with neuronal migration disorders improved on the ketogenic diet (Coppola et al., 2002). 21.7.2. Surgical treatment Curative surgical treatment for intractable epilepsy due to malformations of cortical development is often considered when there is a single seizure semiology with convergent anatomical or functional neuroimaging for the ictogenic focus. Hamiwka et al. (2005) reported a favorable outcome 10 years after surgery in 72% of children with developmental tumors and 32% of children with focal cortical dysplasias. Several authors have shown better surgical results in a small series of children with Taylor-type cortical dysplasia than those with cortical dysplasia and no balloon cells (Palmini et al., 1995; Hildebrandt et al., 2005; Lawson et al., 2005).

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Long-term outcome for children with tuberous sclerosis has been assessed in one series that showed excellent long-term outcome if seizures originated from a single tuber and children were normal or had only mild cognitive delay (Hardiman et al., 1988). Children with tuberous sclerosis can be particularly challenging to investigate for surgical candidacy. They may have multiple tubers that are potentially epileptogenic, their tubers frequently involve functional cortex and there is often rapid synchronization of the EEG at seizure onset with frontal and temporal tubers (Curatolo et al., 2005). Patients with bihemispheric malformations of cortical development are typically medically refractory and focal resection is ineffective. Palliative surgical procedures, including corpus callosotomy and vagal nerve stimulation, may be considered in these difficult cases. Corpus callosotomy has been performed in children with lissencephaly, band heterotopia, perisylvian polymicrogyria and tuberous sclerosis, with the greatest benefit being seen in atonic or tonic drop attacks (Jarrar et al., 2004; Kawai et al., 2004). Improved quality of life due to fewer injuries may also be a benefit. While vagal nerve stimulation may afford improvement in some cases of malformation of cortical development, overall efficacy appears modest at best (Murphy et al., 1999, 2003; Patwardhan et al., 2000). A small case series suggests that this therapy may play a role in the treatment of children with hypothalamic hamartomas who are either not surgical candidates or in whom surgery has failed (Murphy et al., 2000). An immediate and marked improvement in behavior in all children with severe autistic features was seen, although only two experienced significant reduction in seizure frequency.

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Chapter 22

Neuromuscular disorders associated with cerebral malformations JEAN K. MAH* University of Calgary, Calgary, Alberta, Canada

22.1. Skeletal muscle embryology Skeletal muscles in the body originate primarily from the somites, which are paired segmental structures of mesodermal origin (Brand-Saberi and Christ, 1999). The epiblast from the bilayered blastula during the 1st to 2nd gestational week contains primordial mesoderm that subsequently forms a distinct layer by the 3rd week at the gastrulation stage. Mesodermal cells that are destined for myogenic differentiation first appear in the primitive node and streak. These cells migrate in a lateral and rostral direction to form paired columns between the outer ectodermal and the inner endodermal layer. The paraxial mesodermal somitomeres subsequently undergo segmentation and differentiate into a series of somites on each side of the notochord along a rostrocaudal gradient. The myotomal plate on the dorsomedial aspect of the somites gives rise to muscles of the back, and the dorsolateral part of the myotome forms the limbs and ventral body wall musculature. While the somites give rise to the contractile elements of the developing muscles, the connective tissues, tendon, fat and blood vessels are derived from the superficial layer of the lateral mesoderm. In addition, the motor neurons and axons that innervate the muscles are of neural tube origin, and multipotent neural crest cells develop into Schwann cells, dorsal root ganglia and autonomic ganglion cells (Yamada et al., 1991; Zorick and Lemke, 1996). Development of the neuromuscular system requires a series of complex interactions between the myotomal cells, the neurons and the glial elements (Lien et al., 2004). During early embryogenesis, diffusible signals from the notochord and the floor plate, mediated by

the Sonic hedgehog (SHH ) gene, induce the ventral neuroepithelial cells to differentiate into motor neurons (Christ and Brand-Saberi, 2002). The postmitotic motor neurons extend their axons along specific pathways towards the myotomes and premuscle masses and then establish synaptic contacts with the forming myotubes at 9th to 11th week gestation. The interactions of Schwann cell precursors with their target muscles is mediated via a complex array of molecular cues (Padilla et al., 1998; Krull and Koblar, 2000). Terminal differentiation of neural and muscle cells depends on continual contact between Schwann cell precursors and growing motor or sensory axons until birth. In turn, feedback from skeletal muscles and associated motor neurons modulate brain development by influencing the ratio of differentiated neuronal types and the intrinsic properties of their progenitor cells (Kablar and Rudnicki, 2002). The presumptive myoblast around the 5th to 8th gestational weeks is an undifferentiated mesenchymal cell that lacks the distinctive myofilaments and fusional capacity of the more mature muscle cells (Sarnat, 2003). It differentiates into the myoblast by synthesizing myosin and actin and assembling these proteins into thick and thin myofilaments. Fusion of the myoblasts and the addition of more cytoskeletal proteins gives rise to the myotubes. Innervations of the muscle fibers occur during the myotubular stage, between the 9th and 11th weeks. Concurrently, the primitive motor endplates develop around the 9th week and become mature by the 20th week. Although muscle innervation is not required for the formation of functional acetylcholine receptors, the innervation process leads to a redistribution of the acetylcholine receptors and subsequently a

*Correspondence to: Jean K. Mah MD, MSc, FRCPC, Alberta Children’s Hospital, 2888 Shaganappi Trail NW, Calgary, Alberta T3B 6A8, Canada. E-mail: [email protected], Tel: þ1-403-955-2296; Fax: þ1-403-955-7649.

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remodeling of the neuromuscular junction. During the 14th gestational week, synaptic gutters and primary synaptic clefts are formed, and secondary synaptic clefts are present by the 16th week. Further differentiation of the myotubes into myocytes involves the addition of more contractile myofilaments, sarcoplasm and organelles, with displacement of the central nuclei to the periphery. Neural influence leads to histochemical differentiation of the muscles between the 20th and the 28th gestational weeks. Alignment of Z-band from the adjacent myofibrils and further development of the sarcotubular system occurs at the 30th week of gestation. A number of muscle developmental genes including those for myogenic factor 5 (MYF5), myogenin (MYF1), myogenic regulatory factor 4 (MRF4) and myoblast differentiating factor (MYOD) are important for the proliferation and differentiation of striated muscle precursor cells, and this topic has been recently reviewed (Sabourin and Rudnicki, 2000; Muntoni et al., 2002). These myogenic regulatory genes encode transcription factors, and they are members of the basic helix–loop–helix (bHLH) family. MYF5 is the earliest muscle-specific bHLH gene that is activated in the developing embryo, and it induces the proliferation of myoblast precursors (Ott et al., 1991). Similarly, MYOD is required for myogenic determination of the somitic cells. Activation of myogenin and MRF4 leads to myoblast fusion and further differentiation of the skeletal muscles (Arnold and Braun, 1996). Although the myogenic regulatory factors (MRF) can substitute for one another to some degree to preserve skeletal muscle development (Weintraub, 1993), they are not truly redundant, as MYOD and MYF5 have different functions in the formation of trunk and limb muscles (Kablar et al., 1997). Other genes that influence the MRF include the paired homeobox gene (PAX3), the bone morphogenic protein 2 gene (BMP2), and the muscle LIM protein gene. Similarly to MYF5, PAX3 induces myoblast proliferation and is essential for the migration of limb muscle precursors (Relaix et al., 2005). BMP2 inhibits terminal differentiation of the myogenic cells by suppressing MYOD and myogenin (Katagiri et al., 1997), and LIM protein promotes myogenesis by enhancing the activity of MYOD (Kong et al., 1997).

nance of either type I or II muscle fibers (Fig. 22.1), congenital muscle fiber type disproportion with type I fiber predominance and hypotrophy, and maturational arrest. The observed relationship between cerebral malformations and altered fetal muscle development may be the result of a common genetic defect, the effect of perinatal denervation during muscle development, or the influence of upper motor neuron diseases on the developing motor unit. 22.2.1. Multiple expression of a genetic defect Combined central and peripheral nervous system abnormalities are noted in patients with congenital muscular dystrophy, Duchenne muscular dystrophy and myotonic dystrophy, due to the manifold expressions of a common genetic mutation. The association between brain malformation and congenital muscular dystrophy was first reported in 1971 (Fowler and Manson, 1971). Subsequently, merosin deficiency due to mutations of the LAMA2 gene on 6q22–23 was identified in patients with congenital muscular dystrophy type 1A (MDC1A). Merosin deficiency affects the basal lamina of striated muscles and Schwann cells, cerebral blood vessels and glial-limiting membranes of the CNS, leading to muscular dystrophy, demyelinating polyneuropathy and white matter abnormalities (MiyagoeSuzuki et al., 2000). Other CNS abnormalities in MDC1A may include occipital agyria, pontocerebellar hypoplasia and ventriculomegaly. More severe CNS malformations have also been reported in congenital muscular dystrophy due to defects of a-dystroglycan glycosylation (see section 22.4, below). The genes encoding fukutin, fukutin-related protein and other glycosyltransferases have diffuse tissue distribution and

22.2. The effects of cerebral malformations on fetal muscle development Abnormal suprasegmental input from the brainstem and cerebellum around the 20th to 28th gestational weeks may be associated with maturational delay, histochemical alterations and/or aberrant morphology in the developing muscles (Sarnat, 1986). Morphological changes include greater than 80% fiber type predomi-

Fig. 22.1. Vastus lateralis muscle of a 4-year-old boy with global developmental delay showed massive type I fiber predominance without grouping of the minority type II (dark) fibers. Frozen section, myofibrillar ATPase preincubated at pH 10.4, 100.

NEUROMUSCULAR DISORDERS ASSOCIATED WITH CEREBRAL MALFORMATIONS are expressed in the developing muscles, brain, and eyes (Muntoni et al., 2004). Other types of muscular dystrophy can also be associated with CNS malformations but prominent myopathic features may overshadow the CNS involvement. In boys with Duchenne muscular dystrophy, mutations of the dystrophin gene on Xq21.2 may be associated with cerebral atrophy, ventricular dilation and microscopic CNS abnormalities such as heterotopias and disorganized cytoarchitecture (Anderson et al., 2002). These changes are attributed to altered dystrophin (particularly the Dp140 isoform) expression in the cerebellum, hippocampus and other regions of the central nervous system (Muntoni et al., 2003). Nonspecific subcortical white matter changes, cerebral atrophy and focal dysplasia has been reported in patients with myotonic dystrophy type 1 (DM1), presumably because of the multisystem effect of trinucleotide expansions on 19q13 (Bachmann et al., 1996; Kornblum et al., 2004). 22.2.2. The influence of perinatal denervation on muscle development The initial development of the motor neurons occurs independently from the muscle fibers. However, once the axons of the motor neurons reach their target muscle fibers and the neuronal connections between the motor neurons and the upper motor neuron system are established, survival of the motor neurons is critically dependent on their continual contact with targeted muscles. Unlike adult muscle fibers, immature muscles do not undergo reinnervations from nearby motor neurons. If the neural connections are disrupted early, the majority of the motor neurons will die and there will be massive muscle fiber loss (Greensmith and Vrbova, 1997). The severity of perinatal denervation is inversely related to the gestational age of the developing fetus and directly related to the duration of target deprivation. Such denervation can be seen in infants with spinal muscular atrophy, perinatal polyneuropathy, neuromuscular blockade or spinal cord lesions (Drachman, 2003). In addition, severe denervation due to the neonatal form of spinal muscular atrophy (SMA-0) was associated with the development of centronuclear myopathy (Nadeau et al., 2005). 22.2.3. Influence of suprasegmental disease on the developing motor unit The motor unit is capable of developing normally at the segmental spinal level without input from the upper motor neuron system (Sarnat, 1985). However, malformations of the cerebrum, cerebellum and brainstem can alter the discharge pattern of motor neurons and

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affect fetal muscle development. In particular, abnormal suprasegmental input from the descending bulbospinal pathways during the 20th to 28th gestational weeks may change the proportions of fiber types and the size of muscle fibers and/or delay muscle maturation. In contrast, the corticospinal tract is still unmyelinated at this stage and has little or no effect on the developing motor unit (Sarnat, 1989). Among children with cerebellar hypoplasia, the muscle histochemical changes may include type I fiber type predominance or congenital fiber type disproportion (Sarnat, 1986). Other examples of central nervous system malformations associated with neuromuscular disorders are summarized in Table 22.1. Table 22.1 Neuromuscular disorders associated with central nervous system malformations 1. Ventral horn cell disease a. Spinal muscular atrophy and pontocerebellar hypoplasia type 1 2. Hereditary motor and sensory neuropathy a. Peripheral neuropathy with agenesis of the corpus callosum 3. Neuromuscular junction transmission defect a. Neonatal myasthenia gravis 4. Congenital muscular dystrophies a. CMD due to laminin a2 deficiency b. CMD due to a-dystroglycanopathies i. Fukuyama type congenital muscular dystrophy ii. Walker–Warburg syndrome iii. Muscle–eye–brain disease iv. Congenital muscular dystrophy type 1C v. Congenital muscular dystrophy type 1D c. Other forms of congenital muscular dystrophy 5. Other myopathies with central nervous system malformations a. Metabolic disorders with secondary muscle fiber type disproportion i. Mitochondrial cytopathy ii. Zellweger syndrome iii. Pompe disease iv. Congenital disorders of glycosylation (CDG) CDG-1a CDG-IId b. Genetic syndromes with myopathy and cerebral dysgenesis i. Pena–Shokeir syndrome ii. Marden–Walker syndrome iii. Carey–Fineman–Ziter syndrome iv. Marinesco–Sjo¨gren syndrome v. Congenital fibrosis of the extraocular muscles vi. Secondary amyoplasia Spinal cord dysplasia Sacral agenesis

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22.3. Specific neuromuscular disorders associated with central nervous system malformations 22.3.1. Ventral horn cell disease 22.3.1.1. Spinal muscular atrophies and pontocerebellar hypoplasia type 1 This autosomal recessive syndrome of early-onset spinal muscular atrophy and pontocerebellar hypoplasia (PCH type 1) was first described by Norman (1961) and was further delineated by Barth in 1993. Type 2 PCH differs from type 1 by the absence of ventral horn cell pathology and by the presence of extrapyramidal dyskinesia (Grellner et al., 2000). Infants with PCH type 1 may present with polyhydramnios, decreased fetal movement, pulmonary hypoplasia and multiple contractures at birth, as seen with the Pena–Shokeir sequence (Moerman and Barth, 1987). There is associated prenatal hypoplasia of the pons, olives and neocerebellum (Fig. 22.2), plus postnatal cerebellar atrophy (Go¨rgenPauly et al., 1999). Other brain abnormalities such as partial agenesis of the corpus callosum, delayed myelination, polymicrogyria and cerebral atrophy may be present. Neuromuscular manifestations include severe weakness, muscle wasting, hypotonia, fasciculation, areflexia and respiratory insufficiency due to ventral horn cell involvement. However, the brainstem and upper motor neuron signs may be more prominent initially before the lower motor neuron disease becomes evident (Goutieres et al., 1977). Progressive microcephaly,

seizures, autonomic instability, visual impairment and mental retardation are common in PCH type 1, and extraneurological manifestations include failure to thrive, dysmorphism, visceromegaly and laryngomalacia (Ryan et al., 2000). Extensive genetic and metabolic studies are usually nondiagnostic, as PCH type 1 is not related to mutations of the survival motor neuron gene (Dubowitz et al., 1995). Nerve conduction study demonstrates a mixed sensorimotor demyelinating and axonal polyneuropathy, with chronic denervation changes seen during electromyography exam (Ryan et al., 2000). Abnormal electroretinogram due to progressive retinal dystrophy has also been described (Salman et al., 2003). Muscle biopsies showed group fiber atrophy in muscles and loss of myelinated nerves in selected cases, and postmortem examination revealed a loss of neurons and reactive gliosis in the cerebellum, brainstem, basal ganglia and ventral horns of the spinal cord. The genetic defect of this rare disorder is still unknown. Treatment consists of supportive care only. Death usually occurs during the first year of life because of respiratory complications but milder phenotypes with longer survival to early childhood are possible (Rudnik-Scho¨neborn et al., 2003). 22.3.2. Hereditary motor and sensory neuropathy 22.3.2.1. Peripheral neuropathy with agenesis of the corpus callosum This autosomal recessive disorder of hereditary motor and sensory neuropathy plus absent corpus callosum

Fig. 22.2. Newborn infant with pontocerebellar hypoplasia type I. (A) Sagittal and (B) coronal T-weighted brain MRI showed severe cerebral and pontocerebellar atrophy, ventriculomegaly and partial agenesis of the corpus callosum.

NEUROMUSCULAR DISORDERS ASSOCIATED WITH CEREBRAL MALFORMATIONS (HSMN/ACC) was first described by Leblanc et al. (1966) and then further delineated by Andermann (1981). It is now commonly known as the Andermann syndrome, and is most prevalent among French-Canadians living in the Saguenay–Lac-St-Jean and Charlevoix county of Quebec, with an estimated incidence of 1:2000 live births (De Braekeleer et al., 1993). Non-FrenchCanadian cases have also been reported worldwide (Dupre´ et al., 2003). HSMN/ACC is characterized by global developmental delay, hypotonia, progressive sensorimotor neuropathy, areflexia, muscle wasting and variable degrees of corpus callosum agenesis. Additional features include ptosis, upward gaze palsy, facial asymmetry, tremor, seizures, scoliosis and minor dysmorphic features such as high arched palate (Mathieu et al., 1990). Most affected individuals are mentally retarded and a third of the patients may experience atypical psychosis during adolescence (Filteau et al., 1991). Electrophysiological studies reveal a mixed axonal and demyelinating motor polyneuropathy, with absent sensory nerve action potentials. In addition to partial or complete callosal agenesis, CNS abnormalities may include diffuse cerebral and/or cerebellar atrophy. Some cases of HSMN/ACC may have normal corpus callosum on imaging studies but callosal axonal loss is evident on histological examination. Other pathological features include enlarged axons in the spinal and cranial nerve roots with onion bulb formations. Sural nerve biopsies show severe reduction in large myelinated fibers, swollen axons and partial axonal regeneration (Fig. 22.3). Howard et al. (2002) identified mutations of the SLC12A6 gene on chromosome 15q as the genetic basis for HSMN/ACC. The SLC12A6 gene codes for a potassium and chloride cotransporter 3 (KCC3) protein. The KCC3 protein facilitates cell volume regulation and

Fig. 22.3. A 7-year-old boy with Andermann syndrome. Sural nerve biopsy showed a reduction of large and small myelinated axons, endoneural fibrosis and axonal degeneration. 1 mm plastic embedded section stained with toluidine blue, 400.

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transepithelial transport of potassium and chloride within cells or interstitial space (Race et al., 1999). As KCC3 is widely expressed in the brain and spinal cord, it may also play an important role in the development of the central nervous system (Pearson et al., 2001). Individuals with HSMN/ACC experience loss of independent ambulation and scoliosis due to progressive muscle weakness, leading to wheelchair dependency during adolescence. Life expectancy is reduced, with death usually occurring around the third or fourth decade of life. 22.3.3. Neuromuscular junction transmission defect 22.3.3.1. Neonatal myasthenia gravis Myasthenia gravis is usually an acquired autoimmune disorder (Drachman, 1994) due to antibodies directed against the nicotinic acetylcholine receptor (AChR). Approximately 10–15% of myasthenic patients do not have detectable anti-AchR antibodies; instead, they may have antibodies against the muscle-specific tyrosine kinase (MuSK) or other neuromuscular junction target (Evoli et al., 2003). Myasthenia gravis is associated with impaired neuromuscular junction transmission and fatigable muscle weakness, with an estimated incidence of 1:10 000 in the general population (Polizzi et al., 2000). About 10% of neonates born to mothers with myasthenia gravis may experience transient neonatal myasthenia gravis due to placental transfer of maternal IgG antibodies against fetal AChR (Namba et al., 1970). Occasionally, the diagnosis of maternal myasthenia gravis has been made only after the birth of an affected infant, as the mother was asymptomatic (Brueton et al., 2000). These infants typically present with hypotonia, muscle weakness, respiratory distress and feeding difficulties, which resolve after 4–6 weeks. A more severe clinical spectrum may be associated with reduced fetal movement, polyhydramnios, craniofacial dysmorphism, arthrogryposis multiplex congenita and multiorgan anomalies, as seen in fetal akinesia sequence (Vincent et al., 1995; Riemersma et al., 1996). The severity of arthrogryposis is variable and does not correspond to the severity of maternal myasthenia gravis. Additional CNS anomalies including cerebral atrophy, cerebellar atrophy and spina bifida have been observed (Polizzi et al., 2000), and similar outcomes were noted in chick embryos injected with curare and in fetal mice with human AChR antibodies (Drachman and Coulombre, 1962; Barnes et al., 1995). The CNS anomalies may be due to maternal antibodies directed against other fetal-specific antigens. The recurrence risk of neonatal myasthenia gravis is high; recognition and treatment of

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mothers with myasthenia gravis may improve outcome in subsequent pregnancies (Holmes et al., 1980). Neonates with transient myasthenia gravis require supportive intervention for respiratory and feeding difficulties. Acetylcholinesterase inhibitors such as neostigmine or physostigmine may also be helpful in these infants. 22.3.4. Congenital muscular dystrophies Congenital muscular dystrophy (CMD) was first described by Batten (1903), and was reviewed by Muntoni and Voit (2004) and Jimenez-Mallebrera et al. (2005). It encompasses a spectrum of autosomal recessive muscle disorders of varying severity, with an overall incidence of 4.65 105 and a prevalence of 8 106 (Mostacciuolo et al., 1996). Congenital muscular dystrophy results in severe muscle weakness, hypotonia and contractures at birth or within the first year of life, and may include CNS involvement such as leukodystrophy, structural brain malformations, mental retardation and ocular abnormalities. Serum creatine kinase levels may be normal or markedly elevated. Muscle biopsies show a dystrophic process, with variation in fiber size, prominent muscle fiber necrosis and proliferation of endomysial connective tissues (Fig. 22.4). The diagnosis of CMD subtypes relies on the combination of clinical features, electrophysiological findings, muscle biopsies and identification of the underlying genetic and biochemical defects. To date, 11 genes associated with specific forms of CMD have been identified. According to Muntoni and Voit (2004), CMD with known genetic defects affecting the skeletal muscle fibers can be classified into three categories:

1. CMD due to mutations of the basal membrane or extracellular matrix proteins. Examples include CMD with laminin a2 deficiency, Ullrich CMD and CMD due to integrin a7 deficiency. CMD with laminin a2 deficiency is associated with white matter changes, occipital polymicrogyria and pontocerebellar hypoplasia. 2. CMD due to mutations of the glycosyltransferases. This includes the Walker–Warburg syndrome, muscle–eye–brain disease, Fukuyama type CMD, CMD with secondary merosin deficiency type 1 and 2 (MCD1B and MCD1C) and CMD with mental retardation and pachygyria (MCD1D). Most of these are associated with CNS malformations. 3. CMD due to mutations of the endoplasmic reticulum proteins. This includes CMD with spinal rigidity (RSMD1); brain imaging studies and intelligence are usually normal. 22.3.4.1. Congenital muscular dystrophy due to laminin 2 deficiency Laminin a2 (or merosin) deficiency was first identified by Tome et al. (1994) as a cause of CMD, and this disorder is known as congenital muscular dystrophy type 1A (MDC1A). It accounts for approximately 30–40% of all patients with CMD. Laminins are extracellular proteins that are essential for maintaining the integrity of the muscle basement membrane, by binding to other anchoring molecules such as dystroglycan, agrin, integrin and collagen VI (Jimenez-Mallebrera et al., 2005). Laminin is comprised of an a, b, and g chain, each encoded by different genes (Gullberg et al., 1999).

Fig. 22.4. Vastus lateralis muscle of a 9-month-old infant with congenital muscular dystrophy. She presented with generalized axial hypotonia and muscle weakness at birth, with elevated creatine kinase of 1170 IU/l. Muscle biopsy showed variation in fiber size, scattered muscle fiber necrosis and proliferation of endomysial and perimysial connective tissues. (A) Paraffin section, hematoxylin & eosin (H&E) 100. (B) Frozen section, H&E, 250.

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Fig. 22.5. Electron microscopy of (A) normal control and (B) 2-year-old girl with a merosin-deficient congenital muscular dystrophy. The basal lamina (basement membrane) as indicated by the black arrows was present in the control, while the white arrows showed a disruption of the basal lamina surrounding the sarcolemma in the affected child ( 800).

Subsequent studies confirm that mutations of the laminin a2 (LAMA) gene on chromosome 6q2 is responsible for MDC1A (Hillaire et al., 1994; Helbling-Leclerc et al., 1995). Absence of laminin a2 leads to massive muscle fiber necrosis and fibrosis. Antibodies show reduced or absent expression of laminin a2 and disruption of the basal lamina (Figs. 22.5 and 22.6) surrounding the muscle fibers (Minetti et al., 1996). Secondary reduction of a-dystroglycan, laminin b2 and integrin a7 has also been reported (Cohn et al., 1997, 1999). Children with laminin a2 deficiency present at birth or soon after with hypotonia and proximal greater

Fig. 22.6. A 10-year-old boy with merosin-deficient congenital muscular dystrophy. Transverse frozen section of his sural nerve showing absent immunoreactivity for merosin and loss of myelinated axons ( 250).

than distal weakness, contractures, feeding difficulties and/or respiratory distress (Philpot et al., 1995). Laminins are also expressed in the basal lamina of cerebral blood vessels, in the glia limitans of brain surface, in Schwann cells and along developing white matter tracts of the central and peripheral nervous system (Shorer et al., 1995). Thus, brain MRI studies invariably show diffusely increased white matter signal intensity on T2-weighted MRI after 6 months of age (Fig. 22.7). Additional structural brain changes such as occipital polymicrogyria/agyria and hypoplasia of the pons and/or cerebellum have been reported and are associated with epilepsy and mental retardation (Sunada et al., 1995; Herrmann et al., 1996; Philpot et al., 1999). Muscle biopsies reveal a dystrophic process, with absent or reduced expression of the laminin a2 protein by immunostaining (Sewry et al., 1997). Nerve conduction studies show a motor demyelinating neuropathy and somatosensory revoked responses may also be abnormal (Mercuri et al., 1995). Most affected individuals have severe motor delay and fail to progress beyond the ability to sit independently (Muntoni and Voit, 2004). Increased joint contractures and scoliosis occurs over time. Frequent complications include respiratory insufficiency, failure to thrive and aspiration pneumonia. For severely affected individuals, conservative management such as noninvasive positive pressure ventilation and gastrostomy is usually recommended. Occasionally some patients with partial laminin a2 deficiency may experience a milder phenotype, with the ability to ambulate independently.

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Fig. 22.7. A 2-year-old girl with merosin-deficient congenital muscular dystrophy. (A) Sagittal T1-weighted brain MRI at 21 months of age showed no cerebellar or cerebral malformations. (B) On axial T2-weighted MRI, symmetrical hyperintense white matter changes (black arrows) were present, consistent with dysmyelination.

22.3.4.2. Congenital muscular dystrophy due to -dystroglycanopathies Dystroglycans are glycoproteins that are essential for the formation of the basement membrane during early development and are expressed in many epithelial, muscle and neural cell types (Henry and Campbell, 2001). The polypeptides encoded by the dystroglycan gene (DAG1) undergo post-translational cleavage and glycosylation to produce a- and b-dystroglycan (Michele and Campbell, 2003). These two glycoproteins connect the extracellular matrix with the cytoskeleton of the muscle fibers (Henry and Campbell, 2001; Michele and Campbell, 2003). In particular, the addition of carbohydrate moieties via O-mannosylation enables a-dystroglycan to bind to laminin-a2 and agrin (Chiba et al., 1997). Complete disruption of dystroglycan is lethal in mice and partial disruption leads to a loss of the dystrophin–glycoprotein complex and muscular dystrophy (Cohn et al., 2002). Similar disruption in the brain results in overmigration of the neurons into the subarachnoid space and loss of cortical layering, as seen in type II lissencephaly (Michele et al., 2002). Mutations of a-dystroglycan produce CMD variants such as Fukuyama type CMD, Walker–Warburg syndrome, muscle–eye–brain disease, congenital muscular dystrophy type 1C (MDC1C) and congenital muscular dystrophy type 1D (MDC1D). These types of CMD are collectively referred to as a-dystroglycanopathies. The extent of reduction of a-dystroglycan broadly correlates with disease severity. 22.3.4.2.1. Fukuyama type congenital muscular dystrophy Fukuyama type congenital muscular dystrophy (FCMD) was first described in 1960 (Fukuyama et al.,

1960) and is caused by mutations of the fukutin gene on chromosome 9q31. It is the second most common form of muscular dystrophy in Japan, after Duchenne muscular dystrophy. The majority of FCMD is due to a retrotransposal insertion into the 30 UTR of fukutin mRNA (Kobayashi et al., 1998). Muscle biopsies demonstrate a loss of a-dystroglycan and disruption of the basal lamina (Hayashi et al., 2001). Similar to other a-dystroglycanopathies, brain abnormalities include type II lissencephaly (Figs. 22.8 and 22.9), pontocerebellar hypoplasia, cerebellar cystic lesions and/or delayed myelination (Barkovich, 1998). Children with FCMD present in the neonatal period with generalized muscle weakness, hypotonia, seizures and encephalopathy. About 50% of patients with FCMD have eye abnormalities and mental retardation is common (Muntoni and Voit, 2004). Affected individuals usually improve over time and some may eventually be able to walk with support. Progressive weakness, contractures, scoliosis, dilated cardiomyopathy and respiratory failure in mid to late teen years are common complications. Previous life expectancy was about 15 years but prolonged survival is now possible through supportive care (Osawa et al., 1997). 22.3.4.2.2. Muscle–eye–brain disease Muscle–eye–brain (MEB) disease was first described by Santavuori et al. (1977), and it is due to mutations of the O-linked mannose b-1, 2-N-acetylglucosaminyltransferase (POMGnT1) gene on chromosome 1p3 (Yoshida et al., 2001). Muscle biopsies from affected individuals show dystrophic changes, severe loss of a-dystroglycan (Kano et al., 2002), and reduced POMGnT1 enzymatic activity (Manya et al., 2003).


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Fig. 22.8. A 2-year-old girl with Fukuyama-type congenital muscular dystrophy. Her brain MRI showed lissencephaly, open operculum (white arrows) and bilateral colpocephaly (black arrows). (A) Axial T1-weighted MRI. (B) Sagittal T1-weighted MRI.

This enzymatic assay has been proposed as a screening test for MEB disease (Zhang et al., 2003). Patients with MEB disease have severe hypotonia, profound weakness, seizures and poor visual attention at birth. The severity of CNS involvement depends on the types of POMGnT1 mutation, as more severe phenotypes are associated with mutations towards the

50 end of the gene (Taniguchi et al., 2003). Severely affected infants have the pachygyria/polymicrogyria/ agyria complex, with or without partial absence of the corpus callosum, hypoplasia of the corticospinal tracts and obstructive hydrocephalus (Haltia et al., 1997). Other CNS malformations include vermal hypoplasia, cerebellar cysts and dysmyelination

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Fig. 22.9. A 32-month-old girl with Fukuyama-type congenital muscular dystrophy. MRI showed widened gyri, band heterotopia (black arrows) and symmetrical white matter changes (white arrows). (A) Axial FLAIR MRI. (B) Axial T2-weighted MRI.

(Fig. 22.10). Severely affected patients are bedridden and usually die during early childhood. Less affected individuals may be able to sit unsupported or speak a few words. Progressive contractures, spasticity and ataxia may pose additional functional impairment over time. The majority of patients can survive to adulthood with supportive care (Muntoni and Voit, 2004). 22.3.4.2.3. Walker–Warburg syndrome The Walker–Warburg syndrome (WWS) was first described by Walker (1942), and then further delineated by Warburg (1978). Mutations in the O-mannosyltransferase 1 (POMT1) gene on chromosome 9q34 have been identified in some patients with WWS, with significant reduction in a-dystroglycan immunolabeling (BeltrańValero de Bernabe et al., 2002). The fukutin and fukutin-related protein (FKRP) genes have also been implicated but the majority of WWS remains unidentified (Jimenez-Mallebrera et al., 2003). WWS represents the most severe variant of the a-dystroglycanopathies. Infants with WWS may be identified prenatally due to the detection of encephaloceles and/or severe hydrocephalus (Figs. 22.11 and 22.12). Other characteristic brain anomalies include type II lissencephaly/agyria, pontocerebellar hypoplasia, diffuse white matter changes, absent corpus callosum, posterior fossa arachnoid cysts and eye malformations leading to blindness (Dobyns et al., 1989). In addition to these CNS malformations, the presence of muscular atrophy, joint contractures and underdevelopment of the pyramidal tracts all contribute to severe immobility and

feeding difficulties. The prognosis of WWS is poor, with death usually before 3 years of age (Muntoni and Voit, 2004). 22.3.4.2.4. Congenital muscular dystrophy type 1C Mutations of the fukutin-related protein (FKRP) gene on chromosome 19q13 give rise to several phenotypes of muscular dystrophies, including congenital muscular dystrophy type 1C (MCD1C) and limb girdle muscular dystrophy type 2I (LGMD2I). a-dystroglycan is abnormal in all patients with the FKRP mutations and secondary deficiency of laminin a2 is common (Mercuri et al., 2003). The FKRP gene encodes a protein that is homologous to members of the phosphosugar transferase family (Brockington et al., 2001). The mildest end of the FKRP mutations presents as LGMD2I, with an incidence of approximately 1:400 in the UK (Poppe et al., 2003). Most patients with LGMD2I maintain the ability to walk for life; however, they may develop dilated cardiomyopathy, respiratory failure and anesthetic complications (Frosk et al., 2002). The milder form of MCD1C is phenotypically similar to MDC1A, except that brain imaging studies and intelligence are normal (Mercuri et al., 2000). The more severe end of MDC1C is associated with structural brain abnormalities such as agyria, pontocerebellar hypoplasia and microphthalmia. It resembles MEB or WWS in phenotype, with severe mental retardation and reduced life expectancy (Driss et al., 2003; Topaloglu et al., 2003; Beltrań-Valero de Bernabe et al., 2004).


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C Fig. 22.10. Infant boy with muscle–brain–eye disease. (A) Axial T1-weighted and (B) T2-weighted MRI showed pachygyria, occipital polymicrogyria (white arrows), ventricular dilatation, and periventricular white matter hyperintensity. (C) Sagittal T1-weighted MRI showing dysgenesis of the corpus callosum (black arrows) and pontocerebellar hypoplasia. (Courtesy of Dr Xingchang Wei, Calgary, Alberta.)

22.3.4.2.5. Congenital muscular dystrophy type 1D Mutation of the LARGE gene in the myodystrophic mouse produces a severe form of muscular dystrophy with retinopathy, neuroblast migratory defect, cardiomyopathy, and peripheral neuropathy. The LARGE gene presumably encodes an unspecified glycosyltransferase (Muntoni and Voit, 2004). Recently an adolescent girl with CMD, mental retardation, pachygyria, hypoplastic brainstem, and white matter abnormalities was discovered to have a missense

mutation in the LARGE gene on chromosome 22q (Longman et al., 2003). Her muscle biopsy showed reduced immunoreactivity of a-dystroglycan. This single case is known as congenital muscular dystrophy type 1D (MCD1D). 22.3.4.3. Other forms of CMD associated with cerebral malformation There are several types of CMD with unspecified genetic or biochemical defects and some of them have

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C Fig. 22.11. Term newborn with Walker–Warburg syndrome. (A) Sagittal T1-weighted MRI showed severe hydrocephalus with cerebellar hypoplasia, callosal agenesis and kinking of the brainstem (black arrows). (B, C) Axial T2-weighted MRI showing type II lissencephaly (white arrows) with markedly reduced white matter. (Courtesy of Dr Xingchang Wei, Calgary, Alberta.)

associated mental retardation and CNS malformations such as cerebellar hypoplasia and giant cisterna magna (Muntoni and Voit, 2004). A type of CMD with abnormal glycosylation of a-dystroglycan and secondary

merosin deficiency (MCD1B) was isolated to chromosome 1q4, but the gene product has not yet been discovered (Brockington et al., 2000). The nature of these forms of CMD awaits further elucidation.


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Fig. 22.12. A 6-month-old infant with Walker–Warburg syndrome. Coronal sections of the left hemisphere revealed type II lissencephaly-pachygyria (black arrows), thin cortical mantle and severe hydrocephalus (white arrows).

22.4. Other myopathies with central nervous system malformations 22.4.1. Metabolic disorders with secondary muscle fiber-type disproportion Myopathy with congenital muscle fiber-type disproportion (CMFTD) was first described by Brooke (1973) as a distinct entity, and it was recently reviewed by Clarke and North (2003). The term CMFTD refers to a type of congenital myopathy characterized by type I fiber predominance and hypotrophy, in the absence of other primary neuromuscular or upper motor neuron disorders (Fig. 22.13). In cases of pure CMFTD myopathy, presenting features include generalized weakness and hypotonia at birth, or nonprogressive proximal muscle weakness noted during infancy or middle childhood (Eisler and Wilson, 1978; Sobrido et al., 2005). Rare cases of CMFTD may require ventilatory support and gavage feeding due to severe facial, bulbar, and respiratory weakness at birth, with high mortality rate (Torres and Moxley, 1992). Cardiomyopathy is uncommon (Banwell et al., 1999) but other complications such as contractures and scoliosis may occur. The pathogenesis of CMFTD is presently unknown and either sporadic, autosomal recessive or autosomal dominant patterns of transmission have been described. A number of other neuromuscular, skeletal, genetic or metabolic disorders may produce a similar histological pattern as CMFTD, and these disorders are considered as secondary causes of muscle fiber type disproportion (Clarke and North, 2003). Examples of metabolic

disorders that may produce a combination of fiber type disproportion and CNS malformations include mitochondrial cytopathies, Zellweger’s disease, Pompe’s disease, and carbohydrate deficiency glycoprotein syndrome. In addition, genetic disorders such as Marden–Walker syndrome, Carey–Fineman–Ziter syndrome (CFZ), and Marinesco–Sjo¨gren syndrome (MSS) can be associated with myopathy and cerebral dysgenesis (Sarnat and Alcala´, 1980; Simon et al., 1991). 22.4.1.1. Mitochondrial cytopathies Mitochondrial diseases are a heterogeneous group of metabolic disorders that can affect every organ system the body (Sarnat and Marıń-Garcıá, 2005). In particular, the brain, heart and striated muscles are most frequently involved. Chronic mitochondrial cytopathies produce neurogenic atrophy in muscles due to axonal alterations of the peripheral nerves. Myopathic features may include the presence of ragged-red fibers, increased lipids and absence of oxidative enzymes such as nicotinamide adenine dinucleotide-tetrazolium reductase (complex I), succinate dehydrogenase (complex II) and cytochrome C oxidase (complex IV) in the myofibers. Cerebellar hypoplasia and secondary muscle fiber-type disproportion have been reported in patients with mitochondrial dysfunction (Naumann et al., 1995; Lincke et al., 1996). Additional CNS malformations in mitochondrial cytopathies include polymicrogyria, leukodystrophy, focal dysplasia of the brainstem and septo-optic dysplasia (Schuelke et al., 2002; Keng et al., 2003; Betts et al., 2004; Finsterer,

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Fig. 22.13. A 6-month-old infant with hypotonia, nonprogressive muscle weakness and normal creatine kinase. Vastus lateralis muscle biopsy showed congenital muscle fiber type disproportion, with type I (dark) fiber predominance and hypertrophy. Frozen section: myofibrillar ATPase preincubated at pH 4.7. (A) 100. (B) 400.

2004; Sarnat and Marıń-Garcıá, 2005). However, the correlations between clinical phenotypes, pathological findings and mitochondrial mutations are often imprecise. The prognosis is highly variable. Treatment of mitochondrial diseases may include a trial of l-carnitine, creatine monohydrate, antioxidants (vitamins C and E) and electron transport agents such as coenzyme Q10, quinones, niacin, thiamine and other vitamin B complex compounds (Longo, 2003). 22.4.1.2. Cerebro-hepato-renal (Zellweger) syndrome Inborn errors of metabolism can affect the nervous system during early embryological development, during proliferation and differentiation stages near birth or later in life by altering the maturation of myelin (Steinlin et al., 1998). Metabolic diseases such as Zellweger syndrome (ZS), Pompe disease or congenital disorders of glycosylation (CDG) can have associated CNS malformations and neuromuscular involvement. Zellweger syndrome is a group of disorders due to defects in peroxisome biogenesis, with elevated serum very-long-chain fatty acids and absence of peroxisomes in hepatocytes and renal tubule cells (Lazarow and Moser, 1995). Features of ZS include facial dysmorphism, hypotonia, muscle weakness, seizures, encephalopathy and hepatorenal dysfunction shortly after birth. Examples of cerebral dysgenesis in ZS include pachygyria, lissencephaly, heterotopia, white matter dysmyelination, colpocephaly, partial agenesis of the corpus callosum and dysplasia of the inferior olive (Nakai et al., 1995; Barkovich and Peck, 1997). Neuromuscular manifestations including muscle fiber-type disproportion and peripheral neuropathy have been reported

(Iannaccone et al., 1987; Baumgartner et al., 1998). Defective peroxisomal oxidation due to mutations of the PEX genes and/or reduced expression of doublecortin in ZS may lead to abnormal neuroblast migration, proliferation and differentiation (Qin et al., 2000; Faust, 2003; Steinberg et al., 2004). The prognosis is poor as the majority of infants with ZS die within the first year of life. 22.4.1.3. Pompe’s disease Pompe’s disease (glycogenosis type II) is a lysosomal disorder due to deficiency of a-1,4-glucosidase, leading to the accumulation of glycogen in all organs, particularly in the heart, liver, skeletal muscles and CNS (Hirschhorn, 1995). Complete deficiency of a-1,4glucosidase results in progressive hypotonia, muscle weakness, hepatomegaly and death during infancy as a result of cardiomyopathy and/or respiratory insufficiency (van den Hout et al., 2003). In addition to muscle fiber type disproportion, vacuolar myopathy and glycogen accumulation in the ventral horn cells (Martin et al., 1973, 1976), cerebral dysgenesis such as hydrocephalus, open operculum and focal pachygyria have been reported (Lee et al., 1996; Sahin and du Plessis, 1999). Recombinant a-glucosidase is currently under evaluation as a potential replacement therapy for patients with infantile Pompe’s disease (Klinge et al., 2005). 22.4.1.4. Congenital disorders of glycosylation Congenital disorders of glycosylation (CDGs) were first described by Jaeken et al. (1980), and reviewed in 2002 (Grunewald et al., 2002). The term refers to a large family of inherited metabolic disorders due to abnormalities in the biosynthesis of glycans within

NEUROMUSCULAR DISORDERS ASSOCIATED WITH CEREBRAL MALFORMATIONS the cytosol, the endoplasmic reticulum or the Golgi apparatus. Defects in the assembly of the lipid-linked oligosaccharides or the transfer of the oligosaccharides to the proteins are designated as type I CDG, while type II CDG refers to defects in the processing of the protein-bound glycans. Most CDGs involve multiple systems and neurological manifestations may include hypotonia, developmental delay, psychomotor retardation, seizures, ataxia and stroke-like episodes (Patterson, 1999). CDG with abnormal O-glycosylation was discussed above under Congenital muscular dystrophies. Disorders of N-glycosylation such as CDG type Ia (CDG-Ia) and type IId (CDG-IId) have combined CNS malformations and neuromuscular defects, including cerebellar hypoplasia and peripheral neuropathy, or Dandy–Walker malformation and myopathy. CDG-Ia is the most common type of CDG and is due to a deficiency of phosphomannomutase on 16p13 (Marquardt and Denecke, 2003). Common presenting features of CDG-Ia include hypotonia, strabismus, failure to thrive, inverted nipples, abnormal subcutaneous fat pads and elevated liver enzymes. Cerebellar hypoplasia is usually present from birth but may be difficult to diagnose clinically in infancy because of associated hypotonia and motor developmental delay. Cerebellar signs such as ataxia, intentional tremor and cognitive impairment may become evident as the child matures. Additional neurological features such as retinitis pigmentosa, seizures, peripheral neuropathy and strokelike episodes may also be present in CDG-Ia. CDG-IId is due to a deficiency of b-1,4-galactosyltransferase I on 9p13 (Hansske et al., 2002). Peters reported a young boy with CDG-IId who presented with hypotonia, developmental delay, myopathy with elevated creatine kinase and progressive hydrocephalus due to Dandy–Walker malformation (Peters et al., 2002). The diagnosis of CDG can be considered with abnormal serum transferrin isoelectric focusing tests and then confirmed by determination of the specific enzymatic defect (Marklova and Albahri, 2004). Oral mannose supplementation is helpful for patients with CDG type Ib due to phosphomannose isomerase deficiency (Alton et al., 1997). There is currently no cure for CDG type Ia or IId. 22.4.2. Genetic disorders with myopathy and cerebral dysgenesis 22.4.2.1. Pena–Shokeir syndrome A number of genetic disorders have associated myopathy, cerebellar hypoplasia and/or other CNS malformations. Reduced fetal movements due to prenatal neurological dysfunction may explain the myopathic changes seen in the Pena–Shokeir or fetal akinesia–

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hypokinesia deformation sequence (FADS) syndrome. The FADS syndrome refers to a heterogeneous group of disorders characterized by maternal polyhydramnios, multiple joint contractures, facial anomalies and pulmonary hypoplasia (Pena and Shokeir, 1974). Both upper and lower motor neuron disorders have been associated with FADS (Rodrı´guez and Palacios, 1991; Yamanouchi et al., 1999). Examples of upper motor neuron disorders include intrauterine infection, prenatal brain injury and neuroblast migrational defect. Lower motor neuron disorders such as severe spinal muscular atrophy or myotonic dystrophy may similarly result in FADS. In one autopsy study of Pena– Shokeir syndrome, fetal immobility during the critical stage of joint development (15th gestational week) contributed to developmental immaturity of the skeletal muscles (Torii et al., 2002). Other specific examples of genetic disorders associated with combined central and peripheral neurological features include Marden–Walker syndrome, Carey–Fineman–Ziter syndrome, Marinesco–Sjo¨gren syndrome and congenital fibrosis of the extraocular muscles (CFEOM). 22.4.2.2. Marden–Walker syndrome Marden–Walker syndrome was first described in 1966 (Marden and Walker, 1966) and is characterized by congenital contractures, hypotonia, myopathic face, prominent forehead, blepharophimosis, failure to thrive, global developmental delay and mental retardation in affected infants. Approximately 30 cases have been reported over the last 40 years, and the etiology is still unknown. Muscle biopsy in one case showed fiber-type disproportion and CNS anomalies such as hypoplastic corpus callosum, cerebellar vermal hypoplasia, Dandy– Walker malformation, colpocephaly and brainstem hypoplasia have been reported (Garcia-Alix et al., 1992; Ozkinay et al., 1995; Garavelli et al., 2000). Either sporadic or autosomal recessive mode of inheritance has been implicated in Marden–Walker syndrome. The joint contractures appear to improve over time, while mental retardation remains severe in a few surviving patients (Orrico et al., 2001). 22.4.2.3. Carey–Fineman–Ziter syndrome The Carey–Fineman–Ziter syndrome is a rare autosomal recessive syndrome characterized by the combination of Pierre Robin’s complex, Mo¨bius’ sequence, hypotonia, congenital myopathy, contractures and variable degrees of CNS malformations (Carey et al., 1982). Isolated pontine hypoplasia, generalized cerebral, cerebellar and/or brainstem hypoplasia, dentato-ponto-olivary dysplasia and neuronal heterotopias have been described in association with CFZ (Maheshwari et al., 2004; Verloes

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et al., 2004). Myopathic features include muscle fiber type disproportion, type II fiber predominance (Ryan et al., 1999), scattered necrotic fibers, increased endomysial connective tissue (Dufke et al., 2004) and other nonspecific changes (Schimke et al., 1993). Reduced fetal movement due to the CNS anomalies may explain the presence of cleft palate and congenital myopathy in CFZ. Most affected individuals have normal intelligence. The genetic basis for CFZ remains unknown; potential candidate genes include the Hoxb1 gene on 17q and the calcium channel CACNL1A3 gene on 1q32 (Carey, 2004). 22.4.2.4. Marinesco–Sjo¨gren syndrome Marinesco–Sjo¨gren syndrome is an autosomal recessive disorder characterized by developmental delay, cerebellar ataxia, bilateral cataracts, short stature, mental retardation, hypogonadotrophic hypogonadism and skeletal deformities secondary to associated myopathy and/or neuropathy (Slavotinek et al., 2005). Recent homozygosity mapping in two large consanguineous families identified a locus on chromosome 5q31 but other families with MSS did not share the same mutation (Lagier-Tourenne et al., 2003). CNS malformations in MSS include cerebellar hypoplasia, vermal atrophy, cortical atrophy and periventricular white matter abnormalities, with or without agenesis of the corpus callosum (Georgy et al., 1998; Reinhold et al., 2003). Myopathic changes such as variation in fiber size, muscle necrosis, rimmed vacuoles and dense membranous structures surrounding the myonuclei on electron microscopy have been reported (Sasaki et al., 1996; Suzuki et al., 1997). Acute rhabdomyolysis may develop in patients with MSS following an intercurrent viral infection (Muller-Felber et al., 1998). The cause of the syndrome is presently unknown. 22.4.2.5. Congenital fibrosis of the extraocular muscles Congenital fibrosis of the extraocular muscles is an inherited disorder characterized by ptosis and external ophthalmoplegia (Laughlin, 1956). It has been subdivided into three main types based on the identified genetic loci: CFEOM1 on chromosome 12 (Engle et al., 1994), CFEOM2 on 11q13 (Wang et al., 1998), and CFEOM3 on 16q24 (Doherty et al., 1999). Although CFEOM was originally considered as a myopathic or neurogenic disorder, recent evidence suggests that it may be a familial disorder related to abnormal CNS development. Cases of CFEOM with cerebellar hypoplasia, fusion of the caudate nucleus and widespread cortical dysplasia have been reported (Parmeggiani et al., 1992; Flaherty et al., 2001). The association of CFEOM and widespread cerebral structural abnormalities will require further characterization of the identified genes.

22.4.2.6. Secondary amyoplasia Amyoplasia congenita refers to a primary defect in muscle development associated with replacement of muscles by adipose or fibrous tissues (Hall et al., 1983). It is a common cause of arthrogryposis with multiple joint contractures (Sells et al., 1996). Similar histological findings of muscular atrophy can also be secondary to a number of neuromuscular diseases, including spinal muscular atrophy and muscular dystrophy, or CNS disorders, such as spinal dysraphism (Parmar et al., 2003) and sacral agenesis (Bernstein, 2002). Sacral agenesis is a rare congenital malformation syndrome characterized by partial or complete absence of the sacrum and caudal spine, with variable degrees of genitourinary, anorectal and spinal cord abnormalities (Lynch et al., 2000; Catala, 2002). It is associated with muscle atrophy and arthrogryposis in affected infants and animals (Tihansky and Hafeez, 1984; Jones, 1999). The ventral part of the spinal cord (including the ventral horn cells) is abnormal but the dorsal horns, dorsal roots and ganglia remain well preserved (Sarnat et al., 1976). This difference is related to deficiency of SHH, a gene with a strong ventrodorsal gradient in the vertical axis, resulting in defective or absent notochord in the involved region. The autosomal dominant form of sacral agenesis with presacral mass and anorectal stenosis was first described by Currarino et al. (1981). Mutations in the homeobox gene HLXB9 on chromosome 7q36 have been shown to cause most of the familial and occasionally sporadic cases of Currarino syndrome, as a result of a loss of function in the homeodomain-containing transcription factor (Ross et al., 1998; Hagan et al., 2000). Sacral dysgenesis can also occur as a result of embryopathy in infants of diabetic mothers, or as part of other malformation syndromes (Alles and Sulik, 1993; Rojansky et al., 2002). Early recognition of sacral dysgenesis, surgical corrections of associated anomalies including tethered cord and multidisciplinary approach are important to ensure optimal outcomes in these patients (Pang, 1993; Wilmshurst et al., 1999).

22.5. Conclusion Myogenic development and differentiation is regulated by a number of developmental genes. Abnormal suprasegmental input from the brain and spinal cord can lead to maturational delay, histochemical alterations and/or aberrant morphology in the developing muscles. Similarly, disorders of the ventral horn cells, neuromuscular junction and peripheral nerves can be associated with CNS malformations. The presence of upper and lower motor neuron signs or coexisting

NEUROMUSCULAR DISORDERS ASSOCIATED WITH CEREBRAL MALFORMATIONS features such as encephalopathy, mental retardation, micro- or macrocephaly, seizures, dysmorphism, ataxia and ocular abnormalities in infants with severe muscle weakness and hypotonia may suggest a combined neuromuscular and central nervous system disorder. On the other hand, upper motor neuron signs due to cerebral malformations may initially overshadow the lower motor neuron involvement. Recognition of this unique group of disorders and appropriate investigations (Table 22.2) will facilitate the diagnosis and management of these patients.

Table 22.2 Investigations for children with combined upper and lower motor neuron disorders Neuroimaging Brain magnetic resonance imaging (MRI) and spectroscopy (MRS) Spine MRI Electrophysiological studies Electroencephalogram Nerve conduction study and electromyography Auditory and/or visual evoked potentials Electroretinogram Biochemical investigations Full blood count Blood Electrolytes, glucose, creatinine, and urea Liver function tests, creatine kinase Ammonia, lactate, and pyruvate Very long chain fatty acids and phytanic acid Transferrin isoelectric focusing Lysosomal enzymes Serum amino acids Urine metabolic screen Urine Urine for organic acids Urine for mucopolysaccharides and oligosaccharides Cerebrospinal CSF protein, glucose, lactate and fluid pyruvate Genetic testing Chromosomal studies Molecular genetic Spinal muscular atrophy testing Myotonic dystrophy Specific genetic tests Others Muscle, skin, and/or nerve biopsies Abdominal ultrasound X-ray of long bones Maternal acetylcholine receptor antibodies Ophthalmological examination Endocrine evaluation

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Acknowledgments I thank Dr Harvey Sarnat and Dr Xingchang Wei for supplying some of the figures.

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Chapter 23

Neuroendocrine complications of central nervous system malformations STEFANO CIANFARANI* Tor Vergata University of Rome, Rome, Italy

23.1. The hypothalamic–pituitary unit The hypothalamus is one of the most evolutionarily conserved regions of the mammalian brain, playing a key role in the maintenance of homeostasis (Reichlin, 1967; Cone et al., 2002). Indeed, the hypothalamus is an integration site that orchestrates coordinated endocrine, autonomic and behavioral responses to both exogenous and endogenous stimuli (Swaab, 2004a). Hypothalamic neurons receive sensory inputs from external environment (e.g. light, pain, temperature) and information from the internal environment (e.g. blood pressure, blood osmolality). In addition, hormones (e.g. growth hormone, thyroid hormone, sex steroids, glucocorticoids) exert negative feedback directly on the hypothalamus (Reichlin, 1967; Lechan, 1987; Ganong, 2000). Therefore, the hypothalamus integrates the inputs, providing coordinated responses through outputs to target organs. These include the anterior and posterior pituitary glands, the cerebral cortex, brain stem and spinal cord, and autonomic (parasympathetic and sympathetic) preganglionic neurons. The hypothalamus comprises a large number of distinct nuclei (Swaab, 2004a), each with its own complex pattern of connections and functions (Fig. 23.1). The nuclei, which are intricately interconnected, can be grouped in three longitudinal regions referred to as periventricular, medial and lateral. They can also be grouped along the anterior–posterior dimension, being referred to as the anterior (or preoptic), tuberal and posterior regions. The anterior periventricular group contains the suprachiasmatic nucleus, which receives direct retinal input and drives circadian rhythms. More scattered neurons in the periventricular region (located

along the wall of the third ventricle) manufacture peptides known as releasing or inhibiting factors that control the secretion of a variety of hormones by the anterior pituitary. The axons of these neurons project to the median eminence, a region at the junction of the hypothalamus and pituitary stalk where the peptides are secreted into the portal circulation that supplies the anterior pituitary. Nuclei in the anterior-medial region include the paraventricular and supraoptic nuclei (Swaab, 2004a), which contain the neurosecretory neurons whose axons extend into the posterior pituitary. With appropriate stimulation, these neurons secrete oxytocin or vasopressin (antidiuretic hormone) directly into the bloodstream. Other neurons in the paraventricular nucleus project to the preganglionic neurons of the sympathetic and parasympathetic divisions in the brainstem and spinal cord. It is these cells that are thought to exert hypothalamic control over the visceral motor system and to modulate the activity of the poorly defined nuclei in the brainstem tegmentum that organize specific autonomic reflexes such as respiration and vomiting. The paraventricular nucleus, like other hypothalamic nuclei, receives inputs from the other hypothalamic zones, which are in turn related to the cortex, hippocampus, amygdala and other central structures that, as noted in the text, are all capable of influencing visceral motor function. The medial–tuberal region nuclei (‘tuberal’ refers to the tuber cinereum, the anatomical name given to the middle portion of the inferior surface of the hypothalamus) include the dorsomedial and ventromedial nuclei (Swaab, 2004a), which are involved in feeding, reproductive and parenting behavior, thermoregulation and water balance. These nuclei receive inputs from

*Correspondence to: Stefano Cianfarani MD, ‘Rina Balducci’ Center of Pediatric Endocrinology, Department of Public Health and Cell Biology, Room E-178, ‘Tor Vergata’ University, Via Montpellier 1, 00133, Rome, Italy. E-mail: [email protected], Tel: þ39 06 51002314 (Hosp.)/þ39 06 72596178 (Lab.), Fax: þ39 06 5917415.

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Fig. 23.1. Diagram of the human hypothalamus, illustrating its major nuclei. (Modified from Purves et al., 2001.)

structures of the limbic system, as well as from visceral sensory nuclei in the brainstem (e.g. the nucleus of the solitary tract). Finally, the lateral region of the hypothalamus is really a rostral continuation of the midbrain reticular formation. Thus, the neurons of the lateral region are not grouped into nuclei but are scattered among the fibers of the medial forebrain bundle, which runs through the lateral hypothalamus. These cells control behavioral arousal and shifts of attention, especially as related to reproductive activities (Fig. 23.1). The pituitary is developmentally, anatomically and functionally closely linked to the hypothalamus, the pituitary stalk serving as an anatomical and functional connection to the hypothalamus. The pituitary gland comprises the anterior lobe, the posterior lobe and a vestigial intermediate lobe (Fig. 23.2). The gland is situated within the sella turcica and is overlain by the dural diaphragma sellae, through which the stalk connects to the median eminence of the hypothalamus (Cone et al., 2002; Melmed and

Kleinberg, 2002). The dural roofing protects the gland from compression by cerebrospinal fluid (CSF) pressure. The optic chiasm, located anterior to the pituitary stalk, is directly above the diaphragma sellae. The intimate relationship of the pituitary and chiasm is borne out in optic chiasmal hypoplasia associated with developmental pituitary dysfunction seen in patients with septo-optic dysplasia (SOD). The predominant blood supply to the anterior pituitary gland originates from vessels that form the hypothalamic-portal circulation (Swaab, 2004b). They deliver hypothalamic releasing and inhibiting hormones to the trophic hormone-producing cells of the adenohypophysis, thus allowing the hypothalamic modulation of pituitary secretion (Table 23.1). Indeed, the hypothalamus contains nerve cells that synthesize hypophysiotropic releasing and inhibiting hormones as well as the neurohypophyseal hormones of the posterior pituitary (arginine vasopressin and oxytocin). Five distinct hormone-secreting cell types are present in the anterior pituitary gland. Corticotroph

NEUROENDOCRINE COMPLICATIONS OF CENTRAL NERVOUS SYSTEM MALFORMATIONS 435

Fig. 23.2. Anatomy of the functional connections between the hypothalamus and pituitary gland. AL, anterior lobe; MB, mammillary body; OC, optic chiasm; PL, posterior lobe. (Modified from Nussey and Whitehead, 2001.)

Table 23.1 Principal human hypothalamic peptides directly related to pituitary secretion Vasopressin Oxytocin Thyrotropin-releasing hormone (TRH) Gonadotropin-releasing hormone (GnRH) Corticotropin releasing hormone (CRH) Growth hormone releasing hormone (GHRH) Somatostatin Vasoactive intestinal peptide Prolactin releasing hormone (PrRP) Ghrelin

cells express pro-opiomelanocortin (POMC) peptides, including adrenocorticotropic hormone (ACTH); somatotroph cells express growth hormone (GH); thyrotroph cells express the common glycoprotein a subunit and the specific thyrotropin (thyroid-stimulating hormone, TSH) b subunit; gonadotrophs express the a and b subunits for both follicle-stimulating hormone (FSH) and luteinizing hormone (LH); the lactotroph cells express prolactin (PRL). All the hypothalamic–pituitary regulating hormones are peptides with the exception of dopamine, which is a biogenic amine functioning as an inhibitor of prolactin secretion. The posterior pituitary gland is made of neural tissue and consists only of the distal axons of the

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hypothalamic magnocellular neurons that make up the neurohypophysis. These axon terminals are in close association with a capillary plexus and secrete arginine vasopressin and oxytocin into the hypophyseal veins and into the general circulation (Fig. 23.3). The median eminence lies in the center of the tuber cinereum (Swaab, 2004a). It is composed of an extensive array of blood vessels and nerve endings and is the functional link between the hypothalamus and the anterior pituitary gland (Green and Harris, 1947; Flerko, 1980). The median eminence contains the vascular complex linking the hypothalamus to the anterior pituitary gland. The vessels are fenestrated, thus allowing diffusion of the peptide-releasing factors from hypothalamus to pituitary. This vascular network composes a circulatory system analogous to the portal vein system of the liver, hence the term hypophyseal–portal circulation (Fig. 23.3).

The median eminence comprises three zones: the ependymal layer forming a barrier between the CSF and the vascular complex, the internal zone and the external zone. The internal zone contains the axons coming from the supraoptic and paraventricular magnocellular neurons en route to the posterior pituitary. The external zone contains terminals from peptidergic neurons (e.g. thyrotropin releasing hormone, corticotropin releasing hormone, LH releasing hormone) and neurons containing bioamines (e.g. dopamine and serotonin) (Elde and Hokfelt, 1979).

23.2. Central nervous system control of endocrine function Endocrine cells and neurons are secretory cells characterized by the ability to be stimulated to cause the release of their products.

Fig. 23.3. Hypothalamus-pituitary connections. The median eminence is the functional link between hypothalamus and anterior pituitary. AHA, anterior hypothalamic area; AR, arcuate nucleus; DMN, dorsomedial nucleus; MB, mammillary body; ME, median eminence; MN, medial nucleus; OC, optic chiasm; PHN, posterior hypothalamic nucleus; POA, preoptic area; PVN, paraventricular nucleus; SCN, suprachiasmatic nucleus; SO, supraoptic nucleus; VMN, ventromedial nucleus. (Modified from Nussey and Whitehead, 2001.)

NEUROENDOCRINE COMPLICATIONS OF CENTRAL NERVOUS SYSTEM MALFORMATIONS 437 23.2.1. Neurosecretion Neurons are specialized secretory cells that send their axons throughout the nervous system to release neurotransmitters and neuromodulators into synapses (Cone et al., 2002). A particular subset of neurons comprises the neurosecretory cells, such as neurohypophyseal and hypophyseotropic cells. The neurons of the hypothalamic paraventricular and supraoptic nuclei are neurohypophyseal cells, while neurons that secrete their products (e.g. releasing and inhibiting factors) into the pituitary portal vessels at the median eminence are hypophyseotropic cells (Fig. 23.4). Neurosecretory cells are hence neurons capable of secreting substances in the blood stream to act as hormones. 23.2.2. Autonomic nervous system control of endocrine function The nervous system regulates the function of both endocrine and exocrine glands, not only through neurosecretion (typically the hypothalamic control of pituitary function) but also through the autonomic nervous system via cholinergic and noradrenergic pathways (e.g. central control of pancreas and adrenal glands) (Loewy, 1990). The autonomic nervous system comprises the sympathetic and parasympathetic systems. Both are characterized by a preganglionic neuron that innervates a postganglionic neuron that targets an end organ. Pre- and postganglionic parasympathetic neurons are cholinergic. Preganglionic sympathetic neurons are cho-

linergic, whereas postganglionic sympathetic neurons are noradrenergic (except for those innervating sweat glands, which are cholinergic). Furthermore autonomic neurons coexpress several neuropeptides, such as somatostatin, neuropeptide Y and vasoactive intestinal polypeptide (Cone et al., 2002). The importance of coordinated autonomic control of endocrine organs is illustrated by the innervation of the pancreas (Edwards, 1990). The cholinergic innervation is provided by the vagus nerve, which modulates insulin secretion. Noradrenergic stimulation inhibits insulin secretion. Although glucose can induce insulin secretion even in the absence of neural input, brain stem and hypothalamus contain cells that, like the beta cells, have the ability to sense glucose concentrations in the blood stream. This information is integrated in the hypothalamus and ultimately results in an autonomic mediated modulation of pancreas secretion.

23.3. Development of the anterior pituitary gland The pituitary gland consists of anterior, intermediate and posterior lobes and is a central regulator of growth, metabolism and development (Melmed and Kleinberg, 2002). Its complex functions are mediated via hormone signaling pathways that regulate the finely balanced homeostatic control in vertebrates by coordinating signals from the hypothalamus to peripheral endocrine organs (thyroid, adrenals and gonads). The origins of the anterior and posterior lobes of the pituitary gland are embryologically distinct. Rathke’s

Fig. 23.4. Neurosecretion in the hypothalamic–pituitary unit. (Reproduced from Shier et al., 2002, with permission from McGraw-Hill.)

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pouch, a primitive ectodermal invagination anterior to the roof of the oral cavity, is formed by the 4th to 5th week of gestation and gives rise to the anterior pituitary gland (Lazzaro et al., 1991; Takuma et al., 1998; Etchevers et al., 2001). The pouch is directly connected to the stalk and hypothalamic infundibulum and ultimately becomes distinct from the oral cavity and nasopharynx. Rathke’s pouch proliferates towards the third ventricle, where it fuses with the diverticulum and subsequently obliterates its lumen, which may persist as Rathke’s cleft. The anterior lobe is formed from Rathke’s pouch, whereas the diverticulum gives rise to the adjacent posterior lobe. In mice, anterior pituitary development takes place in four distinct stages: pituitary placode formation; the development of a rudimentary Rathke’s pouch; the

formation of a definitive pouch; and finally the terminal differentiation of the various cell types in a temporally and spatially regulated manner (Fig. 23.5). The apposition of Rathke’s pouch and the diencephalon, which later develops into the hypothalamus, is maintained throughout the early stages of pituitary organogenesis and is critical for normal anterior pituitary development (Takuma et al., 1998). Several signaling molecules and transcription factors that are expressed in the neural ectoderm and not in Rathke’s pouch – FGF8, BMP4, and NKX2.1 (Takuma et al., 1998; Ericson et al., 1998) – are thought to play a significant part in normal anterior pituitary development, as shown by the phenotype of mouse mutants that are either null or hypomorphic for these alleles (Dattani and Preece, 2004). These signaling molecules can then activate or repress

Fig. 23.5. Pituitary development. Midsagittal or parasagittal section drawings of rat embryos showing pituitary development. (A) (1) Growth of preinfundibular portion of neural plate and establishment of presumptive Rathke’s pouch area; (2) formation of a rudimentary pouch – with absence of mesoderm between pouch and floor of diencephalons; (3) formation of definitive pouch and posterior lobe with invasion of neural crest and mesenchymal tissue, and separation of brain and oral cavities; (4) nascent pituitary gland. Corresponding stages in mouse and rat development indicated. (B) Cascade of transcription factors that regulate anterior pituitary development: the approximate timing of mRNA expression in the mouse is shown for transcription factors (green bar) and other marker genes (blue bars). Arrows represent activation and truncated lines represent repression. Black solid arrows indicate effects supported by analysis of pituitary mutants. Broken arrows hypothetical interactions that might be revealed by analysis of expression in other pituitary mutants. AL, anterior lobe; AN, anterior neural pore; E, embryonic day; F, forebrain; H, heart; HB, hindbrain; I, infundibulum; IL, intermediate lobe; MB, midbrain; NP, neural plate; O, oral cavity; OC, optic chiasma; OM, oral membrane; P, pontine flexure; PL, posterior lobe; PO, pons; PP, prechordal plate; RP, Rathke’s pouch; SC, sphenoid cartilage. (Modified from Dattani and Preece, 2004, with permission from Elsevier.)

NEUROENDOCRINE COMPLICATIONS OF CENTRAL NERVOUS SYSTEM MALFORMATIONS 439 key regulatory genes encoding transcription factors, such as HESX1, LHX3 and LHX4, within the developing Rathke’s pouch that are essential for subsequent development of the pituitary (Takuma et al., 1998; Watkins-Chow and Camper, 1998). The terminal differentiation of the progenitor cells into the distinct cell types found within the mature pituitary gland is tightly regulated by extrinsic factors (FGF8, BMP2, BMP4, and BMP7) from the surrounding infundibulum and the juxtapituitary mesenchyme. These factors then establish gradients of transcription factors (LHX3, SIX3, PROP1, PIT1, NKX3.1, ISL1, LHX4, SIX1, BRN4 and P-FRK) which in turn lead to a wave of cell differentiation (Simmons et al., 1990; Sheng et al., 1997; Treier et al., 1998; Sheng and Westphal, 1999; Rosenfeld et al., 2000; Dattani and Preece, 2004).

Disorders involving GH and one or more additional pituitary hormones are caused by mutations in the homeodomain transcription factors that direct embryological development of the anterior pituitary gland (Table 23.2).

23.4. Mutation of transcription factors: a link between brain abnormalities and endocrine diseases 23.4.1. HESX1 The HESX1 gene plays an important role in the development of the optic nerves as well as the anterior pituitary gland. Its name denotes ‘a homeobox gene expressed in embryonic stem cells’ (Thomas et al., 1995). The same gene is also referred to as RPX, denoting ‘Rathke’s

Table 23.2 Genes implicated in isolated growth hormone deficiency and combined pituitary hormone deficiency

Gene (mouse/human) hesx-1/HESX1 (paired-like family)

sox-3/SOX3 (Sox family) lhx-3/LHX3 (LIM family)

Loss of function phenotype in mice Anophthalmia or microphthalmia, agenesis of corpus callosum, absence of septum pellucidum, pituitary dysgenesis or aplasia Unknown Hypoplasia of Rathke’s pouch

lhx-4/LHX4 (LIM family)

Mild hypoplasia of anterior pituitary

prop-1/PROP1 (paired-like family)

Hypoplasia of anterior pituitary with reduced somatotrophs, lactotrophs, thyrotrophs, and gonadotrophs

pit-1/POU1F1 (PIT1) (POU-family)

Anterior pituitary hypoplasia with reduced somatotrophs, lactotrophs, and thyrotrophs

ghrhr/GHRHR

Reduced somatotrophs with anterior pituitary hypoplasia

gh-1/GH1

Human phenotype

Inheritance (mouse/human)

Variable: Septo-optic dysplasia (midline forebrain, eye and pituitary defects), CPHD, IGHD with ectopic posterior pituitary

Dominant or recessive in both. Variable penetrance with dominant inheritance

Isolated GH deficiency with mental retardation GH, TSH, gonadotropin deficiency with pituitary hypoplasia. Corticotrophs spared. Short, rigid cervical spine with restricted rotation GH, TSH, cortisol deficiency, persistent craniopharyngeal canal and abnormal cerebellar tonsils Highly variable GH, TSH, prolactin, and gonadotropin deficiency. Evolving ACTH deficiency. Enlarged pituitary (tumour-like) with later involution Variable GH, TSH and prolactin deficiencies. Anterior pituitary size variable (normal/ hypoplastic) Type 1B GH deficiency with anterior pituitary hypoplasia GH deficiency

X-linked in both Recessive in both

Recessive in mouse, dominant in human Recessive in both

Recessive in mouse, dominant/recessive in human Recessive Recessive (type 1A, 1B), dominant (Type II)

ACTH, adrenocorticotropic hormone; CPHD, combined pituitary hormone deficiency; GH, growth hormone; IGHD, isolated growth hormone deficiency; TSH, thyroid stimulating hormone. Source: from Dattani and Preece, 2004, with permission from Elsevier.

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pouch homeobox’ gene. The mouse gene is located on chromosome 14 and the human gene is located on chromosome 3p21.2 (Sornson et al., 1996). The mouse and human genes contain four exons, span a distance of 1.7kb and encode proteins of 185 amino acids (Fig. 23.6). The proteins contain a putative repressor domain from amino acids 21–27 and a paired-like homeodomain extending from amino acid 110–167 (Parks et al., 1999). Expression of HESX1 begins before that of PROP1 and is more widespread. The mRNA and protein can be found in many organs, including liver and brain. In the developing central nervous system, HESX1 expression begins in a small patch of cells in the anterior midline visceral endoderm at the beginning of gastrulation, spreads to the adjacent ectoderm, then to the rostral neural folds and subsequently the ventral diencephalon. At day 9.5 it is also expressed in a layer of oral ectoderm, which gives rise to Rathke’s pouch. It appears to be expressed in the precursors of all pituitary cell types but expression declines after day 11.5 and is extinguished by day 15, following the appearance of PIT1 (Parks et al., 1999). Embryonic mice lacking rpx have bifurcations in Rathke’s pouch and pituitary dysplasia, reduced prosencephalon, anophthalmia or microphthalmia, defective olfactory development and ventral midline defects in the hypothalamus (Dattani et al., 1998). Neonatal mice have abnormalities in the corpus callosum, anterior and hippocampal commissures and septum pellucidum. In many ways, this phenotype resembles the syndrome of SOD, also known as De Morsier syndrome, in humans. SOD is a heterogeneous condition with any combination of optic nerve hypoplasia, pituitary gland hypoplasia and midline abnormalities of the brain, such as absence of the corpus callosum and septum pellucidum.

Fig. 23.6. HESX1 cDNA, protein domains and mutations. 1, Arg53Cys, loss of DNA binding; 2, Asn125Ser, normal DNA-binding; 3, Ser170Leu, reduced DNA binding. (Modified from Parks et al., 1999.)

Dattani et al. (1999) screened a total of 135 patients with pituitary disorders, including 35 with SOD, for mutations in the HESX1 gene. Two siblings with agenesis of the corpus callosum, optic nerve hypoplasia and panhypopituitarism were found to have a homozygous mutation at codon 53 in the homeodomain of HESX1, resulting in conversion of a highly conserved arginine to a cysteine. The mutant protein failed to bind to DNA-containing sequences that are normally recognized by HESX1 (Fig. 23.6). No mutations were found in the HESX1 gene from patients with SOD. Heterozygosity for a Ser170Leu mutation was demonstrated in two siblings with isolated deficiency of GH. One of the siblings also had optic hypoplasia. The mutant protein had reduced affinity for HESX1-binding elements but did not prevent access of the normal protein. Another patient with isolated GH deficiency and pituitary hypoplasia was heterozygous for an Asn125Ser mutation. This mutation did not alter binding to DNA. Although HESX1 mutations do not provide a general explanation for the syndrome of SOD, the findings of Dattani et al. (1998, 1999) suggest an important potential role for this gene in understanding recessive and dominant forms of hypopituitarism. 23.4.2. Pitx-2 Transcripts of pitx-2 are first detected at e8.5 in the mouse embryo in oral epithelium and oral ectoderm. At e9.5, there is expression of pitx-2 in the nascent Rathke’s pouch, as well as expression in the mesenchyme near the optic eminence, the basal plate of the central nervous system, the base of the forelimbs and domains of the abdominal cavity (Cohen and Radovick, 2002). At e16.5, there is a low level of expression in the intermediate lobe and the rostral tip of the anterior lobe of the pituitary gland in an apparently homogeneous pattern (Muccielli et al., 1996). The pitx-2a and pitx-2b mRNA isoforms are expressed in the adult pituitary gland in the thyrotrophs, gonadotrophs, somatotrophs and lactotrophs but not in the corticotrophs. The pitx2c isoform is expressed in all five of these lineages (Gage et al., 1999). PITX2 appears required for pituitary gland development shortly after formation of the committed pouch (Gage et al., 1999), and PITX2 may be a determinant for one or more anterior pituitary cell types or may function by acting in concert with other transcription factors (Fig. 23.7). In the chick, Xenopus and mouse, pitx-2 expression is on the left side of the embryo in the lateral plate mesoderm and then continues to be expressed asymmetrically in several organs that are asymmetric with respect to the left-right axis of the embryo. Ectopic expression of pitx-2 results in reversed


Fig. 23.7. Model for the multifunctional role of the Pitx-2 C-terminal tail. The Pitx-2 protein is shown as an intramolecular folded species. The folding interferes with DNA binding of Pitx-2. Pit-1 binds to the C-terminal tail of Pitx-2 and disrupts the inhibitory function of the C terminus. This allows for a more efficient homeodomain interaction with the target DNA and transactivation. The Pitx-2 C39 peptide interaction with the N terminus of Pitx-2 displaces the C-terminal tail and increases its binding activity. However, the C39 peptide masks an N-terminal transactivation domain that results in repressed transcriptional transactivation. C, C-terminal end; HD, homeodomain; N, N-terminal end. (Modified from Amendt et al., 1999.)

looping of the heart and intestine and reversed body rotation in chick and Xenopus embryos, suggesting that Pitx-2 may interpret and subsequently execute the left–right developmental program dictated by upstream signaling molecules (Ryan et al., 1998). Pitx-2-knockout mice have been generated (Gage et al., 1999; Lin et al., 1999; Kitamura et al., 1999; Lu et al., 1999). These animals have normal formation of Rathke’s pouch. However, there is a decreased cell content by e10.5. Bmp-2 and a-Gsu are initially expressed, as well as low levels of Gata-2, Prop-1, and Nkx-3.1. There is failure of organ progression with undetectable levels of Pit-1, TSH-b, and Lhx-4, and only a few POMC-positive cells. Thus, pitx-2 may fail to correctly activate target genes that require synergistic activation by pitx-2 and lhx-3 (Lin et al., 1999). RIEG is the human homolog of pitx-2 (Semina et al., 1996). In individuals with Rieger syndrome, an autosomal dominant condition with variable manifestations including anomalies of the anterior chamber of the eye, dental hypoplasia, a protuberant umbilicus, mental retardation and pituitary alterations, several mutations of RIEG have been found. All mutations described to date have been heterozygous. Eight mutations were found to affect the homeobox region: five were missense mutations in the homeodomain (Semina et al., 1996; Priston et al., 2001; Saadi et al., 2001), two were splicing mutations in the intron dividing the homeobox sequence (Amendt et al., 1998), and one was an in-

frame duplication of 21bp causing a 7 amino-acid duplication of threonine 44 to lysine 50 (residues 6–12 of the homeodomain) (Priston et al., 2001). Patients with Rieger syndrome do not have an alteration in organ situs, which may be due to the presence of a wild-type allele (Ryan et al., 1998). There appears to be a differing sensitivity of various organs to Pitx-2 deficiency, and mice that are heterozygous for hypomorphic or null alleles for pitx2 mimic some aspects of Rieger syndrome. GH promoter activity was not evaluated but, because there is GH insufficiency in a subset of affected individuals with Rieger syndrome, RIEG may also have a role in activation of the GH gene (Semina et al., 1996). 23.4.3. Lhx-3/Lhx-4 Lhx-3 is a LIM-type homeodomain protein, where the acronym LIM comes from the original members of the LIM homeobox genes, lin-11, isl-1, and mec-3 (Zhadanov et al., 1995). Lhx-3 is also known as LIM3 (Mbikay et al., 1995) and P-Lim (pituitary LIM) (Bach et al., 1995). The LIM proteins contain two randomly repeated unique cysteine/histidine LIM domains located between the N terminus and the homeodomain (the DNA binding domain) (Zhadanov et al., 1995). The LIM domains do not bind to DNA (Bach et al., 1995) but may be involved in transcriptional regulation (Zhadanov et al., 1995). There is weak expression of one isoform, Lhx-3a, at e8.5 in the mouse embryo, whereas the other isoform,

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Lhx-3b, does not appear until e9.5. At e9.5, Lhx-3 is expressed in Rathke’s pouch and the closing neural tube. During subsequent development, there is Lhx-3 expression in the anterior and intermediate lobes of the pituitary gland, the ventral hindbrain, and the spinal cord. Lhx-3 expression persists in the adult pituitary, suggesting a maintenance function in one or more of the anterior pituitary cell types (Zhadanov et al., 1995; Cohen and Radovick, 2002). Lhx3 is expressed at high levels before the initial detection of a-GSU transcripts and binds to and activates the a-GSU promoter. Lhx-3 and Pit-1 are synergistic in transcriptional activation of the TSH-b and PRL promoters and the pit-1 enhancer (Bach et al., 1995). In lhx3-knockout mice, in which the two LIM domains and some of the homeodomain are deleted, Rathke’s pouch is initially formed but fails to grow. There are also changes in the expression of pituitaryspecific markers. Rpx is detected at e10.5, suggesting that initial specification of Rathke’s pouch occurs and initial expression of Rpx is independent of Lhx-3. However, Rpx expression ceases early at e12.5. a-GSU is undetectable at e12.5 and 15.5. TSH-b and GH are undetectable at e16.5. There are no LH-positive cells at e18.5 but, because there is late activation of LH-b and small numbers of gonadotrophs in wild-type mice, it is unclear whether LH-b is missing or decreased. There are also

no Pit-1 transcripts, so Lhx-3 must be required directly or indirectly for Pit-1 expression (Cohen and Radovick, 2002). In these mice, POMC is detected in the floor of the diencephalon and in a small cohort of cells at the ventral base of the Rathke’s pouch remnant, with placement corresponding to the position of the first presumptive corticotroph cells to differentiate at e13. Specification of the corticotroph cell lineage occurs, confirming that the derivation of the corticotroph lineage must be distinct from that of the other anterior pituitary cell lineages. However, the POMC cells fail to proliferate, suggesting that there is an intrinsic feature common to all pituitary cells, or failure of more than one pituitary cell type to differentiate and produce trophic factors that indirectly affect proliferation of other neighboring cells, or failure to respond to factors produced by adjacent structures or proliferative factors (Sheng et al., 1996; Cohen and Radovick, 2002). Humans have been found to have mutations in the LHX3 gene (Fig. 23.8). These patients have complete deficits of GH, PRL, TSH and gonadotropins and a rigid cervical spine leading to limited head rotation. A tyrosine to cysteine conversion at codon 116 (Y116C) in the LIM2 domain is associated with a hypoplastic anterior pituitary. An intragenic 23 amino-acid deletion, predicting a severely truncated protein lacking the

Fig. 23.8. Localization of described mutations of the LHX3 gene on a schematic representation of the protein and gene. The gene has been mapped to 9q34.3 and consists of at least 6 exons. It is translated in 2 isoforms LHX3a and LHX3b that differ in their amino-terminal sequences (exon 1) and their functional capacity. Two mutations have been described in human: the first one, a homozygous mutation predicted a change from a tyrosine to a cysteine in the LIM2 domain involved in the zinc finger structure of this transcription factor; the second one in another family, a 23bp deletion was responsible for a severely truncated protein lacking the homeodomain. (Modified from Reynaud et al., 2004, with permission from Elsevier.)

NEUROENDOCRINE COMPLICATIONS OF CENTRAL NERVOUS SYSTEM MALFORMATIONS 443 entire homeodomain, is associated with an enlarged pituitary (Netchine et al., 2000). Whereas the intragenic gene deletion mutant protein does not bind DNA, the Y116C mutant does. Both mutant LHX3 proteins have a reduced gene activation capacity (Sloop et al., 2001). A closely related gene, LHX4 (GSH4) is expressed in specific fields of the brain and spinal cord (Li et al., 1994) and, with LHX3, regulates proliferation and differentiation of pituitary lineages (Sheng et al., 1997). As in lhx3-knockout mice, Rathke’s pouch forms in lhx4-knockout mice and therefore Lhx-4 is not required for induction of the pouch. Although both Lhx-3 and Lhx-4 are expressed throughout the invaginating pouch at e9.5, Lhx-4 expression becomes restricted to the future anterior lobe of the pituitary at e12.5, whereas Lhx-3 remains expressed in the whole pouch. Lhx-4 expression diminishes at e15.5, whereas Lhx-3 expression is maintained. Lhx3/lhx4 double-knockout mice display an early arrest of pituitary development that is more severe than either single mutant, suggesting a redundant function by these two factors. Lhx-3 plays a more significant role than lhx-4 during formation of the mature pituitary structures, because at least one functional copy of lhx-3 is required (Sheng et al., 1997). Unlike lhx-3-knockout mice, lhx-4-knockout mice have transcripts for a-GSU, Pit-1, GH, and TSH-b. There are a few LH-positive cells, suggesting that lhx-4 may support, but is not required for, specification of gonadotroph cells. However, all five anterior pituitary cell lineages show reduced numbers (Sheng et al., 1997). Patients with deficiencies of GH, TSH and ACTH (LH, FSH, and PRL were not evaluated), a small sella turcica, a persistent craniopharyngeal canal, a hypoplastic anterior hypophysis, an ectopic posterior hypophysis and a deformation of the cerebellar tonsil into a pointed configuration have a heterozygous intronic point mutation of the splice acceptor site preceding exon 5. This human mutation was transmitted in a dominant manner, affecting only the maternal side. Transmission suggests that the gonadotropin axis is intact. The result is two mutant products from use of two cryptic splice-acceptor sites located within exon 5. Use of the first splice-acceptor site is predicted to lead to an in-frame deletion of four highly conserved amino acids in the third helix of the homeodomain. Use of the second splice-acceptor site should alter the reading frame at position 47 of the homeodomain, leading to a premature termination codon within exon 5 (Machinis et al., 2001). However, the targeted disruption of lhx-4 in mice is asymptomatic in the heterozygous state, so the relationship of this mutation with pituitary hormone deficiencies is not confirmed.

23.5. Endocrinopathies associated with midline cerebral and cranial malformations Congenital midline malformation of the cerebrum is often associated with defective ontogeny of the pituitary gland and the subsequent clinical picture of hypopituitarism (Adamsbaum and Chaussain, 1996; Cameron et al., 1999). Congenital midline brain defects include a spectrum of anomalies (Swaab, 2004b). These vary from conditions that are incompatible with life through disfiguring palatofacial clefts associated with disordered neuroanatomy to incidental radiological findings in otherwise healthy children (Cameron et al., 1999). Holoprosencephaly is a condition in which the early forebrain fails to diverticulate and develops instead into a single, unpaired forebrain termed the holoprosencephalon (DeMyer and Zeman, 1963). It can be lobar (almost complete lobar and interhemispheric fissure formation), semilobar (incomplete, partial formation of the interhemispheric fissure), variant (heterotopic gray matter) or alobar (with a large holoventricle) (Traggiai and Stanhope, 2002). Optic nerve hypoplasia (ONH), unilateral or bilateral, is a congenital anomaly and is characterized by the absence of ganglion cells at about 6 weeks gestation, also by different frequency, size and morphology of the optic nerve, optic disc and retinal vessels (with the typical ‘double ring sign’) (Frisen and Holmegaard, 1978; O’Dwyer et al., 1980). When there is an association between ONH and cerebral hemispheric abnormality, two distinct pathogenic mechanisms (involving neuronal migration and axonal degeneration) coexist (Barkovich et al., 1992). De Morsier (1956) described the association between ONH and the absence of septum pellucidum (ASP), calling it septo-optic dysplasia. SOD is defined as a combination of two of these features: ONH, ASP and hypopituitarism. The condition is considered to be sporadic but, in one family, an abnormality in the pituitary development gene HESX1 has been described (Dattani et al., 1998). Some patients with De Morsier syndrome appear to retain the ability to secrete gonadotrophins in the face of loss of other hypothalamic releasing factors (Nanduri and Stanhope, 1999). Children with SOD may lose height potential because of the combination of GH deficiency producing a reduced growth velocity and precocious/premature puberty permitting an increased rate of epiphysial closure. Such children therefore need careful monitoring not only of their growth rate but also of their pubertal development and bone maturation; where it is appropriate, treatment with a gonadotropin releasing hormone (GnRH) analog may be necessary in addition to GH treatment.

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This is an analogous situation to children with acute lymphoblastic leukemia who develop GH deficiency and precocious/premature puberty secondary to lowdose cranial irradiation (Leiper et al., 1987). GnRH neurons differentiate in the nasal mucosa in the roof of the mouth and migrate to the hypothalamus in early fetal life (Schwanzel-Fukuda and Pfaff, 1989). It has been demonstrated that this migration is completed at approximately 13 weeks in the human fetus (Bugnon et al., 1977). The central nervous system midline developmental insult that results in SOD develops between the 5th and 8th week of pregnancy (Futz, 1994). The migration of GnRH neurons after the development of the midline defect may explain the dissociation between gonadotrophin function and the rest of hypothalamic–pituitary function (Nanduri and Stanhope, 1999). In children with single malformations, GH is the most frequent pituitary hormone affected. Multiple pituitary hormone deficiency is much more common in patients with ONH combined with ASP and this explains why neonatal hypoglycemia as well as genital abnormality from hypogonadotrophic hypogonadism are more common in this group (Traggiai and Stanhope, 2002) (Fig. 23.9). Patients with holoprosencephaly have a greater percentage of combined anterior and posterior hormone defects. Midline cleft lip and palate are associated with both GH deficiency and diabetes insipidus. Holoprosencephaly and ONH with ASP show a similar incidence of GH deficiency and diabetes insipidus. ONH with ASP has the highest incidence of multiple pituitary endocrinopathies and neonatal hypoglycemia. Unilateral, although more commonly bilateral, ONH can be associated with GH deficiency. The extent of the cerebral malformation does not always correlate with the severity of the endocrinopathy. Therefore, patients with midline cranial and intracranial malformations should be carefully assessed and followed from early childhood because of the risk of developing hormonal dysfunction. Hormone dysfunction may evolve with time. Patients with cleft lip and palate or unilateral ONH should be assessed and followed in the same manner, although the risk of an associated endocrinopathy is lower. In a neuroradiological survey of 35 children with isolated GH deficiency and multiple pituitary hormone deficiencies, a high frequency of midline CNS malformations was found (Hamilton et al., 1998), 37% showing medial deviation of the carotid arteries, 20% Chiari type I malformations, and 9% ONH (Hamilton et al., 1998). Finally, brain imaging shows that posterior pituitary ectopia is a sensitive and specific neuroradiological

Fig. 23.9. Percentage of patients with midline cerebral malformations having diabetes insipidus, MPHD, GHD, neonatal hypoglycemia (NH) and genital anomalies. ACC, absence of corpus callosum; ASP, absence of septum pellucidum; BNH, Bilateral optic nerve hypoplasia, HOL, Holoprosencephaly, ONH, Optic nerve hypoplasia, UNH, unilateral optic nerve hypoplasia. DI, diabetes insipidus, GHD, growth hormone deficiency; HH, hypogonadotrophic hypogonadism; MPHD, multiple pituitary hormone deficiency (two or more deficiencies out of GH, TSH, ACTH, HH). (Modified from Traggiai, and Stanhope, 2002, with permission from Elsevier.)

marker for anterior pituitary hormone deficiency (Traggiai and Stanhope, 2002).

23.6. Auxological and endocrinological abnormalities in children with hydrocephalus and spina bifida Many children with meningomyelocele (MMC) and hydrocephalus fail to attain normal stature and this has originally been attributed to orthopedic abnormalities only (Hayes-Allen, 1972; Greene et al., 1985). Because of interdisciplinary long-term follow-up programs, children with MMC have improved life expectancy and quality of life. Therefore, more attention has been placed on disturbances of growth and pubertal development of MMC patients (Greene et al., 1985; Duval-Beaupere et al., 1987; Roberts et al., 1991; Atenico et al., 1992; Rotenstein et al., 1995; Trollman et al., 1996; Hochhaus et al., 1997). Several reasons for disturbed growth and development have been found (Trollman et al., 2000). First, spinal cord lesion, vertebral anomalies and various skeletal deformities reduce the growth of the lower limbs and the spine. Second,

NEUROENDOCRINE COMPLICATIONS OF CENTRAL NERVOUS SYSTEM MALFORMATIONS 445 due to complex central nervous system anomalies, including hydrocephalus, Chiari malformation, midline defects, neural migration abnormalities and morphological anomalies of the pituitary gland, patients are at risk of hypothalamopituitary dysfunctions (Swaab, 2004b) such as central precocious puberty (Elias, 1994; Trollman et al., 1996) and GH deficiency (Rotenstein et al., 1989; Leveratto et al., 1993; Rotenstein and Reigel, 1996; Satin-Smith et al., 1996; Trollman et al., 1998). Various spinal and orthopedic as well as infectious and nutritional factors contribute to short stature in MMC patients. Adults reach final heights between 142 cm (women) and 152 cm (men) (Atenico et al., 1992; Rotenstein et al., 1995; Trollman et al., 1996). Additionally, hypothalamus–pituitary dysfunction resulting in central precocious puberty and GH deficiency limit growth development and final height in MMC patients (Rotenstein et al., 1989; Leveratto et al., 1993; SatinSmith et al., 1996; Hochhaus et al., 1997; Trollman et al., 1998). As 50–60% of MMC patients are of short stature, it has recently been proposed to use IGF-I and IGFBP-3 serum levels and arm span as screening methods for GH deficiency in these patients (Trollman et al., 1998) in addition to growth parameters such as supine length, growth velocity and bone age. Short stature and obesity in children with MMC measured as supine length or standing height was reported by Hayes-Allen (1972). Normal arm span in children with MMC was found by Rosenblum and colleagues (1983). Short stature was shown by measuring lower limbs, sitting height and subischial leg length but arm span was normal, suggesting that short stature was not the result of hormonal disorders (Greene et al., 1985). However, more recent evidence demonstrates that short stature of single individuals does not only result from shortened lower limbs and sitting height but can be also found in arm span. An accurate auxological and endocrinological evaluation of children with hydrocephalus and/or MMC shows that more than 40% of patients suffer from growth deficiency measured as arm span below –2.0SD when arm span measurements are taken as a substitute for standing height. Assuming that this reflects short stature in these children, it has been found that more than 20% of these growth deficient patients are GH-insufficient. An endocrine study done by Perrone and colleagues (1994) in children with MMC showed no endocrine abnormalities for thyroid hormones, gonadotropins, adrenocorticotropin and gonadal steroids in relation to pubertal stage. The endocrine disorders might be caused by increased cerebrospinal fluid pressure that damages the hypothalamus or the pituitary gland. Endocrine abnormalities and

precocious puberty or early sexual maturation in association with small pituitary gland after birth injury in children with Chiari malformation was shown by Fujita and colleagues (1992). Hydrocephalus in patients with MMC is usually caused by Chiari malformation, and the neuroendocrine dysfunction has been claimed to be caused by disproportionate pressure from the enlarged third ventricle on the pituitary gland (Hier and Wihel, 1977) (Fig. 23.10). In addition, the hypothalamus itself seems to be very sensitive to damage by increased pressure, local edema or altered blood flow with impaired oxygen supply. GH deficiency in patients with myelomeningocele leads to the question of whether these disabled patients should be treated with human GH. To date, only a few short-term reports of GH therapy are available in the literature, and long-term data for final height are lacking. GH treatment has been proved to significantly improve the arm span. However, the increase in length SD score seems not to be significant (Trollman et al., 2000). Another common problem in children with hydrocephalus and/or MMC is precocious puberty (DuvalBeaupere and Soulignac, 1975; Meyer and Landon, 1984; Brauner et al., 1987). Gonadotropins and sex hormones are stimulated early in children with hydrocephalus and/or MMC. This most likely reflects damage to the hypothalamus which in animals has been shown to induce sexual precocity (Ma et al., 1994).

23.7. Empty sella Empty sella is characterized by the herniation of the subarachnoid space within the sella turcica, which is often associated with some degree of flattening of the pituitary gland (Bergland et al., 1968; McLachlan et al., 1968). An empty sella may develop as a consequence of a primary congenital weakness of the diaphragm in patients in whom no secondary cause is evident. A secondary empty sella may develop after infarction of a pituitary adenoma or surgical or radiation-induced damage of the sellar diaphragm. In the case of primary empty sella, several etiopathogenetic hypotheses have been proposed, including a congenital incomplete formation of the sellar diaphragm, suprasellar factors such as stable or intermittent increase in intracranial pressure, and volumetric changes in the pituitary (as observed in pregnancy) (Bergland et al., 1968; McLachlan et al., 1968; Jordan et al., 1977). Magnetic resonance imaging (MRI) usually shows demonstrable pituitary tissue compressed against the sellar floor with lateral stalk deviation (Fig. 23.11).

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Fig. 23.10. Chiari malformation type 1 (ACM1).

Fig. 23.11. MRI scan of (A) normal hypothalamic–pituitary unit; (B) empty sella. (Modified from Besser and Thorner 2002.)


Fig. 23.11. Cont’d

Although an empty sella is usually an incidental finding, if more than 90% of pituitary tissue is compressed or atrophied, pituitary failure occurs. An empty or partially empty sella turcica may be found in patients with short stature or delay in sexual maturation, precocious puberty or hypoparathyroidism. Some short children with empty sella show decreased growth hormone secretion as determined by subnormal GH secretory responses to provocative tests or assessment of endogenous 24-hour GH secretion. Multiple pituitary hormone deficiencies have also been described. Precocious puberty is another isolated endocrine disorder frequently associated with empty sella.

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Greene SA, Frank M, Zachmann M, Prader A (1985). Growth and sexual development in children with meningomyelocele. Eur J Pediatr 144: 146–148. Kitamura K, Miura M, Miyagawa-Tomita S, et al. (1999). Mouse Pitx2 deficiency leads to anomalies of the ventral body wall, heart, extra- and periocular mesoderm and right pulmonary isomerism. Development 126: 5749–5758. Hamilton J, Blaser S, Daneman D (1998). MR imaging in idiopathic growth hormone deficiency. Am J Neuroradiol 19: 1609–1615. Hayes-Allen MC (1972). Obesity and short stature in children with meningomyelocele. Dev Med Child Neurol 172–14: 59–64. Hier DB, Wiehl AC (1977). Chronic hydrocephalus associated with short stature and growth hormone deficiency. Ann Neurol 2: 246–248. Hochhaus F, Butenandt O, Schwarz HP, Ring-Mrozik E (1997). Auxological and endocrinological evaluation of children with hydrocephalus and/or meningomyelocele. Eur J Pediatr 156: 598–601. Jordan RM, Kendall JW, Kerber CW (1977). The primary empty sella syndrome: analysis of the clinical characteristic, radiographic features, pituitary function and cerebral fluid adeno-hypophysial concentrations. Am J Med 62: 569–580. Lazzaro D, Price M, De Felice M, Di Lauro R (1991). The transcription factor TTF-1 is expressed at the onset of thyroid and lung morphogenesis and in restricted regions of the foetal brain. Development 113: 1093–1104. Lechan RM (1987). Neuroendocrinology of pituitary hormone regulation. Endocrinol Metab Clin North Am 16: 475–501. Leiper AD, Stanhope R, Kitchin GP, Chessells JM (1987). Precocious and premature puberty associated with treatment of acute lymphoblastic leukaemia. Arch Dis Child 62: 1107–1112. Leveratto L, Picco P, Cama A, et al. (1993). Insulin-like growth factor I in children with neural tube closure defects: A preliminary report. European Journal of Pediatric Surgery 3 (Supplement 1): 19–20. Li H, Witte DP, Banford WW, et al. (1994). Gsh-4 encodes a LIM-type homeodomain, is expressed in the developing nervous system, and is required for early postnatal survival. EMBO J 13: 2876–2885. Lin CR, Kloussi C, O’Connell S, et al. (1999). Pitx2 regulates lung asymmetry, cardiac positioning and pituitary and tooth morphogenesis. Nature 401: 279–282. Loewy AD (1990). Anatomy of the autonomous nervous system: an overview. In: AD Loewy, KM Spyer (Eds.), Central regulation of autonomic functions. New York, NY: Oxford University Press, 3–16. Lu M-F, Pressman C, Dyer R, Johnson RL (1999). Function of Rieger syndrome gene in left-right asymmetry and craniofacial development. Nature 401: 276–278. Ma YJ, Costa ME, Ojeda SR (1994). Developmental expression of the genes encoding transforming growth factor alpha and its receptor in the hypothalamus of female rhesus macaques. Neuroendocrinology 60: 346–359.

NEUROENDOCRINE COMPLICATIONS OF CENTRAL NERVOUS SYSTEM MALFORMATIONS 449 Machinis K, Pantel J, Netchine I, et al. (2001). Syndromic short stature in patients with a germline mutation in the LIM homeobox LHX4. Am J Hum Genet 69: 961–968. McLachlan MSF, Williams ED, Doyle FH (1968). Applied anatomy of the pituitary gland and fossa: a radiological and histopathological study based on 50 necropsies. Br J Radiol 41: 782–788. Mbikay M, Tadros H, Seidah NG, Simpson EM (1995). Linkage mapping of the gene for the LIM-homeoprotein LIM3 (locus Lhx3) to mouse chromosome 2. Mamm Genome 6: 818–819. Melmed S, Kleinberg D (2002). Anterior pituitary. In: PR Larsen, HM Kronenberg, S Melmed, KS Polonsky (Eds.), Williams Textbook of Endocrinology. Philadelphia, PE: Saunders, 177–279. Meyer S, Landon H (1984). Precocious puberty in meningomyelocele, and usefulness of arm-span measurement. J Pediatr Orthop 4: 28–31. Muccielli ML, Martinez S, Pattyn A, et al. (1996). Otlx2, an Otx-related homeobox gene expressed in the pituitary gland and in a restricted pattern in the forebrain. Mol Cell Neurosci 8: 258–271. Nanduri VR, Stanhope R (1999). Why is the retention of gonadotrophin secretion common in children with panhypopituitarism due to septo-optic dysplasia? Eur J Endocrinol 140: 48–50. Netchine I, Sobrier M-L, Krude H, et al. (2000). Mutations in LHX3 result in a new syndrome revealed by combined pituitary hormone deficiency. Nat Genet 25: 182–186. Nussey SS. Whitehead SA Endocrinology. An Integrated Approach and Oxford, UK (2001). O’Dwyer JA, Newton TH, Hoyt WF (1980). Radiologic features of septo-optic dysplasia: de Morsier syndrome. Am J Neuroradiol 1: 443–447. Parks JS, Brown MR, Hurley DR, et al. (1999). Heritable disorders of pituitary development. J Clin Endocrinol Metab 84: 4362–4370. Perrone L, Del Gaizo D, D’Angelo E, et al. (1994). Endocrine studies in children with myelomeningocele. J Pediatr Endocrinol 7: 219–223. Priston M, Kozlowski K, Gill D, et al. (2001). Functional analyses of two newly identified PITX2 mutants reveal a novel molecular mechanism for Axenfeld-Rieger syndrome. Hum Mol Genet 10: 1631–1638. Purves D, George J, Augustine J, David Fitzpatrick, et al. Neuroscience, 2nd Edition. 2001 by Sinauer Associates. Reichlin S (1967). Function of the hypothalamus. Am J Med 43: 477–485. Reynaud R, Saveanu A, Barlier A, et al. (2004). Pituitary hormone deficiencies due to transcription factor gene alterations. Growth Horm IGF Res 14: 442–448. Roberts D, Shepherd RW, Shepherd K (1991). Anthropometry and obesity in myelomeningocele. J Paediatr Child Health 27: 83–90. Rosenfeld MG, Briata P, Dasen J, et al. (2000). Multistep signaling and transcriptional requirements for pituitary organogenesis in vivo. Rec Prog Horm Res 55: 1–13. Rosenblum MF, Finegold DN, Charney EB (1983). Assessment of stature of children with meningomyelocele, and

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Chapter 24

Cerebral dysgeneses associated with chromosomal disorders JOSEPH D. PINTER* UC Davis Medical Center, Sacramento, CA, USA

24.1. Introduction Abnormalities of chromosomes are seen in 1:150 live births and in 10% of newborns with a congenital malformation. Because 15–20% of children with mild to moderate mental retardation are found to have chromosomal disorders (Carey, 2003), they are frequently encountered in pediatric neurology practice. Individuals with trisomy 21 (Down syndrome) make up a third of all chromosomal abnormalities while about 1:300 children have some type of sex chromosome aneuploidy. All the other autosomal disorders of number (aneuploidies) and structure (deletions, translocations, etc.) have a combined frequency of less than 1:1000. However, balanced rearrangements of chromosomal material, such as translocations and inversions, are seen in 1:500 individuals. (Carey, 2003) Most of these individuals are normal but their offspring are at increased risk of inheriting unbalanced chromosomal abnormalities with resulting problems and abnormalities depending on the specific genes included within these pieces of missing or extra genetic material. About half of all first-trimester spontaneous abortions are due to chromosomal abnormalities, most frequently aneuploidy (Korenberg and Mohandas, 2002). The clinical features, including the brain abnormalities, seen in chromosomal disorders are often nonspecific. For example, many of the described disorders are noted to involve mental retardation, hypotonia and microcephaly. Within many syndromes, a broad spectrum of central nervous system abnormalities may be seen but certain patterns of brain malformation are seen with greater frequency. This, of course, is due to the fact that common genes are affected in specific syndromes and it is the disturbance of normal gene expression (either through over- or underexpression)

that ultimately results in the observed malformations. In the case of aneuploidy, where a great number of genes are either over- (such as Down syndrome, trisomy 21) or underexpressed, the complex interplay of these many gene products naturally leads to a very broad spectrum of malformations in the brain and other organs. More uniform findings might be expected in microdeletion syndromes, which have only recently been appreciated as major causes for mental retardation. Recent studies indicate that submicroscopic, subtelomeric deletions may account for as much as 5% of cases of moderate to severe mental retardation (De Vries et al., 2003; Yu et al., 2005). Several excellent reviews of the general features of chromosomal syndromes are available but descriptions of the observed neuropathological and neuroimaging features are quite difficult to find. (Korf, 1999; Korenberg and Mohandas, 2002; Carey, 2003; Menkes and Falk, 2006) The goal of this chapter is to provide an overview of the brain abnormalities reported in chromosomal disorders commonly seen in a pediatric neurology practice as well as in a few rare disorders of special interest. The reader is encouraged to refer to the more general information supplied in the reviews cited above, to key articles referenced in the discussion of each syndrome and to the outstanding summaries and links to multiple databases maintained by the National Center for Biotechnology Information (NCBI) at the Online Mendelian Inheritance in Man (OMIM) website (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi? db¼OMIM). The University of Kansas also maintains a very useful website, ‘Support Groups for Chromosomal Conditions’, which contains links to family, support and information resources for many chromosomal syndromes at http://www.kumc.edu/gec/support/chromoso.html. Other chapters in this book deal with specific brain

*Correspondence to: Joseph D. Pinter MD, Division of Child Neurology and MIND Institute, UC Davis Medical Center, 2825 50th Street, Sacramento, CA 95817, USA. E-mail: [email protected], Tel: þ1-916-703-0258, Fax: þ1-916-703-0242.

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malformations, including holoprosencephaly and lissencephaly, for which the molecular-genetic underpinnings are now well understood. Refer to those chapters for details on those specific genetic brain malformation disorders, as well as for examples of magnetic resonance images (MRI) showing the characteristic findings in each. Human somatic cells normally contain 23 pairs of chromosomes: 22 homologous somatic chromosomes and two sex chromosomes, XX (46, XX) in females and XY (46, XY) in males. Chromosome abnormalities are usually divided into two groups: 1) disorders of number (aneuploidy) and 2) disorders of structure, which include chromosomal rearrangements such as deletions, duplications and translocations. In this chapter, only a few important disorders in each category will be discussed. Note that mosaic conditions, which tend to have less severe phenotypes as a rule, and many important but rare rearrangements are not reviewed here. The interested reader should consult one of several outstanding reviews of basic principles of all aspects of chromosomal disorders, including techniques used in diagnosis (Dobyns, 1999; Korenberg and Mohandas, 2002; Chen and Carey, 2003).

24.2. Disorders of chromosome number (somatic and sex chromosome aneuploidy) Aneuploidy is seen in 3–4% of all clinically recognized pregnancies and in most cases is lethal (Korenberg and Mohandas, 2002). Among somatic aneuploidies, only the three seen frequently in liveborn infants are considered here. 24.2.1. Somatic aneuploidy Trisomy 21 (47, XX, þ21 or 47, XY, þ21), or Down syndrome, is the most common complete trisomy that results in a viable infant. It is also the most common genetic cause of mental retardation (Nadel, 1999) with a recent Centers for Disease Control study estimating the frequency at 1:733 live births (Canfield et al., 2005). In 95% of cases, it is caused by complete trisomy of chromosome 21, most due to maternal non-disjunction at the first meiosis (Antonarakis, 1991). Affected individuals have a constellation of congenital malformations variably affecting multiple organ systems. They are microcephalic and hypotonic, have characteristic facies and small stature. While the physical phenotype is easily recognized in most affected children, mental retardation (with particular impairment of language and memory) is the most consistent feature (Coyle, 1986). Gross examination of the brain reveals: decreased brain weight; brachycephaly; small cerebellum, brain-

stem, frontal and temporal lobes; a simplified appearance of the sulci; and a narrow superior temporal gyrus (Coyle, 1986; Wisniewski, 1990; Becker et al., 1991). The external appearance of selective hypoplasia of the superior temporal gyrus bilaterally is largely an illusion, despite histological abnormalities in lamination and microscopic architecture that are real; the true anomaly is the abnormal depth of the superior temporal sulcus that separates the superior and middle temporal gyri and causes the superior gyrus to become ‘buried’ so that a smaller area is visible at the surface of the uncut brain (Sarnat et al., 2006). Histological evaluation has revealed abnormalities of dendritic spine morphology in the neocortex (Takashima, 1981; Wisniewski, 1990) and adults with Down syndrome uniformly have senile plaques and tangles typical of Alzheimer’s disease, with a majority of adults with Down syndrome eventually developing clinical Alzheimer’s dementia (Lott and Head, 2005). Clinical MRI studies typically reveal the smaller brain size and brachycephaly but in most cases no structural abnormalities are noted. High-resolution volumetric MRI studies have revealed a syndromespecific pattern of regional volume abnormalities in both adults (Kesslak et al., 1994; Raz et al., 1995; Aylward et al., 1997a, 1997b, 1999) and children with Down syndrome (Jernigan et al., 1993; Pinter et al., 2001b). These include reduced volumes of cerebellum, frontal and temporal lobes, including the hippocampus. Hippocampal volume has been noted to be specifically reduced in children with Down syndrome at times when the amygdala is normal in size, when normalized for overall brain volume (Pinter et al., 2001a). This is in contrast to the observed decreased volumes in both hippocampal and amygdala volumes in older adults with Down syndrome, especially in those with clinical Alzheimer’s dementia (Aylward et al., 1999). Normal volumes have been noted in both adults and children in the subcortical gray matter, including thalamus and lenticular nuclei (Jernigan et al., 1993; Raz et al., 1995; Pinter et al., 2001a, 2001b). These findings, in combination with decreased volumes of cortical regions, suggest that brain development through the first two trimesters of prenatal development may be relatively undisturbed, while interference with development of increasing cortical synaptic complexity and connectivity in the third trimester may be important determinants of the observed cognitive deficits (Schmidt-Sidor et al., 1990; Pinter et al., 2001b). Trisomy 18 (47, XX, þ18 or 47, XY, þ18), or Edwards syndrome, is the second most common somatic aneuploidy seen at birth, occurring in about 1:4000 live births (Canfield et al., 2005). Incidence in conceptuses is much higher but only 2.5% survive

CEREBRAL DYSGENESES ASSOCIATED WITH CHROMOSOMAL DISORDERS to birth (Menkes and Falk, 2006) and 90% die by 1 year of age (Baty, 1994). Cardiac, renal and extremity abnormalities are common. Characteristic facies include a long, narrow skull with prominent occiput. Microcephaly and mental retardation are always noted. Beyond microcephaly, no consistent brain malformations are noted and on gross examination most appear normal (Gullotta et al., 1981; Menkes and Falk, 2006). Malformations sometimes noted on neuroimaging include agenesis of the corpus callosum and heterotopia (Inagaki et al., 1987). Choroid plexus cysts are very common, seen in about half of second and third trimester fetal ultrasounds (Ville et al., 2004). Trisomy 13 (47, XX, þ13 or 47, XY, þ13), or Patau syndrome, occurs in about 1:7500 live births (Canfield et al., 2005). Multiple severe congenital defects are frequently noted. Holoprosencephaly, frequently with variably severe accompanying midfacial abnormalities, is noted in about two-thirds of affected infants. Those with holoprosencephaly have less than a 5% chance of living to 1 year of age (Hahn and Pinter, 2002; Tolmie, 2002). For further details on holoprosencephaly, see Chapter 2. 24.2.2. Sex chromosome aneuploidy As noted above, about 1:300 individuals have some type of sex chromosome aneuploidy (Carey, 2003). While not due to aneuploidy, fragile X syndrome is included below in the discussion of sex chromosome abnormalities because it is commonly seen by pediatricians and pediatric neurologists. Klinefelter syndrome (47, XXY karyotype in about 80% of cases) is defined as having two or more X chromosomes in addition to one or more Y chromosomes (Kumar et al., 2005). It is seen in between 1:500 and 1:1000 male live births (Allanson and Graham, 2002). Clinically, affected boys are often tall with long limbs and hypogonadism (Korf, 1999). Mental retardation is not typically seen. However, mild cognitive impairments, including language and learning disabilities, and behavior problems are frequently seen (Ratcliffe et al., 1982). Clinical brain MRI is usually normal. Recent volumetric MRI studies have reported global decrease in brain size with ventricular enlargement as well as specific regional volume abnormalities (Shen et al., 2004). Among areas with reduced volume reported are left temporal lobe gray matter, a finding that is consistent with verbal and language deficits sometimes associated with Klinefelter syndrome. Interestingly, relative preservation of gray matter in this region and increased verbal fluency scores were associated with exposure to exogenous testosterone during develop-

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ment (Patwardhan et al., 2000). Reduced amygdala volumes have been reported; these may be related to the observed increased incidence in psychiatric illness (Patwardhan et al., 2002; Shen et al., 2004). Turner syndrome (45, X), caused by monosomy of the X chromosome, is noted in 1.5% of all conceptuses and has been reported in up to 18% of spontaneous abortions (Moore et al., 2000). Because of a greater than 90% rate of fetal loss, however, it is seen in only about 1:2500 to 1:5000 liveborn females (Allanson and Graham, 2002; Carey, 2003). Turner syndrome is the only viable monosomy. This unique survivability is probably due to the fact that random X chromosome inactivation (‘lyonization’) results in single copy expression of most X chromosome genes even in the normal diploid state. Heart and endocrine abnormalities, including short stature and abnormal sexual development, as well as a characteristic physical appearance, usually lead to diagnosis (Carey, 2003). Most affected girls are not mentally retarded. While no consistent patterns of gross or clinical MRI brain abnormalities are noted, recent volumetric MRI studies have revealed decreased regional gray matter volume in the bilateral parietal lobes, with IQ scores positively correlated with postcentral gyrus tissue volume. These regional volume abnormalities could be related to observed visuospatial and visuomotor deficits (Brown et al., 2004). Girls with Turner syndrome have also been reported to have reduced volume of the genu of the corpus callosum, which was postulated to reflect abnormal connectivity between the inferior parietal regions. Smaller pons and portions of the cerebellar vermis and increased size of the fourth ventricle have been noted (Fryer et al., 2003). A recently published functional MRI study of arithmetic processing in girls with Turner syndrome revealed decreased fronto-parietal activations during arithmetic tasks compared with controls, possibly reflecting abnormalities of functional connectivity relating to the parietal lobe and callosal structural differences (Kesler et al., 2006). Females with an extra X chromosome (47, XXX) constitute the largest number of individuals with sex chromosome aneuploidy. While 47, XXX occurs in about 1:1000 female births, affected girls are typically normal neurologically or have mild motor and language delays and so do not often come to the attention of the pediatric neurologist (Linden et al., 1988). No clinical MRI abnormalities are typically seen. An increased risk of psychiatric illnesses has been reported and one recent volumetric MRI study reported reduced size of the amygdala (Patwardhan et al., 2002). While future studies may provide important evidence for amygdala size being correlated with

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psychiatric morbidity in this syndrome, as of now the relationship of this finding to clinical outcomes is speculative. Fragile X syndrome is the second most common genetic cause of mental retardation after Down syndrome (trisomy 21). Caused by an expanded trinucleotide (triplet) repeat of CGG in the FMR1 gene on the X chromosome, the full mutation (more than 200 repeats, with normal individuals usually having fewer than 40) is estimated to occur in 1:4000 boys and in 1:8000 girls (Crawford et al., 1999). The premutation (between 55 and 200 repeats) prevalence in boys is estimated to be 1:1000 (Crawford et al., 2001). Besides mental retardation, many recognizable physical and behavioral features are noted. Also noted is a very high incidence of seizures and autism, with both occurring in about a fourth of affected individuals (Hagerman, 2002). Gross appearance of the brain is typically normal but microscopic histological abnormalities, mostly of dendritic spine morphology, have been described in autopsy studies (Beckel-Mitchener and Greenough, 2004; Hessl et al., 2004). Heterotopia are frequently cited as being seen in this disorder, but have only been reported in a couple of autopsy cases (Dunn et al., 1963; Desai et al., 1990). While clinical MRI studies are mostly normal, recent volumetric MRI studies have indicated a consistent pattern of abnormal regional brain volumes. These include decreased size of the superior temporal gyrus and cerebellar vermis, the latter with an associated increased volume of fourth and lateral ventricles (Hessl et al., 2004). Increased ventricular volumes have been positively correlated with age and inversely correlated with IQ (Reiss et al., 1995). Increased volumes of hippocampus, caudate and thalamus have been reported (Hessl et al., 2004). About a third of older men who are premutation carriers (with between 55 and 200 repeats, and who often come to attention because they are maternal grandfathers of children with diagnosed fragile X syndrome) develop severe tremor and ataxia. This has been termed the fragileX-associated tremor/ataxia syndrome or FXTAS (Hagerman and Hagerman, 2004). Characteristic white matter abnormalities are noted on MRI and neuropathological findings include intranuclear inclusions in both neurons and astrocytes throughout the central nervous system (Greco et al., 2006).

24.3. Disorders of chromosome structure: selected deletion syndromes As noted above, microdeletions (i.e. those not visible on routine cytogenetic studies (karyotype)) may be

found in up to 5% of cases of moderate to severe mental retardation. Identification of microdeletions requires advanced techniques, including fluorescence in situ hybridization (FISH), subtelomeric probes and comparative genomic hybridization. The reader is referred to one of several outstanding overviews for further background (Dobyns, 1999; Korenberg and Mohandas, 2002; Chen and Carey, 2003). The deletions responsible for the syndromes discussed below are sometimes identifiable by routine cytogenetics but in most cases are discovered using available specific FISH probes. 22q11.2 deletion syndrome is also commonly referred to as velocardiofacial syndrome, previously known variably as DiGeorge or Sprintzen syndrome. It occurs in 1:4000 births (Burn and Goodship, 1996). Common manifestations include conotruncal heart defects, neonatal hypercalcemia, cleft palate, characteristic facies and mild to moderate mental retardation (Simon et al., 2005b). Recent studies have reported visuospatial and numerical cognitive deficits in affected children, which is thought to be due to posterior parietal dysfunction (Simon et al., 2005a). Clinical MRI studies often show no abnormalities, although cavum septum pellucidum is frequently noted (Shashi et al., 2003; Simon, personal communication). Recent volumetric MRI studies have reported several structural abnormalities, including reduced size of posterior fossa structures and superior temporal lobe gray matter, as well as a decrease in left parietal lobe gray matter (Eliez et al., 2000). Enlarged sylvian fissures and caudate head volume have been reported (Bingham et al., 1997; Eliez et al., 2002). A recent study has reported posterior displacement of the corpus callosum in addition to these findings and suggests that the pattern of cognitive deficits and anatomic differences may reflect structurally and functionally disturbed connectivity of the posterior parietal regions (Simon et al., 2005b). Williams syndrome is caused by a deletion on the long arm of chromosome 7 at 7q11.23. Frequency is estimated at 1:20 000 to 1:50 000. Clinical findings include congenital cardiovascular abnormalities, most commonly supravalvular aortic stenosis, infantile hypercalcemia and growth retardation, as well as characteristic ‘elfin’ facies (Spinner and Emanuel, 2002). Affected children have mild to moderate mental retardation but a fascinating cognitive profile with typically relatively spared (and often extraordinarily good) language and very poor visuospatial and numerical cognitive skills. Further, a bubbly and outgoing personality is frequently noted (Mervis et al., 2000). While clinical MRI studies are usually normal, research studies using volumetric MRI have consistently reported reduced

CEREBRAL DYSGENESES ASSOCIATED WITH CHROMOSOMAL DISORDERS volume of parieto-occipital gray matter in both adults and children (Eckert et al. 2005; Boddaert et al., 2006). Cerebral and callosal shape differences have also been noted, with less bending than normal noted in Williams syndrome subjects, possibly because of decreased parieto-occipital mass (Schmitt et al., 2001). Abnormalities of extent and depth of the dorsal parietal and occipital sulci have also been reported (Galaburda et al., 2001; Kippenhan et al., 2005). Taken together, these results support the popular hypothesis that abnormalities in the dorsal parietal cortex might be responsible for much of the observed visuospatial difficulties, while allowing for preserved language, which is subserved primarily by more ventral pathways. Chromosome 15q microdeletions: Deletions of a small portion of the long arm of chromosome 15 are associated with two distinct syndromes. Whether a deletion of 15q11–q13 results in Prader-Willi syndrome or Angelman syndrome is determined by whether the chromosome bearing the deletion is maternally or paternally derived. Differential modification of genes depending on the parent of origin, most notably by methylation, leading to differential expression of genetic material is termed ‘genetic imprinting’. Maternal deletions result in Angelman syndrome, while paternal deletions result in Prader-Willi syndrome. Deletions account for a majority of both syndromes, with some cases of Prader-Willi syndrome due to maternal uniparental disomy (two copies of the maternal chromosome region and no normal paternal copy) and some cases of Angelman syndrome due to a mutation in a single gene within the region of the typical deletion, UBE3A. Only rare cases of Angelman syndrome are caused by paternal uniparental disomy. A small percentage are caused by mutations in the imprinting center and in a small percentage no genetic etiology can be determined (Menkes and Falk, 2006). The deletions are usually submicroscopic, undetectable on routine karyotype, but in the majority of cases are detected by FISH. For further discussion of the molecular basis of imprinting, including the uncommon defects that can be responsible for each of these syndromes, refer to one of several excellent reviews (Nicholls and Knepper, 2001; Korenberg and Mohandas, 2002; Clayton-Smith and Laan, 2003; Menkes and Falk, 2006). Angelman syndrome (deletion of maternal 15q11– q13) occurs in fewer than 1:10 000 births. Clinically, affected children are severely mentally retarded, microcephalic and have jerky (puppet-like) movements and ataxia. Frequently noted unprovoked smiling and laughter led to use of the term ‘happy puppet syndrome’ in the past. Expressive language is usually severely limited or lacking. Seizures of multiple types

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are characteristic, as is the electroencephalogram (EEG), which typically includes large-amplitude slow spike-wave discharges (Williams, 2005; Menkes and Falk, 2006). Pathological study of one case revealed cerebral and cerebellar atrophy, with loss of cerebellar Purkinje and granule cells and decreased dendritic arborization in the visual cortex (Jay et al., 1991). Clinical MRI does not typically reveal any specific findings beyond the microcephaly (Williams, 2005). Prader-Willi syndrome (deletion of paternal 15q11– q13) occurs in about 1:25 000 births (Butler, 1990). Clinical findings include severe hypotonia in infancy, then hyperphagia and obesity after infancy, obsessive-compulsive behavior, mild to moderate mental retardation and hypogonadism (Spinner and Emanuel, 2002; Dimitropoulos et al., 2006). No consistent findings have been noted on clinical MRI. A single report of apparent abnormal gyral folding pattern has not been replicated (Yoshii et al., 2002). Wolf-Hirschhorn syndrome (4p) is caused by partial monosomy of the short arm of chromosome 4 and is seen in about 1:50 000 births (Carey, 2003). Affected individuals have characteristic dysmorphic features including frontal bossing and ‘Greek helmet’ facies, ocular hypertelorism, cleft lip and palate (Spinner and Emanuel, 2002). Microcephaly, hypotonia, seizures and severe mental retardation are uniformly seen. Thus far, no typical MRI features have been reported but prenatal ultrasound studies have shown several cases with intrauterine growth retardation, typical facies, multifocal white matter lesions and periventricular cystic lesions (De Keersmaecker et al., 2002; Boog et al., 2004). A mouse model has been developed by creating radiation-induced deletions in a region of mouse chromosome 5 syntenic with the human Wolf-Hirschhorn critical region, 4p16.3 (Naf et al., 2001). The mice developed seizures and were noted to have cerebellar hypoplasia and shortened cerebral cortex. These findings, along with the human ultrasound data, suggest that a consistent neuroradiological phenotype might be identifiable in the near future. Cri du chat (cat cry) syndrome (5p) is caused by deletion of the short arm of chromosome 5. It is seen in between 1:15 000 and 1:50 000 births (Tolmie, 2002). Low birth weight, hypertelorism and a cat-like cry are noted, as are microcephaly, hypotonia and severe mental retardation. One MRI study has reported very small brainstem, cerebellum, middle cerebellar peduncles and apparently reduced cerebellar white matter in seven affected patients (Tamraz et al., 1993). Abnormalities in cranial base development (Kjaer and Niebuhr, 1999) suggest the possibility that a notochordal developmental defect could underlie both these and the observed posterior fossa abnormalities.

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Miller-Dieker syndrome is caused by a deletion of the distal end of the short arm of chromosome 17 (17p.13.3). It is extremely rare, estimated to occur in only 11.7 per million births (Pilz, 2005) but because of its unique features is likely to be encountered by pediatric neurologists. The deleted region includes LIS1, the gene found to be responsible for classical (type 1) lissencephaly. In addition to lissencephaly, characteristic dysmorphic facies are noted, with high forehead, bitemporal hollowing, upturned nose and midface hypoplasia. Affected individuals are profoundly mentally retarded (Dobyns, et al., 1991; Spinner and Emanuel, 2002). For a full discussion of lissencephaly, including its pathological and neuroradiological appearance, see Chapters 13 and 14.

24.4. Conclusion From this brief review of brain malformations associated with chromosomal abnormalities one can appreciate not only the variety and complexity of the brain malformations that may be seen but also the enormous challenges that lie ahead in understanding how these lead to the observed and often widely varying structural and functional abnormalities. Even in the more common disorders, much of what is known about brain structural abnormalities continues to come from often very old autopsy reports or from individual case reports. Wider use of high-resolution MRI in the clinical evaluation of children with chromosomal disorders will dramatically improve our understanding but, because these disorders are relatively rare, collaborative studies collecting patients and imaging data from many centers will be necessary. Advances in neuroimaging, along with new techniques being developed in the fields of gene expression and proteomics, promise to revolutionize our understanding of the mechanisms responsible for brain malformations. High-resolution MRI can already be done not only in very young infants but also in fetuses, which will allow us to learn not only what is abnormal about the brains of children with chromosomal abnormalities but also when the changes are first noted (Coakley et al., 2004). Soon it will be clinically feasible not only to obtain highresolution structural neuroimaging studies but also to routinely use diffusion tensor imaging sequences to identify white matter organizational and structural abnormalities and to trace white matter tracts (Albayram et al., 2002). Functional MRI, currently used more in research than clinical studies, will enable routine functional assessment of both primary cortical functions (motor and sensory activations during spontaneous movement or with stimulation-tactile, auditory, visual) and higher cortical functions, including

activations related to language or emotion. These new modalities will be useful in categorizing degrees of abnormality and providing further phenotypic data to allow finer dissection of the molecular-genetic and environmental contributions to congenital brain malformations in chromosomal syndromes.

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Chapter 25

Cerebral dysgeneses secondary to metabolic disorders in fetal life WILLIAM D. GRAF* Children’s Mercy Hospitals and Clinics and University of Missouri, Kansas City, Missouri

25.1. Introduction Perturbation of cellular metabolism during critical periods of embryonic or fetal brain development can often explain both cause and effect in cerebral dysgeneses. Prenatal metabolic disturbances may result from the effects of nutritional deficits, environmental toxins and genetic mutation (also called ‘inborn errors’). The mechanisms by which cellular metabolic alterations disrupt sequential stages in cerebral morphogenesis are the study of biochemical genetics, developmental neurobiology, pediatric neuropathology, teratology and clinical child neurology. The large amounts of data produced in these diverse areas of study create ongoing challenges for clinical classification, model building and outcome analysis. This chapter provides an overview of both clinical and experimental observations that link several prenatal-onset metabolic disorders to cerebral dysgeneses.

25.2. General considerations about brain metabolism in fetal life Metabolism can be defined as the sum of the physical and chemical changes affecting the cell, including uptake and distribution of chemical compounds, synthesis (anabolism) of molecules, biotransformation of such substances, breakdown (catabolism) of the compounds and elimination of these components and their metabolites. Cerebral metabolism includes the anabolism and catabolism of neurotransmitters, hormones, neurotrophic molecules and other brain-specific chemicals. Clearly, not all metabolic disorders lead to cerebral dysgeneses, and often metabolic disturbances are

vaguely associated with a wide spectrum of abnormal organogenesis. Most inborn errors of metabolism remain either undetected or clinically insignificant during fetal life because of maternal hepatic and placental compensation. Such ‘silent’ metabolic disorders generally include disorders of the urea cycle, protein synthesis, protein degradation, glycogen metabolism, purine and pyrimidine metabolism, bile acid synthesis, lipid metabolism and lysosomal enzymes. Other circumstances pertinent to prenatal metabolic errors and their effect on brain development are summarized below. 25.2.1. Critical periods of vulnerability (timing) Brain development depends on an orderly sequence of events that are susceptible to disruption at critical time points. Any alteration of this sequence by metabolic processes greatly depends on the timing of the disruption. The developing brain is most vulnerable to environmental agents and major metabolic disturbances during the first two trimesters of pregnancy when critical stages of neuronal proliferation and migration are at their peak. However, neurodevelopmental disruption from altered metabolism also occurs during later processes of neuronal organization and synaptogenesis. One of the best documented models of ‘timing’ is thalidomide embryopathy, which is associated with an autism spectrum disorder by exposure before neural tube closure and limb development defects by exposure during a slightly wider window of vulnerability during later stages of development (Stromland et al., 1994; Trottier et al., 1999). Other time-dependent models of dysgenesis include thorough animal studies

*Correspondence to: William D. Graf, MD, Section of Neurology, Children’s Mercy Hospitals and Clinics, 2401 Gillham Road, Kansas City, MO 64108, USA. E-mail: [email protected], Tel: þ1-816-234-3090.

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of isotretinoin, phenytoin and methamphetamine (Koren et al., 1998). 25.2.2. Substrate or toxin load (dose and duration) In addition to critical time periods in metabolic dysgenesis, the induction of injury depends on dosage and duration of excess substrate, metabolite or product exposure. Acknowledging the clear limitations of clinical medicine, it is often not possible to prospectively or retrospectively determine the exact quantity of potential toxic exposure to an embryo or fetus. Neuronal and glial dose-dependent mechanisms of action include changes of membrane permeability and axonal transport, inactivation of specific enzymes in cell respiration and protein synthesis, and interference with neurotransmitters or their receptors. 25.2.3. Substrate or toxin inactivation (maternal factors) The effects of a neurotoxicant may be reversible or irreversible depending upon additional factors such as maternal hepatic function. Neurotoxicants can be categorized on the basis of their metabolic activation. Metabolic activation of some xenobiotic chemicals is mediated by cytochrome P450 enzymes (e.g. CYP2E1) and other enzymes including glutathione-Stransferase. The cytochrome P450 enzymes are involved in phase 1 of the detoxification process. These enzymes add an oxygen atom to the parent molecule, thereby permitting the formation of an epoxide or ketone. Phase 2 enzymes then form conjugates with glutathione or other molecules for excretion in the urine. The epoxide or ketone also can react with cellular macromolecules, which results in neurotoxic effects. Neurotoxic chemicals that require metabolic activation to induce effects include N-hexane, methyl N-butyl ketone, styrene, trichloroethylene and perchloroethylene. Neurotoxicants that do not require metabolic activation to induce CNS injury include lead, arsenic, ethylene oxide, carbon disulfide and organophosphate insecticides. Less commonly, metabolic disturbances in the fetus can affect maternal health. For example, pregnancies with fetal deficiency of long-chain 3-hydroxyacyl coenzyme A dehydrogenase are associated with the HELLP syndrome (hemolysis, elevated liver enzymes and low platelets) or acute fatty liver during the third trimester of pregnancy (Wilcken et al., 1993). This enzyme resides in the alpha subunit of the mitochondrial trifunctional protein and catalyzes the third step in long-chain fatty acid beta-oxidation. A single gene mutation (E474Q) for trifunctional protein is commonly present in the fetus (Yang et al., 2002).

25.2.4. Toxin type (specificity) Determining the specific types of prenatal neurotoxic exposures and their neurodevelopmental consequences remains an important diagnostic issue for clinicians and individuals as well as a public health policy issue for governments. Even natural elements such as lead, mercury, aluminum, arsenic and cadmium, which have no known biochemical benefits in human metabolism, are of ongoing concern as neurodevelopmental toxins. Other common environmental toxins associated with neurodevelopmental disorders include perchloroethylene, toluene, carbon tetrachloride, ethylene glycol, methyl alcohol, organophosphates, formaldehyde and carbon monoxide. Teratology is the study of the effects that drugs, medications, chemicals and other exposures may have on the embryo or fetus. Teratology Information Services (TIS) are comprehensive, interdisciplinary resources for medical consultation on prenatal exposures that interpret information regarding known and potential reproductive risks into risk assessments. The Organization of Teratology Information Services (OTIS) provides regional telephone consultation for professionals and internet resources (http://www.otispregnancy.org). Over 20 current prescription drugs (e.g. isotretinoin) with proven teratogenic effects are in use in the USA. Because there has been insufficient human exposure, testing or reporting, newer pharmaceutical drugs may have teratogenic effects that are not yet recognized and it can be assumed that yet unidentified adverse effects of both therapeutic and illicit drugs are impending (Honein et al., 2004). 25.2.5. Effects of macronutrient deficiency (intrauterine growth retardation) Macronutrient deficiency secondary to malnutrition remains a significant cause of cerebral dysgeneses worldwide. According to the World Health Organization, malnutrition affects approximately 792 million people. Several million pregnant women lack sufficient nutrients or suffer infections that contribute to further malabsorption of nutrients needed for fetal brain development. Although diet plays a vital role in cellular metabolism, the optimal nutrition for prenatal brain growth remains uncertain because of the intricacy of innumerable genetic and environmental factors. Studies of maternal nutrition are typically confounded by the overlapping environmental deprivation and other adversities that accompany malnutrition (Wauben and Wainwright, 1999; Liu et al., 2003). Prenatal malnutrition results in a range of cerebral dysfunction including intellectual disabilities, attentional deficits,

CEREBRAL DYSGENESES SECONDARY TO METABOLIC DISORDERS IN FETAL LIFE mood disorders, and schizophrenia (Morgane et al., 1993; Neugebauer et al., 1999; St Clair et al., 2005). Certain essential nutrients, such as fatty acids, are needed to complete cell membrane formation (Cockburn, 2003). Choline is the best example of an essential nutrient for brain growth in fetal life and in early infancy. As a quaternary amine, choline has multiple functions in lipid transport, cell signaling, cholinergic neurotransmission and methyl-group donation through its metabolite trimethylglycinc (betaine) in the S-adenosylmethionine synthesis pathways (Zeisel, 2000; Li et al., 2004). In studies of human and other animal brain development, maternal malnutrition causes a reduced rate of neuronal cell division and reduced learning ability that may persist throughout life (Morgane et al., 2002). Better cognition and visual function is reported in breast-fed infants when compared to formula fed infants (Jain et al., 2002; Hoffman et al., 2003; Rey, 2003). Breast milk contains both docosahexaenoic acid and arachidonic acid, which are essential for normal brain development and are often reduced in formula feeds (Gordon, 1997). 25.2.6. Effects of micronutrients deficiency (nutritional deficit hypothesis) Many health consequences are associated with micronutrient deficiency in the developing brain, including premature birth, low birth weight and a higher rate of infections and death (Grantham-McGregor and Ani, 1999). However, research examining the effects of micronutrient deficiencies on early brain development suffers from the same methodological problems inherent to research on the effects of malnutrition. Micronutrient deficiencies most often occur in the context of poverty, frequently with confounding physical and social stressors that may interfere with health and optimal development. If pregnant mothers and their children are deficient in multiple macro- and micronutrients, it is difficult to interpret the effects of individual nutrient deficits or the meaning of dietary supplementation trials. Numerous micronutrients have essential roles in brain development, but deficits in some appear to have a greater impact than others. Humans require at least 60 elements and minerals including those that perform essential functions as integral components of metalloenzymes or as activating cofactors for other enzymes. Elements are considered to be essential if their deficiency consistently results in impairment of function. Trace elements are those minerals present in picogram-to-microgram quantity per gram of tissue and constitute less than 0.01% of total body weight. The 13 elements believed to be nutritionally important,

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in order of importance, are: iron, zinc, copper, iodine, fluoride, selenium, manganese, chromium, cobalt, molybdenum, nickel, silicon and vanadium. Iron-deficiency anemia is the most common nutritional disorder in the world, affecting all age groups. Some 2 billion people, or approximately one-third of the world population, are anemic because of iron deficiency. The biological basis of the cerebral disorders observed in iron-deficient persons is presumed to involve changes in neurotransmitter metabolism, disturbances in myelin formation and alterations in brain energy metabolism (Beard, 2003; Ortiz et al., 2004). The CNS has highly regulated, age-dependent mechanisms for iron acquisition (transferrin receptor), mobilization (transferrin and ceruloplasmin) and cellspecific iron storage (H and L isoforms of ferritin). Magnetic resonance imaging (MRI) can track iron distribution in the brain during postnatal development (Aoki et al., 1989). The basal ganglia, deep cerebellar nuclei and substantia nigra are particularly rich in iron, although substantia nigra iron does not fully evolve until puberty. The total concentration of iron is highest in the brain at birth, decreases during early postnatal life and then rises coincident with the increase in myelination. Early iron deficiency in experimental animals results in altered behavior and neurotransmitter disorders, including altered postsynaptic responses to serotonin and dopamine, and these disorders are irreversible if uncorrected (Scrimshaw, 1991). In human studies, it is not certain whether iron deficiency is the complete cause of such neurodevelopmental disorders or whether they are primarily due to other factors surrounding global malnutrition, environmental factors such as lead poisoning, or social deprivation. Infants of diabetic mothers frequently have polycythemia, elevated serum erythropoietin concentrations and decreased serum iron and ferritin concentrations, probably representing a redistribution of fetal iron into erythrocytes to support augmented fetal hemoglobin synthesis. Severely affected infants of diabetic mothers have reduced liver, heart and brain iron concentrations (Petry et al., 1992). Term infants born to mothers with uteroplacental vascular insufficiency related to severe hypertension, and term and preterm infants with intrauterine growth retardation, have a higher prevalence of reduced cord serum ferritin concentrations, which is suggestive of low fetal iron stores (Georgieff et al., 1995). Infants of diabetic mothers with suspected brain iron deficiency (e.g. cord ferritin < 34 mg/l) have impaired neonatal auditory recognition memory and lower developmental motor skills at 1 year of age than non-iron-deficient controls (Siddappa et al., 2004).

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W. D. GRAF

Zinc deficiency is common in both developing and developed countries and is particularly relevant to brain development because of its fundamental roles in cell division and maturation (Pfeiffer and Braverman, 1982; Halas et al., 1983). The brain has the highest zinc content of all organs, and its highest concentrations are in the cortex, certain forebrain regions, hippocampus and amygdala (Frederickson, 1989). Zinc is an essential cofactor for many enzymes, structural proteins and transcription factors and has unique vesicular localization in presynaptic terminals, where it may play a modulatory role in synaptic transmission, but the physiological significance of this phenomenon is poorly understood (Weiss et al., 2000). Several studies on zinc-deficient infants and children have shown beneficial effects of zinc repletion on growth, health and neuropsychological function (Friel et al., 1993; Sazawal et al., 1996; Bentley et al., 1997; Ashworth et al., 1998; CastilloDuran et al., 2001; Brown et al., 2002). 25.2.7. Effects of cofactor deficiency (vitamin deficit hypothesis) Through decades of interdisciplinary study, it became evident that brain development is critically dependent upon a cluster of folic-acid-dependent processes and that perturbation of these by nutritional deficiency or by metabolic defect can induce various serious developmental disorders, beginning with neural tube defects. Folic acid deficiency may result from depletion in folic acid reserves due to increased demands during pregnancy, decreased dietary intake, decreased absorption due to gastrointestinal disturbances or the action of certain drugs (including many antiepileptic drugs). Folic acid deficiency can be exacerbated by deficiencies in iron and in vitamins C and B12. Epidemiological evidence supports intervention with folic acid fortification of flour and supplementation during pregnancy (MRC Vitamin Study Group, 1991; Czeizel and Dudas, 1992). Follow up studies of neural tube defect populations have demonstrated a higher frequency of abnormal folic acid assimilation and methyl group metabolism during pregnancy. Two enzymes that are potentially involved at this level are methyl-tetrahydrofolate homocysteine-methionine methyltransferase and 5,10-methylenetetrahydrofolate reductase (MTHFR). A thermolabile isoform of MTHFR is associated with risk for neural tube defects in humans (van der Put et al., 1995) and also in certain forms of cardiovascular disease (Kang et al., 1991). The clinically observed metabolic perturbations related to these enzyme variants are resolved by dietary supplementation of folic acid, pyridoxine and cobalamine.

Part of the protective effect of folic acid may reflect tissue-specific expression and temporal patterns of gene expression, established through gene imprinting, gene silencing and gene activation (Graf and Oleinik, 2000). However, the exact biological mechanisms of vitamin-deficiency-related cerebral dysgeneses under ordinary dietary conditions are less clear. As previously discussed, measuring the effects of suboptimal vitamin status on human brain development and separating these effects from the wide-ranging signs of global malnutrition is often difficult. Nonetheless, vitamins of the B complex are vital for optimal neuronal metabolism, and both vitamin E and vitamin C appear to be potent antioxidants in the brain. Vitamin deficiency states may also result from the adverse effects of medications and alcohol. Prenatal alcohol exposure can influence embryological and fetal development, resulting in a range of functional deficits and structural malformations described as fetal alcohol spectrum disorders (Burd and Wilson, 2004). Classic fetal alcohol syndrome, a relatively severe syndrome in infants born to mothers who abuse alcohol during pregnancy, is characterized by dysmorphic facial features and relative microcephaly (Streissguth et al., 1980). Fetal alcohol syndrome may be related to nutrient and vitamin deficiency states through displacement of food in the diet, the relative excess of carbohydrate calories and the malabsorption of vitamins. Lifelong CNS sequelae of milder prenatal alcohol teratogenicity include learning disabilities, attention deficits and other similar behavioral disorders (Spohr et al., 1994; Streissguth and O’Malley, 2000). 25.2.8. Energy metabolism disorders linked to cerebral dysgeneses (impaired energy hypothesis) The fetus is dependent upon glucose and all other nutrients from maternal circulation for its oxidative metabolism and growth. The two primary substrates for energy production are carbohydrates and fatty acids. Carbohydrate metabolism is initiated in the cytosol, where glycolysis generates pyruvate from glucose. Pyruvate subsequently is metabolized in mitochondria to form acetyl-coenzyme A (CoA), which enters the Krebs cycle. Fatty acids undergo b-oxidation to produce acetyl-CoA, which also enters the Krebs cycle. Oxidation of these products of carbohydrate and fatty acid metabolism in the Krebs cycle provides electrons to the mitochondrial ETC, a complex system of cytochromes, nonheme iron proteins and quinones that ultimately produces adenosine triphosphate (ATP) by oxidative phosphorylation.

CEREBRAL DYSGENESES SECONDARY TO METABOLIC DISORDERS IN FETAL LIFE Cerebral glucose utilization is initially lower in the fetus but rises with advancing age as a result of increasing functional activity and regional cerebral energy demands. The most reliable information regarding the cell- and region-specific detection of the glucose transporters derives from in situ hybridization and autoradiographic 2-deoxyglucose analysis (Vannucci and Vannucci, 2000). Facilitative brain glucose transporter proteins, especially GLUT3 for neurons and GLUT1 for the blood–brain barrier and glia, show a pattern of increasing expression with age and maturation. Despite the major contribution of glucose to fetal energy metabolism, the immature fetal and neonatal brains maintain the ability to utilize alternative intermediate substrates for oxidative metabolism such as lactic acid and the ketone bodies, b-hydroxybutyrate and acetoacetate. These compounds are transported across the blood– brain barrier by the monocarboxylate transporter (Pierre and Pellerin, 2005). During perinatal hypoglycemia, lactic acid uptake and its use as cerebral metabolic fuel is high (Hellman et al., 1982). Nevertheless, glucose is the primary energy substrate in the developing brain for energy production and physiological biosynthetic processes. Cerebral dysgeneses have been associated with various primary energy metabolic disorders, such as hypoglycemic syndromes, glycolytic enzymopathies, hypoketonemic syndromes associated with fatty acid oxidation defects (e.g. glutaric aciduria type 2 and carnitine palmitoyl transferase type 2 deficiency), peroxisome biogenesis disorders and deficiencies of the housekeeping mitochondrial enzymes (e.g. pyruvate dehydrogenase or fumarase deficiency) (Table 25.1). 25.2.9. Energy allocation or mobilization disorders linked to cerebral dysgeneses (impaired hormone hypothesis) The hypothalamic–pituitary–thyroid (HPTh) axis and the hypothalamic–pituitary–adrenocortical (HPA) axis share many common features, namely the release of short-chain peptides by the median eminence of the hypothalamus into the hypophyseal portal system triggering the release of tropic protein hormones from the anterior pituitary before circulating to the thyroid and adrenal cortex to secrete the small, lipid soluble thyronines or corticosteroids hormones, respectively. Through complementary mechanisms, thyroid hormones and corticosteroids act as global energy regulation systems. The HPTh and HPA axes in the fetus are both thought to involve adaptive adjustments of energy turnover to match environmental conditions by shunting energy to support brain development at the

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expense of other tissues. Significant interaction of thyroid hormone with catecholamines in the reticular activating system act to regulate electrolytes and blood flow (Loosen, 1992). Brain triiodothyronine (T3) receptors are expressed in a developmentally specific pattern with strict, age-dependent requirements (Bernal et al., 2003). Concentrations of both thyroxine (T4) and T3 are tightly regulated within narrow ranges; small alterations in thyroid hormone concentrations may lead to significant changes in cerebral function. In congenital hypothyroidism, secondary cerebral dysfunction can result from iodine deficiency or from defects in thyroid hormone biosynthesis (Calaciura et al., 1995; Delange, 2000). The effects of congenital hypothyroidism have decreased conspicuously because of newborn screening, early recognition and thyroxine replacement therapy. However, long-term outcome studies in adolescents with congenital hypothyroidism reveal persistent memory, attention and visuospatial skill deficits, all of which correlate with severity of early hypothyroidism (Rovet, 1999). These clinical findings demonstrate the critical role of thyroid hormone in brain development. Primary iodine deficiency can lead to a severe diffuse developmental encephalopathy, which was termed ‘cretinism’ in the early 19th century. Iodine deficiency constitutes the world’s most prevalent single cause of preventable fetal metabolic encephalopathy (Delange, 2000). According to the World Health Organization, iodine deficiency affects over 740 million people, or about 13% of the world’s population. It is still a public health concern in parts of Africa and Asia where water and food lack iodine or where the consumption of iodized salt is inadequate (Sankar et al., 1998). As would be expected with any deficiency disorder, there is a wide range of severity in the clinical features within affected populations. Serious iodine deficiency during pregnancy results in miscarriage, stillbirth and cerebral malformations such as severe cerebellar hypoplasia (Mauceri et al., 1997; Ramos and Weiss, 2006). Conversely, because the fetal thyroid is particularly susceptible to iodine-induced goiter, iodine should not be given in large doses during pregnancy and pregnant women should not receive radioactive iodine. 25.2.10. Effects of altered cell cycle (hypoproliferation hypothesis) Proliferative cells lining the cerebral ventricles generate the phenotypically diverse neurons of the mature cerebral cortex. The differentiation of these periventricular neuroepithelial embryonic cells are influenced by numerous signaling factors, neurotransmitters and

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Table 25.1 ‘Single gene’ metabolic disorders associated with cerebral dysgeneses in fetal life

Metabolic category

Specific metabolic disorder/deficiency

Detected by MS/MS*

Postnatal changes

PAA; biopterin

ACC; brain atrophy

# cerebral WM/ brain atrophy; VM

(Levy et al., 1996)

X

CSF serine

# cerebral WM/brain atrophy

(De Koning and Klomp, 2004)

Non-ketotic hyperglycinemia

X

CSF glycine

ACC, PMG, cerebellar hypoplasia

2-Hydroxyglutaric aciduria (L-2 and D2 forms)

X

UOA

ACC; subependymal cysts; abnormal opercularization; occipital agyria

CFEA; UOA; GC/MS

Temporal lobe hypoplasia; abnormal opercularization

# cerebral WM/brain atrophy; congenital microcephaly # cerebral WM/brain atrophy, " intracerebral glycine, lactate, creatine; # glutamine, citrate # cerebral WM; posterior ventriculomegaly, putamina and dentate nuclei degeneration, cerebellar atrophy Cerebral atrophy, subependymal pseudocysts, striatal degeneration, and delayed myelination

Maternal hyperphenylalaninemia (phenylketonuria) Serine deficiency syndromes

Glutaric acidemia-type I

Pyruvate metabolism and tricarboxylic acid (TCA) cycle disorders

Reference

No{

X

X

3-Hydroxyisobutyric aciduria

X

UOA

ACC; LISS, CB-DYS

# cerebral WM, VM

Fumarase deficiency (fumeric academia /aciduria)

X

UOA; CFEA

ACC; PMG; abnormal opercularization

Angulation of ventricle frontal horns; # periventricular WM volume; small brainstem structures

(Dobyns, 1989)

(van der Knaap et al., 1999; Wang et al., 2003) (Kimura et al., 1994; MartinezLage et al., 1994; Brismar and Ozand, 1995; Bjugstad et al., 2000) (Chitayat et al., 1992; Sasaki et al., 1998) (Kerrigan et al., 2000)

W. D. GRAF

Organic acidopathies

MRI, MRS or neuropathology findings Prenatal dysgenesis

Yes Amino acidopathies

Other diagnostic methods/tests

Peroxisome biogenesis/ assembly disorders/ Zellweger syndrome spectrum

Cholesterol biosynthesis disorders

Glycoprotein metabolism

X

Pyruvate dehydrogenase deficiency

X

Carnitine palmitoyl transferase type II

X

Glutaric acidemia-type II

X

Zellweger (most severe)

Neonatal adrenoleukodystrophy (intermediate) Infantile Refsum (least severe) Bifunctional enzyme deficiency Chondrodysplasia punctata Smith–Lemli–Opitz (SLO) syndrome

Other rare cholesterol biosynthesis defects: Desmosterolisis Lathosterolosis Conradi–Hu¨nermann CHILD syndrome Greenberg dysplasia Congenital disorder of glycosylation

CFEA; gene mutatation analysis

PVL with diffuse edema ACC, C-HT; LISS; CB-DYS

Severe diffuse WM spongiosis and volume loss Cerebral atrophy with large cisterna magna C-HT, PMG

CFEA

LISS, C-HT, CB-DYS

X

" plasma VLCFA;

X

" pipecholic acid; # plasmalogens; #

ACC, LISS, PMG, CHT, OND, CB-DYS ACC, CB-DYS

X

DHA production; PEX gene screen

X X

Cerebral atrophy and hypomyelination Dysmyelination

(Brun et al., 1999) (Shevell et al., 1994; Michotte et al., 1993) (Hug et al., 1991; North et al., 1995) (Shevell et al., 1995) (Barkovich and Peck, 1997)

Dysmyelination

ACC, OND, CB-DYS Hypomyelination and microcephaly

(Kaufmann et al., 1996)

Cerebral atrophy and microcephaly; no universal pattern of CNS malformation is recognized in SLO syndrome Cerebral atrophy and microcephaly, demyelination, gliosis, multifocal calcifications

(Caruso et al., 2004; Hennekam, 2005)

" cisterna magna and superior cerebellar cistern cerebral atrophy; DandyWalker malformation; white matter cysts; # myelination

(Stibler et al., 1993; Holzbach et al., 1995; Peters et al., 2002)

X

# cholesterol biosynthesis; " CSF cholesterol precursors

ACC, LISS, PMG, CHT, CB-DYS, HPE

X

Multiple cholesterol biosynthesis enzyme defects related to hedgehog or Sonic hedgehog signaling

ACC, PMG

X

Isoelectric focusing transferase

ACC; CB-DYS

465

ACC, LISS, PMG, CHT, OND OND

CEREBRAL DYSGENESES SECONDARY TO METABOLIC DISORDERS IN FETAL LIFE

Fatty acid oxidation disorders

Pyruvate carboxylase

(continued)

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Table 25.1 (continued)

Metabolic category

Specific metabolic disorder/deficiency

Detected by MS/MS* Yes

Menkes disease

No{ X

# copper and ceruloplamin; " CSF DOPA, dihydrophenylacetic acid, and dopamine; hair Pili torti; gene testing

MRI, MRS or neuropathology findings Prenatal dysgenesis

Postnatal changes

C-HT; CB-DYS; ACC

Impaired myelination, diffuse atrophy, ventriculomegaly, and tortuosity of cerebral blood vessels

Reference

(Bjugstad et al., 2000; Liu et al., 2002)

These metabolic disorders are infrequently encountered in clinical practice but serve as models for the understanding of cerebral genesis and maintenance. Many of these metabolic disorders may not be detected by tandem mass-spectroscopy (MS/MS) newborn screening.*Inherited metabolic disorders typically detected and not detected/reported by tandem mass spectrometry (MS/MS). This list represents a constantly growing field with variation between states and laboratories. The clinical significance of some conditions detected by MS/MS in asymptomatic individuals is uncertain. { Disorders listed under NOT detected/reported are either not reliably detected or reported due to 1) high false-positive rates, 2) high false-negative rates or 3) limitation of technology. Disorders that are typically not detected/reported include disorders of glycogen metabolism, purine, pyrimidine, bile acid synthesis and lysosomal disorders (mucopolysaccharidoses and oligosaccharidoses). ACC, agenesis/dysplasia of the corpus callosum; AR, autosomal recessive; CB-DYS, cerebellar dysplasia; CFEA, cultured fibroblast enzyme activity; C-HT, cortical heterotopia; CSF, cerebrospinal fluid; GC/MS, gas chromatography/mass spectrometry; HPE, holoprosencephaly; HPLC, high-pressure liquid chromatography; LISS, lissencephaly/pachygyria; MRI, magnetic resonance imaging; MRS, magnetic resonance spectroscopy; MS/MS, tandem mass spectroscopy; OND, olivary nuclei dysplasia; PAA, plasma amino acids; PMG, polymicrogyria; PVL, periventricular leukomalacia; UAA, urine amino acids; UOA, urine organic acids; VM, ventriculomegaly; WM, white matter.

W. D. GRAF

Trace metal metabolism

Other diagnostic methods/tests

CEREBRAL DYSGENESES SECONDARY TO METABOLIC DISORDERS IN FETAL LIFE environmental metabolic factors (i.e. alcohol, drugs, maternal diabetes) in the early phases of neurogenesis (Kriegstein, 1996). Iron-containing enzymes and hemoproteins are involved in the control of the cell cycle through critical cellular enzymes such as ribonuclease reductase. Maternal insulin-dependent diabetes mellitus has been associated with severe fetal iron deficiency with a 40% reduction in brain iron concentration. In perinatal animal models, a similar degree of iron deficiency results in diminished hippocampal iron content and decreased cytochrome c oxidase activity, with persistent hippocampal biochemical and dendritic structural changes (Kriegstein, 1996; Siddappa et al., 2004). 25.2.11. Toxic accumulation of metabolite (radical injury hypothesis) Circulating intermediate metabolite disorders are thought to be an unlikely cause of cerebral malformations because of the ability of the placenta to filter toxic metabolites. The close interrelationship between maternal phase I and phase II hepatic enzymes facilitates the detoxification and excretion process. Disruption of the activity of any maternal enzymes in this process could potentially increase the effect of toxic exposure from a toxic compound or its metabolites but limited research has been performed in this area of cerebral dysgenesis research. Toxic injury from endogenous free radicals may play a role in dysgenesis. Free-radical-mediated injury has been hypothesized to be the final common pathway to oligodendroglial cell death in cerebral white matter injury in the premature infant (Volpe, 2001). Indirect biomarkers of reactive oxidant production and lipid peroxidation can be measured as specific markers of free radical reaction with lipids. Elevated cerebrospinal fluid (CSF) concentrations of 8-isoprostane and other oxidative products are associated with cerebral white matter injury in premature infants (Inder et al., 2002). 25.2.12. Neurotoxic elements and cerebral dysgeneses (environmental toxin hypotheses) Several environmental toxins can affect brain development. Neurotoxic actions can be expressed either as developmental disorders or as an increased risk of neurodegenerative diseases in later life. The major metals and minerals that are considered to be toxic include lead, mercury, aluminum, arsenic and cadmium. No clear beneficial biochemical effect of these metals in humans is known. Environmental toxins associated with neurological disorders include perchloroethylene, toluene, carbon tetrachloride, ethylene glycol, methyl

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alcohol, organophosphates, formaldehyde and carbon monoxide. It is likely that the most prominent developmental effects of these neurotoxins are nonspecific intellectual and learning disability syndromes. For example, effects of the developmental neurotoxin methylmercury in large populations of people include mixtures of developmental delays, learning disabilities, attention deficits, seizures and mental retardation, depending on the size, timing and duration of exposure. The combination of studies in experimental animals and in major human poisoning episodes in Japan and in Iraq has firmly established methylmercury as a severe neurotoxic agent. The effects of methylmercury are particularly toxic to the developing fetus and are associated with cerebral dysgenesis after exposure at higher concentrations (Choi et al., 1978; National Research Council, 2000). Mercury occurs in nature in three forms (metallic elemental mercury, inorganic mercury salts and organic mercury compounds) and each form has its own profile of toxicity. Organic mercury compounds (methylmercury, ethylmercury and phenylmercury) are most toxic to the CNS, although the extent of toxicity is dependent on the type of compound, route of exposure, dose and age of the person at exposure (Goldman and Shannon, 2001). Exposure to organic mercury typically occurs by ingestion. Infants are exposed to methylmercury through consumption of human milk. Children and pregnant women who consume excessive amounts of either freshwater or ocean fish can have a significant exposure to organic mercury. Methylmercury is demethylated to mercury in the nervous system, where it maintains a long half-life (National Research Council, 2000). Methylmercury is toxic to the developing cerebral and cerebellar cortex. In sufficient dosage, it leads to focal necrosis of neuronal and glial tissue (Choi et al., 1978). In the Minamata Bay disaster and the Iraq seed grain epidemic, mothers were either asymptomatic or had transient paresthesias but their infants developed a range of cognitive deficits, blindness, deafness and seizures (Amin-Zaki et al., 1979). Prospective studies conducted in the Faroe islands and the Seychelles assessed mercury concentrations in the hair of pregnant women who consumed various fish diets and the neurodevelopmental outcomes in their children. The Faroe islands study suggested that low level prenatal exposure is associated with subtle neurodevelopmental disorders, in which memory, attention and language scores were inversely associated with higher methylmercury exposures in children up to 7 years of age (Grandjean et al., 1997). Adverse effects on development or IQ were not found in the Seychelles study at up to 66 months of age, although exposures were in the

468

W. D. GRAF

same range as the Faroe islands study (Davidson et al., 1998). Prior to autumn 1999, thimerosal was used as a preservative for most diphtheria and tetanus toxoids and acellular pertussis vaccines as well as some Haemophilus influenzae type b, influenza, meningococcal, pneumococcal, hepatitis B and rabies vaccines. Thimerosol contains up to 50% mercury and is metabolized to ethylmercury and thiosalicylate. Because of the concern that cumulative doses of up to 187 mg mercury by 6 months of age are considered to be potentially toxic, no new vaccines in the recommended childhood immunization schedule contain thimerosal as a preservative (Centers for Disease Control, 2000). Clinical studies of lead exposure demonstrate an agedependent selective vulnerability to neuronal circuitry involved in learning and memory (Alfano and Petit, 1982; Alkondon et al., 1990). Lead appears to affect three interrelated steps in synaptic neurotransmission: through interruption of presynaptic neurotransmitter release from nerve terminals, through blockade of excitatory amino acid receptors and through alteration of protein kinases downstream from synaptic receptors (Bressler and Goldstein, 1991).

25.3. ‘Single gene’ disorder models of metabolic cerebral dysgeneses Description of cerebral malformation in association with individual metabolic disorders provides models for understanding the impact of metabolism in neurodevelopment. Although the exact frequency of discrete metabolically based cerebral dysgeneses is uncertain, up to one-sixth of known metabolic disorders begin their injurious effects during fetal life (Bamforth et al., 1994). Examples of these disorders, categorized by type of molecule, are summarized below and in Table 25.1. 25.3.1. Amino acid metabolism disorders linked to cerebral dysgeneses Disorders of amino acid catabolism are among the most frequent and best recognized inborn errors in humans. Their clinical phenotypes are variable but are often characterized by acute encephalopathy (e.g. from glycine encephalopathy in nonketotic hyperglycinemia), or acquired microcephaly with progressive mental retardation (e.g. from untreated phenylketonuria). The biochemical diagnosis of these aminoacidopathies is made through detection of characteristic metabolites accumulating in body fluids. Most amino acids act as a primary supply of brain nutrients or as neurotransmitters

or as precursors to neurotransmitters. In postnatal life, the regulation of circulating amino acids across the blood–brain barrier depends upon multiple factors, including the dietary content of amino acids and other nutrients, plasma amino acid concentrations, inherent regulation of protein synthesis, individual enzyme kinetics and blood–brain barrier transport mechanisms. Three model amino acid disorders related to cerebral dysgenesis are summarized below. 25.3.1.1. Maternal phenylketonuria or secondary hyperphenylalaninemia Offspring of mothers with the autosomal recessive disorder phenylketonuria acquire a severe developmental encephalopathy if maternal phenylalanine concentrations are persistently elevated during pregnancy (Levy et al., 1992). This metabolic cerebral dysgenesis is preventable if mothers with phenylketonuria adhere to the recommended diet before conception and throughout pregnancy. The major neurodevelopmental signs of secondary prenatal hyperphenylalaninemia are microcephaly (73–100%), cognitive deficiency (92–94%), seizures and autistic-like behaviors, as well as both prenatal and postnatal growth retardation. Hyperphenylalaninemia embryopathy is demonstrated by mild craniofacial dysmorphic features, corpus callosum hypoplasia and cerebral atrophy (Levy et al., 1992, 1996). Although the exact mechanism of excess fetal phenylalanine toxicity is uncertain, experimental exposure of human or rat astroglial cells to phenylalanine results in decreased cell proliferation and cell cycle arrest. Phenylalanine and its metabolites are not directly cytotoxic to neurons but phenylalanine metabolites may have an adverse effect on glial cells (Oberdoerster et al., 2000). 25.3.1.2. Serine-deficiency syndrome Defects in the biosynthesis of the ‘non-essential’ amino acid L-serine results in dysgenesis of cerebral white matter (de Koning and Klomp, 2004). The abnormalities in these disorders result from deficiency of CNS serine rather than excess accumulation of an amino acid, as exemplified by phenylketonuria. Two genetically distinct disorders, namely 3-phosphoglycerate dehydrogenase (3-PGDH) deficiency and 3-phosphoserine phosphatase (3-PSP) deficiency, lead to errors in L-serine biosynthetic pathways. The neurological phenotype in the majority of reported patients comprises congenital microcephaly, neurodevelopmental disabilities and seizures, including infantile spasms. MRI may show profound white matter reduction and hypomyelination (de Koning et al., 2000). The diagnosis is best determined by CSF amino acid analysis. Low concentrations

CEREBRAL DYSGENESES SECONDARY TO METABOLIC DISORDERS IN FETAL LIFE of CSF glycine and 5-methyltetrahydrofolate may be present (Ramaekers and Blau, 2004). Oral glycine and L-serine supplementation may be helpful in treating both seizures and prenatal brain growth (de Koning et al., 2004). 25.3.1.3. Nonketotic hyperglycinemia (also known as glycine encephalopathy) This amino acid disorder stems from an error in the glycine cleavage system secondary to mutations in either the GLDC, AMT, or GCSH gene encoding glycine decarboxylase (P-protein), aminomethyltransferase (T-protein) and glycine cleavage system hydrogen-carrier protein (H-protein), respectively. Postnatal clinical manifestations begin as neonatal lethargy, marked hypotonia, hiccups and myoclonic seizures progressing rapidly into coma. Laboratory findings show accumulation of glycine in body fluids without ketosis and decreased 13CO2 exhaled after administration of 13C-glycine (Kure et al., 2006). Nonketotic hyperglycinemia has been associated with dysgenesis of the corpus callosum, polymicrogyria, colpocephaly, cerebellar hypoplasia and signs of impaired myelination (Dobyns, 1989; Press et al., 1989; Fletcher et al., 1995). Certain sodium-benzoate-responsive forms of the disorder, such as that homozygous for a novel GLDC mutation (A802V) may produce transient clinical signs and normal neurodevelopmental outcome (Korman et al., 2004).

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25.3.2.1. Glutaric acidemia type 1 The cause of this organic acidopathy is a deficiency of glutaryl CoA dehydrogenase and an error in the catabolism of lysine, hydroxylysine and tryptophan. The prenatal effects of glutaric acidemia type 1 (GA1) typically result in severe hypoplasia of the temporal lobes with widening of the sylvian fissures. This malformation includes an absence of operculation during the last trimester of gestation. The association of GA1 with cerebellar heterotopia further suggests metabolic injury beginning in utero (Kimura et al., 1994). Postnatal clinical manifestations include macrocephaly, dystonia and choreoathetosis (Martinez-Lage et al., 1994). Progressive pathological changes include striatal degeneration with necrosis and diffuse CNS atrophy (Brismar and Ozand, 1995). The mechanism of prenatal injury in GA1 is uncertain but glutaric acid itself may have a toxic effect on brain tissues. 25.3.2.2. 3-hydroxyisobutyric aciduria This is a rare organic acidemia associated with a range of dysmorphic features and neurodevelopmental disabilities. CNS malformations include agenesis of the corpus callosum, abnormal neuronal migration, congenital intracerebral calcification and brain atrophy. Cerebral dysgenesis associated with this disorder appears to be related to the direct toxic effects of high intrauterine concentrations of 3-hydroxyisobutyrate (Chitayat et al., 1992).

25.3.2. Organic acid metabolism disorders linked to cerebral dysgeneses

25.3.2.3. D-2-hydroxyglutaric aciduria

Disorders of organic acid metabolism are a group of inherited amino and fatty acid oxidation disorders that cause nonamino organic acids to accumulate in tissues. Each organic acid disorder is associated with a specific enzyme or transport protein deficiency. The mechanism of metabolic injury leading to clinical signs is most probably secondary to the toxic effects of the accumulated organic acid compounds or their metabolites. Patients with many organic acidemias may not show clinical signs for many months postnatally but may present along with a metabolic crisis during an infection mimicking conditions such as encephalitis, Leigh syndrome (subacute necrotizing encephalomyelitis) or a Reye-like syndrome. Nonspecific MRI changes in patients with organic acidemias include: widening of the CSF spaces (more than two-thirds of patients); white matter changes (about half of all patients) and high T2-signal or volume loss in the basal ganglia or upper brainstem (one-third of patients) (Brismar and Ozand, 1994). Three organic acidemias that show affect fetal brain development are summarized below.

2-hydroxyglutaric acid is found in low concentrations in human urine and occurs in D and L configurations. D-2hydroxyglutaric aciduria stems from an error in the conversion of D-2-hydroxyglutarate to 2-ketoglutarate, usually because of a deficiency of one of the mitochondrial enzymes D-2-hydroxyglutaric acid dehydrogenase or transhydrogenase. Clinical manifestations and disorder progression are highly variable in patients with D-2hydroxyglutaric aciduria and range from minimal neurological signs to severe neonatal-onset developmental disabilities with intractable seizures and early death (van der Knaap et al., 1999). The most significant signs of prenatal onset in this disorder are agenesis of the corpus callosum, subependymal cysts, abnormal opercularization and delayed cerebral maturation affecting processes of gyral development including occipital agyria with a smooth and slightly thickened cortex (van der Knaap et al., 1999; Wang et al., 2003). Marked deficiency in postnatal myelination is a common finding. The exact pathophysiology of D-2-hydroxyglutaric acidurias is not known but may be related to elevated GABA concentrations, changes in the mitochondrial

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tricarboxylic acid cycle, or respiratory chain function (van der Knaap et al., 1999). 25.3.3. Pyruvate metabolism and tricarboxylic acid cycle disorders linked to cerebral dysgeneses Deficient production of the high-energy metabolites of intermediary metabolism can lead to subsequent disturbances in cellular function during critical phases of brain development. The best models of this mechanism for cerebral dysgeneses are elevated tissue concentrations of fumaric acid and congenital deficiency of pyruvate dehydrogenase and carboxylase. 25.3.3.1. Fumarase deficiency Fumarase is a key enzyme in the tricarboxylic acid (Krebs) cycle. Deficiency of this enzyme can lead to consistent signs of cerebral dysgenesis including diffuse polymicrogyria, decreased white matter, ventriculomegaly, open operculum, agenesis of corpus callosum with angulations of the frontal horns (colpocephaly), small brainstem or cerebral and cerebellar heterotopias (Walker et al., 1989; Gellera et al., 1990; Kerrigan et al., 2000). The postnatal clinical presentation typically demonstrates initial macrocephaly with polymicrogyria, followed by eventual microcephaly (Kerrigan et al., 2000). The exact biochemical mechanisms by which fumarase deficiency leads to cerebral malformation are unknown but the major hypotheses may revolve around the effects of decreased production of high-energy intermediate metabolites or toxicity of elevated fumarate concentrations disturbing processes in neuronal migration between 18 and 35 weeks gestation. 25.3.3.2. Pyruvate dehydrogenase complex deficiency Pyruvate dehydrogenase (PDH) is a key regulatory enzyme in mitochondrial energy production. PDHE1a-subunit deficiency, the most common enzyme defect, is an X-linked recessive disorder that is lethal in utero in hemizygote males with no enzyme activity. Heterozygote females typically manifest severe neonatal lactic acidosis, subtle dysmorphic features and evidence of cerebral dysgenesis, including dysgenesis of the corpus callosum, along with a spectrum of other migration abnormalities, such as the absence of the medullary pyramids, ectopic olivary nuclei, abnormal Purkinje cells in the cerebellum, dysplasia of the dentate nuclei, enlarged cisterna magna, subcortical heterotopias and pachygyria (Michotte et al., 1993; Shevell et al., 1994). The mechanism of dysgenesis may revolve around the critical role of PDH in neuronal and glial cell energy metabolism in complexes I and IV of the respiratory chain (Shevell et al., 1994).

25.3.3.3. Pyruvate carboxylase deficiency Pyruvate carboxylase (PC) is a biotin-dependent, nuclearencoded mitochondrial enzyme that catalyzes the conversion of pyruvate to oxaloacetate, supplying the Krebs cycle with an essential intermediate. Besides its role in gluconeogenesis, PC is also involved in lipogenesis and neurotransmitter synthesis. PC deficiency is a rare autosomal recessive disorder that typically comes to attention through persistent primary lactic acidemia. Neuroimaging, even in the prenatal period, demonstrates ischemialike periventricular lesions. Pathological findings include spongiform degeneration, neuronal loss, gliosis and delayed myelination. The ischemia-like lesions may develop during a vulnerable gestational period in a region where brain tissue demands increased energy and oxygen. Such processes may trigger an irreversible excitotoxicity, overwhelming the capacity of astrocytes to buffer glutamate or autoregulate the microvasculature (Brun et al., 1999). Clinically, PC deficiency should be included in the differential diagnosis of unexplained cystic periventricular leukomalacia. 25.3.4. Fatty acid oxidation disorders linked to cerebral dysgeneses Mitochondrial oxidation of fatty acids involves essential metabolic processes via Krebs cycle intermediates either to produce acetyl CoA for subsequent degradation for energy or for amino acid anabolism. It is hypothesized that impairment of fatty acid oxidation may alter the energy-intensive processes of neuronal proliferation and migration through various mechanisms. 25.3.4.1. Carnitine palmitoyl transferase type 2 deficiency Carnitine palmitoyl transferase type 2 (CPT2) is an enzyme in the internal mitochondrial membrane that acts to transport long-chain fatty acids from the cytosol into mitochondria. Milder and more common forms of CPT2 deficiency manifest from childhood to early adulthood with muscle pain and weakness after strenuous exercise. In contrast, a severe form of CPT2 deficiency may present in the early postnatal period with metabolic acidosis, hyperammonemia, ketotic hypoglycemia, diffuse muscle hypotonia, cardiomyopathy and elevation of serum long-chain acylcarnitines (Hug et al., 1991). In severe CPT2 deficiency, cerebral dysgenesis has been reported to include glial nodules or glia-lined paraventricular cysts combined with neuronal migration abnormalities. As in other metabolic disorders, the exact mechanism remains uncertain but plausible mechanisms include the lowered availability of acyl-CoA for cell membrane phospholipid synthesis, decreased mitochondrial production of ATP or the

CEREBRAL DYSGENESES SECONDARY TO METABOLIC DISORDERS IN FETAL LIFE direct toxic effects of intermediary metabolites on periventricular ependyma (North et al., 1995; Bonnefont et al., 1999). 25.3.4.2. Glutaric acidemia type 2 ( multiple acyl–CoA dehydrogenase deficiency) Glutaric acidemia type 2 (GA2) is a disorder of fatty acid oxidation that typically presents in the early postnatal period with metabolic acidosis, nonketotic hypoglycemia and progressive muscle hypotonia with cardiomyopathy (Bohm et al., 1982). In addition, enlargement of the kidneys, liver, and spleen may be present with an increased echogenic pattern on ultrasound (al-Essa et al., 2000). Urine organic acid analysis demonstrates high concentrations of glutaric acid, dicarboxylic acids and glycine derivatives. Numerous cerebral malformations have been described in GA2, including gray matter heterotopias, temporal lobe hypoplasia, cortical pachygyria, corpus callosum hypoplasia and cerebellar vermis agenesis. In the postnatal period, white matter changes can mimic primary leukodystrophy. Brain MR spectroscopy shows elevated concentration of intracerebral lactate and a high choline:creatine ratio associated with diffuse dysmyelination (Shevell et al., 1995; Takanashi et al., 1999). Neurological deficits may improve after initiation of low fat and protein diet, riboflavin and, in some forms of GA2, carnitine supplementation (Uziel et al., 1995). 25.3.5. Peroxisome biogenesis disorders (Zellweger syndrome spectrum) linked to cerebral dysgeneses The peroxisome is an ubiquitous organelle involved in the cellular metabolism of plasmalogens and verylong-chain fatty acids, cholesterol, bile acids, docosahexaenoic acid, eicosanoids and hydrogen peroxide. The peroxisome biogenesis disorders (PBD) are deficiencies in the function of peroxisome enzymes and defects in the assembly of peroxisomes (Brown and Baker, 2003). Most PBD are caused by errors in one of the 15 known human PEX genes, which encode proteins such as receptors for peroxisomal proteins, accessory factors required for receptor function, receptor docking factors in the peroxisome membrane and components of the protein translocation apparatus. These proteins play a role in lipid metabolism pathways such as fatty acid b-oxidation, fatty acid a-oxidation and etherphospholipid biosynthesis. Neuropathological findings vary greatly between the PBDs but generally involve a nonspecific reduction in myelin volume (hypomyelination); noninflammatory dysmyelination; or inflammatory demyelination (Powers, 2005). Certain PBDs cause cerebral dysgeneses, including pachymicrogyria, heterotopias, aberrant

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myelination, dysgenesis of the corpus callosum, hypoplastic olfactory lobes and abnormal cerebellar histogenesis (Faust, 2003) (Table 25.1). Brain MRI in PBD often demonstrates hypomyelination, abnormal cortical gyral patterns and germinolytic cysts best visualized in the caudothalamic groove on coronal or sagittal T1-weighted images (van der Knaap and Valk, 1991; Barkovich and Peck, 1997). PBD neuropathology is best represented by classic cerebro-hepato-renal syndrome of Zellweger, which displays ependymal abnormalities, heterotopias and disordered cortical lamination (Evrard et al., 1978; Sarnat et al., 1993). 25.3.6. Cholesterol biosynthesis disorders linked to cerebral dysgeneses Cholesterol is a precursor of bile acids, lipoproteins and steroid hormones and is also a major constituent and regulator of cellular membrane properties. A group of distal cholesterol biosynthesis disorders is also related to defects in the pathway of the signaling molecule of the Sonic hedgehog gene (shh). The Shh protein is a critical ‘cell fate switch’ that triggers proliferation of neuronal stem cells. This group of disorders illustrates the interactions between disorders of intermediary metabolism in cerebral dysgenesis (Kelley et al., 1996). 25.3.6.1. Smith–Lemli–Opitz syndrome The Smith–Lemli–Opitz (SLO) syndrome is caused by a deficiency of 7-dehydroxycholesterol reductase (Tint et al., 1994). Downstream effects of cholesterol metabolism errors act on several development genes responsible for neurogenesis and neuronal migration. A range of abnormalities have been described in SLO syndrome including cortical polymicrogyria, corpus callosum dysgenesis, brainstem hypoplasia and small cerebellum with abnormal foliation, and inferior cerebellar vermis hypoplasia. Holoprosencephaly type 3, a genetically heterogeneous disorder that affects midline forebrain and midfacial development, has been considered to come within the severe end of the SLO clinical spectrum (Cunniff et al., 1997). A holoprosencephaly–SLO-like phenotype can be created in rats through the administration of 7-dehydrocholesterol inhibitors (Roux et al., 2000). Clinical diagnosis is suggested by low cholesterol and high 7-dehydroxycholesterol concentrations in serum. 25.3.7. Glycoprotein metabolism disorders linked to cerebral dysgeneses Glycoprotein synthesis disorders involve the biochemical reactions in glycoprotein metabolism and include any protein containing covalently bound monosaccharide

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(glycose) or oligosaccharide residues other than a nucleic acid moiety. 25.3.7.1. Congenital disorders of glycosylation Congenital disorders of glycosylation (CDG) represent a heterogeneous group of genetically and biochemically distinct conditions that result from abnormal synthesis of N-linked oligosaccharides. Formerly known as the ‘carbohydrate-deficient glycoprotein’ (also CDG) syndromes, the first cases were reported as a new syndrome in 1980 by Jaeken et al., (1980) in monozygotic twins with severe developmental disabilities, decreased nerve conduction velocities, increased CSF protein and thyroid binding globulin deficiency. Depending on the specific type of glycosylation defect, variable cerebral dysgeneses may be present. For example, olivopontocerebellar atrophy, along with neonatal-onset hepatic steatosis and microcystic renal changes, can be found in a disialotransferrin developmental deficiency syndrome (Horslen et al., 1991). Major clinical signs of CDG include congenital ‘inverted nipples’, severe hypotonia, failure to thrive, strabismus, abnormal distribution of subcutaneous fat and MRI evidence of severe cerebellar hypoplasia. Isoelectric focusing of serum transferrin and, more recently, affinity chromatography–electronspray ionization mass spectroscopy are the best screening tests for these abnormalities of N-linked glycosylation (Vakhrushev et al., 2006). 25.3.8. Trace metal metabolism disorders linked to cerebral dysgeneses Reduced concentration of plasma ceruloplasmin is a key diagnostic biochemical marker of Menkes (kinky hair) syndrome and Wilson’s disease (hepatolenticular degeneration) (Menkes, 1997). Both these conditions involve genes that encode P-type copper-transporting ATPases (Bull et al., 1993). The gene for Wilson’s disease encodes 7.5 kb mRNA for a protein synthesized in hepatocytes as a single-chain polypeptide that is localized to the trans-Golgi network. 25.3.8.1. Menkes disease (Menkes kinky hair syndrome) Menkes kinky hair syndrome is associated with severe cerebellar vermis hypoplasia, cerebral cortex neuronal loss and signs of progressive degenerate of Purkinje cells (Erdohazi et al., 1976; Okeda et al., 1991). The finding of widespread mitochondrial enlargement and swelling in cerebellar Purkinje cells, molecular and granule cell neurons, cortical neurons, globus pallidus, thalamus, caudate nucleus and myelinated axons of white matter demonstrate mitochondrial abnormality

as an essential manifestation of this metabolic disorder (Yoshimura and Kudo, 1983).

25.4. Conclusion The identification of cerebral dysgeneses has progressed rapidly since the routine use of prenatal ultrasound and postnatal MRI in the diagnostic evaluation of children with cerebral growth abnormalities, seizures and nonspecific developmental delays. Metabolic screening technologies, such as prenatal a-fetoprotein and neonatal tandem mass-spectroscopy testing, contribute to the earlier recognition of metabolic disorders. Progress in the recognition of prenatal-onset metabolic disorders is expected in the coming decades. As contemporary technologies become increasingly implemented for the screening of metabolic neurodevelopmental disorders, many legal and ethical dilemmas will remain in evolution. For many disorders, no definitive guidelines will be available to direct parents about unambiguous reasons for providing or withholding care for a particular infant with signs of cerebral dysgenesis and a metabolic disorder. Early diagnosis and treatment of many inherited inborn errors of metabolism may allow the reduction of secondary neurological dysfunction in later childhood. Traditional therapies for metabolic diseases include dietary therapy such as protein restriction, avoidance of fasting or cofactor supplements. Evolving therapies for metabolic disorders now include organ transplantation, enzyme replacement, pharmacological intervention or other strategies. Efforts to provide treatment through somatic gene therapy are still in early stages but there is hope that this approach will provide additional therapeutic possibilities. Even when no effective therapy exists or an infant dies from a metabolic disorder, an accurate diagnosis is still imperative for family clarification, reassurance, genetic counseling and future prenatal screening.

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Section III Diagnostic methods


Chapter 26

Imaging malformations of cortical development INGRID E. B. TUXHORN*,1 AND FRIEDRICH WOERMANN2 1

Epilepsy Center in the Neurology Institute, Cleveland, Ohio, USA

2

Epilepsy Center Bethel, Evangelisches Krankenhaus Bielefeld Campus – Mara Hospital, Bielefeld, Germany

26.1. Introduction Abnormal brain development leading to dysplasia or malformations is a common finding in the neuroimaging studies of adults and children with developmental delay, mental retardation or epilepsy. Over the past decade the imaging characteristics of many malformations have become well established as a direct result of various technical advances, including the increased resolution of images, developments in data acquisition, imaging sequences and methods of analysis of structural and functional neuroimaging. Classification systems for malformations of cortical development that are strongly driven by imaging data have been established by a number of physicians who are active in the everyday evaluation and care of children and adults with this condition to provide a useful and logical framework both in the clinical setting and for related research activities (Mischel et al., 1995; Barkovich et al., 2001, 2005a). However, besides recent developments in imaging technology of malformations of cortical development, many newer discoveries of the genetics and molecular biological basis of some of these disorders requires continued updating and reclassification of these entities (Barkovich, 2005b). Cerebral cortical development is an extremely complex process, which may be divided into three broad sequential and overlapping steps of cell proliferation, neuronal migration and cortical organization and malformations of cortical development may result in the disruption of any of these events. In this chapter we will describe the role and basic approaches of different neuroimaging techniques and discuss the value of advanced imaging techniques that are helpful to identify and char-

acterize the ‘functional anatomy’ of malformations of cortical development, thus forming the basis for treatment and prognosis. We will present the imaging features of a select number of malformations with reference to the disruption of various developmental steps. The pivotal role of imaging in patients with epilepsy will be discussed in some detail. Further we will outline newer imaging techniques that allow assessment of their inherent neuronal function and intrinsic epileptogenicity. Finally we will refer to some well defined syndromes and imaging features of genetically determined malformations of cortical development.

26.2. The role and basic approach of imaging in identifying malformations Magnetic resonance imaging (MRI) is generally the imaging technique of choice for identifying malformations of cortical development while positron emission tomography (PET) or single-photon-emission computed tomography (SPECT) images normal cerebral function and functional impairment sometimes yielding additional information to reanalyze the structural MRI. In epilepsy patients with normal ‘standard’ MRI where there is a high index of suspicion for an underlying malformation of cortical development, the use of three-dimensional (3D) MRI, the application of surface coils, ultrathin MRI cuts (exploiting reduced motion artifacts during general anesthesia), as well as quantitative morphometric methods (voxel-based morphometry, texture analysis) may enhance the degree of visualization of a cryptogenic malformation of cortical development – again offering a guide for post hoc reanalysis of the structural MRI. In the epilepsy surgery

*Correspondence to: Dr Ingrid E. B. Tuxhorn, Epilepsy Center in the Neurologic Institute, Cleveland Clinic, 9500 Euclid Avenue/S51, Cleveland, OH, USA. E-mail: [email protected], Tel: þ1 216-444-8827, Fax: þ1 216-445-6814.

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work-up of a patient these methods may give valuable information to optimize the placement of invasive EEG electrodes for epilepsy surgery monitoring. Functional MRI (fMRI) is useful in delineating eloquent cortex adjacent to or within a malformative cortical region. The application of PET with various ligands and magnetic resonance spectroscopy (MRS) is at this point mainly a research tool for identifying and characterizing chemical processes and receptor systems in dysplastic cortical areas and related networks but shows promise for more widespread use in the clinical setting. However, changes shown with functional imaging techniques (PET, SPECT, fMRI, MRS, diffusion-weighted imaging, perfusion MRI) may be useful in clinical scenarios if they yield additional information beyond the structural changes picked up with anatomical imaging techniques. The role of multimodal imaging in the diagnosis and management of patients with malformations of cortical development is becoming increasingly important to: 1. improve the detection of overt and subtle cryptogenic malformation of cortical development 2. allow counseling of patients and families with genetically determined malformative sequences 3. guide epilepsy surgery patient selection by defining surgically amenable dysplastic substrates with greater sensitivity 4. modify and optimize presurgical invasive monitoring strategies to map the structure–function relationships of the epileptogenic zone for surgical resection.

26.2.1. Routine MRI Clinical studies applying MRI neuroimaging in patients with epilepsy have shown a high detection of abnormalities in most patients with localization-related epilepsy and in a sizable number of patients with first seizure as well as epilepsy in remission (Wieshmann et al., 2003). In various series of patients undergoing evaluation for epilepsy surgery the incidence of malformations of cortical development has been reported to range between 12% and 25% of cases (Li et al., 1995; Raymond et al., 1995; Tassi et al., 2002). As a consequence of the increased awareness of the presence of malformations of cortical development coupled with improved imaging techniques, malformations are discovered with increasingly greater frequency in patients with developmental delay and partial epilepsy. As a corollary to this it follows that cortical malformations need to be ruled out in essentially every patient with developmental delay and/or focal epilepsy.

‘Proper imaging technique and a high index of suspicion are crucial for the identification of cortical malformations’ (Barkovich, 2005b). Features of the clinical examination of the patient including specific nevi and other ectodermal changes, cardiac, renal, pulmonary findings, facial–cranial dysmorphisms and skeletal changes may point to the diagnosis of specific genetic and nongenetic syndromes, neurocutaneous disorders or neurometabolic disorders associated with malformations of cortical development. Infants with early-onset catastrophic focal epilepsy are a subgroup of children with severe epilepsy invariably caused by malformations of cortical development if no other etiology is apparent. Magnetic resonance is the imaging method of choice as it provides high-contrast resolution for the analysis of cerebral structures and the cortex. Conventional computed tomography (CT) has been reported to miss abnormalities evident on MRI in more than 30% of patients (Raymond et al., 1995). Special imaging protocols including 1) T1-weighted volumetric 3D spoiled gradient echo sequences using 1.0 mm or 1.5 mm partition size (and reformatting in three orthogonal planes) (Barkovich, 1995b), 2) T2weighted 3D volumetric acquisition (using 1.5 mm partition size and multiplanar reformation) or 2–3 mm spin echo or fast spin echo sequences in two planes (usually axial and coronal orientations) and 3) protondensity-weighted or fluid-attenuated inversion recovery (FLAIR) sequences obtained by using a standard quadrature head coil or phased array surface coils to improve the sensitivity for visualization of small or subtle dysplasias have been reported in detail by authors evaluating children with epilepsy or developmental delay (Grant al., 1997; Barkovich, 2005b). The routine MRI protocol at the Bethel Epilepsy Center starts with a sagittal, T1-weighted sequence for anatomical reference as this orientation allows the assessment of midline structures (corpus callosum, pituitary gland, hypothalamus, posterior fossa). Abnormalities in these areas (callosal agenesis and hypogenesis, Dandy–Walker and Chiari malformations, interhemispheric cysts, callosal lipomas, nasal dermoids and gliomas, frontoethmoidal encephaloceles, etc.) may be associated with malformations of cortical development. The lateral sagittal cuts allow the visual identification of perisylvian abnormalities (opercular polymicrogyrias, schizencephaly) and abnormal gyral patterns, which have been reported to be a useful marker in the search for malformations of cortical development (Sisodiya et al., 1996). While they are not that helpful in patients younger than 24 months of age, we routinely use axial and coronal FLAIR sequences in older children and adults.

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C Fig. 26.1. Sagittal images to detect abnormalities associated with malformations of cortical development. (A) Callosal malformation associated with malformations of cortical development: agenesis of the corpus callosum (left) associated with left fronto-orbital subcortical heterotopia and pachygyria (axial T1-weighted image, right). (B) Posterior fossa abnormalities associated with malformations of cortical development: here Dandy–Walker malformation in a patient with epilepsy and subependymal heterotopias (coronal T1-weighted image on the right). (C) Abnormality around the sylvian fissure associated with abnormal ratio between face and anteroposterior axis: perisylvian polymicrogyria and microcephaly (in the presence of a relatively normal-sized cerebellum).

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In principle, these axial images allow the inspection of the frontal lobes (the largest lobes of the brain, including the periventricular areas) and coronal images are used for the inspection of the temporal lobes. FLAIR images are used as a screening sequence for pathological signal changes related to macroscopic lesions (e.g. malformations of cortical development, tuberous sclerosis, hemimegalencephaly) and for a visual search of more subtle changes in the gray–white matter interface to determine the presence of cortical thickening, subcortical signal blurring and hyperintensity, which is frequently seen in focal cortical dysplasias (Kuzniecky et al., 2001). It needs to be kept in mind that these sequences are very prone to various artifacts, including movement and pulse artifacts. FLAIR signal changes are best visible on relatively thick slices (3–5 mm) and need confirmation by T2-weighted images, either in axial or coronal orientation, preferably as a 3D acquisition. In the past and on older MRI machines, 3D acquisitions were only available as T1-weighted images. Three-dimensional T1-weighted sequences may be acquired in very thin ( l mm) contiguous images covering the entire brain in a relatively short imaging time. The superior resolution and thus superior visualization of the gyral patterns and of the gray–white matter interface has led to the importance of a 3D T1weighted acquisition in the visual and quantitative detection of malformations of cortical development with subtle gyral abnormalities. 3D acquisitions offer the possibility for reformatting the images into planes other than the original orientation that the images were acquired in, which may for example include curvilinear multiplanar reformatting to inspect the cortex in the depth of the sulcal patterns (Bastos et al., 1999). These postprocessing techniques are the basis for more quantitative morphometric methods, including the recently reported results of texture analysis of malformations of cortical development (Bernasconi et al., 2001; Huppertz et al., 2005). We find the combination of both T1- and T2-weighted images quite helpful to differentiate heterotopic gray matter nodules from subependymal nodules or tumors based on the different signal features of these lesions. When searching for cerebral calcifications it needs to be remembered that T2*-weighted images are not as sensitive as CT in detecting calcifications, which may be seen in tumors, malformations of cortical development and the subependymal nodules in patients with tuberous sclerosis. Contrast enhancement with gadolinium may be useful to further characterize low grade tumors, some of which may be of developmental origin, showing imaging and pathological overlap with malformative

lesions as emphasized by some authors (Barkovich, 2005b). Enhancement of abnormal vascular structures may further exclude a developmental venous anomaly, e.g. venous angioma with juxtacortical perilesional gliosis manifesting with a blurred gray–white matter interface. Abnormal cortical vasculature has however also been reported in malformative lesions of the cortex and searching for these is a helpful marker for the presence of an associated malformation of cortical development, as reported by Barkovich (1988). However the sensitivity or specificity of these findings has not been studied systematically. In children younger than 6 months with immature but ongoing myelination, the gray–white matter interface is best assessed on T2-weighted images, which display an inverse cortical (dark)–white matter (light) signal contrast compared to images of mature brains where the myelination process has been completed and the white matter is darker than the cortical ribbon. The process of active myelination changes and reduces the T2 contrast most between the 6th and the 18th months of age so that large dysplasias may be missed on imaging during this stage. During this period the MRI search for malformations of cortical development should therefore be based on T1-weighted images too. Repeating the MRI early before the myelination effect on the T2-weighted contrast results in a more isointense cortical white matter interface, and repeating it later once myelination nears completion may increase the detection and assessment of the extent of malformations of cortical development in infants where the index of suspicion for an occult malformation of cortical development remains high. At this point we would like to emphasize that, when viewing images, abnormalities that are depicted only on a single image in a single orientation of a single sequence need to be interpreted with the utmost caution and considered an artifact until proven otherwise – for example if the finding is reproducible on images in other planes and can be visualized in contiguous thinner slices in multiple orientations and differently weighted sequences of the MR images. 26.2.2. Advanced structural and functional imaging of malformations (advanced MRI, PET, SPECT, MRS) 26.2.2.1. Advanced MRI In pediatric patients with extratemporal partial epilepsy that does not fit the classification of an idiopathic (e.g. benign) syndrome it is especially important to search for malformations of cortical development. In cases with normal conventional MRI further developments in MRI techniques – such as diffusion tensor imaging (Eriksson

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C Fig. 26.2. FLAIR images to detect focal cortical dysplasia (FCD) (A) Left fronto-lateral FCD close to eloquent cortex. Cortical signal change (T2-hyperintensity) and circumscript thickened cortex in the frontal lobe best visualizable on axial FLAIR or T2weighted images (upper row). Note the subtle subcortical transmantle hyperintensity suggesting FCD with balloon cells. The fMRI activity (lower row), associated with movements of the right hand, centers over the central sulcus and identifies the FCD as lying within the precentral gyrus. (B) Search strategy: screening test FLAIR in more than one orientation (axial on the left, coronal in the middle), followed by further confirmation on T2 (on the right). FCD in the region where the left-sided superior frontal sulcus joins the precentral sulcus, a region difficult to inspect because of partial volume effects. (C) In a patient with right-sided mesiotemporal sclerosis (here hippocampal hyperintensity more markedly than hippocampal atrophy, plus hyperintensity in the parahippocampal gyrus; right side of the panel), the anterior temporal lobe shows a blurred gray–white matter interface (left side of the panel) which might represent associated FCD, gliosis or edema.

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C Fig. 26.3. In tuberous sclerosis, imaging appearance changes during myelination. In this case, tuberous sclerosis with multiple tubers (including a later calcified left pericentral tuber) was imaged at age 3 months with MRI and at 36 months with MRI and CT. (A) Prior to 6 months, malformations of cortical development resulting from a disturbances of neuronal and glial proliferation are best imaged exploiting the gray–white matter contrast on T2-weighted images. The left precentral tuber/FCD returns a signal as if the involved white matter was ‘prematurely’ myelinated. Note the subependymal nodules, typically bilateral in the groove between head of the caudate nucleus and the thalamus. (B) FLAIR images increase the conspicuity of pre-existing bilateral tubers/FCD. (C) The left precentral tuber/FCD is clearly calcified on CT, as are the subependymal nodules (right, middle). Calcification can be detected using MRI and T2*-weighted imaging (left).

IMAGING MALFORMATIONS OF CORTICAL DEVELOPMENT et al., 2001; Rugg-Gunn et al., 2001), magnetization transfer imaging (Rugg-Gunn et al., 2003), fast flair T2 imaging or double inversion recovery – have enabled identification of abnormalities in about half of those patients in whom conventional MRI did not isolate a cause for focal seizures. Three-dimensional acquisitions offer the possibility for reformatting the images into additional planes of orientation from the original acquisition plane, which may, for example, include curvilinear multiplanar reformatting to inspect the cortex in the depth of the sulcal patterns (Bastos et al., 1999). These postprocessing techniques have allowed the development of a number of quantitative morphometric methods, including the recently reported results of texture analysis and voxelbased morphometry of malformations of cortical development (Bernasconi et al., 2001; Huppertz et al., 2005) with increased sensitivity of detection of subtle ‘microdysgenesis’ and small malformative lesions. In patients with malformations of cortical development, and intractable epilepsy in particular, the identification and resection of the whole malformation is closely related to freedom from seizures after epilepsy surgery (Edwards et al., 2000; Kloss et al., 2002; Tassi et al., 2002). To improve lesion detection and delineation of the extent of the malformation, newly developed MR morphometric methods referred to above have been applied more systematically. Automated voxel-based detection methods have been developed that further delineate specific MRI features of focal cortical dysplasia (Antel et al., 2003) in contrast to the normal variability in brain and cortical structure (Richardson et al., 1997, Wilke et al., 2003). Widespread or bilateral changes, typically excluding patients from further presurgical assessment, were identified in patients with apparently focal malformations of cortical development by applying the use of manual MR morphometry as well as automated voxel-based analysis of MRI (Sisodiya et al., 1995; Woermann, Free et al., 1999). Methods of texture analysis of the neocortex, utilizing the histopathological characteristics of malformations of cortical development (e.g. increased cortical thickness transposed into grayscale maps), may increase the sensitivity for identification of focal cortical dysplasia (Bernasconi et al., 2001; Bonilha et al., 2003). Such voxel-based image postprocessing methods, including texture analysis and morphological processing modeled on known MRI features of focal cortical dysplasia on T1-weighted 3D images (thickened gray matter, blurred gray–white matter interface, signal hyperintensity), were developed and tested on patients with histologically proven malformations of cortical development, including so-called ‘MRI-

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negative’ patients (Bernasconi et al., 2001; Huppertz et al., 2005). Analyzed on a voxel-by-voxel basis, MR magnetization transfer imaging identified areas of abnormal magnetization transfer ratio in patients with malformations of cortical development without clear correlation to seizure outcome or tissue histology after surgery (Rugg-Gunn et al., 2003). Similarly, using MR diffusion tensor imaging and a voxel-based analysis, anisotropy and diffusivity changes were seen in tissue outside the malformations of cortical development as defined by conventional MRI, i.e. tissue that appeared normal on routine anatomical imaging (Eriksson et al., 2001). The clinical relevance and prognostic value of these findings for postsurgical outcome remain unclear at this point but warrant further investigation. On a cautionary note, we need to realize that increasing the sensitivity of malformations of cortical development detection will increase the likelihood of false-positive findings. Larger studies using appropriate gold standards, including the seizure outcome after epilepsy surgery and tissue histology, are needed to address these issues. Further limitations are placed on our efforts to improve the visual detection of subtle malformations of cortical development, e.g. in studies involving higher field strengths (3 T MRI) and other techniques of optimized MRI resolution (e.g. with phased array surface coils), by the restricted availability of these technologies, their technical limitations (e.g. the difficulty of separating the effects of 3 T imaging, surface coil imaging) and the limited experience of ‘expert’ readers with the skills to interpret these complex epilepsy imaging findings (Knake et al., 2005). In addition the overall ‘standard’ quality of the neuroradiological detection of malformations of cortical development, especially in patients with intractable epilepsy, can be improved on, as was suggested by a recent study (von Oertzen et al., 2002). 26.2.2.2. PET and SPECT imaging of malformations of cortical development In patients with extratemporal partial epilepsy with normal MR imaging studies, PET and SPECT may be helpful in finding small areas of dysplasia (Chugani et al., 1990; Richardson et al., 1996, 1997). PET studies are usually performed with 18F-fluorodeoxyglucose or 11C-flumazenil, while SPECT uses 99mTc-HMPAO or 123I-iodoamphetamine. More recently, other compounds including 11C-methionine, which may be more concentrated in a variety of lesions including malformations of cortical development, tumors and abscesses, have been studied (Madakasira et al., 2002).

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The mechanism of increased uptake of this tracer in dysplasias is unknown and is probably nonspecific for the etiology. Using semiquantitative analysis with region of interest templates the sensitivity and specificity of 18 F-fluorodeoxyglucose PET in the evaluation of frontal lobe lesions was reported markedly improved from 50% to 90% (Swartz et al., 1995). Decreased flumazenil binding has been observed in patients with focal cortical dysgenesis and up to 72% of patients with focal epilepsy, and normal MRI may have PET abnormalities with this tracer. In a number of studies 11C-flumazenil PET, which reflects changes in GABAA/benzodiazepine receptor binding, showed a higher yield in detecting abnormalities in MRI-negative patients than advanced MRI techniques. In 45 patients with temporal lobe epilepsy and normal MRI (Henry et al., 1993; Debets et al., 1997; Ryvlin et al., 1998; Koepp et al., 2000; Lamusuo et al., 2000; Hammers et al., 2002) 11C-flumazenil PET scans were abnormal in 38; however, only half of the abnormalities shown were considered useful for surgical treatment. Similarly in 102 MRI-negative patients with extratemporal seizure origin who showed abnormalities of 11C-flumazenil binding these findings were of use for surgery in a quarter of patients (Savic et el., 1995; Ryvlin et al., 1998; Richardson et al., 1996; Hammers et al., 2003; Salmenpera et al., 2004). Increased flumazenil binding was found in the subcortical white matter in 11 of 18 patients with temporal lobe epilepsy and in seven of 44 patients with extratemporal epilepsy, most probably reflecting an increased density of heterotopic neurons in the underlying white matter (Hammers et al., 2002). This study showed that the increased 11C-flumazenil binding in the white matter in ‘microdysgenesis’ strongly correlated with the number of heterotopic neurons, measured semiquantitatively ex vivo in the resected specimens. As microdysgenesis is not readily detectable on MRI this new PET imaging finding of white matter abnormalities representing the pathological basis of medically refractory patients with temporal lobe epilepsy and a normal MRI may improve selection for surgery, tailoring of the surgical procedure and subsequent seizure outcome. Using 18F-fluorodeoxyglucose (Kim et al., 2000), 11 C-flumazenil (Richardson et al., 1996; Hammers et al., 2001a) and carbon-11-labeled-diprenorphine (Hammers et al., 2001b) PET in malformations of cortical development, abnormal regions of tracer binding were commonly more extensive than the abnormality seen with MRI, and were also noted in distant sites that were unremarkable on MRI.

Detection of neurochemical abnormalities by use of PET might help in differentiating epileptogenic from nonepileptogenic lesions. a-11C-methyl-L-tryptophan uptake, which reflects serotonin synthesis, was found in children with tuberous sclerosis and differentiated between epileptogenic and nonepileptogenic tubers in patients with tuberous sclerosis complex (Chugani et al., 1998; Fedi et al., 2003). The resection of tubers with increased a-11C-methyl-L-tryptophan uptake seems to be most promising, as this approach led to seizure-free surgical outcome in children with tuberous sclerosis complex and intractable epilepsy (Kagawa et al., 2005). The interpretation of these highly sensitive but nonspecific mapping methods in the clinical management of MRI-negative patients for epilepsy surgery selection will become more relevant once we better understand the underlying pathophysiology by correlations with histology and outcome (Koepp and Woermann, 2005). Ictal SPECT studies may be sensitive for demonstrating extratemporal foci of focal cortical dysplasia, particularly if located in the frontal lobe (Newton et al., 1995; Chiron et al., 1996) while postictal studies are not reliable in these cases. Technical issues such as timing of tracer injection and seizure duration are highly relevant for image quality and pick-up rate. 26.2.2.3. MRS in malformations of cortical development Proton MRS imaging may useful in localizing epileptogenic foci; however, this technique is still in an early stage of development. Proton MRS imaging (1HMRSI) studies permit the noninvasive measurement of concentrations of a variety of cerebral metabolites implicated in cerebral structure and function. To date there is a dearth of quantitative 1H-MRSI studies of malformations of cortical development. Readily available on modern MR scanners, proton MRS (1H-MRS) measures metabolites in vivo either within single voxels or within a slice with numerous voxels (MRS imaging (MRSI) or chemical shift imaging (CSI)). N-acetyl aspartate (NAA), one of the main metabolites measured, is believed to be located primarily within neurones, so that reduced NAA suggests either neuronal loss or neuronal dysfunction. A number of proton MRS studies have been performed in patients with malformations of cortical development. A decrease in NAA was the most frequent finding in individual malformations of cortical development patients and in group comparisons (Kuzniecky et al., 1997; Li et al., 1998; Simone et al., 1999; Kaminaga et al., 2001; Woermann et al., 2001; Mueller et al., 2005). An in vitro MRS study has also shown

IMAGING MALFORMATIONS OF CORTICAL DEVELOPMENT a reduction of NAA in areas of malformations of cortical development that were confirmed by subsequent biopsies (Aasly et al., 1999). Woermann et al. have shown abnormal metabolite levels in up to 90% of patients with malformations of cortical development, which however varied among patients regarding levels and metabolic patterns (Woermann et al., 2001). This metabolic heterogeneity may reflect the step at which fetal cortical development was probably first disturbed according to the classification framework for malformations of cortical development that has been proposed by Barkovich (Barkovich et al., 2001; Barkovich, 2005a). A significantly reduced NAA: creatine ratio was shown in malformations of cortical development secondary to early disturbances in stem cell formation (dysplastic neurones), normal or variably reduced in heterotopias (later disturbance of migration with variably dysmature neurones and abnormal synaptogenesis) and normal in polymicrogyria (late disturbance of cortical organization with mature neurones but abnormal gyration) (Li et al., 1998; Widjaja et al., 2003). Using absolute quantification and short echo time 1 H-MRSI, abnormal absolute metabolite concentrations in the remote malformations of cortical development, perilesional tissue and brain tissue remote were demonstrated in patients with localization-related epilepsy (Woermann et al., 2001). These findings support the concept of widespread abnormalities in patients with apparently focal malformations of cortical development in some patients. However these spectroscopic abnormalities do not necessarily represent widespread structural changes, as described earlier in malformations of cortical development (Sisodiya et al., 1995; Woermann, McLean et al., 1999), but might demonstrate remote areas of dysfunction. Measurements of individual metabolites were abnormal in some malformations and normal in others, suggesting metabolic heterogeneity of malformations of cortical development as already discussed above in detail (Woermann et al., 2001). A more recent study of the metabolic profile within a single malformation showed an even more complex heterogeneous pattern, with metabolically normal regions interspersed with metabolically abnormal regions (Mueller et al., 2005). MRS may contribute as a diagnostic tool to distinguish between low-grade gliomas and focal malformations of cortical development. Promising results from group comparisons showing less NAA in tumors compared to malformations of cortical development await replication and prospective translation into clinical practice (Vuori et al., 2004). A further application of MRS is suggested by results of a recent study in patients with tuberous scle-

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rosis. In the presence of multiple and bilateral cortical lesions it may be difficult to identify a single epileptogenic tuber amenable to surgical resection. Using MRS, a lactate peak was detected in the regions corresponding to an epileptic focus in some patients (Yapici et al., 2005). When considering the feasibility of this technique for general clinical use it should be mentioned that most MRS studies have been performed on magnetic resonance scanners with high field strengths not commonly used in the clinical setting (Kuzniecky et al., 1997), in single voxels restricting the regions investigated (Castillo et al., 1993; Hanefeld et al., 1995; Simone et al., 1999; Widjaja et al., 2003), with long echo times restricting the number of measured metabolites (Marsh et al., 1996; Li et al., 1998; Simone et al., 1999; Widjaja et al., 2003; Mueller et al., 2005) or without absolute metabolite quantification (Castillo et al., 1993; Marsh et al., 1996; Kuzniecky et al., 1997; Li et al., 1998). Although this technique appears promising, further quantitative studies need to be performed and the results validated with other in vivo and in vitro methods to understand their significance. 26.2.3. Value of imaging in patients with epilepsy caused by malformations of cortical development The second most common cause of refractory epilepsy in adults is malformation of cortical development (Sisodiya, 2004). High-resolution MRI, ideally with multiplanar study, is needed for diagnosis of malformation of cortical development with imaging. Routine MRI cannot detect or identify the extent of all pathologies. Freedom from seizures after surgery is closely related to resection of the whole malformation (Edwards et al., 2000; Tassi et al., 2002); identification of widespread or bilateral changes typically excludes patients from further presurgical assessment (Sisodiya et al., 1995). Surgery is less successful in patients with widespread lesions than in patients with discrete lesions, most probably because of anatomical and functional abnormalities extending beyond the visible lesions. In patients with malformations of cortical development, antiepileptic drug treatment does not control seizures in 25% of those who develop epilepsy. The success of medication largely depends on the cause of the epilepsy. Patients without an identifiable lesion have the best prognosis for seizure freedom (50%); despite adequate drug treatment, this chance is lower in patients with malformation of cortical development, unilateral hippocampal sclerosis (another frequent pathology in partial epilepsy; 11%), or dual pathology, e.g. malformation of cortical development and

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hippocampal sclerosis; 3% (Semah et al., 1998). However, clear identification and complete resection of an epileptogenic lesion leads to seizure freedom in up to 90% of patients with unilateral mesial temporal lobe epilepsy (Wiebe et al., 2001) or malformations of cortical development associated tumors (Wyllie et al., 1998) and up to 70% of patients with malformations of cortical development (Cohen-Gadol et al., 2004) or dual pathology (Salanova et al., 2004). In patients with malformations of cortical development and epilepsy, antiepileptic drugs alone rarely prevent seizures. However, the success of epilepsy surgery is improved when a structural lesion has been identified. Malformations of cortical development, with their intrinsic epileptogenicity, often overlap with an extended area of the cortex generating seizures and may contain or continue into areas sustaining normal functions. To prevent postsurgical morbidity, the

Fig. 26.4. Left sided temporo-parietal focal cortical dysplasia. Around age 6 months myelination affects the gray–white matter contrast on T2-weighted images so that T1-weighted images become more important.

spatial relation between functionally important areas and the epileptogenic lesion must be assessed before epilepsy surgery.

26.3. Imaging focal cortical dysplasia with and without balloon cells The imaging characteristics of balloon-cell-positive focal cortical dysplasia are quite characteristic and have been well described (Barkovich et al., 1997; Colombo et al., 2003). Based on the first histological study by Taylor, this malformation is frequently also called the Taylor type of focal cortical dysplasia (Taylor et al., 1971), ‘forme fruste of tuberous sclerosis’ because of the shared pathological features with the cortical hamartomas seen in patients with the tuberous sclerosis complex genotype and phenotype, or transmantle cortical dysplasia because the abnormality extends through the whole cortical mantle (Barkovich et al., 1997). On imaging, the cortical gyral pattern is abnormal with broad gyri and large, irregular sulci. From the cortex, an abnormal signal will extend radially in a cone-shaped fashion to the ventricular surface. There is blurring of the cortical–white matter junction and the region of abnormal signal intensity is heterogeneous and contains portions that are isointense to gray matter, isointense to white matter and regions that are hyperintense both to gray and white matter (Tassi et al., 2002). The transmantle tract of abnormal T2 signal hyperintensity correlates well with location of balloon cells, hypomyelination and astrogliosis of the white matter (Urbach et al., 2002). Small areas of focal cortical dysplasia may be difficult to detect on routine imaging and may require the special imaging techniques for visualization described in this chapter. Focal cortical dysplasia without balloon cells has been divided into three histoarchitectural subtypes based on the severity of disruption of cortical lamination and neuronal dysplasia (Colombo et al., 2003): 1) abnormal lamination, dysmorphic and giant neurones (Taylor type); 2) cytoarchitectural dysplasia (abnormal cortical lamination and giant neurones); and 3) architectural dysplasia (abnormal cortical lamination only). The MRI appearance is usually one of cortical thinning with hyperintensity and volume loss of the underlying white matter resembling the findings seen in ischemic injuries. In mild lesions only reflecting architectural dysplasia (focal dyslamination), the focal blurring at the gray–white matter junction may easily be missed or nondetectable even with high-resolution scanning. A helpful finding may be a ‘dimple’ in the cortex, a point where the subarachnoid space is focally increased in volume (Bronen et al., 2000).

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Fig. 26.5. Right frontal malformations of cortical development consisting of pachygyria and ‘blurred gray–white matter interface’ – better visualized on T2-weighted images prior to age 6 months (A) as compared to T2-weighted image at age 12 months (B) – i.e. MRI as early as possible.

Fig. 26.6. Gyral abnormalities on T1-weighted images with thin slices (A) Localized gyral abnormality – left frontal polymicrogyria. (B) Left fronto-temporal schizencephaly.

26.4. Imaging hemimegalencephaly and schizencephaly Unilateral hemispheric enlargement is termed hemimegalencephaly and probably reflects a number of het-

erogeneous disorders following defective proliferation, migration and organization of the affected hemisphere. It may be a multisystem disorder with cutaneous abnormalities, hemihypertrophy of the whole ipsilateral body as well as part and parcel of well defined

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neurocutaneous disorders – tuberous sclerosis complex, hypomelanosis Ito, Proteus syndrome, epidermal nevus syndrome, neurofibromatosis, Klippel–Trenaunay– Weber syndrome to name a few. MRI features show the bulky overgrowth of the hemisphere, sometimes also the ipsilateral cerebellum and brainstem, various grades of gyral anomaly from minimally to grossly pachygyric or lissencephalic. The white matter signal intensity abnormalities reflect heterotopias, increased number of glial cells, dysmyelination and other hamartomatous changes while the lateral ventricle is characteristically enlarged (but may be small), with the frontal horn being abnormally straight and pointed (Barkovich and Chuang, 1990). Schizencephaly describes a malformation characterized by a transhemispheric cleft lined with gray matter extending from the subependymal lining of the lateral ventricle to the pia covering the cortex. The etiology may be genetic, with a mutation of the EMX2 homeobox gene on chromosome 10q26 being described (Gulisano et al., 1996) or acquired in utero following an injury, most probably in the second trimester. It is a malformation that probably develops from disturbances in proliferation, migration and cortical organization and may be unilateral (60%), bilateral (40%), with fused ‘closed’ lips (15–20%) or separated ‘open’ lips, which may appear as small or large clefts (Barkovich, 2005). These anatomical features are easily visible on MR; however, the clefts with fused lips where the CSF space is obliterated by the apposing lips may be more difficult to appreciate than the wide open clefts where the CSF fills the cleft from the outer subarachnoid space to the ventricles, resembling porencephaly, which does however not have the pial–subpial cortical lining seaming the cleft.

26.5. Limitations of advanced imaging in ‘MRI-negative’ patients with epilepsy In about 20% of patients with chronic focal epilepsy, pathology is not seen on visual inspection of highresolution MRI (Li et al., 1995; Wiebe et al., 2001). Much current imaging research is directed at such patients. The usefulness of the different MRI and PET techniques for assessment of MRI-negative patients is not clear because few studies have directly compared the different imaging techniques in large samples. The risk of type I errors – the identification of abnormalities that are not present – increases in diagnostic methods with a high sensitivity. Type I errors could result in unnecessary invasive EEG explorations with possibly harmful procedures or even surgical resections. For patients with normal MRI, only postoperative follow-up will provide information about the specificity of the

imaging findings. However a successful postoperative outcome is less likely when patient’s imaging is negative (Berkovic et al., 1995). Whether a patient is ‘negative’, as measured by a certain imaging tool, depends on the expertise of its users, which varies according to the technique of the research center. Standard MRI assessment in clinical practice – interpretation of axial images by a radiologist who does not work in an epilepsy center – did not detect 57% of focal epileptogenic lesions and led to false ‘MRI-negativity’ (von Oertzen et al., 2002). When even expert assessment did not detect lesions on MRI but electrophysiological findings guided surgery, lesions were not commonly found on histopathological assessment of the resected tissue (Urbach et al., 2004). Only a few studies have assessed the contribution of multimodality imaging in MRI-negative patients (Spencer et al., 1995). The comparisons are generally biased towards the technique with which the researchers have the most experience. In a study comparing hippocampal volumetry, T2 relaxometry, MRS and 18-fluoro deoxyglucose PET in patients with temporal lobe epilepsy who were MRI-negative, all methods increased sensitivity but only hippocampal atrophy was associated with postoperative seizure-free outcome (Knowlton et al., 1997). However, the findings obtained from new MRI techniques may not always be concordant with the localization results from the EEG evaluation, as was noted in only one-quarter of patients studied (Salmenpera et al., 2004), while in a further study of hippocampi that were structurally normal, quantitative short-echo-time MRS showed high concentrations of glutamate and glutamine (Woermann, McLean et al., 1999). Therefore when coregistering various anatomical and functional imaging findings with results of studies defining the epileptic focus, congruence of results should remain the cornerstone for planning any treatment intervention.

26.6. Tumors associated with malformations of cortical development Benign tumors commonly underlie refractory partial seizures. About 20% of adults and children who have surgery for chronic epilepsy have a benign tumor, most commonly in the temporal lobe. Complete resection of dysembryoplastic neuroepithelial tumors is an effective epilepsy treatment and adjunctive chemotherapy or radiotherapy is not needed. Gangliogliomas and dysembryoplastic neuroepithelial tumors are benign developmental glial-neuronal tumors that cause focal epilepsy with onset frequently before age 20 years. They are classified as benign because they typically lack mass effect and are stable on serial imaging (Daumas-Duport et al., 1999).

IMAGING MALFORMATIONS OF CORTICAL DEVELOPMENT Dysembryoplastic neuroepithelial tumors are hypometabolic lesions on 18F-fluorodeoxyglucose PET with low binding on 11C-flumazenil PET (Kaplan et al., 1999; Richardson et al., 2001). PET with 11C-methionine can help to differentiate between low-grade gliomas, which manifest as increased uptake lesions (Derlon et al., 1997) and dysembryoplastic neuroepithelial tumors, which demonstrate negative uptake (Maehara et al., 2004). Dysembryoplastic neuroepithelial tumors may be associated with cortical dysplasia, which may not be seen on MRI (Stanescu et al., 2001). SPECT studies may reveal circumscribed hyperperfusion in dysembryoplastic neuroepithelial tumors without cortical dysplasia, which is limited to the location of the tumor on MRI, whereas in patients with cortical dysplasia hyperperfusion spreads more extensively into normal-looking perilesional regions (Valent et al., 2002).

26.7. Imaging epileptogenic networks: functional connectivity The activity maps produced from neuroimaging studies suggest that cortical cerebral functions arise from the action of distributed networks in the brain. On the basis of meticulous investigations with multiple depth electrodes in patients with epilepsy, Bancaud and Talairach (1992) proposed a similar concept of interconnected regions composed of a pacemaker and relay as well as sub-relay areas essential for producing individual ictal symptoms and signs. Corticocortical facilitatory connections allow propagation of mesiotemporal discharges in temporal lobe epilepsy after a preferential route from the amygdala and hippocampus toward the cingulate gyrus and orbitofrontal cortex. Propagation toward the insular cortex is common and happens early during the seizure, suggesting a specific insular network, which might develop progressively with repeated seizures (Isnard et al., 2000). The results of various imaging studies support the idea that the epileptogenic zone is not confined to the temporal lobe in some patients with mesial temporal lobe epilepsy. Quantitative MRI, PET and SPECT studies suggest widespread extrahippocampal and even extratemporal abnormalities in patients with unilateral hippocampal sclerosis (Newton et al., 1992; Henry et al., 1993; Koepp et al., 1996; DeCarli et al., 1998; Dupont et al., 1998; Juhasz et al., 2001; Knowlton et al., 2001; Bouilleret et al., 2002; Blumenfeld et al., 2004; Chassoux et al., 2004). Ictal SPECT studies suggest that temporal lobe seizures may result from focal abnormal activity in temporal and subcortical networks linked to widespread impaired function of the association cortex (van Paesschen et al., 2003).

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Diffuse and widespread changes were associated with poor outcome after anterior temporal lobectomy (Sisodiya et al., 1997). Cross-sectional and longitudinal studies reported progressive hippocampal atrophy and widespread neocortical changes within an interconnected network of cortical and subcortical areas remote from the putative epileptic focus (Liu et al., 2003). Cortical and subcortical networks causing focal seizures might explain some of the surgical failures and the common occurrence of simple auras persisting after resections and diminishing later (Yoon et al., 2003). These observations are consistent with good surgical outcome initially but a substantially lower long-term success rate – around 30% of patients relapse when antiepileptic drugs are withdrawn. Unlike other lesions, malformations of cortical development have intrinsic epileptogenicity, suggesting that they are highly interconnected with normal brain regions (Palmini et al., 1995). Advanced imaging techniques are very sensitive in detecting subtle lesions but generally do not provide information on the epileptogenicity of these abnormalities – this is important in the surgical treatment of bilateral and multicentric malformations of cortical development such as tuberous sclerosis. Detection of neurochemical abnormalities by use of PET might help in differentiating epileptogenic from nonepileptogenic lesions. Increased serotonin metabolism has been found in samples taken from patients with epilepsy (Chugani et al., 2000). High 11C-labeled-methyl-Ltryptophan uptake, which reflects serotonin synthesis, was found localized in the epileptogenic tubers of children with tuberous sclerosis. (Chugani et al., 1998; Asano et al., 2000; Shoaf et al., 2000; Fedi et al., 2001). Similarly 11Cmethyl-L-tryptophan uptake was high in the presumed epileptogenic zone in four of seven adult patients with focal cortical dysplasia and was correlated with the frequency of interictal epileptiform discharges both in patients with visible malformations of cortical development and in those with cryptogenic epilepsy (Fedi et al., 2003). In a further study, malformations of cortical development verified by histology was associated with increased 11C-methyl-L-tryptophan uptake, and subdural electrodes placed adjacent to the area of increased tracer uptake recorded the region of seizure onset (Juhasz et al., 2003). This methodology may prove very helpful in the presurgical diagnostic work-up of patients with tuberous sclerosis complex and a leading epileptogenic tuber despite the presence of multiple cortical tubers. An important precondition for surgical resection of an epileptogenic malformation of cortical development

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is that this area is not involved in normal neurological functions (Marusic et al., 2002). Combinations of fMRI during motor activity, continuous EEG-correlated fMRI, and diffusion-weighted imaging has demonstrated how the epileptogenic focus and lesion may displace neuronal function (Diehl et al., 2003). Examination of cerebral function provides further evidence for widespread microanatomical disorganization (Richardson et al., 1998a). Following a PET study in a larger group of malformations of cortical development patients with heterogenous results (Richardson et al., 1998b), a fMRI study of different types malformations of cortical development classified by time of origin of the disturbance leading to the malformation demonstrated that all later disturbances of cortical organization (polymicrogyria, schizencephaly and mild-type focal cortical dysplasia) showed fMRI activity in the lesioned cortex, whereas early disturbances of proliferation and migration (hemimegalencephaly, Taylortype focal cortical dysplasia and heterotopia) showed activity in a significantly smaller proportion of cases (Janszky et al., 2003). In addition to this finding that malformations caused by disturbances of cortical organization were activated on fMRI during simple tasks, which indicated preserved neuronal functions, but malformations of cortical development caused by disturbances of neuronal proliferation and migration were activated less commonly, MRI activations within malformation of cortical development were less frequent when patients were imaged during complex cognitive tasks involving activity in both or one hemisphere, suggesting functional or neuronal reorganization (Janszky et al., 2003). In patients with malformations of cortical development, atypical language representation has been shown to occur less frequently than in patients with epileptogenic lesions acquired in infancy, suggesting further that a malformation may be involved in normal physiological function related to its cortical topography (Duchowny et al., 1996; Adcock et al., 2003). Numerous case reports support the hypothesis that malformations of cortical development may be activated by external stimuli or motor tasks (Hatazawa et al., 1996; Achten et al., 1999; Pinard et al., 2000; Iannetti et al., 2001; Innocenti et al., 2001; Spreer et al., 2001) and may participate also in complex cognitive brain functions (Muller, 1998). Activation studies examining cases of heterotopia and focal cortical dysplasia (Achten et al., 1999; Riedel et al., 2004) or polymicrogyria (Innocenti et al., 2001; Calistri et al., 2004) demonstrated activation of the both neuronal networks within the dysplasias and in addition abnormal patterns of activation including ipsilateral or

contralateral displacements of activation suggesting plasticity and reorganization of networks. In a single case, fMRI activation within the malformations of cortical development during movements of a paretic hand appeared functionally irrelevant (Vandermeeren et al., 2002). This indicates a major shortcoming in the clinical use of fMRI activity: especially in the presence of bilateral fMRI activity it remains unclear which activated area is primarily essential for the task. Using a combination of fMRI and a transcranial magnetic stimulation, different types of malformations of cortical development revealed various degrees of participation in motor functions, ranging from corticospinal (‘primary’) motor control, to putative participation as ‘nonprimary’ motor areas, to no clear evidence for any functional participation (Staudt et al., 2004). Despite these limitations fMRI may be useful in the presurgical evaluation of malformations of cortical development patients because it allows us to visualize intrinsic functions of malformations of cortical development and their relationship to the adjoining eloquent cortex. From the above described results, surgery in patients with malformations of cortical development due to cortical organization disturbances should be performed with caution because these malformations usually participate in brain function. The association between the type of malformations of cortical development and its functionality emphasizes that the malformations of cortical development type should be clearly defined by MRI before considering epilepsy surgery. The association of reflex epilepsies with malformations of cortical development suggests that external stimuli sometimes activate the neurons within the malformation (Grosso et al., 2004; Palmini et al., 2005). Ictal fMRI and PET studies in patients with focal seizures induced by reading suggest a network of hyperexcitable cortical and subcortical areas overlapping with areas that are active during reading (Koepp et al., 1998; Archer et al., 2003; Noppeney and Price, 2004). This provides further circumstantial evidence of altered functional connectivity in dysplastic cortical regions and offers a basis to continue investigating neuronal network function in malformations of cortical development with e.g. noninvasive techniques such as PET to measure activation of cerebral blood flow and glucose metabolism (Grafton, 2000) and bloodoxygenation level dependent fMRI (Binder et al., 2002). Further study of epileptogenicity has been made possible with newer techniques utilizing a combination of electroencephalography (EEG) and fMRI allowing visualization of bold changes within the irritative zone,

IMAGING MALFORMATIONS OF CORTICAL DEVELOPMENT which has been defined as the cortical area generating interictal epileptiform discharges. In a study of patients with polymicrogyria, which is usually widespread and frequently bilateral, EEG-fMRI responses have shown a maximum activation involving the lesion, but are often limited to a small fraction of that lesion, suggesting intrinsic epileptogenicity in small areas of the polymicrogyria (Kobayashi et al., 2005). A further EEG-fMRI study in areas of heterotopia have revealed ‘metabolic responses’ within the ectopic gray matter lesions while the spike activity was clearly generated in the adjoining neocortical areas (Kobayashi et al., 2006).This study demonstrated a variable correlation of ‘metabolic activity’ between the lesion and surrounding or distant cortex. The responses appeared in the form of activation reflecting intense neuronal activity or excitation and deactivation implying distant extralesional inhibition, again supportive evidence for more widespread functional and perhaps also structural abnormalities in patients with circumscript-appearing malformations of cortical development (Kobayashi, 2006). These findings may in part explain why patients with extratemporal dysplasias have a lower seizure-free outcome after surgery for epilepsy than patients with other etiologies.

26.8. Imaging of structural connectivity The idea of interconnected cortical areas led to the use of tractography in recent studies. Tractography is derived from diffusion tensor imaging, an MRI technique that assesses brain structure through the threedimensional measurement of diffusion of water molecules in tissue. Obstructions to diffusion, such as cell membranes, cause the water molecules to be arranged in a highly directional manner in white-matter fibers. This information can be used to assess connectivity between voxels, show connections between brain areas in vivo and provide information on the integrity of white-matter tracts. For the first time with a noninvasive technique, optic radiation (Toosy et al., 2004), primary-motor-cortex connectivity and connectivity between the parahippocampal gyrus and orbitofrontal areas was shown (Guye et al., 2003). Tractography also showed direct connectivity between the parahippocampal gyrus and the hippocampus itself (Powell et al., 2004). These results are consistent with those of histological tract-tracing studies in animals. The connections shown between neocortical areas and the hippocampus via the parahippocampal gyrus may provide the structural basis for theoretical models of memory and visual processing as well as seizure propagation. In addition, this technique might be of use in evaluating patients with epilepsy for resective brain

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surgery to minimize the risks of cognitive, motor or visual-field impairment. The application of these techniques in malformations of cortical development appears promising to further define the ‘functional anatomy’ of these lesions in relation to normal brain structures.

26.9. Imaging of genetically determined malformations It is becoming increasingly clear that many malformations of cortical development are caused by chromosomal mutations making counseling of parents with affected children important in the clinical management. The identification of malformations of cortical development and its specific morphology by imaging will influence and guide appropriate genetic counseling significantly. Prenatal and postnatal development of neuroanatomical structures implicated in the cause of epilepsy is affected by genetic as well as environmental factors in a multifactorial scenario. Use of both MRI and molecular genetic analyses in patients with malformations of cortical development and in their families will help to identify genes that affect prenatal brain development (Dobyns et al., 1999). Subcortical band heterotopia and lissencephaly (‘smooth brain’) – two neuroradiologically distinct malformations of cortical development that lead to epilepsy and variable cognitive impairment – can be inherited alone or together in a single pedigree. Both can be caused by either mutation of a single gene on the X chromosome called XLIS or doublecortin, with the milder phenotype of band heterotopia expressed in heterozygotic females, or by mutation of a single gene on chromosome 17 called LIS1. A subgroup of patients with chromosome 17 mutations has a characteristic facies classified as the Miller–Dieker syndrome. Malformation of cortical development due to XLIS (also DCX) mutations are severe over the frontal cortex, whereas LIS1 mutations lead to an anterior–posterior gradient with the malformations of cortical development affecting mostly parietal and occipital regions. Typically patients with classical lissencephaly caused by an XLIS mutation are boys whose mothers have a double cortex malformation and therefore a good family history should be obtained in these cases. Thus MRI characteristics can guide genetic counseling (Dobyns et al., 1999). The complex genetics of ‘smooth brain disorders’ is gradually being further unraveled with the help of imaging. While approximately 75% of patients with lissencephaly will have mutations of either the 17p13.3 or Xq22.3–23 genes, other mutations have been found. These include the RELN gene coding for

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the extracellular matrix protein reelin, which is important for the release of migrating neurons from the radial glial cells, and an X-linked lissencephaly with callosal agenesis and ambiguous genitalia (XLAG) resulting from mutations of the ARX gene on chromosome Xp22.13 (Dobyns et al., 1999). The relative frequency of these disorders is as yet unknown. A paracentral dominance of gyral abnormalities has also been described involving mainly the precentral and postcentral gyri and may be inherited as an autosomal disorder or be part of the socalled Baraitser–Winter syndrome characterized by ocular anomalies, short stature, dysmorphic features and mental retardation (Rossi et al., 2003). In the group of children with microcephaly and simplified gyral pattern (MSG) and true microlissencephaly, which are malformations secondary to abnormal stem cell proliferation or apoptosis, one cause of MSG type 1 appears to be the Nijmegen breakage syndrome, an autosomal recessive disorder belonging to the group of DNA repair disorders (Lammens et al., 2003). Rare cases of schizencephaly may be familial, the etiology being genetic secondary to a mutation of the EMX2 homeobox gene located on chromosome 10q26, which is a gene expressed in the germinal matrix of the developing cortex (Gulisano et al., 1996). Polymicrogyria may be seen in several specific syndromes that occur sporadically or familially. The familial congenital bilateral perisylvian syndrome probably results from heterogeneous mutations of different genes. One mutation located on Xq28 has been identified (Villard et al., 2002). Seizures, mental retardation, oropharyngeal dysfunction secondary to pseudobulbar palsy and congenital arthrogryposis are typical clinical manifestations, which may be mild in some familial cases. Several other syndromes of region-specific bilateral symmetrical polymicrogyria have been described and linked to specific genetic loci (Barkovich et al., 1999; Villard et al., 2002). A distinct clinical syndrome characterized by bilateral frontoparietal polymicrogyria, global developmental delay, seizures, disconjugate gaze with esotropia and bilateral pyramidal and cerebellar signs has been linked to chromosome 16q (Chang et al., 2003). The genes for these disorders may be critical for proper development of the regionspecific architecture of the cerebral cortex. The potential value of a candidate gene approach to studying human developmental malformation by MRI is shown in studies of patients heterozygous for defined PAX6 mutations. These patients can have widespread structural abnormalities, including unilateral polymicrogyria and two previously unknown human brain malformations, namely the absence of

the pineal gland and absence of the anterior commissure without callosal agenesis (Mitchell et al., 2003). In addition, a number of nongenetic forms of polymicrogyria – bilateral mesial parieto-occipital polymicrogyria (Guerrini et al., 1997) bilateral lateral polymicrogyria (Barkovich, 2005), unilateral polymicrogyria (Pascual-Castroviejo et al., 2001) and megalencephaly with polymicrogyria and hydrocephalus – have been described (Barkovich et al., 2001). Animal studies on epileptogenesis suggest that seizures arise mainly from the cortical rim adjoining the polymicrogyric cortex, as there is a paucity of axonal connections in the malformation as well as an upregulation of excitatory and downregulation of inhibitory receptors in the cortex (Jacobs et al., 1999). Subtle structural abnormalities were found in relatives of patients with malformation of cortical development by use of quantitative morphometric tools, suggesting common genes shared by patients and relatives that result in similarities of cerebral development (Merschhemke et al., 2003). Heterotopia of gray matter comprises disorders of arrest of radial neuron migration and manifest on imaging with collections of nerve cells in abnormal locations. Invariably patients present with seizure disorders. For the purpose of clinical and prognostic evaluation a division into three groups has been proposed: a) subependymal heterotopia; b) focal subcortical heterotopia; and c) band heterotopia (Barkovich and Kjos, 1992). Familial forms are usually X-linked or autosomal recessive and mutations of several genes may cause heterotopia. Mutations of the filamin-1 (FLN1) gene located on chromosome Xq28 usually result in diffuse subependymal heterotopia lining the walls of the body of the lateral ventricles while some patients with missense mutations only have peritrigonal subependymal heterotopia (Sheen et al., 2001). In addition to these gene disorders ‘blueprinting’ specific cerebral malformations, a number of syndromes featuring neocortical neuronal malformations with extracerebral dysmorphic features have been described (Hennekam and Barth, 2003). These varied mutations may include chromosomal inversions, e.g. 2p–12–q14 ( pachygyria); deletions of chromosomes, e.g. 4p (heterotopia, microgyria); deletions of chromosome 4q (heterotopia, pachygyria); deletions of chromosome 17p13.3 (lissencephaly type 1 in the Miller–Dieker syndrome); deletions of chromosome 22q11 (polymicrogyria); duplications of chromosomes, e.g. 3q (polymicrogyria, microcephaly, hypoplasia of olfactory bulbs); trisomies, e.g. trisomy 9 (band heterotopia), trisomy 13, trisomy 18, trisomy 19, trisomy 21; as well as a ring-structured chromosome 17 manifesting with heterotopia and variable

IMAGING MALFORMATIONS OF CORTICAL DEVELOPMENT midline dysgeneses and specific dysmorphic features (Hennekam and Barth, 2003; Barkovich, 2005b). A number of inherited metabolic disorders including peroxisomal disorders, e.g. Zellweger syndrome featuring perirolandic pachygyria and occipital polymicrogyria, neonatal adrenoleukodystrophy and peroxisomal bifunctional protein deficiency, mitochondrial disorders, organic acidurias (specifically 3-hydroxyisobutyric acidemia associated with polymicrogyria and D-2-hydroxy-glutaric aciduria – rarely associated with occipital agyria) and disorders of cholesterol synthesis, such as the Smith–Lemli–Opitz syndrome may manifest with malformations of cortical development (Barkovich and Peck, 1997; Trasimeni et al., 1997; Barkovich, 2005). In addition, Rett syndrome, a

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disorder of transcriptional regulation secondary to a mutation in the MECP2 gene on the X chromosome, may manifest with nodular heterotopia and perisylvian cortical dysplasia (Geerdink et al., 2002). Oculo-cerebral syndromes such as the X-linked Aicardi syndrome (chorioretinopathy) and the autosomal dominant micro syndrome (microphthalmia, cataracts and optic atrophy) are associated with heterotopia, polymicrogyria and callosal agenesis, in addition to glioependymal cysts and infantile spasms in the case of patients with Aicardi syndrome (Donnenfeld et al., 1989; Nassogne et al., 2003). In children with congenital neuromuscular disorders, which are all inherited as autosomal dominant disorders (merosine/laminin-a2-negative congenital

Fig. 26.7. Left-sided hemimegalencephaly with a posterior preponderance; at age 4 months on T2-weighted images (A), very subtle volume asymmetry and ‘blurred gray–white matter junction’; at age 3 after a hemispherotomy, the remaining occipital lobe displays most abnormal gray and white matter signal (B).

A

B

C

Fig. 26.8. Right-sided hemimegalencephaly, with a posterior preponderance of hyperintensities on FLAIR images (A) and of gyral abnormalities (polymicrogyria) best seen on sagittal T1-weighted images (B); typically the ipsilateral ventricle is enlarged; note the signal loss on T2*-weighted images indicating right occipital, periventricular calcification (C).

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A

B Fig. 26.9. (A) Bilateral frontoparietal polymicrogyria associated with a mutation on chromosome 16, in a 6-day-old girl who was born to consanguineous parents; T2 weighted images displaying the contrast prior to myelination (cortex darker than white matter). (B) X-linked band heterotopia in a 33 year old patient with epilepsy who had multiple miscarriages before diagnosis was reached; note the diffuse distribution of the band with a tendency to a more pronounced appearance anteriorly.

muscular dystrophy, Walker–Warburg syndrome, Fukuyama congenital muscular dystrophy, muscle– eye–brain disease, myotonic dystrophy), associated cerebral malformations including heterotopia, polymicrogyria (cobblestone malformations), occipital lissencephaly and white matter lesions have been described (van der Knaap et al., 1997; Barkovich, 1998, 2005b).

26.10. Conclusion MRI, PET and SPECT are noninvasive imaging techniques that are widely available. In the clinical situation, a combination of multiple imaging modalities

increases the specificity and sensitivity of detection of malformations of cortical development, improving the clinical management of patients with epilepsy and mental delay caused by these dysplasias. In surgical cases, postoperative seizure control may be improved on and postoperative impairments reduced through more accurate delineation of the epileptogenic zone, surrounding eloquent cortex and vital connections between cortical areas. Clinical studies using newer structural and functional imaging methodologies highlight the link between developmental pathologies, abnormal epileptiform activity, network brain activity and cognitive function.

IMAGING MALFORMATIONS OF CORTICAL DEVELOPMENT In addition to their use for presurgical planning, reducing the need for invasive techniques, a combination of multiple imaging modalities could help with monitoring the course of treatment and definition of prognosis. Specific anatomical features of malformations of cortical development defined by imaging may serve as a ‘blueprint’ for underlying gene mutations, aiding in family counseling. For all these reasons, clinical neuroimaging will continue to improve the quality of health care. The strengths and weaknesses of any clinical imaging tool must be fully appreciated by the health care professional before it can be used routinely and influence clinical management.

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Chapter 27

Clinical neurophysiology of cortical malformations: magnetoencephalography and electroencephalography HIROSHI OTSUBO* AND KATSUMI IMAI The Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada

27.1. Introduction The classification of cortical malformations depends on the developmental stage during which cortical development is disturbed and falls into three stages as follows: 1) cell proliferation and differentiation, 2) neuronal migration and 3) cortical organization (Barkovich et al., 2001). Corroborative studies from neurological, neuroradiological, neuropathological, genetic and neurophysiological fields are necessary for understanding the pathophysiology of cortical malformations (Guerrini et al., 2002). Neurophysiological findings alone may not be sufficient to diagnose a specific cortical malformation but they may simply identify the epileptogenic region. This chapter focuses on the current neurophysiological studies of scalp electroencephalography (EEG), intracranial EEG using stereotaxic EEG (SEEG) and electrocorticography (ECoG), and magnetoencephalography (MEG) in patients with cortical malformations to understand their specific patterns, histopathology correlates, intrinsic epileptogenesis and regions of the epileptogenesis.

27.2. Magnetoencephalography correlates to magnetic resonance imaging MEG is a relatively new clinical technique that uses superconducting quantum interference devices (SQUIDS) to measure and localize sources of extracranial magnetic fields generated by intraneuronal electric currents. MEG measures the magnetic field component that is perpendicular to the skull surface and is produced by the tangentially aligned neurons, which probably account for two-thirds of the cortical surface, in fissure

walls. MEG typically uses the equivalent current dipole to model the source of synchronized neuronal activities (Barth et al., 1982; Sato et al., 1991). MEG dipole localizations are then coregistered with the patient’s magnetic resonance images (MRIs), producing a combined functional and structural image called a magnetic source image (MSI) (Lewine and Orrison, 1995; Otsubo and Snead, 2001c; Chuang et al., 2006). MSI localizes the interictal MEG dipoles for patients with intractable localization-related epilepsy to evaluate the potential of cortical excision. In patients with intrinsic epileptogenic cortical malformations, MSI provides threedimensional information about locations, distributions and characteristics of the MEG dipoles estimated from the interictal spikes (Paetau et al., 1994; Morioka et al., 1999; Otsubo et al., 2005). We classified the MEG dipoles into five groups by number and density: ‘clusters’, consisting of 20 or more dipoles with 1 cm or less between adjacent sources; ‘small clusters’, consisting of 6–19 dipoles with 1 cm or less between adjacent sources; ‘scatters’, consisting of fewer than 6 dipoles regardless of the distance between sources; ‘diffuse scatters’, consisting of 6 or more dipoles with 1 cm or more distance between sources; and ‘no MEG dipoles’ (Iida et al., 2005a; Oishi et al., 2006b). 27.2.1. MEG clustered dipoles of focal cortical dysplasia on MRI Patients with focal cortical dysplasia had frequent, polyphasic, various amplitude magnetic and electrical bursting epileptiform discharges projecting clusters of MEG dipole sources representing interictal spike sources localized within and extending from MRI lesions

*Correspondence to: Hiroshi Otsubo, Director of Neurophysiology, Division of Neurology, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada. E-mail: [email protected], Tel: þ1-416-813-6295, Fax: þ1-416813-6334 or 1-416-598-2092.

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(Otsubo et al., 2001a; Ishibashi et al., 2002; Header et al., 2004; Iida et al., 2005a). Header et al. (2004) reported on 39 patients who underwent surgical treatment for cortical dysplastic lesions. In ten patients whose preoperative imaging showed cortical dysplastic lesions and MEG dipole sources, seven patients experienced good outcomes after resection of lesions and areas with MEG-clustered dipole sources. Intracranial EEG has confirmed the correlation of MEG dipole sources with the ictal onset zone (Knowlton et al., 1997; Minassian et al., 1999; Morioka et al., 1999; Iida et al., 2005a; Oishi et al., 2006b). Clusters of MEG sources outlined the extensive focal cortical dysplasia, which was partially defined on MRI (Otsubo et al., 2001a). When MEG and ECoG data delineated the extended epileptogenic zone, complete resection of the focal cortical dysplasia relieved seizures (Iida et al., 2005a). In nonlesional epilepsy, complete resection of the interictal zone delineated by MEG sources was highly predictive of seizure relief and good outcome (Smith et al., 1995).

Compared with other lesions, focal cortical malformations have pronounced MEG activity (Fig. 27.1). This finding supports the concept that focal cortical malformation is intrinsically epileptogenic (Morioka et al., 1999; Otsubo et al., 2001a; Bast et al., 2004). The extensive epileptogenic zone contiguous to focal cortical malformation differs from the remote epileptic cortex present with tumors because epileptogenic dysplastic neurons that can produce seizures exist in this zone. In addition, focal cortical dysplasia is often located around the frontal and central regions and, in some cases, causes status epilepticus (Desbiens et al., 1993; Otsubo et al., 2001a; Otsubo et al., 2001b; Otsubo et al., 1993; Sheth et al., 1997; Kuzniecky et al., 1988; Kuzniecky and Jackson, 1995). When MEG indicates epileptic focal cortical malformation adjacent to eloquent cortex, extraoperative ECoG is required for precise localization of the epileptogenic zone and for functional mapping. Complete resection of cortical dysplastic lesions results in good outcomes in the majority of children with cortical malformations

Fig. 27.1. In a 17 year-old boy with right frontal focal cortical dysplasia and nocturnal tonic seizures since 6 years of age, (A) axial T2-weighted MRI and (B) coronal FLAIR show a subtle thickened gyrus in the right middle frontal gyrus (arrows). (C, D) Axial T1-weighted MRI showing a cluster of MEG dipoles (closed triangles, locations of spike sources; tails, orientation and strength of dipole moments) along with the abnormal gyrus.

CLINICAL NEUROPHYSIOLOGY OF CORTICAL MALFORMATIONS (Header et al., 2004). Multiple subpial transection, in conjunction with incomplete cortical resection, does not improve seizure outcome in patients with cortical dysplastic lesions within or adjacent to the functional cortex. Location of a cortical dysplastic lesion outside eloquent cortex and ability to completely excise the lesion are important predictors for seizure relief and good outcome. 27.2.2. MEG dipoles with occult focal cortical dysplasia on MRI Edwards et al. (2000) reported that complete resection of MRI-apparent lesions resulted in seizure-free outcomes in 35 patients (49%) with intractable focal epilepsy due to cortical malformations, which were identified by MRI. Focal cortical dysplasia, however, is sometimes occult on MRI (Desbiens et al., 1993; Otsubo et al., 2001b). SEEG study found that seizures occurred outside the dysplastic lesion in subset of patients with focal cortical dysplasia. The epileptogenic zone may involve histologically proven normal tissue; incomplete lesion resection could give a good outcome (Francione et al., 2003a). Therefore, surgical planning for extensive removal of the epileptogenic zone extending beyond the cortical dysplastic lesion on MRI, using information from MEG dipole sources, can achieve better control of intractable seizures. In patients with no apparent lesions on preoperative imaging MEG showed clustered dipole sources (Oishi et al., 2006a). MEG dipoles, with corroborating subdural grid results, confirmed the epileptogenic zone, which was ultimately resected and was shown histologically to contain focal cortical dysplasia (Fig. 27.2). MEG, therefore, was useful in localizing the focal cortical dysplasia, even when the condition was not detected by MRI. In a certain case whose MRI is reported normal, the MEG-clustered region

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indicates the subtle cortical malformation. The further advanced modality such as curvilinear reformatting techniques, higher magnetic MRI can realize the focal cortical dysplasia (Bastos et al., 1999; Antel et al., 2003; Knake et al., 2005). MEG results may indicate a surgical option for patients who previously may not have been considered for resective surgery. 27.2.3. Brain tumors and dysembryoplastic neuroepithelial tumor Tumors other than dysembryoplastic neuroepithelial tumor had associated MEG spike sources beyond their borders. This corroborates a demonstrated frequent association of dysembryoplastic neuroepithelial tumor with cortical dysplasia and the importance of excision of both to seizure outcome (Sakuta et al., 2005). MEG clustered spike sources within lesions were only found with cortical dysplasia (Otsubo et al., 2001a; Iida et al., 2005a). No epileptic sources were located deep within the lesions and there were an increasing number of spikes localizing near or along the tumor border with gliomas when compared to other neoplasms. This has suggested that neurons immediately along the border of gliomas become more epileptogenic as a result of alterations in excitability caused by interactions with and the presence of infiltrating tumor cells. 27.2.4. Tuberous sclerosis complex In children with tuberous sclerosis complex, MEG showed multiple patterns of spike sources, including a single clustered MEG spike source, bilateral or multiple clustered MEG spike sources and bilateral scattered MEG spike sources without clusters (Iida et al., 2005b). MEG added informative data to EEG and MRI studies for evaluation of epileptic zones in tuberous sclerosis complex (Wu et al., 2006).

Fig. 27.2. In a 14-year-old boy with asymmetric epileptic spasms of possible left frontal origin, MRI was reported to be normal. (A) Coronal and (B) sagittal T1-weighted MRI showing a cluster of MEG dipoles (closed triangles, locations of spike sources) over the left inferior to superior frontal gyri accompanied by left fronto-centro-temporal EEG interictal epileptic discharges. The MEG dipoles were overlaid on to the single MRI slice. Examination of pathology specimens from left fronto-temporal cortical excision revealed microdysgenesis.

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H. OTSUBO AND K. IMAI tigate interactions between the cortex and underlying heterotopic structures. 27.3.1. Focal cortical dysplasia

Fig. 27.3. Three dimensional MRI shows various frequency epileptiform discharges using synthetic aperture magnetometry. Relatively fast epileptiform discharges (5–35 Hz) are seen around the cortical tubers. Slow waves (0–5Hz) indicate the areas of the cortical tubers.

Synthetic aperture magnetometry (SAM) is a new technique based on spatially filtered MEG (Vrba and Robinson, 2001). It allows three-dimensional estimation of magnetic fields. The strength of the SAM technique in epilepsy is its ability to identify the frequency-dependent volumetric distribution of neuromagnetic activation. SAM for relatively slow frequency epileptiform discharges detected irritable zones for 74% of anatomical tubers on MRI in comparison to conventional dipole modeling, which detected 45% (Xiao et al., 2006) (Fig. 27.3). MEG dipoles were commonly near a small tuber or a corner of a big tuber but not in the center of a big tuber. It would be interesting to see whether surgical data confirm the usefulness of SAM in the clinical management of tuberous sclerosis complex and other cortical malformations.

27.3. Intracranial EEG Intracranial EEG uses subdural and depth electrodes to record on the cortex and subcortical tissues to probe the mesial temporal regions or certain intracerebral regions. When placed either intraoperatively under anesthesia or extraoperatively, corticography and depth electrodes can localize the ictal onset zone, interictal zone and functional cortex for epilepsy surgery. As SEEG accurately placed electrodes to record the interictal and ictal discharges in the lesion of neuronal migrations guided by high resolution MRI and PET, distinctive neurophysiological findings correlated to the histopathology. Especially for heterotopic neurons, including periventricular and subcortical nodular heterotopia, double cortex or band heterotopias, SEEG seems to be a tool to record ictal discharges and inves-

In the lesion of Taylor-type cortical dysplasia characterized by giant dysmorphic neurons and balloon cells, interictal SEEG showed complete disruption of background activity, high-frequency fast spikes and polyspikes, occasionally of high amplitude, interspersed with flattenings, fast, lower-amplitude activity and short bursts of fusiform micropolyspikes (Tassi et al., 2002, 2005; Francione et al., 2003b). In both architectural dysplasia characterized by abnormal cortical neurons and ectopic neurons in white matter, and cytoarchitectural dysplasia characterized by giant neurofilament-enriched neurons and altered cortical lamination, there were no distinctive features observed in Taylor-type cortical dysplasia (Tassi et al., 2002). Chassoux (2003) reported continuous, rhythmic or pseudorhythmic spikes or polyspikes, with frequencies ranging from 0.5–10 Hz, but usually 1–3 Hz. The pseudoperiodic spikes or burst of spikes, interrupted by depression or suppression of activity, resembled suppression burst patterns. These rhythmic spike discharges were simultaneously recorded on scalp EEG. The site of maximal rhythmic spike discharges correlated with dysplastic cortex in which intravenous diazepam had no effect on spike discharges. Seizure frequency in Taylor-type focal cortical dysplasia was significantly greater than in architectural dysplasia (Tassi et al., 2002). The ictal pattern was recognized as medium-amplitude 14–18 Hz rhythmic activity followed by a low-amplitude recruiting fast activity in 12 of 21 patients with Taylor-type cortical dysplasia (Francione et al., 2003b). Boonyapisit et al. (2003) analyzed 163 seizures in patients with focal cortical dysplasia on intracranial video EEG and defined three ictal onset patterns: 1) paroxysmal fast (faster than 10 Hz with evolution in amplitude and/or frequency); 2) high-amplitude repetitive spiking (3–10 Hz with evolution in amplitude and/or frequency); 3) paroxysmal fast with repetitive spiking. Taylor-type cortical dysplasias showed fast activity at the seizure onset significantly more commonly than repetitive spikes in nondysplastic lesions (Turkdogan et al., 2005). These authors found that seizure onset was focal in both dysplastic and nondysplastic patients, although seizures in the dysplastic patients propagation occurred more rapidly than in the nondysplastic patients. They claimed no significant difference in the morphological type of interictal epileptiform abnormalities between groups.

CLINICAL NEUROPHYSIOLOGY OF CORTICAL MALFORMATIONS Hippocampal sclerosis was often associated with cortical dysplasia in the temporal neocortex (Fauser and Schulze-Bonhage, 2006). Seizure onset from temporal neocortical dysplasia was significantly more frequent in patients with mild hippocampal sclerosis than in those with severe hippocampal sclerosis. The contribution of the hippocampus to seizure generation corresponded with the severity of hippocampal sclerosis. However, the type of cortical dysplasia alone did not predict a neocortical or mesial seizure onset. Intra-lesional stimulation using either high frequency (50 Hz) or low frequency (1 Hz) elicited seizures similar to the spontaneous seizures (Chassoux, 2003). The thresholds after discharge and provoking seizures by electrical stimulation were closer to the motor cortical thresholds and lower than those in patients without cortical malformations (Chitoku et al., 2001, 2003). Paired-pulse stimulation of the focus revealed abnormally enhanced intracortical inhibition at interstimulus interval of 1–10 ms (maximum 22%) compared with control stimulation of the hand somatosensory area (interstimulus interval of 1–2 ms, maximum 18%) during the interictal state (Matsumoto et al., 2005). SEEG recorded peculiar interictal activity on intralesional recordings, colocalization of the epileptogenic zone and dysplastic areas, and favorable outcome correlating with complete resection of focal cortical dysplasias (Chassoux, 2003). In one patient with MRI occult parietal focal cortical dysplasia, the initial subdural electrodes failed to localize seizure onset; 2 years later the second depth electrodes, 2 cm below the surface of the parietal cortex, showed a discrete site of continuous interictal spiking and seizure onset (Privitera et al., 2000). The intracranial electrodes explore only a very small volume of brain tissue. The amplitude can be misleading when electrodes mislocate a few millimeters away from the generators. Intracranial electrodes are a unique and powerful tool in the evaluation of refractory epilepsy, although they are also a tool that is potentially dangerous because they easily give the illusion of precision and that they are exploring large brain regions (Gotman, personal communication). In several studies using high-sampling digital intracranial EEG, very fast activities ranging from 20–400 Hz were observed at the beginning of partial seizures in the ictal onset zone (Allen et al., 1992; Fisher et al., 1992; Alarcon et al., 1995; Traub et al., 2001; Worrell et al., 2004; Akiyama et al., 2005; Jirsch et al., 2006; Ochi et al., 2007). High-frequency oscillations reported at the onset of partial seizures have varied and included 20–80 Hz (Alarcon et al., 1995), 40–120 Hz (Fisher et al., 1992), 60–100 Hz (Worrell et al., 2004),

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70–90 Hz (Traub et al., 2001), 80–110 Hz (Allen et al., 1992), 60–150 Hz (Akiyama et al., 2005), 64–220 Hz (Ochi et al., 2007) and 120–400 Hz (Jirsch et al., 2006) in patients with cortical malformations. Despite the variety of faster frequencies, these reports demonstrated that high frequency oscillations are related to seizure onset (Fig. 27.4). 27.3.2. Periventricular nodular heterotopia Tassi et al. (2005) reported epilepsy surgery in 10 patients with periventricular nodular heterotopia. By SEEG in nine patients, interictal activity within nodules was similar: low-voltage, low-frequency interrupted by frequent high-voltage spikes followed by positive waves in eight patients. The interictal spikes were sustained almost pseudorhythmic, and always asynchronous between different nodules in one patient, though morphofunctional organization of all nodules was considered similar because of background and interictal nodule activity was similar in all patients. They never observed these types of SEEG activity in patients with other lesions or cortical malformation.

A Fig. 27.4. (A) Right hemispheric brain of the same patient as in Figure 27.1 showing cortical excision area (open circle), motor function (open triangles) and sensory function (open squares). The large square encloses 13 electrodes showing (B) high-frequency oscillations (HFOs) at the onset of a seizure (low-frequency filter, 40 Hz). (C, top) One single electrode, 34, of the 13 electrodes shows amplitude changes in HFOs during 20 s at seizure onset. (C, below) Multiple band frequency analysis shows dynamic changes of power spectra of HFOs on electrode 34. Wide-band HFOs increases up to 160 Hz at seizure onset, with following sustained isolated single band (open triangle) associated with secondarily generalized seizures. Power ranges 0–15 mV2. Epilepsy surgery revealed cortical dysplasia type IIb with balloon cells in the right frontal cortical excision, without any signs of tuberous sclerosis complex.

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H. OTSUBO AND K. IMAI G21 G22 G23 G24 G32 G33 G34 G35 G36 G44 G45 G46 G47 400 uV 2 sec

B 200 uV 2 sec

15

160Hz

12

120 0

9

6 3

60 0

0

C

5

10

15

20sec

Fig. 27.4. (Continued).

Ictal activity never started from nodules alone (Tassi et al., 2005). In five cases simultaneous activation of one nodule and the overlying cortex was observed. In three cases, ictal discharges originated from the cortex. Kothare et al. (1998) showed 30–50 Hz low-voltage beta activity starting from heterotopia in two of three patients and from both hippocampi and heterotopia in one patient. Dubeau et al. (1995) had four patients with seizures starting temporo-parieto-occipital involving mesial temporal structures and two with both these and heterotopia. The same study showed generalized, unilateral or bilateral temporal, unilateral, bilateral or multilobar onsets on the scalp EEG. Battaglia et al. (2005) suggested that seizures were generated by abnormal anatomical circuitries, including the heterotopic nodules and adjacent cortical areas. 27.3.3. Double cortex Bernasconi et al. (2001) reported eight patients (five women) with double cortex syndrome and intractable epilepsy. Six patients underwent invasive EEG recordings, three of them with subdural grids and

three with SEEG. Although EEG showed multilobar epileptic abnormalities, regional or focal seizure onset was recorded in all. Surgical procedures included multiple subpial transections, frontal lesionectomy, temporal lobectomy and an additional anterior callosotomy resulted in no significant improvement in five patients. In one patient with subcortical band heterotopia, interictal SEEG showed the regional different sharp waves, spikes, slow waves and frequent spikes in the bilateral heterotopias without correlates to the ictal onset zone (Mai et al., 2003). Ictal SEEG had two focal origins in either outer and heterotopic cortices in the middle and inferior temporal gyri or fusiform and lingual gyri in 12 seizures. The limited resection reduced seizures by 90% and spared neurological status. Bernasconi et al. (2001) suggested that the time courses of propagation were nonspecific, perhaps from the result of spreading or of multifocal generators, thus representing a misleading pseudotemporal lobe localization similar to that found in patients with hypothalamic hamartoma or periventricular nodular heterotopia.

CLINICAL NEUROPHYSIOLOGY OF CORTICAL MALFORMATIONS

27.4. Scalp EEG As a valuable clinical tool, EEG helps physicians 1) to localize and lateralize cortical abnormalities, 2) to characterize cortical malformations, 3) to determine appropriate medical or surgical treatment of epilepsy and epileptic syndromes and 4) to select candidates for epilepsy surgery. Specific cortical malformations produce identical EEG patterns (Bernardina et al., 1996). These patterns include background activities and interictal epileptiform discharges on scalp EEG, ictal epileptiform discharges on prolonged video EEG. Current digital EEG techniques can provide multichannel prolonged recordings and time-locked video monitoring of patients for reviewing seizure semiology and identifying the epileptiform discharges that occur with second by second resolution. 27.4.1. Interictal epileptiform discharges on scalp EEG 27.4.1.1. Focal epileptiform discharges There were focal, rhythmic, repetitive sharp waves or spikes, at times continuous, and stereotypical epileptiform discharges seen on scalp EEG in patients with focal cortical dysplasia. Of 34 patients with focal cortical dysplasia, 15 had either of two patterns of focal, rhythmic, stereotypical (Fig. 27.5), repetitive sharp waves or spikes lasting more than 1 s without any clinical manifestations (Gambardella et al., 1996). In eight of the 15 patients the rhythmic pattern was a train of repetitive, rhythmic, 4–10 Hz sharp waves or spikes lasting 1–4 s; the remaining seven patients had a pattern that consisted of quasicontinuous, slower, rhythmic,

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2–7 Hz sharp wave activity. The presence of rhythmic epileptiform discharges on scalp EEG correlated with the occurrence of continuous rhythmic epileptiform discharges on ECoG and suggested the epileptogenic zone in 12 of the 15 patients. In two patients, repeated MRIs detected no lesions but EEG and ECoG showed rhythmic epileptiform discharges. These observations indicated that focal, rhythmic, stereotyped, sharp wave or spike patterns represent intrinsic epileptogenic discharges, even in the absence of MRI abnormalities. Epilepsia partialis continua can be seen not only in Rasmussen’s encephalitis but also in cortical dysplasia involving the motor area and hemimegalencephaly (Fusco et al., 1992; Kuzniecky and Powers, 1993; Ochi et al., 2002; Misawa et al., 2004). Epilepsia partialis continua happens in patients with tumors, vascular lesions and trauma around the central cortex. When first reported, five of 15 children with cortical malformations had positive epileptiform discharges (Otsubo et al., 1997). These positive discharges were attributed to projections by either upside-down heterotopic neurons or horizontal/oblique layered epileptic neurons located in the lateral fold in a deep fissure, such as the rolandic or sylvian fissure. The positive epileptiform discharges correlated to early age of seizure onset, the presence of dysplastic cortical lesions on MRI around the rolandic fissure, hemiparesis and a less favorable postsurgical seizure outcome. 27.4.1.2. Hemispheric or diffuse epileptiform discharges Patients with cortical malformations including hemimegalencephaly, polymicrogyria and focal cortical dyspla-

Fp1-F7 F7-T3 T8-T5 T5-O1 Fp2-F8 F8-T4 T4-T6 T6-O2 Fp1-F3 F3-C3 C3-P3 P3-O1 Fp2-F4 F4-C4 C4-P4 P4-O2 Fz-Cz Cz-Pz

100 uV 1 sec

Fig. 27.5. Anteroposterior bipolar EEG shows continuous, focal, rhythmic and stereotyped low-to-medium amplitude polyspikes and slow waves (closed triangle) over the right fronto-central region at F4 and Cz in the same patient as in Figure 27.1.

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sias frequently present with early infantile epileptic encephalopathy (Ohtahara syndrome) (Ohtsuka et al., 1999), although a combination of early myoclonic epilepsy and cortical malformations is rare (Aicardi and Ohtahara, 2002). Neonates with severe hemimegalencephaly (Vigevano et al., 1996) or focal cortical dysplasia (Pedespan et al., 1995; Komaki et al., 1999) had early infantile epileptic encephalopathy with a suppression burst pattern (Fig. 27.6). Usually, suppression burst patterns associated with cortical malformations initially predominate on the side with the malformation but later involve the opposite hemisphere as well (Vigevano et al., 1989). Suppression burst patterns change to hypsarrhythmia in infancy and diffuse pseudorhythmic spike-waves or multifocal spikes in early childhood (Vigevano et al., 1996). In rare cases, suppression burst patterns continue to appear decades after infancy (Ohtsuka et al., 1999). Patients with hemimegalencephaly refractory to antiepileptic drugs commonly had focal and subclinical EEG seizures (Vigevano et al., 1996). Focal seizures were common in patients with cortical malformations, especially focal cortical dysplasia (Raymond et al., 1995). Recurrent focal electrographic seizures occurred in 15 of 34 patients with intractable epilepsy secondary to focal cortical dysplasia (Palmini et al., 1995). Four of 12 patients with hemimegalencephaly had asymmetrical, high-amplitude triphasic complexes (Paladin et al., 1989). The triphasic complexes consisted of high-amplitude positive slow spikes preceded by small negative waves and followed by high-amplitude negative very slow waves. Diffuse bi- or triphasic, pseudoperiodic complexes were observed in three of 14 patients with diffuse cortical dysplasia (Bureau et al., 1996).

Patients with both diffuse cortical malformations, such as diffuse pachygyria, lissencephaly and focal cortical dysplasia, polymicrogyria or hemimegalencephaly present with epileptic spasms, including infantile spasms (Kobayashi et al., 2001). Patients with focal cortical malformations can have asymmetric spasms (Lortie et al., 2002). Ictal EEGs of patients with focal cortical malformations show asymmetric slow waves with superimposed fast waves. Epileptic spasms often precede, follow or intermix with focal seizures (Pachatz et al., 2003). Patients with tuberous sclerosis complex or cortical dysplasia (Gobbi et al., 1987) and microdysgenesis (Akiyama et al., 2005) can have periodic spasms. Epileptic spasms have also been seen in adults with cortical dysplasia and correlate on EEG to positive, diffuse, high-amplitude slow wave activity with superimposed fast activity during spasms, followed by a diffuse flattening. These EEG findings in adult epileptic spasms are similar to those in infantile spasms (Cerullo et al., 1999). A hypsarrhythmia pattern of chaotic, random, asynchronous high-voltage slow waves and spikes (Gibbs and Gibbs, 1952) often occurs in infancy with infantile spasms secondary to cortical malformations, such as lissencephaly, focal cortical dysplasia and tuberous sclerosis complex (Boyd and Dan, 2001). Chugani et al (1990) reported cases of infantile spasms with focal or lobar cortical malformations. While EEG showed hypsarrhythmia during infantile spasms, subsequent regional temporo-parieto-occipital slow waves or spike and waves appeared and corresponded to PET abnormalities. Cerebral malformations may be present in 30% of West syndrome patients (Dulac et al., 2002). Infantile spasms occur in tuberous

Fp2-F8 F8-T4 T4-T6 T6-O2 Fp1-F7 F7-T3 T3-T5 T5-O1 F4-C4 C4-P4 P4-O2 F3-C3 C3-P3 P3-O1 T4-Cz Cz-T3

A

B

50 µV 1 sec

Fig. 27.6. In a 2-month-old girl with left hemimegalencephaly and early infantile epileptic encephalopathy, anteroposterior bipolar EEG shows asymmetric suppression and burst pattern consisting of a period of suppression lasting 2–3 s (closed triangle) between high-amplitude epileptic bursts lasting 1–2 s over the left hemisphere during wakefulness (A) and sleep (B).

CLINICAL NEUROPHYSIOLOGY OF CORTICAL MALFORMATIONS sclerosis complex, but typical hypsarrhythmia is rare (Curatolo et al., 2001). EEGs of patients with hemimegalencephaly have asymmetrical or unilateral hypsarrhythmia (Tjiam et al., 1978). Patients with diffuse or bilateral cortical malformations (Boyd and Dan, 2001), band heterotopia (Ricci et al., 1992), bilateral perisylvian polymicrogyria (Kuzniecky et al., 1994), diffuse gray matter heterotopia and microdysgenesis present with diffuse slow spike-wave bursts similar to those seen in Lennox– Gastaut syndrome. Some 20% of patients with electrical status epilepticus during slow sleep had extensive fronto-parietal polymicrogyria, left temporal polymicrogyria or perisylvian polymicrogyria (Guerrini et al., 1998). Electrical status epilepticus during slow sleep occurred in 25% of pediatric epilepsy patients with unilateral and 18% with bilateral focal cortical dysplasia but was not present in patients with diffuse cortical malformations and schizencephaly (Bernardina et al., 1996; Caraballo et al., 2004). 27.4.2. Abnormal background activities Abnormal rhythmic scalp EEG patterns with various amplitude, frequency and region combinations are associated with cortical malformations. These abnormal rhythmic discharges alter with age and have to be compared to the age specific normal background rhythm in children. In such lissencephaly, abnormal rhythmic discharges were most prominent at 4 months of age and often gradually diminished after one year (Bernardina et al., 1996).

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27.4.2.1. High-amplitude slow waves Extremely high-amplitude (> 400 mV) slow waves (theta and delta, 1–7 Hz) originated bilaterally in patients with lissencephaly (smooth lissencephaly) characterized by absence of (agyria) and decreased (pachygyria) gyri (Fig. 27.7) (de Rijk-van Andel et al., 1992). Patients with hemimegalencephaly had asymmetrical, high-amplitude slow rhythms in the hemisphere with the lesion but normal background activity in the opposite hemisphere (Paladin et al., 1989; Vigevano et al., 1989). (Note that in cases with marked asymmetrical cerebral malformations, neuroradiological imaging must confirm the correlation of the electrode position with the anatomical structure because the malformed occipital lobe often crosses the midline, especially in cases with hemimegalencephaly (Vigevano et al., 1996).) Relatively high-amplitude ( 400 mV), diffuse or bilateral, frequent or continuous mixed patterns of abnormal fast and slow activities occurred in patients with lissencephaly (Gastaut et al., 1987), agyria-pachygyria (de Rijk-van Andel et al., 1992; Quirk et al., 1993) and holoprocencephaly (Hahn et al., 2003). Among patients with type I lissencephaly who showed anterior dominant (Miller–Dieker syndrome) and posterior dominant cortical abnormalities (X-linked lissencephaly) on MRI, the topographic pattern of EEG abnormalities did not differ (Boyd and Dan, 2001). 27.4.2.2. Fast waves Medium- to high-amplitude (> 50 mV) fast waves (alpha and beta, 8–18 Hz), particularly evident during

FP1-T3 T3-O1 FP1-C3 C3-O1 FP2-T4 T4-O2 FP2-C4 C4-O2 T3-C3 C3-Cz Cz-C4 C4-T4

750 uV 1 sec

A Fig. 27.7. In a 4-month-old boy with Miller–Dieker syndrome consisting of lissencephaly type 1, (A) neonatal bipolar montage EEG shows extreme high amplitude up to 800 mV irregular slow waves 0.5–1 Hz (closed triangle) over left central and bilateral temporo-occipital regions. (B) Computed tomography shows bilateral diffuse agyria.

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H. OTSUBO AND K. IMAI Fp1-F7 F7-T3 T3-T5 T5-O1 Fp2-F8 F8-T4 T4-T6 T5-O2 Fp1-F3 F3-O3 C3-P3 P3-O1 Fp2-F4 F4-C4 C4-P4 P4-O2 Fz-Cz

100 uV 1 sec

Cz-Pz

A Fp1-Fz Fz-T3 T3-T5 T5-O1 Fp2-F8 F8-T4 T4-T6 T6-O2 Fp1-F3 F3-C3 C3-P3 P3-O1 Fp2-F4 F4-C4 C4-P4 P4-O2 Fz-Cz 100 uV 1 sec

Cz-Pz

B Fig. 27.8. In an 11-week-old boy with right hemimegalencephaly and a seizure history of body jerks with extension of both arms and crying, (A) anteroposterior bipolar EEG shows asymmetrical 7–9 Hz abnormal alpha-like activities (closed triangle) over the right central region and posterior dominant 4–5 Hz superimposed 2 Hz delta slow waves (open circles) in the normal left hemisphere during wakefulness. (B) During sleep, asynchronous 12–14 Hz sleep spindles (open circles) are seen over the bilateral central regions. Intermittent low-to-medium amplitude asymmetric 6–8 Hz theta to alpha range discharges (closed triangle) occur over the right hemisphere with central predominance.

REM sleep, occurred in 11 of 14 patients with diffuse cortical dysplasia (de Rijk-van Andel et al., 1992). This pattern located over the lesion in two of seven patients with focal cortical dysplasia (Bureau et al., 1996) and was located over less than 50% of the cortex in patients with focal pachygyria (Liang et al., 2002). Lower amplitude (< 50 mV) bursts of fast rhythmic activity (alpha or beta, 8–18 Hz) occurred symmetrically in patients with lissencephaly type I, asymmetrically in those with hemimegalencephaly (Fig. 27.8) and hemispheric polymicrogyria and focally in cases of focal cortical dysplasia (Boyd and Dan, 2001).

27.5. Conclusions MEG can directly observe the neurophysiological activity of cortical malformations with exquisite temporal detail and accurately localize epileptogenic sources on high-resolution MRI. Intracranial EEG precisely localizes the intrinsically epileptogenic cortical malformations and ictal propagations between cortical malformations and either neighboring or remote cortices in the epileptic network. Diffuse, hemispheric and focal abnormal background activity and epileptiform EEG discharges highly correlate to patterns of

CLINICAL NEUROPHYSIOLOGY OF CORTICAL MALFORMATIONS cortical malformation. MEG, intracranial and scalp EEG probably cannot distinguish all cortical malformations purely on the basis of the physiological findings, but simply they can identify the epileptogenic zone.

Acknowledgment We thank Mrs Carol L Squires for her editorial assistance and Drs Yoko Ohtsuka and Tomoyuki Akiyama for the EEG figures.

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lography and electrocorticography. Neurol Res 24: 525–530. Jirsch JD, Urrestarazu E, LeVan P, et al. (2006). Highfrequency oscillations during human focal seizures. Brain 129: 1593–1608. Knake S, Triantafyllou C, Wald LL, et al. (2005). 3T phased array MRI improves the presurgical evaluation in focal epilepsies: a prospective study. Neurology 65: 1026–1031. Knowlton RC, Laxer KD, Aminoff MJ, et al. (1997). Magnetoencephalography in partial epilepsy: clinical yield and localization accuracy. Ann Neurol 42: 622–631. Kobayashi K, Ohtsuka Y, Ohno S, et al. (2001). Clinical spectrum of epileptic spasms associated with cortical malformation. Neuropediatrics 32: 236–244. Komaki H, Sugai K, Sasaki M, et al. (1999). Surgical treatment of a case of early infantile epileptic encephalopathy with suppression-bursts associated with focal cortical dysplasia. Epilepsia 40: 365–369. Kothare SV, VanLandingham K, Armon C, et al. (1998). Seizure onset from periventricular nodular heterotopias: depth-electrode study. Neurology 51: 1723–1727. Kuzniecky R, Jackson G (1995). Neuroimaging in epilepsy. In: Magnetic Resonance in Epilepsy. Raven Press, New York, pp. 27–48. Kuzniecky R, Powers R. (1993). Epilepsia partialis continua due to cortical dysplasia. J Child Neurol 8: 386–388. Kuzniecky R, Berkovic S, Andermann F, et al. (1988). Focal cortical myoclonus and rolandic cortical dysplasia: clarification by magnetic resonance imaging. Ann Neurol 23: 317–325. Kuzniecky R, Andermann F, Guerrini R (1994). The epileptic spectrum in the congenital bilateral perisylvian syndrome. CBPS Multicenter Collaborative Study. Neurology 44: 379–385. Lewine JD, Orrison WW (1995). Magnetoencephalography and magnetic source imaging. In: Orrison WW, Lewine JA, MF Hartshorne (Eds.), Functional Brain Imaging. Mosby Year Book, St Louis, pp. 369–417. Liang JS, Lee WT, Young C, et al. (2002). Agyria– pachygyria: clinical neuroimaging and neurophysiologic correlations. Pediatr Neurol 27: 171–176. Lortie A, Plouin P, Chiron C, et al. (2002). Characteristics of epilepsy in focal cortical dysplasia in infancy. Epilepsy Res 51: 133–145. Mai R, Tassi L, Cossu M, et al. (2003). A neuropathological, stero-EEG, and MRI study of subcortical band heterotopia. Neurology 60: 1834–1838. Matsumoto R, Kinoshita M, Taki J, et al. (2005). In vivo epileptogenecity of focal cortical dysplasia: a direct cortical paired stimulation study. Epilepsia 46: 1744–1749. Minassian BA, Otsubo H, Weiss S, et al. (1999). Magnetoencephalographic localization in pediatric epilepsy surgery: comparison with invasive intracranial electroencephalography. Ann Neurol 46: 627–633. Misawa S, Kuwabara S, Hirano S, et al. (2004). Epilepsia partialis continua as an isolated manifestation of motor cortical dysplasia. J Neurol Sci 225: 157–160.

CLINICAL NEUROPHYSIOLOGY OF CORTICAL MALFORMATIONS Morioka T, Nishio S, Ishibashi H, et al. (1999). Intrinsic epileptogenicity of focal cortical dysplasia as revealed by magnetoencephalography and electrocorticography. Epilepsy Res 33: 177–187. Ochi A, Otsubo H, Honda Y, et al. (2002). Electroencephalographic dipoles of spikes with and without myoclonic jerks caused by epilepsia partialis continua. J Child Neurol 17: 127–131. Ochi A, Otsubo H, Donner EJ, et al. (2007). Dynamic changes of ictal high-frequency oscillations in neocortical epilepsy: using multiple band frequency analysis. Epilepsia 48: 286–296. Ohtsuka Y, Ohno S, Oka E (1999). Electroclinical characteristics of hemimegalencephaly. Pediatr Neurol 20: 390–393. Oishi M, Kameyama S, Masuda H, et al. (2006a). Single and multiple clusters of magnetoencephalographic dipoles in neocortical epilepsy: significance in characterizing the epileptogenic zone. Epilepsia 47: 355–364. Oishi M, Otsubo H, Iida Y, et al. (2006b). Preoperative simulation of intracerebral epileptiform discharges: synthetic aperture magnetometry virtual sensor analysis of interictal magnetoencephalography data. J Neurosurg 105 (1 suppl.): 41–49. Otsubo H, Hwang PA, Jay V, et al. (1993). Focal cortical dysplasia in children with localization-related epilepsy: EEG, MRI, and SPECT findings. Pediatr Neurol 9: 101–107. Otsubo H, Steinlin M, Hwang PA, et al. (1997). Positive epileptiform discharges in children with neuronal migration disorders. Pediatr Neurol 16: 23–31. Otsubo H, Ochi A, Elliott I, et al. (2001a). MEG predicts epileptic zone in lesional extrahippocampal epilepsy: 12 pediatric epilepsy surgery cases. Epilepsia 42: 1523–1530. Otsubo H, Chitoku S, Ochi A, et al. (2001b). Diagnosis and treatment of malignant rolandic-sylvian epilepsy in children: magnetoencephalography, intracranial invasive video-EEG and surgery. Neurology 57: 590–596. Otsubo H, Snead OCIII (2001c). Magnetoencephalography (MEG) and magnetic source imaging (MSI) in children. J Child Neurol 16: 227–235. Otsubo H, Iida K, Oishi M, et al. (2005). Neurophysiologic findings of neuronal migration disorders: intrinsic epileptogenicity of focal cortical dysplasia on electroencephalography, electrocorticography, and magnetoencephalography. J Child Neurol 20: 357–363. Pachatz C, Fusco L, Vigevano F (2003). Epileptic spasms and partial seizures as a single ictal event. Epilepsia 44: 693–700. Paetau R, Hamalainen M, Hari R, et al. (1994). Magnetoencephalographic evaluation of children and adolescents with intractable epilepsy. Epilepsia 35: 275–284. Paladin F, Chiron C, Dulac O, et al. (1989). Electroencephalographic aspects of hemimegalencephaly. Dev Med Child Neurol 31: 377–383. Palmini A, Gambardella A, Andermann F, et al. (1995). Intrinsic epileptogenicity of human dysplastic cortex as

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suggested by corticography and surgical results. Ann Neurol 37: 476–487. Pedespan JM, Loiseau H, Vital A, et al. (1995). Surgical treatment of an early epileptic encephalopathy with suppressionbursts and focal cortical dysplasia. Epilepsia 36: 37–40. Privitera MD, Yeh H-S, Blisard K, Sanchez N (2000). Detection of epileptogenic focal cortical dysplasia by depth, not subdural electrodes. Neurosurg Rev 23: 49–51. Quirk JA, Kendall B, Kingsley DP, et al. (1993). EEG features of cortical dysplasia in children. Neuropediatrics 24: 193–199. Raymond AA, Fish DR, Sisodiya SM, et al. (1995). Abnormalities of gyration, heterotopias, tuberous sclerosis, focal cortical dysplasia, microdysgenesis, dysembryoplastic neuroepithelial tumour and dysgenesis of the archicortex in epilepsy: Clinical, EEG and neuroimaging features in 100 adult patients. Brain 118: 629–660. Ricci S, Cusmai R, Fariello G, et al. (1992). Double cortex. A neuronal migration anomaly as a possible cause of Lennox–Gastaut syndrome. Arch Neurol 49: 61–64. Sakuta R, Otsubo H, Nolan MA, et al. (2005). Recurrent intractable seizures in children with cortical dysplasia adjacent to dysembryoplastic neuroepithelial tumor. J Child Neurol 20: 377–384. Sato S, Balish M, Muratore R (1991). Principles of magnetoencephalography. J Clin Neurophysiol 8: 144–156. Sheth RD, Gutierrez AR, Riggs JE (1997). Rolandic epilepsy and cortical dysplasia: MRI correlation of epileptiform discharges. Pediatr Neurol 17: 177–179. Smith JR, Schwartz BJ, Gallen C, et al. (1995). Utilization of multichannel magnetoencephalography in the guidance of ablative seizure surgery. J Epilepsy 8: 1191–1203. Tassi L, Colombo N, Garbelli R, et al. (2002). Focal cortical dysplasia: neuropathological subtypes, EEG, neuroimaging and surgical outcome. Brain 125: 1719–1732. Tassi L, Colombo N, Cossu M, et al. (2005). Electroclinical, MRI and neuropathological study of 10 patients with nodular heterotopia, with surgical outcomes. Brain 128: 321–337. Tjiam AT, Stefanko S, Schenk VW, de Vlieger M. (1978). Infantile spasms associated with hemihypsarrhythmia and hemimegalencephaly. Dev Med Child Neurol 20: 779–798. Traub RD, Whittington MA, Buhl EH, et al. (2001). A possible role for gap junctions in generation of very fast EEG oscillations preceding the onset of, and perhaps initiating, seizures. Epilepsia 42: 153–170. Turkdogan D, Duchowny M, Resnick T, Jayakar P (2005). Subdural EEG patterns in children with Taylor-type cortical dysplasia: comparison with nondysplastic lesions. J Clin Neurophysiol 22: 37–42. Vigevano F, Bertini E, Boldrini R, et al. (1989). Hemimegalencephaly and intractable epilepsy: benefits of hemispherectomy. Epilepsia 30: 833–843. Vigevano F, Fusco L, Granata T, et al. (1996). Hemimegalencephaly: clinical and EEG characteristics. In: R Guerrini, F Andermann, R Canapicchi, et al. (Eds.), Dysplasias of

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Chapter 28

Molecular genetic testing and genetic counseling A. MICHEIL INNES* Alberta Children’s Hospital, Calgary, Alberta, Canada

28.1. Introduction With improvement in noninvasive neuroimaging modalities and advances in neuropathological methods of examining brain tissue, an increasing number of disorders associated with CNS malformations have been clinically delineated. The genetic component of many of these disorders was previously underestimated because the parents of the affected children were typically themselves clinically unaffected. The Human Genome Project has facilitated a rapid explosion in information and techniques that have allowed for the identification of genes associated with many of these malformation syndromes. This is currently an area of intense study and interest and many recent reviews have been published on the subject of genetics and CNS malformations (Clark, 2003; Gaitanis and Walsh, 2004; Mochida and Walsh, 2004; Parisi and Dobyns, 2004; Sarnat and Flores-Sarnat 2004; Guerrini, 2005; Guerrini and Filippi, 2005; Sarnat, 2005). Identification of these disease genes has allowed for an enhanced understanding of the biological mechanisms of normal and abnormal CNS development and human evolution (Gilbert et al., 2005). A long-term goal is improved treatment or prevention of congenital CNS anomalies through a better understanding of the pathways involved. In the short term, however, identification of causative genes has allowed physicians and their patients to have access to testing that can help clarify diagnosis and allows for carrier testing. Molecular testing for germline mutations in patients with CNS malformations is the major focus of this review.

28.2. Genetic counseling and genetic testing Genetic counseling is defined by the American Society of Human Genetics as ‘a communication process which

deals with the human problems associated with the occurrence or risk of occurrence of a genetic disorder in a family’. The key to providing appropriate genetic counseling is identifying the correct diagnosis. There are a number of reasons why an accurate diagnosis is important to families, including the provision of appropriate anticipatory care and advocacy for the child and family (Lemay et al., 2003) Also of importance to families is provision of accurate recurrence risk counseling. Many of the genetic conditions associated with CNS malformations are inherited in an autosomal dominant fashion. However, in the majority of the conditions being reviewed here, the parents are not obviously clinically affected. The most common explanation for this is the presence of a de novo mutation that is not present in either parent, as would be the case for example in LIS1 mutations causing lissencephaly (Cardoso et al., 2002). However some autosomal dominant conditions manifest either variable expression (e.g. tuberous sclerosis) or reduced penetrance (seen in some holoprosencephaly pedigrees). For these reasons careful clinical, radiographic and occasionally molecular investigation of parents is indicated prior to providing definitive genetic counseling. Finally, in known autosomal dominant conditions, the possibility of germline mosaicism can never be excluded and is a well recognized phenomenon in several of the conditions under review here, including tuberous sclerosis (Verhoef et al., 1995). Several of these syndromes, including the majority of the Joubert-syndrome-related disorders (JSRDs), cobblestone (type 2) lissencephalies and primary microcephalies are inherited as autosomal recessive traits. In these cases, the parents are typically assumed to be carriers and providing a recurrence risk of 25% is appropriate. Parental consanguinity is encountered with increased frequency, particularly for rare conditions.

*Correspondence to: A. Micheil Innes, Assistant Professor, Department of Medical Genetics, University of Calgary, Alberta Children’s Hospital, 2888 Shaganappi Trail NW, Calgary, Alberta T3B 6A8, Canada. E-mail: [email protected], Tel: þ1-403-955-7588, Fax: þ1-403-555-9100.

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In addition certain autosomal recessive disorders are over-represented in different ethnic groups, which often implies a single, or few, founder mutations. This can help to streamline genetic investigations. Examples include Andermann syndrome in French Canadians (Howard et al., 2002) and Amish lethal microcephaly (Rosenberg et al., 2002). Identification of an X-linked genetic condition can, of course, have major implications for genetic counseling. In some X-linked ‘dominant’ conditions such as Rett syndrome, the recurrence risk is negligible in future pregnancies because the mothers are typically not carriers. In other examples, such as the filamin-A-related disorders, the recurrence risk is higher, with increased mortality in male infants. In other X-linked ‘recessive’ conditions such as oligophrenin-related X-linked mental retardation or the L1CAM disorders, the mothers are often clinically unaffected and the risk for future affected offspring depends on the carrier status of the mother. This can occasionally be determined by review of the family history alone, although genetic testing is often required. Recently, some authors (Dobyns et al., 2004) have argued that the distinction between X-linked recessive and X-linked dominant diseases is arbitrary and that these two terms should be abandoned. Identification of a specific familial mutation for a known CNS malformation disorder can also help facilitate prenatal diagnosis via either amniocentesis or chorionic villus sampling. In some cases preimplanta-

tion diagnosis may even be possible. A full discussion of this topic is beyond the scope of this chapter. Trying to identify germline mutations in specific genes is only one component of the work-up undertaken to arrive at a specific diagnosis for a malformation syndrome. In addition to a detailed history and physical examination, other modalities that may be used include radiographic investigations, routine cytogenetics and molecular cytogenetic studies (Table 28.1) and routine or specialized metabolic tests (Table 28.2). The disorders covered in this chapter are caused by germline mutations that can be detected by DNA extracted from blood. Analysis of cerebrospinal fluid or other tissues is not necessary for the molecular confirmation of the conditions discussed in this chapter. There are two broad types of molecular test that can be pursued: clinical tests and research tests. Clinical laboratories are usually approved by local standards (e.g. the Clinical Laboratory Improvement Act in the USA), charge for testing, report results in writing and complete testing in a timely manner. Research laboratories usually provide testing free of charge but are not obligated to report results or perform the research in a timely manner. In some instances partnerships exist between clinical and research laboratories that allow for confirmation of results originally obtained in a research lab in a clinical setting. Prenatal diagnosis and carrier testing are unlikely to be performed in research laboratories.

Table 28.1 Selected chromosomal disorders associated with human CNS malformation syndromes Chromosomal rearrangement Deletion 1p36 Deletion 4p (Wolf–Hirschhorn) Deletion 6p Deletion 7q36 Inversion duplication 8p Mosaic trisomy 8

Trisomy 13 Deletion 17p (Miller–Dieker) Deletion 22q11 (velocardiofacial) Deletion 22q13 Triploidy *

Brain malformation

Detectable on routine karyotype

Detectable using subtelomeric probes*

Polymicrogyria Dysgenesis of the corpus callosum

Occasionally Occasionally

Yes Occasionally

Dandy–Walker malformation Holoprosencephaly, sacral agenesis Agenesis of the corpus callosum Agenesis of the corpus callosum

Occasionally Yes

Yes N/A N/A N/A

Holoprosencephaly Lissencephaly

Yes Occasionally (skin fibroblasts often required) Yes Occasionally

Polymicrogyria

No

No. TUPLE1 probe

Macrocephaly Holoprosencephaly

No Yes

Yes. Or ARSA probe N/A

N/A for disorders readily detectable on routine karyotype

N/A No. LIS1 probe

MOLECULAR GENETIC TESTING AND GENETIC COUNSELING

519

Table 28.2 Selected inborn errors of metabolism associated with human CNS malformation syndromes Inborn error of metabolism

Mode of inheritance

Congenital CNS anomaly

Fumarase deficiency Congenital disorders of glycosylation Desmosterolosis

AR AR

Polymicrogyria Cerebellar atrophy

AR

Nonketotic hyperglycinemia Pyruvate dehydrogenase deficiency Smith–Lemli–Opitz syndrome

AR XL AR

Zellweger syndrome

AR

Microcephaly, gyral and corpus callosum abnormalities Agenesis of the corpus callosum Agenesis of the corpus callosum Microcephaly, holoprosencephaly, agenesis of the corpus callosum, cerebellar hypoplasia Gyral abnormalities

AR, autosomal recessive; XL, X-linked.

28.3. Electronic databases/resources The identification of new genes associated with human disease has led to significant improvements in our understanding of these diseases and our ability to diagnose them. However, it has become confusing (and time-consuming) for clinicians to attempt to locate laboratories that provide testing. In many jurisdictions, finding the funding for such clinical tests is also problematic. The landscape of laboratories offering either clinical or research testing for different conditions is frequently changing and therefore printed material is at risk of being out of date. There are a number of free and comprehensive electronic resources that exist to help physicians and researchers. Online Mendelian Inheritance in Man (OMIM; http://www.ncbi.nlm.nih.gov/Omim) is a continuously updated catalog of human genes and genetic disorders. OMIM focuses primarily, but not exclusively, on heritable genetic diseases. This is a comprehensive resource for obtaining up-to-date information on what is known about the genetics of various human phenotypes. However, specific information on availability of genetic testing is not available through OMIM, although, when appropriate, OMIM is linked to the website GeneTestsTM (www.genetests.org). GeneTests, funded by the National Institutes of Health, provides current (peer-reviewed) information on genetic testing and its use in diagnosis, management and genetic counseling. Detailed clinical and molecular reviews (GeneReviews) on a number of important human diseases (including holoprosencephaly, tuberous sclerosis, Joubert syndrome and many others covered in this chapter) exist on the website. In addition a searchable laboratory directory of laboratories providing

clinical or research testing for a large number of heritable diseases is provided. Participation with this resource is voluntary; however, large numbers of North American, European and International laboratories participate. Similar websites are based in Europe, including the European Directory of DNA Diagnostic Laboratories (http://www.eddnal.com) and Orphanet (http://www.orpha.net). The information included on all these websites is updated frequently. Physicians, families and researchers with an interest in specific conditions are encouraged to visit them frequently. Another potential source of information is the disease-specific website. Often maintained by parent support groups, the majority of high-quality sites will also have clear representation from a medical advisory board. Excellent representative examples include www. tsalliance.org (tuberous sclerosis), www.lissencephaly. org (lissencephaly) and www.joubertsyndrome.org (Joubert Syndrome and related disorders).

28.4. Midline malformations 28.4.1. Holoprosencephaly Holoprosencephaly is a structural anomaly of the brain where the developing forebrain does not entirely cleave into separate hemispheres and ventricles (see Ch. 2). Clinical manifestations of HPE include developmental delay, growth failure, seizures and feeding difficulties. The majority of affected individuals have associated midfacial hypoplasia. Many of the morphological cerebral abnormalities in HPE, including the neural crest induction of craniofacial development, can be attributed to the extent of the gradient of genetic expression in the three axes of the neural tube

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(Sarnat and Flores-Sarnat, 2001). Non-genetic causes of HPE include maternal diabetes (Barr et al., 1983), fetal alcohol syndrome and retinoic acid exposure (Cohen and Shiota, 2002). Many children (25–50%) with HPE will have an identifiable chromosomal abnormality, including trisomy 13, trisomy 18, triploidy and numerous chromosome deletions and duplications such as deletion 7q36 (Horn et al., 2004). Unless the cause of the HPE is apparent on history and physical, karyotype is indicated in all cases as an initial investigation. A number of rare mendelian syndromes can be associated with HPE, including Pallister–Hall syndrome, Smith–Lemli–Opitz syndrome and hydrolethalus syndrome. In these instances, other features should be apparent on physical examination that would suggest the diagnosis. A variety of genes are now known to be implicated in nonsyndromic HPE (Table 28.3). Autosomal dominant mutation in (or deletions or chromosomal rearrangements encompassing) SHH (7q36), ZIC2 (13q32), SIX3 (2p21), TGIF (18p11.3) and PTCH (9q22.3) have all been reported in HPE families. Mutations in SHH account for 30–40% of familial cases and fewer than 5% of isolated cases; mutations in the other genes each account for fewer than 5% of familial cases and 2% of isolated cases (Muenke and Gropman, 2005). Evidence regarding genotype/phenotype correlation is very preliminary at this time. For example mutations in ZIC2 are probably less likely to be associated with an abnormal facial phenotype. Clinical testing for these genes is now available in several laboratories worldwide. However, for many clinicians arranging testing in a timely and affordable manner can still be difficult. As a result careful examination of both parents of a child with HPE is indicated to look for HPE ‘microforms’, including microcephaly, single central incisor, hypotelorism, anosmia, ocular coloboma, absent superior labial frenulum, midface hypoplasia and congenital nasal pyriform aperture stenosis. The presence of these findings would imply a recurrence risk as high as 50%. 28.4.2. Septo-optic dysplasia and pituitary disorders Septo-optic dysplasia (de Morsier syndrome) is diagnosed by the presence of at least two of: absent septum pellucidum, optic nerve hypoplasia and evidence of pituitary dysfunction (see Ch. 3). Patients may present with visual disturbances, seizures, mental retardation, cerebral palsy and endocrine abnormalities. Associated brain malformations often include agenesis of the corpus callosum or schizencephaly. Important nongenetic causes of SOD include maternal diabetes, maternal cocaine or alcohol abuse and fetal anticonvulsant

syndromes. Similar to gastroschisis, there is an epidemiological association between young maternal age and SOD, which suggests a possible vascular etiology for many cases (Stevens and Dobyns, 2004). A small proportion of cases of SOD are, however, associated with heterozygous mutations in the transcription factor HESX-1 (Dattani et al., 1998). The number of patients with SOD with confirmed disease causing mutations in HESX-1 is small, and testing is not routinely available at the time of writing. 28.4.3. Agenesis of the corpus callosum Agenesis or, more commonly, dysgenesis of the corpus callosum occurs in 1–3:1000 live births. As an isolated trait it can be ‘sporadic’ or inherited as an autosomal dominant, autosomal recessive or X-linked recessive trait. This finding can be seen in isolation or in the context of multiple CNS anomalies. Agenesis of the corpus callosum (ACC) is also a component of a large number of multiple congenital anomaly syndromes. A detailed classification scheme for ACC has recently been proposed (Da´vila-Gutie´rrez, 2002). Teratogenic causes of ACC include fetal alcohol syndrome (Riley et al., 2004) and fetal valproate syndrome. ACC or dysgenesis of the corpus callosum is an occasional finding in several inborn errors of metabolism including pyruvate dehydrogenase deficiency, nonketotic hyperglycinemia and Zellweger syndrome. Cytogenetic abnormalities are frequent in patients with ACC; common examples include both mosaic trisomy 8 (Serur et al., 1988) and inverted duplication of chromosome 8p (Guo et al., 1995). A number of genetic multiple congenital anomaly syndromes are associated with ACC. Classic examples, for which the specific gene is not yet identified, include acrocallosal syndrome, Coffin–Siris syndrome, Vici syndrome and X-linked dominant Aicardi syndrome. There are now some examples of disorders associated with ACC for which the gene and mutation are known. One important example is the X-linked syndrome associated with mutations in the L1CAM gene that can cause either X-linked aqueductal stenosis, MASA (mental retardation, aphasia, shuffling gait and adducted thumbs) syndrome or X-linked complicated agenesis of the corpus callosum (Weller and Gartner, 2001). Clinical testing for L1CAM mutations is available and should be considered in males with aqueductal stenosis or in families with either hydrocephalus or ACC in which there is a possible X-linked pattern of inheritance. Andermann syndrome, a disorder common in French Canadians, is an autosomal recessive condition with ACC and peripheral neuropathy. The disease is caused by mutations in the gene SLC12A6 (Howard et al., 2002). Clinical testing should be considered,

Table 28.3 Selected genes associated with human CNS malformation syndromes Testing{C, ClinicalR, Research

Major CNS malformations

Gene

Protein

Inheritance

Holoprosencephaly Holoprosencephaly Holoprosencephaly Holoprosencephaly Holoprosencephaly De Morsier syndrome Andermann syndrome

Holoprosencephaly Holoprosencephaly Holoprosencephaly Holoprosencephaly Holoprosencephaly Septo-optic dysplasia Agenesis of the corpus callosum Agenesis of the corpus callosum

SHH ZIC2 SIX3 TGIF PTCH HESX1 SLC12A6

AD AD AD AD AD AD AR

0–50 0–50 0–50 0–50 0–50 0–50 25

C C C C C C, R C

AR

25

R

Agenesis of the corpus callosum, aqueductal stenosis Cerebellar vermis hypoplasia

L1CAM

Sonic hedgehog Zinc finger protein Sine oculis transcription factor 50-TG-30 interacting factor Patched Homeobox protein HESX1 Solute carrier family 12 member 6 Catalytic subunit of the RAB3 GTPase-activating protein complex Neural cell adhesion molecule 1

XLR

0–25

C

Cerebellar vermis hypoplasia

Warburg micro syndrome

L1CAM syndrome

Joubert syndrome Joubert syndrome (juvenile nephronophthisis) Oligophrenin-related Xlinked mental retardation Dandy–Walker malformation Tuberous sclerosis Tuberous sclerosis Cowden syndrome Filamin-A-related disorders

AHI1

AR

25

R

NPHP1

Abelson helper integration protein 1 Nephrocystin

AR

25

C, R

Cerebellar dysgenesis

OPHN-1

Oligophrenin

XLR

0–25

R

Dandy–Walker malformation

ZIC1/ZIC4

Zinc finger in cerebellum 1 and 4

~0

R

Subcortical tubers Subcortical tubers Lhermitte–Duclos

TSC1 TSC2 PTEN

0–50 0–50 0–50

C, R C, R C

Periventricular nodular heterotopia Microcephaly, periventricular nodular heterotopia Lissencephaly

FMN

Hamartin Tuberin Dual specificity phosphatase PTEN Filamin A

AD (sporadic) AD AD AD XLD

50

C, R

AR

25

R

AD

0

C, R

XL

0–25

C, R

Lissencephaly, band heterotopia

ARFGEF2 LIS1 DBL

ADP ribosylation factor guanine nucleotide exchange factor 2 Platelet activating factor acetylhydrolase 1ba subunit Doublecortin

521

Microcephaly with heterotopia Lissencephaly (includes Miller–Dieker) DCX-associated lissencephaly

RAB3GTPase

MOLECULAR GENETIC TESTING AND GENETIC COUNSELING

Disease or syndrome

Recurrence risk (%)*

522

Table 28.3 (Continued) Testing{C, ClinicalR, Research

Major CNS malformations

Gene

Protein

Inheritance

X-linked lissencephaly with ambiguous genitalia Walker–Warburg syndrome Fukuyama congenital muscular dystrophy Muscle-eye-brain disease

Lissencephaly

ARX

Homeobox protein ARX

XL

0–25

C

Cobblestone lissencephaly Cobblestone lissencephaly

POMT1 FCMD

Protein O-mannisoyl transferase Fukutin

AR AR

25 25

C, R C

Cobblestone lissencephaly

POMGnT1

AR

25

C

Lissencephaly with cerebellar hypoplasia Dysequilibrium syndrome (Hutterite type) Schizencephaly

Lissencephaly, cerebellar hypoplasia Cortical simplification, Cerebellar hypoplasia Schizencephaly

RELN

Protein O-mannose b 1–2-N acetylglucosaminyltransferase Reelin

AR

25

R

AR

25

R

EMX2

Very-low-density lipoprotein receptor Homeobox protein EMX2

0–50

C, R

Bifrontal parietal polymicrogyria Goldberg–Shprintzen syndrome True microcephaly True microcephaly

Polymicrogyria

GPR56

G protein coupled receptor 56

? AD and AR AR

25

C

Generalized polymicrogyria

KIAA1279

Hypothetical protein LOC 26128

AR

25

R

Microcephaly Microcephaly

MCPH1 ASPM

AR AR

25 25

C, R R

True microcephaly

Microcephaly

CDK5RAP2

AR

25

R

True microcephaly Seckel syndrome

Microcephaly Microcephaly

CENPJ ATR

AR AR

25 25

R R

Nijmegen breakage syndrome Amish lethal microcephaly

Microcephaly

NBS1

Microcephalin Abnormal spindle-like microcephaly Cyclin dependant kinase regulatory protein Centromere-associated protein J Ataxia telangiectasia and Rad3related protein Nibrin

AR

25

C, R

Microcephaly

SLC25A19

AR

25

R

Rett syndrome Sotos syndrome

Progressive microcephaly Megalencephaly

MECP2 NSD1

XL AD

~0 0–50

C, R C, R

VLDRL

Solute carrier family 25 member 19 Methyl cytosine binding protein 2 Nuclear receptor Suvar, domaincontaining protein 1

* Recurrence risk figures for autosomal dominant and X-linked disorders depend on the status of the parents. In some cases (e.g. holoprosencephaly or tuberous sclerosis) parents may be mildly or subclinically affected and clinical or genetic investigations may be warranted. The possibility of gonadal mosaicism must also always be considered. { Information based primarily on self-reported data collected from GeneTests (www.genetests.org) and EDDNAL (www.eddnal.com) as of 14 July 2005.

A. M. INNES

Disease or syndrome

Recurrence risk (%)*

MOLECULAR GENETIC TESTING AND GENETIC COUNSELING particularly in French-Canadian patients. Other examples of syndromes with ACC for which the gene is known include the Mowat–Wilson syndrome (Mowat et al., 2003) (ZFHXIB) and the Warburg micro syndrome (Graham et al., 2004) (RAB3GTPase). In most cases, the finding of ACC in itself is nonspecific and careful clinical phenotyping of the patient is required in order to arrive at a specific diagnosis. Rarely, certain syndromes are associated with an enlarged corpus callosum. One example is Cohen syndrome, caused by mutations in COH1, an autosomal recessive condition characterized by microcephaly, hematological disturbances, retinal anomalies and truncal obesity (Kivitie-Kallio and Norvio, 2001; Kolehmainen et al., 2003).

28.5. Malformations of the posterior fossa Malformations of the posterior fossa are an important group of conditions that have been studied in less detail than those of the forebrain. Nevertheless much progress is being made in understanding these disorders (Niesen, 2002; Parisi and Dobyns, 2004; Boltshauser, 2004). Disorders of the posterior cranial vault such as Chiari I malformations are known to be a component of many genetic syndromes such as 22q11 microdeletion syndrome, Costello syndrome, Williams syndrome and neurofibromatosis type 1. Familial cases of nonsyndromic Chiari I malformation have been reported (Milhorat et al., 1999). To date no genes associated with nonsyndromic Chiari I malformation have been identified (see Ch. 6). Classical Dandy–Walker malformation is a rare, usually sporadic, entity characterized by cerebellar vermis hypoplasia and cystic dilatation of the fourth ventricle. While a variety of syndromes and chromosome rearrangements have been associated with Dandy–Walker malformation, until recently the cause of isolated ‘sporadic’ Dandy–Walker malformation was unknown. Grinberg and colleagues (2004) recently identified heterozygous deletions of two adjacent ‘zinc finger in cerebellum’ genes ZIC1 and ZIC4 in some cases of sporadic Dandy–Walker malformation. In these cases the deletions were de novo and neither parent was affected. These genes map to chromosome 3q and in some cases the patients had visible cytogenetic deletions of that chromosome. The proportion of cases of nonsyndromic Dandy–Walker malformation related to deletions of ZIC1 and ZIC4 remains the subject of further research. In 1969 Joubert and colleagues described a familial syndrome of cerebellar vermis hypoplasia, neonatal hypotonia, abnormal respirations and eye movements, psychomotor retardation and ataxia. The syndrome

523

was presumed to be autosomal recessive and was first reported in French Canadians. More recently the so called ‘molar tooth sign’ comprising cerebellar vermis hypoplasia, deep interpeduncular fossa and thickened cerebellar peduncles was reported as a useful diagnostic marker for Joubert syndrome (Maria et al. 1997). Since the recognition of the molar tooth sign as a diagnostic marker a large number of overlapping syndromes have been reported, which are termed Joubert-syndrome-related disorders (JSRDs or JBTSs) or cerebello-ocular–renal syndromes (CORS) (Chance et al., 1999; Gleeson et al. 2003). This is clearly a clinically and genetically heterogeneous group of conditions. Some progress has been made regarding the genetics of JSRDs (Valente et al., 2005). Two genes that can cause JSRDs are now known. Mutations in AHI1 on chromosome 6 (JBTS3) cause a CNS phenotype with molar tooth sign, polymicrogyria, corpus callosum anomalies and seizures (Ferland et al., 2004). Homozygous deletions of the gene NPHP1, encoding nephrocystin, are typically associated with a genetic kidney disorder known as juvenile nephronophthisis. However, rarely, the same deletions can be associated with a ‘mild’ form of JSRD with molar tooth sign and renal involvement (Parisi et al., 2004). Two other JSRD genes have been mapped. JBTS1 maps to chromosome 9 (Saar et al. 1999). Patients with this gene appear to have a phenotype mostly confined to the CNS. Patients with JBTS2, which maps to chromosome 11, have a more complicated phenotype with renal, eye and limb involvement (Keeler et al., 2003; Valente et al., 2003). There is some clinical overlap with JBTS2 patients and patients with Meckel–Gru¨ber syndrome, another rare genetically heterogeneous recessive disorder characterized by occipital encephalocele, cerebellar vermis hypoplasia, polydactyly and renal cysts. Other families have been reported with a JSRD that does not map to any of the four known loci (Janecke et al., 2004), including a large consanguineous pedigree with features that overlap with Meckel–Gru¨ber syndrome (Boycott et al. 2005a). The area of Joubert syndrome/JSRD genetics is under active investigation. Clinical testing for NPHP1 deletions is widely available for patients with a predominantly renal phenotype. Research testing is available in several laboratories investigating the other genes and loci associated with Joubert syndrome.

28.6. Hamartomatous disorders 28.6.1. Tuberous sclerosis Tuberous sclerosis complex is a well recognized autosomal dominant disorder (Curatolo, 2003; see also

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Ch. 9). Diagnostic criteria are well established for the syndrome (Roach et al., 1998). CNS involvement in tuberous sclerosis complex is an important cause of morbidity and mortality in the syndrome. Findings include cortical tubers, subependymal glial nodules and giant cell astrocytomas. More than 80% of patients have seizures, although this probably reflects an overestimate due to ascertainment bias. Two different genes, TSC1 (encodes hamartin) and TSC 2 (encodes tuberin), are associated with tuberous sclerosis. All the complications can be seen in patients with mutations in either gene; however there may be subtle differences. There is some evidence for a milder phenotype in individuals with TSC1 mutations (Dabora et al., 2001; Lewis et al., 2004). When a new diagnosis of tuberous sclerosis complex is made in a child or infant, close examination of both parents (including skin and Woods lamp examination, eye examination and renal ultrasound) is indicated. This is because variable expression is well recognized. Penetrance of the mutations is said to be 100%, meaning that all carriers of the mutation should have some manifestations of the syndrome. Cases of recurrence in siblings born to unaffected parents probably represent gonadal mosaicism (Verhoef et al., 1995). Clinical testing, by sequence analysis, for the tuberous sclerosis complex genes is available. Approximately 30% of familial cases and 10% of simplex cases have TSC1 mutations and 50% of familial cases and 60–70% of simplex cases have mutations in TSC2 (Northrup and Au, 2005). The remaining cases cannot be classified using currently available methodologies. In most families the diagnosis is clinically straightforward and genetic testing is often not necessary. Knowledge of the specific familial mutation can be of value in the setting of prenatal diagnosis. 28.6.2. Lhermitte–Duclos disease Lhermitte–Duclos disease (dysplastic gangliocytoma of the cerebellum, PTEN hamartoma syndrome) is a rare condition. It has been recognized to occur in the setting of the rare autosomal dominant syndrome Cowden’s disease, but also occurs sporadically. Two previously distinct syndromes, Cowden disease and Bannayan–Riley–Ruvacalba syndrome were found to be caused, at least in the majority of cases, by autosomal dominant mutations in the dual specificity phosphatase PTEN (Liaw et al., 1997; Marsh et al., 1997). In some cases patients with either a Cowden’s or Bannayan–Riley–Ruvacalba phenotype were seen in the same family, suggesting that these syndromes are the same (Zori et al. 1998). This disorder is now best termed PTEN hamartoma tumor syndrome

(PHTS). Recent work suggests that the majority of patients with adult-onset Lhermitte–Duclos disease have germline mutations in the PTEN gene (Zhou et al., 2003). There is an increased risk for malignancy in patients with PTEN mutations (thyroid, breast and uterus) and screening guidelines have been suggested (Pilarski and Eng, 2004). Therefore it is important to confirm the presence of a PTEN mutation when one is suspected and clinical testing for a PTEN mutation is recommended in cases where Lhermitte–Duclos disease has been diagnosed. Interestingly, cases of childhood-onset Lhermitte–Duclos disease have not been associated with PTEN mutations (Zhou et al., 2003; Capone Mori et al., 2003), suggesting a separate etiology in those patients. As the number of children studied to date has been small, consideration could be given to PTEN genetic testing even in cases of childhood-onset Lhermitte–Duclos disease. 28.6.3. Hemimegalencephaly This hamartomatous dysgenesis is now recognized as a primary disorder of cellular lineage rather than a neuroblast migratory disorder (Flores-Sarnat et al. 2003; see also Ch. 10). The cytology of hemimegalencephaly closely resembles that of tuberous sclerosis.

28.7. Disorders of migration and organization 28.7.1. Heterotopia This section focuses on periventricular heterotopia, a neuroblast migrational abnormality (see Ch. 11). The majority of patients with periventricular heterotopia present with seizures and the diagnosis is suggested by findings on computed tomography or magnetic resonance imaging (MRI) scan. Subcortical laminar (band) heterotopia is a feature of some of the lissencephaly syndromes and will be discussed in the next section. The neuroimaging findings in tuberous sclerosis can, and must, be distinguished from periventricular heterotopia. Periventricular heterotopia is a heterogeneous entity. Perinatal insults and chromosome abnormalities are seen in some cases. Patients with mutations in two autosomal recessive microcephaly genes, MCPH1 (Woods et al., 2005) and ARFGEF2 (Sheen et al., 2004), can have periventricular heterotopia. Mutations in the X-linked gene FLNA are the most important cause of periventricular heterotopia (Sheen et al., 2001). In most cases, this disease is prenatally or neonatally lethal in males, and virtually all female mutation carriers will have heterotopia on neuroimaging. Loss of function mutations in FLNA result in X-linked periventricular heterotopia. Clinical testing

MOLECULAR GENETIC TESTING AND GENETIC COUNSELING detects mutations in 19% of females with a negative family history and 83% of females with an X-linked family history of this disorder (Sheen et al., 2001). Distinct gain of function mutations in FLNA causes four different phenotypes: frontometaphyseal dysplasia, Melnick–Needles syndrome, oto-palato-digital syndrome type 1 and oto-palato-digital syndrome type 2. In most cases, patients with these four syndromes do not have periventricular heterotopia. Further work is being done to understand the genotype– phenotype correlations in the fascinating group of FLNA-related disorders (Robertson, 2005). 28.7.2. Lissencephalies Lissencephaly, a smooth brain, refers to the external appearance of the cerebral cortex that leads to a smooth cortical surface. This appearance is a result of abnormal neuroblast migration. In the broadest sense, two distinct types have been identified: classic (type 1) lissencephaly and cobblestone (type 2) lissencephaly. The clinical genetics of these different groups of disorders are dramatically different. Classic lissencephaly can be an isolated finding or can occur in the context of several important genetic syndromes (Kato and Dobyns, 2003). Miller–Dieker syndrome is characterized by craniofacial dysmorphic features (bitemporal narrowing, upturned nares, downturned mouth) and severe lissencephaly. Patients with Miller–Dieker syndrome have chromosomal rearrangements involving chromosome 17 that disrupt the LIS1 locus. In some cases the chromosomal rearrangement is obvious on routine highresolution karyotype but in the majority of cases fluorescent in situ hybridization (FISH) testing with the LIS1 probe is required to confirm the diagnosis. In some instances the parents may be carriers of a balanced translocation that leads to the unbalanced product seen in their offspring. For this reason FISH testing of parents in cases where a child has a deletion is indicated prior to providing genetic counseling. Classic lissencephaly with a normal facial phenotype is often associated with more subtle rearrangements of the LIS1 gene. The presence of isolated lissencephaly with posterior severity greater than anterior suggests the possibility of a smaller deletion or point mutation in LIS1. Again FISH testing, which is widely available, is the first recommended investigation. If it is normal, then consideration should be given to clinical testing for LIS1 mutations. The prognosis for patients with LIS1 mutations is poor. These mutations are typically de novo dominant mutations, as affected individuals do not reproduce. In males with classic lissencephaly with a more severe gradient anteriorly than posteriorly, a mutation

525

in doublecortin is likely. This is an X-linked disorder: female carriers typically manifest with band heterotopia. As routine DCX mutation testing is not always available, neuroimaging of at-risk female family members should be considered. Another X-linked syndrome with lissencephaly is associated with mutations in the gene ARX. Affected males typically have ambiguous genitalia, agenesis of the corpus callosum, epilepsy and severe developmental delay. Carrier females may have isolated agenesis of the corpus callosum. There are a small number of patients with a rare combination of lissencephaly and severe cerebellar hypoplasia. Some patients with this constellation of findings have been shown to have autosomal recessive mutations in the gene reelin (RELN; Hong et al., 2000). Recently a group of Hutterite patients with cerebellar hypoplasia and mild cortical simplification were shown to be deleted for the gene VLDLR, which is one of the receptors for reelin (Boycott et al., 2005b). Other genes in the reelin pathway would remain viable candidates for mutations in patients with the combination of lissencephaly and cerebellar hypoplasia, and further research is required in this area. Cobblestone (type 2) lissencephalies are distinguished clinically from classic lissencephalies by the presence of features such as eye abnormalities, congenital muscular dystrophy and hydrocephalus (see Ch. 14). The ‘cobblestone’ appearance is evident on pathological examination. At least three type 2 lissencephaly syndromes exist: Walker–Warburg syndrome, Fukuyama congenital muscular dystrophy and muscle–eye–brain disease. All three are autosomal recessive in nature and the genes responsible for at least some cases of each are known (van Reeuwijk et al, 2005). There is considerable clinical overlap between these three conditions. Fukuyama congenital muscular dystrophy is most prevalent in Japan and is caused by mutations in FCMD, encoding fukutin. Walker– Warburg syndrome appears to be genetically heterogeneous. In some cases the disease is due to mutations in POMT1, and clinical testing is available for this gene. Research is also underway to identify other genes that can cause Walker–Warburg syndrome. Muscle– eye–brain disease is caused by mutations in POMGnT1. 28.7.3. Schizencephaly Schizencephalies are brain anomalies characterized by full-thickness unilateral or bilateral clefts of the cerebral cortex (see Ch. 15). Schizencephalies can be either bilateral or unilateral and open- or closed-lipped. Clinical manifestations are remarkably variable among patients. The etiology of schizencephaly remains under debate but there is compelling evidence to suggest that

526

A. M. INNES

many cases are the result of disruption of a previously normally developing brain as opposed to a true developmental malformation (Granata et al., 2005). Prenatal infection, hemorrhage and teratogens are all implicated in some cases. The genetics of schizencephaly remain poorly understood, although there are clearly familial cases. Heterozygous mutations of the homeotic gene EMX2 have been demonstrated in some cases of schizencephaly (Brunelli et al., 1996; Faiella et al. 1997); however the biological significance of these mutations and the role of this gene remains highly uncertain. Familial cases that do not link to EMX2 have been reported (Tietjen et al., 2005). In addition, there may be a rare autosomal recessive syndrome associated with schizencephaly and intracranial calcifications (Martin et al., 2000). At present, no standard genetic investigations can be recommended in cases of schizencephaly. Research testing may be appropriate for familial cases, cases with parental consanguinity or cases with atypical CNS or non-CNS features. 28.7.4. Polymicrogyria syndromes Polymicrogyria is an abnormality characterized by unusual folding of the cerebral cortex and replacement of the normal gyral pattern with excessive small gyri. Clinical features depend on the degree of involvement and the underlying etiology but can range from nearly normal in the case of localized polymicrogyria to epilepsy and severe mental retardation in cases of bilateral generalized polymicrogyria. The diagnosis is typically confirmed on MRI scan. Nongenetic causes of polymicrogyria include congenital infection and in utero ischemia. Chromosomal causes of polymicrogyria include the 22q11 microdeletion syndrome (Sztriha et al., 2004) and the 1p36 microdeletion syndrome (Jansen and Andermann, 2005). Other cytogenetic abnormalities have been reported in polymicrogyria patients (Leventer et al., 2001). As karyotyping and FISH testing for 22q11 microdeletion is widely available, this investigation could be considered for most cases of polymicrogyria, particularly if there are suggestive features such as facial dysmorphism, congenital heart disease and cleft palate. A number of inborn errors of metabolism, including nonketotic hyperglycinemia, glutaric acidemia and Zellweger syndrome, are occasionally associated with polymicrogyria. Metabolic studies including creatine kinase, lactate, very-longchain fatty acids and urine organic acids are of value if an inborn error of metabolism is suspected. Niikiwa-Kuroki (Kabuki) syndrome, a multiple congenital anomaly syndrome of unproven etiology is also occasionally associated with polymicrogyria (Di Gennaro et al., 1999; Powell et al., 2003). A variety of

other CNS malformations have been recorded in this syndrome (Ben-Omran and Teebi, 2005). Recently mutations in KIAA1279 were identified in patients with a form of Goldberg–Schprintzen syndrome, an autosomal recessive disorder characterized by Hirschsprung’s disease and bilateral generalized polymicrogyria (Brooks et al., 2005). Multiple types of nonsyndromic polymicrogyria have been reported that are currently distinguished on the basis of neuroimaging and distribution of disease. To date, only one gene has been identified, GPR56, which is mutated in autosomal recessive bifrontal parietal polymicrogyria. In addition to bifrontal parietal polymicrogyria, these patients typically have white matter abnormalities, strabismus, seizures and developmental delay. The syndrome appears to be most prevalent in Middle Eastern and Pakistani families (Piao et al., 2002, 2004). Genetic testing for BFPP is available on a clinical basis and the diagnostic yield appears to be high in typically affected patients. There are forms of bilateral frontal polymicrogyria and bilateral generalized polymicrogyria that appear to be autosomal recessive, as well a syndrome of bilateral perisylvian polymicrogyria (often associated with pseudobulbar signs) that appears to be X-linked (Villard et al., 2002). The genes for these syndromes are not yet known but large or consanguineous families may be suitable for further research studies.

28.8. Disorders of cranial volume 28.8.1. Microcephalies Microcephaly is a clinical finding, typically referring to a head circumference significantly less than expected for patient age and gender, and is often used as a surrogate measurement of brain volume (Woods et al., 2005). Microcephaly, particularly when taken as greater than 3 SD below the mean, is strongly associated with mental retardation. The presence of a small brain volume is termed micrencephaly. Some micrencephalies are a result of a primary disturbance in neuroepithelial cell proliferation. Neuroimaging is strongly recommended in patients with microcephaly and clinical abnormalities, and obviously findings on MRI scan can help direct towards a specific diagnosis. The list of nongenetic causes of microcephaly is extensive and important causes that must be considered include maternal infection (de Vries et al, 2004), fetal alcohol syndrome and maternal phenylketonuria/hyperphenylalaninemia (Levy and Ghavami, 1996). An autosomal recessive phenocopy of congenital infection also appears to exist (Reardon et al, 1994). A large number of genetic, or presumed genetic, multiple congenital anomaly syndromes are

MOLECULAR GENETIC TESTING AND GENETIC COUNSELING associated with microcephaly, including Rubinstein– Taybi syndrome (CPB and EP300; Roelfsema et al., 2005), a-thalassemia X-linked mental retardation syndrome (ATR-X; Gibbons et al., 1995), Seckel syndrome (ATR; O’Driscoll et al., 2003), Njimegen breakage syndrome (NBS-1), Feingold syndrome (MYCN; van Bokhoven et al., 2005) and Mowat–Wilson syndrome (ZFHXIB; Wakamatsu et al., 2001). A complete discussion of syndromes associated with microcephaly is beyond the scope of this chapter. In addition a number of chromosome abnormalities can be seen in patients with microcephaly and a karyotype is indicated, particularly in any child with syndromic microcephaly with no obvious diagnosis. An important group of patients with microcephaly are the patients with autosomal recessive primary microcephaly (MCPH), which has also been termed ‘true microcephaly’ and ‘microcephaly vera’. The defining features of this still heterogeneous group of conditions are: congenital microcephaly at least 4 SD below age and sex means, mental retardation with no other neurological findings and normal height, weight, physical appearance (other than those associated with extreme microcephaly) and results of karyotype and neuroimaging (Jackson et al., 2002; Roberts et al., 2002). These criteria have been modified somewhat as some patients – particularly those with MCPH1 mutations – have moderately short stature or periventricular neuronal heterotopia (Woods et al., 2005). Patients with MCPH typically have essentially normal CNS architecture (Bond et al., 2002) and mild to moderate mental retardation. This diagnosis is important to make, as the recurrence risk implications are significant. Several genes are now known to be associated with MCPH (Table 28.3); however with the exception of MCPH1 clinical testing is not readily available. In most instances the diagnosis of primary microcephaly remains clinical. In patients with classic features of MCPH, particularly if there is parental consanguinity, a recurrence risk of 25% is appropriate. The empiric recurrence risk for idiopathic microcephaly may be closer to 20% (Tolmie et al., 1987). 28.8.2. Macrocephalies Unlike microcephaly, there are few genetic disorders associated with isolated macrocephaly. In addition, not all patients with a large head circumference will have an enlarged brain (megalencephaly). There is a well known association between nonsyndromic macrocephaly and autism that needs further investigation (Miles et al., 2000). In the absence of hydrocephalus, few nongenetic causes of macrocephaly need to be considered. Sporadic disorders of unknown etiology,

527

including macrocephaly cutis marmorata congenita, may be diagnosed on clinical grounds (Lapunzina et al., 2004) Chromosomal disorders associated with macrocephaly are rare, with the important exception of the 22q13 deletion syndrome, which is associated with hypotonia and speech delay and can be detected by FISH testing (Havens et al., 2004). Sotos syndrome is characterized by a typical craniofacial gestalt, generalized overgrowth, advanced bone age and developmental delay. Neuroimaging findings in Sotos syndrome are nonspecific but do include ventricular dilatation, prominence of the trigone and occipital horns, and hypoplasia of the corpus callosum (Schaefer et al. 1997). Sotos syndrome is an autosomal dominant disorder associated with mutations or deletions of the gene NSD1 (Kurotaki et al. 2002). Most cases represent new mutations but many familial cases have been reported. NSD1 mutation testing is clinically available but in most cases the diagnosis is clinically straightforward. The mutation detection rate for classic cases of Sotos syndrome approaches 60–90% but there are important ethnic differences: for example, deletion cases are more common than point mutations in the Japanese population (Kurotaki et al., 2003). Other important causes of macrocephaly for which clinical genetic testing exists include two X-linked disorders, fragile X syndrome (FMR1) and Simpson– Golabi–Behmel syndrome (GPC3); and two autosomal dominant tumor predisposition syndromes, Gorlin syndrome (PTCH) and PTEN hamartoma syndrome (PTEN).

28.9. Inborn errors of metabolism While abnormal neuroimaging findings in inborn errors of metabolism such as infarcts and white matter disease are well known, a number of inborn errors of metabolism can be associated with brain malformations or abnormalities in brain structure. A well known example is corpus callosum agenesis or dysgenesis in patients with the mitochondrial disorder pyruvate dehydrogenase (PDH) deficiency. Cortical dysplasias have been reported in some cases of mitochondrial disease (Brown, 2005). Another well known example is the association of cerebellar atrophy in the autosomal recessive congenital disorders of glycosylation (Grunewald and Matthijs, 2000). A number of congenital CNS anomalies have been reported in patients with cholesterol biosynthesis defects (such as Smith– Lemli–Opitz syndrome), including holoprosencephaly (Hennekam, 2005). As the presentation and investigation of metabolic disorders is often complex, it is important to keep a high index of suspicion regarding the possibility of an inborn error of metabolism even in the context of a congenital CNS malformation.

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A. M. INNES

28.10. Conclusion The genetics of CNS malformations remains an exciting area of research. Over the next few years it is likely that many more novel CNS malformation syndromes will be identified, as will the genes that cause many known CNS syndromes. In the short term it will remain challenging for physicians to arrange timely, affordable genetic testing for their patients. Online information remains an essential tool in this regard. In the long term it is likely that testing strategies will become more straightforward and readily available. Anticipated long-term benefits of the information gained by this research include improved diagnosis, management, prevention and treatment of these often devastating conditions.

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MOLECULAR GENETIC TESTING AND GENETIC COUNSELING Granata T, Freri E, Caccia C, et al. (2005). Schizencephaly: clinical spectrum, epilepsy, and pathogenesis. J Child Neurol 20: 313–318. Grinberg I, Northrup H, Ardinger H, et al. (2004). Heterozygous deletion of the linked genes ZIC1 and ZIC4 is involved in Dandy–Walker malformation. Nat Genet 36: 1053–1055. Grunewald S, Matthijs G (2000). Congenital disorders of glycosylation (CDG): a rapidly expanding group of neurometabolic disorders. Neuropediatrics 31: 57–59. Guerrini R (2005). Genetic malformations of the cerebral cortex and epilepsy. Epilepsia 46 (suppl.): 32–37. Guerrini R, Filippi T (2005). Neuronal migration disorders, genetics, and epileptogenesis. J Child Neurol 20: 287–299. Guo WJ, Callif-Daley F, Zapata MC, Miller ME (1995). Clinical and cytogenetic findings in seven cases of inverted duplication of 8p with evidence of telomeric deletion using fluorescence in situ hybridization. Am J Med Genet 58: 230–236. Havens JM, Visootsak J, Phelan MC, Graham JM (2004). 22q13 deletion syndrome: an update and review for the primary paediatrician. Clin Pediatr 43: 43–53. Hennekam RCM (2005). Congenital brain anomalies in distal cholesterol biosynthesis defects. J Inherit Metab Dis 28: 385–392. Hong SE, Shugart YY, Huang DT, et al. (2000). Autosomal recessive lissencephaly with cerebellar hypoplasia is associated with human RELN mutations. Nat Genet 26: 93–96. Horn D, Tonnies H, Neitzel H, et al. (2004). Minimal clinical expression of holoprosencephaly spectrum and of Currarino syndrome due to different cytogenetic rearrangements deleting the Sonic Hedgehog gene and the HLXB9 gene at 7q36.3. Am J Med Genet A 128: 85–92. Howard HC, Mount DB, Rochefort D, et al. (2002). The K–Cl cotransporter KCC3 is mutant in a severe peripheral neuropathy associated with agenesis of the corpus callosum. Nat Genet 32: 384–392. Jackson AP, Eastwood H, Bell SM, et al. (2002). Identification of microcephalin, a protein implicated in determining the size of the human brain. Am J Hum Genet 71: 136–142. Janecke AR, Muller T, Gassner I, et al. (2004). Joubert-like syndrome unlinked to known candidate loci. J Pediatr 144: 264–269. Jansen A, Andermann E (2005). Genetics of the polymicrogyria syndromes. J Med Genet 42: 369–378. Joubert M, Eisenring JJ, Robb JP, Andermann F (1969). Familial agenesis of the cerebellar vermis: a syndrome of episodic hyperpnea, abnormal eye movements, ataxia, and retardation. Neurology 19: 813–825. Kato M, Dobyns WB (2003). Lissencephaly and the molecular basis of neuronal migration. Hum Mol Genet 12: R89–R96. Keeler LC, Marsh SE, Leeflang EP, et al. (2003). Linkage analysis in families with Joubert syndrome plus oculorenal involvement identifies the CORS2 locus on chromosome 11p12–q13.3. Am J Hum Genet 73: 656–662. Kivitie-Kallio S, Norvio R (2001). Cohen syndrome: essential features, natural history, and heterogeneity. Am J Med Genet 102: 125–135.

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Sarnat HB (2005). CNS malformations: gene locations of known human mutations. Eur J Paediatr Neurol 9: 427–431. Sarnat HB, Flores-Sarnat L (2001). Neuropathologic research strategies in holoprosencephaly. J Child Neurol 16: 918–931. Sarnat HB, Flores-Sarnat L (2004). Integrative classification of morphology and molecular genetics in central nervous system malformations. Am J Med Genet A 126: 386–392. Schaefer GB, Bodensteiner JB, Buehler BA, et al. (1997). The neuroimaging findings in Sotos syndrome. Am J Med Genet 68: 462–465. Serur D, Jeret JS, Wisniewski K (1988). Agenesis of the corpus callosum: clinical, neuroradiological and cytogenetic studies. Neuropediatrics 19: 87–91. Sheen VL, Dixon PH, Fox JW, et al. (2001). Mutations in the X-linked filamin 1 gene cause periventricular nodular heterotopia in males as well as in females. Hum Mol Genet 10: 1775–1783. Sheen VL, Ganesh VS, Topcu M, et al. (2004). Mutations in ARFGEF2 implicate vesicle trafficking in neural progenitor proliferation and migration in the human cerebral cortex. Nat Genet 36: 69–76. Stevens CA, Dobyns WB (2004). Septo-optic dysplasia and amniotic bands: further evidence for a vascular pathogenesis. Am J Med Genet 125A: 12–16. Sztriha L, Buerrini R, Harding B, et al. (2004). Clinical, MRI, and pathological features of polymicrogyria in chromosome 22q11 deletion syndrome. Am J Med Genet 127A: 313–317. Tietjen I, Erdogan F, Currier S, et al. (2005). EMX2-independent familial schizencephaly: clinical and genetic analyses. Am J Med Genet 135A: 166–170. Tolmie JL, McNay M, Stephenson JBP, et al. (1987). Microcephaly: genetic counselling and antenatal diagnosis after the birth of an affected child. Am J Med Genet 27: 583–594. Valente EM, Salpietro DC, Brancati F, et al. (2003). Description, nomenclature, and mapping of a novel cerebellorenal syndrome with the molar tooth malformation. Am J Hum Genet 73: 663–670. Valente EM, Marsh SE, Castori M, et al. (2005). Distinguishing the four genetic causes of Joubert syndrome-related disorders. Ann Neurol 57: 513–519. Van Bokhoven H, Celli J, van Reeuwijk J, et al. (2005). MYCN haploinsufficiency is associated with reduced brain size and intestinal atresias in Feingold syndrome. Nat Genet 37: 465–467. Van Reeuwijk J, Janssen M, van den Elzen C, et al. (2005). POMT2 mutations cause alpha-dystroglycan hypoglycosylation and Walker Warburg syndrome. J Med Genet 42: 907–912. Verhoef S, Vrtel R, van Essen T, et al. (1995). Somatic mosaicism and clinical variation in tuberous sclerosis complex. Lancet 345: 202. Villard L, Nguyen K, Cardoso C, et al. (2002). A locus for bilateral perisylvian polymicrogyria maps to Xq28. Am J Hum Genet 70: 1003–1008.

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Chapter 29

Embryology and neuropathological examination of central nervous system malformations HARVEY B. SARNAT* University of Calgary Faculty of Medicine and Alberta Children’s Hospital, Calgary, Alberta, Canada

29.1. Introduction The impressive advances that have been made in the past decade in neuroimaging and in electrophysiological studies of the brain are matched by equally insightful advances in neuropathology. Embryology is the essence of development; development is the essence of neonatology and pediatric neurology. Yet in recent years both of these clinical disciplines have been delinquent in incorporating a fundamental understanding of embryology into training programmes for residents; comprehension of basic mechanisms of nervous system malformations has suffered both at the academic level of pathogenesis and at the clinical level of patient care. An example of the latter is the unjustified surgical shunting of dilated lateral ventricles in colpocephaly and hemimegalencephaly because of ignorance about normal and abnormal ontogenesis. Another example is the overtreatment of neonates with antiepileptic drugs because of misinterpretation of abnormal postures, tremors and other clinical events related to lesions or dysfunction in the upper brainstem. Yet another is a frequent lack of recognition of cerebral dysgenesis in neonates with facial dysmorphisms or cutaneous facial lesions that clearly indicate a disturbance of neural crest migration, an abnormality of neural induction of non-neural tissues in craniofacial development. Several factors have contributed to the recent downgrading of neuroembryology in postgraduate training curricula. One of the most significant is the low rate of postmortem examination in many institutions compared with previous decades. At times the fault is with clinicians who do not try hard enough to convince parents of the value of an autopsy in a child

who has died; in some cases fault also lies with pathologists who do not provide a timely report to enable the results to be communicated to the clinicians and the parents, or do not perform a meaningful examination for providing information that could not be known during life. Without diminishing the major contributions that the new technologies in neuroimaging have made in identifying cerebral malformations during life, both prenatally and postnatally, the recognition of a malformation pattern is sometimes regarded as more definitive than it actually is. All three grades of severity of holoprosencephaly, alobar, semilobar and lobar, can be recognized by imaging studies and too often infants who do not survive despite the best of clinical care are not studied postmortem in the mistaken belief that the diagnosis is established and there is nothing more to learn. The limitation of all modern imaging techniques, including advanced functional imaging, is that a lesion must be large enough to identify with the naked eye. Neuroimaging is not a microscope and many microscopic changes in the architecture of the brain and cytological abnormalities in cellular lineage remain below the limits of resolution of even the latest generation of magnetic resonance equipment. Another factor that contributes to a low autopsy rate is more sinister; this factor is economic. It is seen in its most exaggerated form in the USA, where many decisions are now taken by business-oriented hospital administrators rather than by physicians and healthcare professionals. When medical care is regarded as a business rather than a basic human right, procedures that do not generate profit are inevitably suppressed or eliminated. The goal no longer includes intellectual knowledge gained, teaching of students and residents,

*Correspondence to: Harvey B. Sarnat MD, FRCPC, Professor of Paediatrics, Pathology (Neuropathology) and Clinical Neurosciences, Alberta Children’s Hospital, 2888 Shaganappi Trail, NW Calgary, Alberta T3B 6A8, Canada, E-mail: [email protected], Tel: 1-403-955-7131, Fax: 1-403-955-2922.

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research for new data, or even confirmation of genetic conditions, which provides a valuable basis for counseling of family members on the risks in future pregnancies. The agenda is both simple and antiintellectual: minimize costs and maximize profits. Hence autopsies are discouraged because of their impact on overall hospital budgets. Private health insurance companies never cover the costs of postmortem examination. Even publicly funded hospitals sometimes find themselves constrained by assigned budgets. The purpose of this chapter is, first, to demonstrate to clinicians caring for patients with malformations of the nervous system why autopsy remains an important final procedure in patients who cannot survive, providing a kind of information that cannot usually be determined during life. The second purpose is to demonstrate to general and pediatric pathologists that the fetal and neonatal autopsy and examination of malformations can be greatly enhanced by the selective supplementary application of immunocytochemistry and histochemistry. With the advent of surgical approaches to the treatment of intractable epilepsy, direct examination of resected cerebral tissue also becomes possible in living patients, using many of the modern techniques summarized below. Modern neuroembryology and modern neuropathology are an integration of traditional descriptive morphology at various stages of maturation, and the application of histochemical and immunocytochemical markers that reveal maturation and function at a level not available from standard histological sections of tissue. These methods provide a precision and insight analogous to the data provided by magnetic resonance imaging (MRI) compared to the data previously provided by pneumoencephalography.

29.2. Criteria of neuronal maturity Neurons are defined by the unique combination of two features that individually are shared in many tissues of the body but coexist in the same cell only in neurons: 1) synthesis of secretory products and 2) electrical polarity and excitability of the plasma membrane. The secretion of neurotransmitters by neurons involves the development of organelles for their biosynthesis, axonal transport to axonal terminals, their packaging in synaptic vesicles in preparation for their release and enzymes for the degradation of an excess. An excitable membrane requires an energy pump to maintain a resting membrane potential, generally Naþ/Kþ adenosine triphosphatase (ATPase) in the case of neurons, ion channels in the plasma membrane and receptors to respond to transmitters produced by other neurons. All these subcellular structures must develop

before function can begin and before the cell is a mature neuron. Though many other morphological and cytological characteristics might be considered criteria of neuronal maturation, none are as universal as the two cited above. For example, chromaffin cells of the adrenal medulla do not form axons and dendrites but are neurons without neurites nevertheless. In the cytological development of neurons with neurites, axons always sprout before dendrites. In the cerebrum, axons emerge from migratory neuroblasts before they have completed their journey to the cortical plate and in the cerebellum. External granule cells establish synaptic contact with Purkinje cell dendrites before migrating through the molecular and Purkinje cell layers to their mature position in the internal granular layer. Most neurons are nonregenerating cells that lack mitotic potential, yet primary olfactory neurons can and do regenerate. Stem cells with a potential for neuronal differentiation are found in the human brain, particularly in the hippocampus (Curtis et al., 2005; Emsley et al., 2005; Ming and Song, 2005). Radial glial cells are transitory in the fetal brain during the period of neuroblast and glioblast migration, and most radial glia then mature as fibrillary astrocytes in the white matter; some radial glia have the potential to become new cortical neurons; however, particularly in these there is a fetal loss of some neurons that previously migrated from the germinal matrix (Tamamaki et al., 2001). Neurons of the lateral geniculate body and inferior olivary nucleus undergo trans-synaptic degeneration if chronically deprived of afferent innervation, but most neurons survive the loss of such continuous stimulation. Descriptive morphogenesis of processes of normal and abnormal development of the fetal brain at the macroscopic level, whether by imaging in living patients or by direct tissue examination, cannot address cellular maturation. Microscopic examination using classical histological stains provides details about the organization of cells to produce a particular normal or abnormal tissue architecture, and also reveals some features of subcellular detail, but cannot address biochemical and functional processes in development. Golgi impregnations enabled Ramoń y Cajal to describe the morphological organization of the human nervous system more than a century ago but are technically difficult, often capricious and always time-consuming techniques not practical to apply routinely to postmortem examinations. Electron microscopy provides further subcellular morphological details about organelles and synaptogenesis but postmortem autolysis often renders the quality of ultrastructure poor and its interpretation difficult. Histochemistry

EMBRYOLOGY AND NEUROPATHOLOGICAL EXAMINATION and immunocytochemistry offer a unique dimension of insight into development by addressing metabolic ontogenesis, as well as being practical and reliable in human autopsy brain tissue.

29.3. A new approach to sampling of immature brain tissue for microscopic sections After the gross description of the brain and associated structures, such as the meninges and major blood vessels, representative sections are taken for paraffin embedding in preparation for histological examination. Samples of the various lobes of the cerebral cortex are usually taken. In examining malformed brains, the selection of samples must now be modified. If the malformation is focal, it should prepared for microscopy but adjacent areas of transition to what appears grossly to be more normal cortex also should be sampled and identified in their relations to the lesion (e.g. gyrus anterior to lesion). Many malformations are now recognized as having gradients of genetic expression in the three major axes of the neural tube (see Ch. 1). Sampling must denote the extent of these gradients. For example, in holoprosencephaly, the mediolateral gradient of cortical dysplasia may extend only to the parasagittal area, with normal cortex lateral to it, or it may extend almost to the lateral extreme. In this type of malformation, therefore, samples should be taken of the frontal midline of noncleaved frontal lobe, the parasagittal region and two or three successive areas laterally, each clearly identified as to their site, often best accomplished with a simple drawing of the brain marking the sites where each sample is taken. This approach is quite different from a single sample of the ‘left frontal lobe’ and another from the ‘right occipital lobe’. In hemimegalencephaly, various regions of dysgenesis should be sampled, as well as corresponding regions in the opposite, more normal hemisphere for comparison. Identification of the site of the tissue samples again is important and a simple line drawing or diagram is often the most effective means. The ependyma lining the ventricles often provides useful information about maturation and the subventricular zone may disclose abnormalities of radial glial cells that explain abnormal patterns of neuroblast migration in focal regions of cortex and subcortical heterotopia. The brainstem is traditionally separated from the forebrain by cutting through the midbrain in the standard neuropathological autopsy. The midbrain is the site of many abnormalities in cerebral malformations and may indicate a long extent of the rostrocaudal gradient and interference with the dorsal mesencephalic site of neural crest generation, explaining abnormal

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neural induction of craniofacial development in some malformations and genetic syndromes. It is important, therefore, that the pathologist ensures that the midbrain, particularly the rostral midbrain at the level of the red nuclei, is included as a section submitted for microscopic examination. The spinal cord is often omitted from the tissue examined at autopsy, particularly in fetuses and neonates, but may provide valuable information. It should be sampled in all regions, cervical, thoracic, lumbar and sacral, for hydromyelia and other abnormalities. The ventral corticospinal tract may be greatly enlarged to include aberrant axons of the corpus callosum (see Chapter 5 by Sarnat HB: Agenesis and dysgenesis of the corpus callosum). In fetuses and young infants, unsuspected conditions such as spinal muscular atrophy may be demonstrated that have important genetic implications to the family. Finally at least one sample of striated muscle should be submitted. Many cerebral malformations are associated with congenital muscular dystrophy, congenital muscle fiber-type disproportion, fiber-type predominance and other muscular conditions (see Ch. 22), and these myopathies might not yet be symptomatic or clinically suspected, yet can be diagnosed at autopsy.

29.4. Markers of morphology in the nervous system Many crucial structures in nervous system development are subtle or not visualized at all in standard hematoxylin and eosin (H&E) histological stains but are easily demonstrated by special immunocytochemical markers. An example is the radial glial cell and its fiber that spans the cerebral mantle from its cell of origin in the subventricular zone, through the white matter and cortical plate to its termination on the pial membrane. This slender fetal astrocytic process guides migratory neuroblasts from the subventricular zone to the cortical plate; disruption of radial glial fibers occurs in both genetic diseases of neuroblast migration and also in acquired lesions in fetal life. Vimentin immunoreactivity is an excellent means of demonstrating these fibers (Fig. 29.1) in most of gestation, and glial fibrillary acidic protein (GFAP) demonstrates them well in late gestation. The technique of demonstrating vimentin is important (see below). Bergmann glial cells of the cerebellar cortex are the radial glia of the cerebellum, the cell bodies residing in the Purkinje cell layer and their process extending through the molecular zone to the pial surface to guide external granule neurons to their mature site in the internal granular layer beneath the Purkinje cells. These Bergmann processes also are well demonstrated by

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Fig. 29.1. Temporal neocortical plate of a midgestation (20-week) normal human fetus. (A) Hematoxylin and eosin stain shows a primitive columnar architecture with incipient lamination, but does not demonstrate radial glial fibers that guide neuroblasts and glioblasts. (B) Vimentin immunoreactivity well demonstrates the radial glial fibers spanning the cerebral mantle and passing through the cortical plate and also better shows the subpial granular glial layer of Brun. The thick radial lines are vimentin-reactive endothelial cells of small blood vessels. (C) Synaptophysin immunoreactivity of the same area shows synapse formation in the molecular zone and in the deep layers of the cortical plate but not yet in the superficial laminae. 25.

vimentin, and also with S-100b protein (see below), whereas GFAP demonstrates Bergmann cell bodies better than their radial processes. Another example of an immunocytochemical reaction being a good marker of histological abnormality is the dysgenesis of the superior lip of the inferior olivary nucleus in Leigh encephalopathy and certain other mitochondrial encephalomyopathies (Sarnat and Marin-Garcia, 2005). The changes in the inferior olive are difficult to recognize in H&E-stained sections but are dramatically highlighted with synaptophysin (Fig. 29.2).

29.5. Markers of cellular maturation in the nervous system Many immunocytochemical markers are now available that not only define the cellular lineage by identifying cell-specific proteins and secretory products but also define the state of maturation. They may be

categorized as markers of maturity and markers of immaturity. Markers of maturity appear only when the cell achieves a degree of maturation that initiates synthesis of the protein to which the specific antibody reacts. Markers of immaturity appear in undifferentiated or incompletely differentiated cells, and regress or disappear as the cell matures. It would be simple if all immunoreactivities could be interpreted exclusively as either markers of maturity or of immaturity. But in reality the majority of the antigens are both, differing at various stages in ontogenesis or in the cellular life cycle. NeuN generally marks only mature neurons and not neuroblasts but a small subpopulation of primitive neuroepithelial cells of the periventricular region are transiently reactive to NeuN antibodies (see below). The same is true of the calcium-binding molecules, such as calbinden-D28k and the neuropeptides, such as somatostatin. MAP2 is expressed in neuroepithelial cells, neuroblasts, some glioblasts and all

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Fig 29.2. Transverse section of hippocampus of a 32-week premature infant. (A) Haematoxylin-eosin stain to show anatomical relations. gm ¼ germinal matrix; th ¼ temporal horn of lateral ventricle. The dentate gyrus and Ammon’s horn are well seen. (B,C) Vimentin immunoreactivity shows radial glia and other astrocytes, as well as endothelium of blood vessels. (D) Neuronal nuclear antigen (NeuN) shows nuclear reactivity of maturity in some, but not all, neurons of the CA2 sector of Ammon’s horn. (E,F) Synaptophysin shows strong immunoreactivity in the molecular layer of the dentate gyrus and less reactivity in Ammon’s horn. (A,B,E X100; C,D,F X250, original magnifications).

mature neurons, but not in mature glial cells or in cells of mesodermal or endodermal lineage at any stage of maturation; it also demonstrates dendritic growth as a marker of maturity and its loss from glial cells as they mature enables it to serve as a negative marker or marker of immaturity. GFAP appears in ependymal

cells, not at the time of differentiation from primitive neuroepithelium but later, persists for a specific period and then regresses and disappears (Sarnat, 1992a, 1998a). Examples of pure markers of immaturity are vimentin, nestin and Ki67. An example of a pure marker of maturity is synaptophysin, a synaptic vesicle

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protein. Table 29.1 is a summary of the most useful markers of neural maturation. Immunocytochemical markers of maturity and immaturity also serve to identify cellular lineage and this application has contributed to an understanding of diseases of abnormal lineage, such as hemimegalencephaly (Takashima et al., 1991; Flores-Sarnat et al., 2003) and tuberous sclerosis (Hirose et al., 1995; Lopes et al., 1996; Mizuguchi and Hino, 2003) and is described in a 20-week fetus with tuberous sclerosis (Park et al., 1997). They also are useful in characterizing many neurodegenerative diseases (Dickson, 2005) and neoplastic lesions of the nervous system, but these conditions are outside the scope of this chapter. 29.5.1. Neuronal nuclear antigen (NeuN) NeuN is a specific marker of neuronal cells, recognizing nucleoproteins rather than cytoplasmic or surface antigens or cytoplasmic organelles, although this difference in cellular localization does not necessarily mean greater specificity. This antibody recognizes the nucleoproteins only of mature or nearly mature neurons (Fig. 29.2) but not neuroblasts or primitive

neuroepithelial cells and also shows no reactivity with glial cells of any type. Not only is it a valuable marker in identifying the ratio of mature and immature neurons in any given structure of the brain or spinal cord during the course of ontogenesis but, because it is primarily a nuclear marker, the sparse cytoplasm of immature neurons does not pose the problem in interpretation that often limits many cytoplasmic markers (Mullen et al., 1992; Wolf et al., 1996; Sarnat et al., 1998). Although the nuclear immunoreactivity is the most important and reliable, an epitope of NeuN often shows cytoplasmic reactivity as well. Mullen et al. (1992) found that immunocytochemically detectable NeuN protein first appears at developmental times that correspond either to the withdrawal of the neurepithelial cell from the cell cycle or to the initiation of terminal differentiation of the neuron. The latter is by far the most frequent time of NeuN appearance but the presence of a few NeuN-positive germinal matrix cells is evidence of the former. In our own studies, we also found that, in the cerebellar cortex at midgestation, external granule cells showed strong immunoreactivity in both the superficial proliferative zone and the deeper premigratory zone, although the internal granule cells, which are postmi-

Table 29.1 Markers of neural maturation in human fetal and neonatal brain and in CNS malformations Marker

Cell type

Maturity

Immaturity

Neuronal nuclear antigen (NeuN) Neuron-specific enolase (NSE) PGP9.5 S-100a protein Neurofilament protein (NFP) Chromogranin-A (CgrA) Calbinden D28k Calretinin Calmodulin GAP43 protein Synaptophysin Acridine orange Neurotransmitter systems Membrane-associated protein-2 (MAP2) Membrane-associated protein-1/5 (MAP1/5) Tubulin, class IIIb Tau protein Nestin Vimentin

Neurons Neurons Neurons Neurons Neurons; axons Neurons Neurons Neurons Neurons Neurite growth Synaptic terminals Neurons; ependyma Neurons Neurons, dendrites Neurons, axons Neurons Mature axons Neuroepithelium Neuroepithelium; neuroblasts; immature glia; ependyma Astrocytes; ependyma Glia; ependyma Myelination Neuroepithelial mitotic cycle

X X X X X X X X X

X X ?

Glial fibrillary acidic protein (GFAP) S-100b protein Myelin basic protein (MBP) Ki67 proliferating cell nuclear antigen

X X X X X X X X

X X X X X X

X X X

X X X


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Fig 29.3. Dysplasia of the superior lip of the inferior olivary nuclei bilaterally in a 3-month-old infant girl born at term with Leigh encephalopathy. Synaptophysin well demonstrates the abnormal and deficient distribution of synapses, compared with the normal appearance of the inferior lip. These changes were poorly appreciated with H&E-stained sections of the same block. 20. (Reproduced from Sarnat and Marıń-Garcıá, 2005 with permission of the Canadian Journal of Neurological Sciences.)

gratory and more mature, showed little or no immunoreactivity until 24 weeks gestation. After that time, they gradually became strongly reactive, as the external granular layer became more weakly reactive; by 36 weeks gestation, only the outermost cells of the external granular layer and a few migratory granule cells within the molecular zone were recognized by NeuN antibodies and most, but not all, of the internal granule cells were immunoreactive. Migratory granule cells within the molecular zone thus seemed to lose the immunoreactivity that they had earlier expressed while still in the external granular layer in young fetuses of less than 24 weeks, and an opposite gradient appeared after that age (Sarnat et al., 1998 and unpublished supplementary data). Strong NeuN immunoreactivity exhibited by external granule cells of the cerebellar cortex is an example of reactivity in cells soon after mitotic cycling is completed and perhaps even during the cell cycle. The reason for their loss of reactivity for a time, later reappearing with maturation of the internal granular layer remains a paradox. In the forebrain of fetuses of 22 weeks gestation, among cerebral neuroblasts within their migratory trajectory along the radial glial fibers to the cortical plate, only occasional cells exhibit reactive nuclei near the germinal matrix but many more marked nuclei are seen distally. Within the cortical plate of fetuses of 22 weeks gestation or less, nearly all neurons of the deep stratum, corresponding to the future layers 4–6, show NeuN immunoreactive nuclei, but the more superficial stra-

tum of future layers 2 and 3 contain only random marked nuclei. Scattered immunoreactive cells of the molecular zone are probably recent arrivals in the process of reversing course to take their position at the surface of the cortical plate, because Cajal–Retzius neurons and sublate neurons are always nonreactive for NeuN at all gestational ages. By 24 weeks gestation, most neurons of the cortical plate, both deep and superficial, exhibit strong reactivity, although many scattered neurons in layer 2 still fail to express NeuN and some cells in this superficial layer are still nonreactive even in the term neonate. Within the deep telencephalic nuclei (basal ganglia), NeuN-reactive neurons appear as early as 14 weeks gestation in the intracapsular part of the caudate nucleus and putamen, and maturation appears to spread as a gradient in both directions away from the internal capsule; this pattern also is seen in the globus pallidus (Sarnat and Born, 1999; Sarnat, unpublished observations). Specific patterns of maturation that correlate reliably with gestational age also are seen in the thalamus and brainstem nuclei but are beyond the scope of this chapter (Sarnat, unpublished observations). Neurons of dorsal root ganglia and cranial nerve ganglia and parasympathetic ganglia within the submucosal and myenteric plexi of the gut are strongly immunoreactive for NeuN as early as 8 weeks gestation (Sarnat et al., 1998). A biological factor of specificity renders certain specific types of neurons unable to recognize anti-NeuN antibodies in either immature or adult neural tissues;

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hence NeuN cannot be used to study these specific neurons. They include: Purkinje cells of the cerebellum (although granule cells are strongly reactive); neurons of the dentate nucleus of the cerebellum and inferior olivary nucleus of the medulla oblongata (although the pontine nuclei, also derived from the rhombic lip of His, are strongly reactive at maturity); mitral cells of the olfactory bulbs; Cajal–Retzius neurons of the molecular zone in the immature cerebral cortex; and retinal photoreceptor cells. Sympathetic chain ganglion cells also are nonreactive but chromaffin cells of the adrenal medulla do react strongly to NeuN antibodies and ganglion cells of the dorsal roots also are strongly reactive as early as 8 weeks gestation. The various types of nonreactive neurons appear to have little in common: they are morphologically diverse, do not share a single neurotransmitter system or any common metabolic feature, do not arise from a common embryological primordium in the brain, and do not have similar functions or similar fiber connections; some are central and others are peripheral, of neural crest origin. Neither immature nor mature glial cells, including radial glia and Bergmann cell nuclei, are reactive for NeuN. The notochord is nonreactive. Outside the nervous system, no immunoreactivity is detected in non-neural tissues. The specificity of NeuN for cells of neuronal lineage may not be total, however, because it recognizes some oligodendrocyte-like nuclei in dysembryoplastic neuroepithelial tumors (Wolf et al., 1996), suggesting that they may be of a different lineage, but neoplastic cells should not confuse the interpretation of normally developing brains or malformations that do not involve cytological dysgenesis, such as in tuberous sclerosis. Normal astrocytes and oligodendrocytes, reactive astrocytes and myelinating oligodendrocytes all fail to express NeuN. 29.5.2. Neuron-specific enolase Enolases are glycolytic enzymes found in many cells of the body, including fibroblasts. They catalyze the interconversion of 2-phosphoglyceric acid and phosphoenolpyruvate. Two principal isozymes are identified in the CNS: neuron-specific enolase (NSE; alpha-enolase) is synthesized by mature neurons and non-neuronal enolase (NNE; gamma-enolase) is produced by glial cells and immature neurons (Schmechel et al., 1978a; Ghandour et al., 1981; Royds et al., 1982). NSE possesses a greater affinity for the cofactor Mg2þ and greater resistance to disruption induced by Cl, urea and temperature than NNE (Marangos et al., 1978). NSE is a cytoplasmic marker of mature neurons of all types but does not show reactivity in neuroblasts and neuroepithelial cells and also is nonre-

active in glial cells. It is one of the earliest neuronal markers discovered but is not as specific as its name implies because it reacts in many other tissues. Nevertheless, within the central and peripheral nervous system it remains specific. During the course of fetal development, immunocytochemical studies of CNS enolases demonstrate that maturing neuroblasts switch from NNE to NSE production after completing migration to the cerebral cortex, in the cerebellum and in brainstem nuclei, in the rat (Schmechel et al., 1980; Hamre et al., 1989), monkey (Schmechel et al., 1980) and human (Nishimura et al., 1985). Measurement of each isozyme by radioimmunoassay during development demonstrates that NSE levels are low in embryonic rat brain and increase at a time corresponding to morphological and functional maturation of neurons; NNE levels are reciprocally high in embryonic brain and decrease when NSE first appears, followed by a gradual increase to adult levels (Marangos et al., 1980). NSE-containing neurons may be demonstrated immunocytochemically in human 17week fetuses in the facial and cochlear cranial nuclei, in neurons of the brainstem reticular formation and in about half the neurons of the globus pallidus and the thalamus (Nishimura et al., 1985). By 21 weeks most Purkinje cells and the majority of dentate nucleus neurons are strongly immunoreactive but by 40 weeks only occasional Purkinje cells were still intensely reactive. This initial increase followed later by a regression in NSE in Purkinje cell soma indicates that NSE is a marker of immaturity as well as of maturity and is similar in the postnatal mouse (Watanabe et al., 1990). No NSE or NNE immunoreactivity is demonstrated in undifferentiated neuroepithelial cells in the cerebral periventricular germinal matrix (subventricular zone) or in the external granular layer of the cerebellar cortex (Nishimura et al., 1985; Watanabe et al., 1990). The development of enolase expression in the cerebral cortex is less well documented. Human data from the striate (primary visual) cortex show immunoreactivity in pyramidal neurons of layer 5 at 21 weeks, followed by those of layer 3 at 24 weeks, but the granule cells, including those of the prominent layer 4 in striate cortex, do not exhibit NSE reactivity until 34 weeks gestation (Nishimura et al., 1985). Enolases thus provide useful markers of neuroblast maturation in sections of human fetal and neonatal brain. Neurons of the peripheral nervous system including chromaffin and other neuroendocrine cells such as the insulin-secreting islet cells of the pancreas also show NSE synthesis and a similar maturational pattern (Schmechel et al., 1978b). In situ hybridization of human adult brain tissue shows that mRNA expression may vary widely among different classes of neurons (Schmechel et al., 1987).


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NSE is of particular value because it can be measured in the cerebrospinal fluid (CSF) of living infants and its elevated concentration in CSF is considered a marker of neuronal death, particularly in hypoxic–ischemic encephalopathy (see below). Its direct examination in the brains of such infants who die can, therefore, be correlated with increased CSF levels of NSE.

of nerve cells (Crino et al., 1997). Nestin is expressed only in undifferentiated neuroepithelial cells and perhaps in the earliest stages of neuronal differentiation (Crino et al., 1997).

29.5.3. PGP-9.5

These four vitamin-D-induced calcium-binding proteins modulate neurotransmitter biosynthesis and secretion in mature neurons, although they are not themselves proper transmitters. They may be well demonstrated by immunocytochemical reactivity in tissue sections and not only serve as markers of maturity, being unexpressed in immature neurons, neuroepithelial cells and glial cells, but also are specific for highly differentiated types of neuron and not all neurons. They therefore share this quality with NeuN, which is not expressed in some types of neuron (see above) and with CgrA, which is also a synaptic neuromodulator (see below). An additional function of calmodulin is in the biosynthesis of steroids, a function so fundamental in nature that calmodulin even plays this role in plants (Du and Poovaiah, 2005). Calbinden D28k is highly specific for cerebellar Purkinje cells, both in normal brain and in cerebellar cortical dysgenesis (Katsetos et al., 1993). The functions of these proteins in development is incompletely understood but calmodulin, calretinin and parvalbumin have been shown to be involved in neuroepithelial cell mitosis (Gotzos et al., 1992; Rasmussen and Means, 1992) and these calcium-binding proteins are also postulated to be important in the control of neurite growth and cell movement, phenomena related to intracellular calcium concentration (Andressen et al., 1993). Parvalbumin in avian cerebellar neurons may be related to the synthesis of membrane components, intracellular transport and fusion of new membrane components into the plasmalemma (Braun et al., 1991). Some of these calcium-binding proteins appear to be induced by vitamin D (Celio and Norman, 1985) but calbindinD28K in the nervous system appears to be independent of vitamin D concentrations (Varghese et al., 1988). Calmodulin has a competitive interaction with its protein kinase, each obstructing the other’s actions when converging because of the embedding of protein kinase phosphorylation sites in shared calmodulin-binding domains of neurons (Chakravarthy et al., 1999). The distribution of these calcium-binding proteins in the mature CNS suggests that they are involved in important neuronal functions and their altered expression may lead to pathological conditions. For example, neurons expressing calbindin are resistant to excitotoxic insults and respond to excitatory amino acid

This antibody was more recently introduced than NSE and was initially thought to be more reliable and specific, but it is not as reliable or specific in fetal and neonatal brain tissue and has a limited use. 29.5.4. Neurofilament proteins and internexin Neurofilament proteins are a family of intermediate filament proteins that serve as cytoskeletal elements in the cytoplasm and neurites of mature and immature nerve cells and do not occur in other cells of the CNS. Their protein triplets are composed of three major subunits of different molecular weights – 68–70 kDa, 150–160 kDa and 200 kDa – that are each distinct biochemically and immunocytochemically. Neurofilament proteins are synthesized in the cytoplasm of the neuronal perikarya and are moved by slow axonal transport (Osborn and Weber, 1982; Lee and Cleveland, 1996). They are the most abundant element in mature axons, particularly those of large diameter. Neurofilament proteins are excellent for demonstrating not only neuronal soma but also mature axons and give reliable results in fetal brain including small, unmyelinated axons. This is a great advantage over the older silver impregnation techniques, such as Bielschowsky, Bodian and Sevier–Munger stains, which effectively demonstrate large myelinated axons of mature brain but are capricious in impregnating small and especially unmyelinated axons even in mature brain and are even less reliable in immature brain. Neurofilament proteins differ during ontogenesis from those of mature neurons by the degree of phosphorylation, being more highly phosphorylated in immature cells. Internexin is a transitional intermediate filament protein that appears in immature nerve cells preceding and transiently coexisting with the earliest expressed neurofilament triplet proteins of mature neurons, which are those of low molecular weight (Kaplan et al., 1988; Arnold and Trojanowski, 1996). As with neurofilament proteins of mature neurons, internexin is specific for cells of neuronal lineage and is not demonstrated in glial cells. Internexin may also be demonstrated in dysplastic neurons and has been used as a marker of developmental maturation

29.5.5. Calbindin D28k, calretinin, calmodulin and parvalbumin

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transmitters (Mattson et al., 1991). Parvalbumin appears to be selective for GABAergic neurons in the rat (Amadeo et al., 1998) and is lacking in neurons of human patients with severe epilepsy (Sloviter et al., 1991). It is coexpressed with GABA in the rat reticular and perireticular thalamic nuclei (Amadeo et al., 1998), whereas calretinin is expressed in the intralaminar and midline thalamic nuclei and is weaker and more restricted in distribution within the reticular nuclei (Frassoni et al., 1998a). Calretinin, also expressed in GABAergic neurons, also shows selective expression in Cajal–Retzius neurons and in a subpopulation of glutamate-containing pyramidal neurons in layer 2 of the rat piriform cortex (Frassoni et al., 1998b). Calbindin D28k is a specific marker for horizontal cells of the retina but correlates well with the coexpression of choline acetyltransferase and acetylcholinesterase activity (Kim et al., 1998). Calbindin D28k, calretinin and parvalbumin expression in human fetal cerebellum at successive gestational ages was studied by Yew et al. (1996, 1997) and Katsetos et al. (1993). Anti-calbindin-D28k antibodies are recognized by human Purkinje cells and Golgi cells and cerebellar nuclear neurons as early as 12– 14 weeks gestation, although both external and internal granule cells failed to show reactivity at any gestational age or in adults. Calbindin D28k delineates the dendritic and somatic spines of Purkinje cells and of a subpopulation of Golgi II cerebellar neurons at various gestational ages. Calbindin and parvalbumin immunoreactivity decreases with advancing fetal age and growth, inversely related to calretinin, which concurrently increases. A similar augmentation of calretinin reactivity was shown in the inferior colliculus of the mouse between 3 months of age postnatally and adulthood (Zettel et al., 1997). In the human fetus, reactivity of parvalbumin and calbindin D28k is detected in neurons of the deep cerebellar nuclei, Purkinje, basket and stellate neurons as early as 14 weeks gestation but then progressively decreases so that only reactivity of calbindin D28K is detected between 21 and 31 weeks gestation and is restricted to Purkinje and basket cells; calretinin-reactive neurons, by contrast, are not detected until 21 weeks and this immunoreactivity increases as the cerebellum matures (Yew et al., 1997). The cerebral cortex, including the hippocampus, has not been systematically studied in humans but the striate cortex of the monkey shows a consistently strong immunoreactivity for parvalbumin and calbindin D28K of cells in all layers in mid- to late gestation (Hendrickson et al., 1991) and parvalbumin reactivity is seen in pericellular clusters of terminal boutons, rather than in neuronal cell bodies, in developing and adult monkey neocortex (Akil and Levis, 1992). The

retina is one of the first regions to show calretinin and calbindin reactivity in the chick, including photoreceptor cones (Ellis et al., 1991; Roman et al., 1988), and the epiphyseal (i.e. pineal) analog of the retina also exhibits a similar expression (Roman et al., 1988). Calbindin D28k and calretinin also are expressed in the superior colliculus of the visual system (Jeon et al., 1998). Calbindin D28k and parvalbumin are expressed in the spinal cord and dorsal root ganglia during development in the rat (Zhang et al., 1990). These calcium-binding proteins have been studied in a variety of birds and mammals but there are considerable interspecific differences. These differences make extrapolation to human brain tenuous, so that studies of human brain are essential. 29.5.6. Acridine orange fluorochrome Acridine orange fluorochrome is a histochemical stain of nucleic acids not an immunocytochemical reaction. All aminoacridine compounds form highly fluorescent complexes with nucleic acids. Acridine orange serves as a ‘marker’ of neuronal maturation in rat and human brain, based on the premise that cytoplasmic RNA abruptly increases at the onset of neurotransmitter synthesis and that RNA remains sparse in glial cells; it is a reliable indirect marker of maturation in both human and rat brain (Sarnat, 1985, 1989; Topaloglu and Sarnat, 1989). Mature neurons show intense orange-red cytoplasmic fluorescence, as do nucleoli, but nucleoplasm shows yellow fluorescence; non-nucleic-acidcontaining proteins fluoresce in green. The RNA fluorescence after staining with acridine orange is weak to barely visualized in germinal matrix cells and during the course of neuroblast migration but becomes strong after neurons have reached their destination and proliferate ribosomes for the synthesis of neurotransmitters, regardless of which specific secretory transmitter is produced, and as synaptic contacts are established. Cajal–Retzius neurons of the molecular zone of the cerebral cortex always exhibit an intense acridine orange-RNA fluorescence, even in young fetuses of 8–12 weeks gestation. Many of the migratory neuroblasts in the subcortical white matter, particularly those approaching the cortical plate, as well as immature neurons within the cortical plate during the stage of lamination, show weak orange-red cytoplasmic fluorescence; these neuroblasts are seen migrating along their green radial glial fibers. In the cerebellar cortex, all neurons show weak RNA fluorescence until 24 weeks gestation, after which time Purkinje and Golgi cells become strongly fluorescent but internal granule cells continue to show only faint orange

EMBRYOLOGY AND NEUROPATHOLOGICAL EXAMINATION cytoplasmic color, at times barely perceptible. The external granule cells are actually more strongly fluorescent for RNA than the internal granule cells. This paradox cannot be ascribed to differences in the sparse amount of cytoplasm; hence acridine orange must be regarded as a marker of immaturity in certain sites in the brain, in addition to its more prevalent role as a marker of maturity. Glial cells of astrocytic and oligodendrocytic lineage do not show orange-red acridine orange-RNA fluorescence either as immature or mature cells, and even reactive astrocytes and gemistocytes do not exhibit this feature, although neoplastic astrocytes do show RNA fluorescence (Sarnat et al., 1986). Ependymal cells show acridine orange-RNA fluorescence as soon as they differentiate from neuroepithelium and this persists into adult life. 29.5.7. S-100a protein This is a cytoplasmic marker of mature neurons but not of glial cells, unlike S-100b protein, which is a glial marker (see below). It is another in the series of calcium-binding small acidic molecules, as with chromogranin and calbindin D28k (see below). There is a precise distribution of expression of the protein in subpopulations of neurons in the rat nervous system, and also of the S-100b protein of glial cells (Yamashita et al., 1999). 29.5.8. Chromogranin-A Chromogranins are small, glycosylated, acidic proteins, widely distributed in neurons, chromaffin cells and other neuroendocrine cells (Nolan et al., 1985). Though localized to secretory granules and the walls of secretory vesicles, as shown by ultrastructural immunocytochemistry (Wilson and Lloyd, 1984; Nolan et al., 1985; O’Connor and Deftos, 1986), they differ from other synaptic vesicle wall proteins such as synaptophysin and synaptobrevin because chromogranins are water-soluble, hence secretory rather than structural proteins. Chromogranins bind calcium, a propensity shared with a few other small, acidic, soluble neuronal proteins such as calbindin D28K, calretinin and S-100 (O’Connor and Deftos, 1986; Lloyd, 1988; Varndell et al., 1985). Chromogranins thus are not themselves neurotransmitter substances that alone mediate depolarization of the postsynaptic membrane but are released at the synaptic cleft from vesicles in the presynaptic axonal terminals in association with a variety of chemical transmitters, especially monoamines but also acetylcholine and several neuropeptides. They also are precursors of neuropeptides,

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particularly in nerve cells unrelated to catecholamine metabolism (Wu et al., 1991). Each of the chromogranins can be well demonstrated by immunoreactivity in tissue sections (Lloyd et al., 1986). Chromogranins are widely distributed within the brain but the distribution and release of chromogranins must be considered in relation to the specific, individual neurotransmitters with which they are associated, such as acetylcholine, monoamines, GABA and neuropeptides, as well as the receptors for these transmitters on the postsynaptic membrane (Munõz, 1990; Munõz et al., 1990; Erickson et al., 1992). Chromogranin may be important in modulating the release of these transmitters, which in turn regulate neuronal excitability. There are three principal chromogranins, designated A, B and C, comprising a family with different molecular weights in various mammalian species. Chromogranin-A (CgrA) is the most extensively studied and the gene regulating it has been identified and its molecular structure characterized (Gratzl, 1987). Chromogranins were originally described in the adrenal medulla and are closely associated with catecholamines and are released together with them (O’Connor and Deftos, 1986; Reiffen and Gratzi, 1986a,b). Chromogranins are demonstrated both in the cytoplasm of the cell bodies of some neurons and in their axonal terminals. Axonal transport of chromogranin to the terminal synaptic vesicles occurs in both the central and peripheral nervous systems (Banks and Helle, 1967). CgrA is found in axons of all sizes but CgrB is restricted to large axons; both are present in dorsal and ventral spinal roots and CgrB is mainly found in cholinergic neurons such as motor neurons, whereas CgrA occurs in these as well as in adrenergic neurons (Banks and Helle, 1967). CgrA and CgrB are not expressed in immature neurons but are demonstrated in neurons in widespread regions of the rat brain in a particular topographical distribution (Banks and Helle, 1967; Blaschko et al., 1967; Winkler et al., 1986; Gibson and Munõz, 1993). An example of specificity is in Ammon’s horn of the hippocampus. In normal subjects, CA2 projects upon CA3 and CA1. CgrA thus shows intense reactivity within the neuronal cytoplasm of CA2 but little reactivity in the neuropil; CA3 and CA1 neurons show only weak cytoplasmic reactivity but the neuropil between individual neurons exhibits numerous terminal axons with CgrA reactivity. Synaptophysin, by contrast, shows synaptic vesicle activity in all regions of Ammon’s horn without distinction. CgrA activity distinguishes monodendritic neurons in the cerebral cortex (Munõz, 1990). In the rat, CgrA is expressed not only in neurons of the CNS but in cerebellar Bermann glial cells as well (Li et al., 1998), though it is not generally demonstrated

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in astrocytes, oligodendrocytes or ependymal cells. Chromogranin-C (CgrC) is secreted, however, by cultured astrocytes (Winkler et al., 1986). 29.5.9. Synaptophysin Synaptophysin is a synaptic vesicle protein and is a valuable marker of synaptogenesis in fetal and neonatal brain tissue (Figs 29.1, 29.4). It is a true marker of maturity and has the advantage of being a very hardy protein that is still detected even after long periods of postmortem autolysis in delayed autopsies. Other synaptic vesicle proteins, such as synaptobrevin (vesicleassociated membrane protein, SV2, VAMP) and SNAP-25, also may be used, although experience with these in human neuropathology is limited (Scranton et al., 1993; Bajjalieh and Scheller, 1995; Su¨dhof, 1995). Synaptobrevin is a keratan sulfate proteoglycan, and this group of compounds also serve as repellants of axonal growth cones when secreted by ependymal processes of the roof and floor plate cells to prevent aberrant axonal decussation in long ascending and descending pathways, such as the dorsal columns in the developing spinal cord (Snow et al., 1990; Scranton et al., 1993). Some authors report that synaptophysin is reliable only in fresh surgical biopsy material and in specially processed postmortem tissue (Snow et al., 1996); in our laboratory we have found the contrary, that this antibody is consistently reliable and hardy, yielding credible results even after 24 hours of postmortem autolysis. Not only is synaptophysin a good marker of synaptogenesis in the developing nervous system but it also

highlights many structures that are difficult to demonstrate in standard H&E-stained histological sections. Examples are the important central respiratory center, the fasciculus/nucleus solitarius of the medulla oblongata, the ventral reticular nucleus of the pons between the tegmentum and basis pontis, the dentate nucleus of the cerebellum and the inferior olivary nucleus. In the latter case, dysplasias that are subtle or ambiguous in H&E preparations are clearly defined using synaptophysin antibodies. In the cerebral cortical plate, synaptophysin reactivity is detected in the molecular zone at 10 weeks gestation and remains strong throughout fetal and postnatal life. In the cortical plate, marked neurons are demonstrated in the deep part of the cortical plate at 19 weeks and, in the more superficial laminae, at 25 weeks (Sarnat and Born, 1999). In the cerebellar cortex, a narrow band of reactivity is seen in the molecular layer at 18 weeks and later becomes a double band within the molecular layer before showing diffuse synaptic activity within the entire layer; synaptic vesicles surround Purkinje cell bodies before 20 weeks, and synaptophysin also is seen in association with internal granule cells at midgestation and demonstrates synaptic glomeruli after 26 weeks (Sarnat and Born, 1999). Ventral horns of the spinal cord and the tegmental gray matter of the brainstem show strong synaptophysin at 12–14 weeks and follow a predictable pattern of acquired axonal terminal synaptophysin reactivity in later gestation, the last area to exhibit complete reactivity being the nuclei of the basis pontis at near-term (Sarnat, unpublished observations). Synaptophysin reactivity may also be demonstrated in the peripheral nervous system, including the dorsal root ganglia and the parasympathetic ganglia of the myenteric plexus (Sobaniec-Lotowska et al., 2004). 29.5.10. Neurotransmitters and their enzymes

Fig 29.4. Synaptophysin reactivity of the region of the head of the caudate nucleus and anterior putamen. (A) 14-week fetus showing incipient synaptogenesis only in the intracapsular part of the corpus striatum. (B) 21-week fetus showing synapse formation in parts of the caudate nucleus and putamen. (C) 40-week term neonate showing extensive synapses throughout the corpus striatum. 250.

Enzymatic activities of choline acetyltransferase and acetylcholinesterase, the enzymes of biosynthesis or degradation of acetylcholine may be demonstrated by both standard histochemistry and immunoreactivity. Most transmitters are themselves simple amino acids that cannot be directly demonstrated in tissue sections as they can with biochemical assays, but many transmitter systems can be indirectly localized in tissue sections by their enzyme systems: acetylcholine, monoamines, serotonin, simple amino acids such as glutamate and glycine, and g-aminobutyric acid (GABA) (Lloyd et al., 1986). In many cases, receptors of these neurotransmitters also may be show by immunoreactivity. In general, the demonstrated neurotransmitter systems in the brain and spinal cord are markers


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Fig 29.5. The strisome pattern seen in the caudate nucleus and putamen (put) is not seen in the globus pallidus (gp) in the midgestational hyman fetus in demonstrating synaptogenesis. Synapse formation is strong in the globus pallidus and in the striosomes of the corpus striatum, but adjacent zones of similar neurons in the putamen show little or no synaptic reactivity. Synaptophysin immunoreactivity. (A) 100; (B) 250.

of maturity, but some associated enzymes, such as acetylcholinesterase, are present in embryonic brain structures long before neurons are mature or any synapses are formed, so they must be serving some other function early in ontogenesis unrelated to neurotransmitter systems; a trophic effect has been suggested. 29.5.11. Microtubule-associated protein-2 Microtubule-associated protein-2 (MAP2) is a highly reliable cytoplasmic marker of cells of neuroepithelial lineage but does not distinguish immature from mature neurons. Neuronal precursor cells of the neuroepithelium and germinal matrix and migratory neuroblasts exhibit a cytoplasmic MAP2 immunoreactivity almost as strong as that seen in mature neurons. All neurons are stained without exception, including Cajal–Retzius cells. Mature glial cells are not reactive but radial glial cells and their centrifugal processes and glial precursors, including many glia of the subventricular zone, also show MAP2 reactivity and cannot always be distinguished from immature neurons. In neurons with distinctive mature morphology, such as pyramidal cells and Purkinje cells, the cytoplasm of the soma and of the dendritic tree, but not of the axon, are reactive; axoplasm of immature neurons also is not recognized by MAP2 antibodies. MAP2 antibodies are widely used in the study of surgically resected brain tissue in epileptic patients (Yamanouchi et al., 1996). MAPs are a family of structurally related but distinct molecules that exhibit different developmental patterns in the CNS. MAP2 exists in high- and lowmolecular-weight subtypes, MAP2a, b and c, and some tubulin-binding repeats are specific for some neurons, such as dorsal root ganglion cells and not for other neurons; the ratio of subtypes also changes during

maturation (Forleo et al., 1996). MAP2 proteins are essential not only for the early neuronal differentiation but also for the maintenance of adult neuronal morphology; changes occur in MAP2 phosphorylation during cerebral development, which may be important for modulating different functions in fetal and adult life and for molecular stability (Schoenfeld et al., 1989). The modification and rearrangement of the MAP2 structural configuration is an early obligatory step in many processes that modify neuronal function at different ages (Johnson and Jope, 1992). MAP2 is localized to the somatic cytoplasm and dendrites of neurons. MAP2 is prevented from entering the axon by its N-terminal projection domain and phosphorylation may also play a role in the microtubule binding domains (De Camilli et al., 1984). MAP2 recognizes the earliest generated neurons in the cerebral cortex, both Cajal–Retzius and subplate neurons, in the cat (Chun and Shatz, 1989) and human (Honig et al., 1996). MAP2 is expressed in immature neurons in all stages of development and even in undifferentiated neuroepithelial cells, as well as in mature neurons, rendering it not useful as a marker of maturation, but it does identify the neuronal lineage from an early stage. Though not a marker of neuroblast maturation because it is expressed in undifferentiated neuroepithelial cells as well as in immature and mature neurons, MAP2 is an immaturity marker for glial cells. Radial glial cells and pseudostratified columnar ependymal epithelium are strongly reactive with MAP2 antibodies but this immunoreactivity is lost as cells mature to become astrocytes, oligodendrocytes and simple cuboidal ependyma. This lack of recognition of the MAP2 antibody by mature glial and ependymal cells leaves neurons as the sole CNS cells expressing MAP2, facilitating the recognition of heterotopic as well as normally situated neurons.

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Immunoreactivity of MAP2 in some non-neural tissues may be nonspecific binding. Cells of the notochord are intensely reactive in 8–10-week fetuses; chondroblasts of the surrounding sclerotome also are reactive but less intensely so. Outside the nervous system, many epithelial cells are variably immunoreactive and even nucleated red blood cells at times react with MAP2 antibody. 29.5.12. Microtubule-associated protein-1/5 MAP1 and MAP5 are nearly identical and show reactivity similar to MAP2, but are reactive for neuronal cell bodies and axons rather than dendrites. MAP1a increases during postnatal development, whereas MAP1b is abundant in the newborn rat brain, with particularly high concentrations in developing axonal processes of the cerebellar molecular layer, corticospinal tract, and mossy fibers of the hippocampus and olfactory bulb, but then decreases in expression (Riederer et al., 1995). 29.5.13. Tubulins The tubulins are a family of cytoskeletal structural proteins related to the microtubule MAP proteins. The most useful and most studied of this group, in terms of ontogenesis of the human brain and the cerebellum in particular, are the class III b-tubulins (Katsetos et al., 2003). These tubulins are abundant in the brain during fetal and postnatal development. Transient expression is seen in primitive neuroepithelial neuronal/glial precursor cells but in adult brain class III btubulins are neuron-specific and not expressed in glia or ependyma. They are excellent markers of Purkinje cells but also of other cerebellar granule cells and basket cells. Another tubulin that has been studied, but is not as specific, is T1J1 (Sarnat, unpublished data). 29.5.14. Tau protein By contrast with MAP2, Tau is localized in mature axons (Matus, 1994; Kanai and Hirokawa, 1995); both proteins appear to serve as cytoskeletal elements by making the microtubules in neurites more stable and stiffer than those in non-neuronal cells (Matus, 1994). The abnormal phosphorylation of tau protein is implicated in the pathogenesis of several neurodegenerative diseases (Feng et al., 2005). 29.5.15. Neuropeptides (somatostatin; substance P; neuropeptide Y; cholecystokinin) The neuropeptides that sometimes serve as neurotransmitters and more frequently are modulators of synaptic transmitter activity appear early in gestation and

sooner than transmitter synthesis or synaptogenesis are initiated. They are found only in two tissues of the body: the nervous system and the intestinal mucosa. In the early neural tube, they therefore must serve some other function than synaptic transmission. Neuropeptides thus may be classified both as markers of immaturity and later as markers of maturity corresponding to true neurotransmitter functions. The cerebellum is the part of the mature brain that normally exhibits the least amount of somatostatin and substance P, and almost none is detected by either immunocytochemistry or biochemical assay in the mature cerebellum. Nevertheless, very high concentrations of both compounds are demonstrated in the rat cerebellum corresponding to 16–18 weeks gestation in humans, after which time they regress (Inagaki et al., 1982a,b). Their precise function at that time is speculative. Neuropeptides are seen in the mature cerebral cortex and hippocampus and throughout many brainstem nuclei. After neuronal injury, substance P receptor binding sites are expressed ectopically by glial cells (Mantyh et al., 1989). 29.5.16. GAP43 Antibodies to demonstrate the products of genes that regulate neurite outgrowth and synaptogenesis, such as GAP43, are available. GAP43 immunoreactivity is present in the cerebral cortex throughout all stages of development from 14 weeks gestation and probably earlier; it also is expressed in the dense radial and transverse nerve fiber bundles in the molecular and intermediate zones but not in undifferentiated neuroepithelial cells (Honig et al., 1996). 29.5.17. Glial fibrillary acidic protein For glial cell maturation, particularly the astrocytic lineage, vimentin appears early and glial fibrillary acidic protein (GFAP) appears later, being coexpressed with vimentin for a period of time until the vimentin is no longer seen and only GFAP is expressed. These two markers are thus complementary and GFAP is a true marker of astrocytic maturation. GFAP is also transiently expressed in ependymal cells at midgestation and late gestation and thus is a maturation marker in early gestation and a marker of immaturity in later gestation. GFAP is never expressed in floor plate ependymal cells at any stage of maturation, however, thus serving also to identify cellular lineage (Sarnat, 1992a). The monoclonal antibody is usually more reliable than the polyclonal antibody in immature brain tissue, the opposite of their respective reactivities in adult brain.

EMBRYOLOGY AND NEUROPATHOLOGICAL EXAMINATION 29.5.18. S-100b protein This glial marker of astrocytes, oligodendrocytes and ependymocytes is expressed at a stage of maturity and not earlier. In ependymal cells, it appears and later disappears, and thus is both a marker of maturity and also a marker of immaturity, depending upon the gestational age. It is an excellent marker of Bergmann glial cells of the cerebellar cortex in both the fetus and adult, showing the cell bodies more intensely than their radial processes, and may be complementary to vimentin and GFAP, which show the processes better than the cell bodies of Bergmann cells. S-100b appears transiently in a precise temporal and spatial pattern in the fetal ependyma but is not expressed in the postnatal period or adult life in the ependyma (Sarnat, 1992a, 1998a), although it continues to be expressed in choroid plexus epithelium throughout life (Sarnat, 1998a). Ependymal S-100b protein may be related to neuroblast and glioblast migration or the conversion of radial glial cells to mature astrocytes at the end of migration (Sarnat, 1992b). It may also be involved in endogenous repair following brain injury (Kleindienst et al., 2005). 29.5.19. Nestin Nestin is an intermediate filament protein found in neural stem cells and undifferentiated neuroepithelial cells with mitotic potential; hence it may be demonstrated immunocytochemically in fetal brain cells as a marker of immaturity. The progenitor cells it marks may later differentiate as neurons, glial or ependymal cells (Leone et al., 2005). It thus shares many properties with vimentin. It has been used to identify a subpopulation of precursor cells in the mouse dentate gyrus that receives GABAergic input (Wang et al., 2005). 29.5.20. Myelin basic protein This immunocytochemical marker of myelination is more sensitive than the traditional histochemical stains, such as Luxol fast blue (LFB) and gallocyanin compounds. 29.5.21. Ki67 proliferating cell nuclear antigen Ki67 proliferating cell nuclear antigen (PCNA), present in all cells, is expressed not only in the mitotic M-phase when mitotic spindles are obvious with any histological stain but also in the gap phases (G1, G2) and the resting phase (S phase) of cells still in the mitotic cycle but not histologically evident as cycling cells. It is, therefore, an excellent marker of undifferentiated neuroepithelial

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cells and is no longer expressed in the postmitotic phase of neuroblasts and glioblasts. The quality it demonstrated also is helpful in some malformations, such as the cortical tubers and subventricular nodules in tuberous sclerosis (Lee et al., 2003). Most PCNA antibodies are applicable only to frozen sections but the MIB-1 form can be reliably applied to formalin-fixed, paraffin-embedded sections. Thermal intensification is required, as with all nuclear antibodies.

29.6. Use of maturational markers in perinatal ischemic–hypoxic encephalopathy and traumatic brain injury Dead and dying neurons lose their immunoreactivities to the various markers of the neuronal lineage, so that antibodies to NeuN (Schmidt-Kastner et al., 1995), CgrA, calbindin DK-28a and others no longer recognize the degenerating proteins of former healthy cells. MAP2 has been extensively used in studying acute brain injury and ischemic–hypoxic encephalopathy and loss of immunoreactivity is convincing evidence that the cell is beyond regeneration and recovery (Kitagawa et al., 1989; Dawson and Hallenbeck, 1996; Malinak and Silverstein, 1996; Ku¨hn et al., 2005). Acridine orange is useful in showing an abnormal centrifugal distribution of RNA in the cytoplasm of neurons with abundant cytoplasm or loss of cytoplasmic RNA fluorescence altogether following severe ischemic–hypoxic insults in term neonates (Sarnat, 1987). Hypoxia–ischemia is known to suppress the expression of NeuN antigen, and hypoxic–ischemic encephalopathy also diminishes the expression of MAP2 in adult and neonatal rat brains. Infants who have suffered fetal distress or perinatal asphyxia, therefore, may show less NeuN and MAP2 immunostaining of neurons than infants who have not experienced such insults, although we have not noted this problem in brains of infants who suffered severe intrapartum asphyxia. At the present time, it is difficult to quantify the degree of hypoxia, the duration of the asphyxia or the time interval between the hypoxic event and the time of death in relation to the reliability of immunoreactivity of these two methods. In our experience, terminal or agonal asphyxia of short duration does not affect the quality of immunocytochemistry staining with either NeuN or MAP2. We are presently evaluating longer duration perinatal hypoxic–ischemic encephalopathy with morphological changes in neurons to better define these variables and their effects on immunoreactivity. NSE has a special importance because its elevation in CSF is sometimes used as a clinical marker of neuronal death in hypoxic encephalopathy; correlation with

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its preservation or loss in tissue sections in infants who do not survive and for whom the concentration of NSE in CSF was measured before death could provide valuable insight into the significance of high CSF levels of this glycolytic enzyme. The quantitative measurement of g-NSE in CSF of living human patients has provided data supporting the value of elevated CSF enolase as a marker of hypoxic injury or infarction of the brain (Royds et al., 1983; Steinberg et al., 1984; Van den Doel et al., 1988; Karkela et al., 1993) and possibly in viral encephalitis (Van den Doel et al., 1988). Experimental stroke in the rat also is followed by high CSF enolase (Hatfield and McKernan, 1992). Of equal clinical interest is the elevation of human CSF or serum g-enolase as a marker of neuronal dysfunction without definite evidence of cellular death: in status epilepticus (DeGiorgio et al., 1995, 1996), in children with continuous paroxysmal electrographic activity without convulsive motor activity (O’Regan and Brown, 1998; Hizli et al., 1997). In other studies, it was found that NSE does not increase significantly in CSF after acute seizures, unless the seizures are symptomatic of specific neurological diseases (Wong et al., 2002). The potential application of these CSF and serum measurements in premature and term neonates makes an understanding of enolase development in individual neurons even more relevant for clinical interpretation. Hypoxia or ischemia in the human newborn also affects acridine orange fluorescence because of the redistribution of cytoplasmic ribosomes associated with hypoxic chromatolysis: during acute stages ribosomes become ‘degranulated’ from the endoplasmic reticulum and, because cytoplasmic flow is centrifugal from the nucleus, the orange-red RNA fluorescence becomes marginalized to the periphery of the neuronal cytoplasm; during recovery phases, when new RNA is being transcribed in the nucleus and discharged into the cytoplasm, a rim of orange-red fluorescence surrounds the nucleus, while most of the cytoplasm remains green, the color of protein free of nucleic acids (Sarnat, 1987). Serum concentrations of S100b protein are elevated in patients with severe head injuries, probably because of release from damaged glial cells or increased synthesis (Raabe et al., 1998). The serum half-life of this protein is only 2 hours.

attention has been paid to the maturation of individual neurons, synaptic circuitry and associated glial relations. The present availability of reliable immunocytochemical markers that may be applied to postmortem tissues enables us for the first time to examine this aspect of the distorted architecture of cerebral dysgenesis in a way that neuroimaging and electrophysiological studies cannot do, but these examinations in living patients can be made more meaningful with good correlations of maturational changes in malformed brain tissue. After all the normal developmental criteria have been established, and acquired conditions that might alter them, such as ischemic–hypoxic encephalopathy and metabolic encephalopathies, the data derived from the study of genetically determined malformations of the brain are likely to be the richest in terms of understanding normal and abnormal neural development. Some of these data in human malformations of the brain are already reported but the known data are a sparse part of what can be known from systematic investigations using these neuropathological tools. In the lissencephalies and in cerebro-hepato–renal (Zellweger) disease, the ependyma exhibits a continued overexpression of GFAP and S-100b protein, but not of vimentin, beyond the time when these proteins should have regressed (Sarnat et al., 1993a,b). In Chiari II malformations, by contrast, ependymal GFAP and S-100b protein are normally expressed and regress at the proper time but vimentin continues to be overexpressed in the areas of dysgenesis (Sarnat and MarıńGarcıá, 2005). In Fukuyama congenital muscular dystrophy, associated with pachygyria and other cerebral dysgeneses, tau proteins in the brain are abnormally phosphorylated (Saito et al., 2005). Mixed cellular lineage and maturational aberrations are defined in cerebral lesions in tuberous sclerosis (Hirose et al., 1995; Lopes et al., 1996; Mizuguchi and Hino, 2003) and hemimegalencephaly (Takashima et al., 1991; Flores-Sarnat et al., 2003). Markers of cellular proliferation also are useful in tuberous sclerosis (Lee et al., 2003) and in focal cortical dysplasias of the Taylor type with balloon cells of often ambiguous lineage (Ying et al., 2005).

29.7. Use of maturational markers in congenital malformations of the nervous system

Both biological and technical variables must be considered in interpreting the results of each method. An important biological factor to be considered in studying autopsy tissue, which is not a consideration in surgically resected tissue, is postmortem autolysis. Delay in performing the autopsy or in fixation of tissue may

Most studies of malformations of the brain have focused on tissue architecture, the arrangement of neurons within a particular structure of the brain. Little

29.8. Technical notes about the use of histochemical and immunocytochemical markers in immature brain

EMBRYOLOGY AND NEUROPATHOLOGICAL EXAMINATION diminish the reliability of histochemical staining and of immunoreactivity long before frank liquifaction occurs. Technical factors that affect the demonstration of NeuN, MAP2 and acridine orange include duration of fixation in formalin and possibly long storage of tissue in paraffin blocks. Long periods of fixation in formalin, for many months or years, render NeuN less immunoreactive than expected, in comparison with age-matched fresher tissue that has been fixed for only a few days or weeks. Old tissue embedded in paraffin blocks for years may also not be entirely reliable but generally yields better results than tissue stored in formalin. Acridine orange often exhibits a diffuse red background fluorescence throughout the tissue after prolonged formalin fixation; this artifactual red color of the neuropil and cytoplasm of cells may sometimes be extinguished by exposing a microscopic field to ultraviolet light for 2–3 minutes. The natural orangered RNA fluorescence is more resistant to quenching, although photodecomposition of the natural color also causes loss of its fluorescence with continued ultraviolet irradiation. The red color that nuclei acquire after prolonged formalin fixation is permanent and is due to ‘denaturing’ of the DNA, i.e. the double strands of DNA that normally fluoresce yellow with acridine orange become separated into single strands that fluoresce red. Another technical factor to consider in immunocytochemistry is the importance of thermal treatment of NeuN, MIB-1 (Ki-67 proliferating cell nuclear antigen) and all other nuclear markers, but also some cytoplasmic markers such as MAP2. These antibodies cannot usually be demonstrated in tissue sections by incubation at room temperature and require the augmented antigen retrieval by boiling. Microwave treatment is the simplest and most reliable method. Many other common immunocytochemistry methods used in neuropathology also exhibit greatly enhanced antigen retrieval after thermal intensification, usually by microwave treatment (Shi et al., 1991; Kok and Boon, 1992; Suurmeyer and Boon, 1993; Ainley and Ironside, 1994; Evers and Uyling, 1994; Login and Dvorak, 1994; Marani and Horobin, 1994; Sarnat, 1998b; Sarnat and Born, 1999). Some markers, such as vimentin, demonstrate different aspects with or without thermal intensification; heating is best to demonstrate radial glial fibers but room temperature incubation is better for showing maturational changes in ependymal cells (Sarnat, 1998b). Boiling the incubating solution on a hot plate produces results indistinguishable from those obtained by heating in a microwave oven; hence it is heat and not microwaves per se that enhances the result.

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Some antibodies have a short shelf-life and, after the date of expiration printed on the supplier’s label, they may no longer be reliable, although most are still good for extended periods if preserved in the freezer or the refrigerator, as recommended for each antibody. Finally, a note on terminology may help to clear up ambiguities and improve precision so that words with specific meanings are not degraded by using them as generalities. The terms ‘immunohistochemistry’ and ‘immunocytochemistry’ are synonymous and may be used interchangeably, though the latter is preferred because it usually refers to the detection of subcellular proteins and other products by antibody reaction. This reaction may be demonstrated in tissue sections by light microscopy using immunoperoxidase and avidin-biotin to yield a colored product within the cell (Hsu et al., 1981), or by immunofluorescence. The term ‘immunostaining’ is incorrect, however, because staining is the coloration with a dye of all cells and their products, as used histologically, or the specific coloration by a dye of metabolic products, such as the periodic-acid–Schiff (PAS) reaction to demonstrate glycogen and polysaccharides, oil red O or Sudan black to demonstrate neutral lipids, tetrazolium reduction reactions to demonstrate oxidative enzymatic activities and other true histochemical stains. Immunocytochemical demonstrations in tissue sections, whether by light microscopic or fluorescence microscopic technique, should be called ‘immunoreactivity’ or simply ‘reactivity’, but not ‘staining’. The term ‘marker’ refers to any histochemical stain or immunocytochemical reactivity that is specific in recognizing a cell as belonging to a particular lineage or denoting its state of maturity or immaturity.

29.9. Conclusions In fetuses and neonates who do not survive despite excellent care, autopsy presents another opportunity to learn about CNS maturation and microscopic and subcellular aspects of malformations to compliment neuroimaging, electroencephalography and other techniques during life. Modern neuropathological examination includes methods such as immunocytochemistry that define maturational changes and cellular lineage in brain tissue, offering a level of diagnostic precision not available from standard histological examination. Markers may either be expressed only in maturing or mature cells or may be expressed only in immature cells, regressing as the cell matures. Most markers are both: they are expressed transiently in a subpopulation of undifferentiated neuroepithelial cells, then regress and later are expressed only as the cell matures. Most are highly specific for particular

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types of neuron, glial and ependymal cell. The most useful immunocytochemical markers for fetal and neonatal brain are vimentin, to demonstrate radial glial fibers for neuroblast migration and cellular immaturity, and synaptophysin, to show the maturation of synaptogenesis in various parts of the nervous system and help explain epilepsy in some cases. Many neuronal, glial and ependymal markers of maturation also are useful in determining the extent of injury following perinatal ischemic–hypoxic encephalopathy and reveal additional abnormalities in the various cerebral dysgeneses. Every attempt should be made to secure permission for autopsy and ensure that meaningful postmortem neuropathological study is performed.

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Section IV Management of central nervous system malformations


Chapter 30

Medical treatment in children with central nervous system malformations PAOLO CURATOLO*, ROBERTA BOMBARDIERI, AND CATERINA CERMINARA Tor Vergata University of Rome, Rome, Italy

30.1. General considerations The management of children affected by malformations of the CNS is particularly complex and needs diagnosis, assessment, treatment, care, counseling and periodic diagnostic review and reassessment. In recent years our ability to diagnose developmental disorders of the CNS has greatly improved through the use of high-resolution magnetic resonance imaging (MRI) (Goyal et al., 2004). Clinical features of brain malformations are extremely variable, mainly depending on the extent and location of the abnormality, as well as the presence of associated malformations. Most patients have seizures associated with variable degrees of learning disorders, delayed or abnormal development and behavioral/psychiatric disorders. However, even for the same kind of MRI abnormality, the long term prognosis is highly variable, often unpredictable at an early stage, and remains a challenge for child neurologists. Although developmental disorders of the CNS are usually not progressive, they are often associated with the presence of early-onset seizures refractory to conventional medical treatment. For example, hemimegalencephaly, tuberous sclerosis and focal cortical dysplasia are highly epileptogenic malformations characterized by severe and intractable seizures. The presence of chronic seizures and persistent EEG abnormalities may in itself have a further negative impact on the cognitive functions of children suffering from developmental disorders. However, it is difficult to identify whether the mental impairment is the functional consequence of epilepsy in itself or is due to the presence of underlying developmental abnormalities. Both clinical and laboratory studies

demonstrate that early-onset seizures can result in permanent behavioral and cognitive abnormalities that are paralleled by changes in brain connectivity, dendritic morphology, excitatory and inhibitory receptor subunits, ion channels and neurogenesis (Holmes, 2005). In any case, any attempt should be made to stop seizures as soon as possible. The assessment of epilepsy in children with developmental abnormalities presents particular problems. It is often necessary to admit the child to hospital for serial observations in order to determine the type of seizure and the epileptic syndrome. The use of video EEG recording is often helpful for the classification of the epileptic phenomenon and it is crucial for early definition of intractability and proper decision for treatment. Drug resistance, defined as the persistence of seizures despite maximum tolerated doses of antiepileptic drugs (AEDs) in mono- or polytherapy, is frequent in this population of children with developmental disorders of the CNS. The prognosis for seizure control with AEDs depends on both seizure type and the type of brain abnormality. Focal cortical dysplasia, schizencephaly, hemimegalencephaly and Aicardi syndrome often present with early-onset focal seizures and spasms that remain intractable to available AEDs. Unfavorable prognostic factors include early onset of seizures, presence of several seizure types, high initial seizure frequency and the occurrence of status epilepticus; a poor response to the first AED is another predictor of refractory epilepsy (de Saint Martin and Hirsch, 2004; Semah and Ryvlin, 2005). Most patients with refractory epilepsy will undergo multiple drug trials, most often without any noteworthy reduction in seizure frequency. The mechanisms

*Correspondence to: Dr Paolo Curatolo, Paediatric Neurosciences, ‘Tor Vergata’ University of Rome, Via Montpellier 1, 1-00133, Rome, Italy, E-mail: [email protected]

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underlying this ‘multidrug resistance’ in epilepsy are not well understood. Recent evidences show that multidrug efflux transporters such as P-glycoprotein are involved in the emergence of multidrug resistance, which plays an important role in the failure of antiepileptic treatment. Brain expression of MDR1, which encodes P-glycoprotein in humans, is markedly increased in the majority of individuals with medically intractable partial epilepsy (Tishler et al., 1995; Kwan and Brodie, 2005; Lo¨scher and Potschka, 2005; Schmidt and Lo¨scher, 2005). P-glycoprotein may play a clinically significant role by limiting access of AEDs to the brain parenchyma. Therefore, the decrease in AED levels at brain targets may contribute to the refractoriness of seizures in patients with treatmentresistant epilepsy secondary to malformations of cortical development. The overexpressed multidrug transporters lower the extracellular concentration of AEDs in the vicinity of the epileptic pathology, thereby rendering the epilepsy caused by these pathologies resistant to AED treatment (Sisodiya et al., 2002). Genetic susceptibility factors might participate in the development of this condition. Gene polymorphisms involving the GABAB receptor or MDR1 could explain why apparently similar brain lesions may result in the development of drug resistance in some patients but not in others (Siddiqui et al., 2003). Assessment of the child with brain malformations involves looking at all aspects of development, comparing functions with the expected norms for age. In particular, gross and fine motor functions, vision, speech and language, perceptual and intellectual functions, and social and emotional development should be considered. When counseling a family about treatment, a physician should remain sensitive to the family’s need to find solutions to their seemingly incurable situation, and should openly discuss with parents and caregivers what is known about the benefits of a specific treatment approach and any possible adverse effects. In general, in this special population of children, medication is considered as one component in a broad treatment model with the selection of the drug based on a comprehensive assessment and diagnosis of the developmental disorder and associated problems. In younger children it is a standard practice to combine pharmacological treatment with rehabilitation, and behavioral and other interventions. Treatment often implies cures, so it is important to emphasize to the parents that, while one can improve the clinical manifestations of a developmental disorder, as yet there is no curative treatment for it and the child will have to live with this persistent abnormality. Thus, the main goal of the global management

program is to ameliorate the condition, allowing children to develop the best possible quality of life and offering early support and help for the parents and family, who have to face a persistent chronic condition. Nutritional, dietary and hormonal treatments are also important in improving the child’s quality of life and should be taken into consideration (Ellis et al., 1999). The provision of optimal care to promote both general health as well as those issues specific to children with CNS malformations should be emphasized to parents and caregivers. Pharmacotherapy is commonly used in children with malformations of the CNS for the treatment of seizures, behavioral/ psychiatric problems and spasticity. The use of drugs in this population is influenced by a number of factors, with increasing drug use correlated with increasing age, decreasing intellectual impairment and increasing number and severity of behavior and psychiatric problems. The rational indications and the management principles that guide drug therapy in individuals with developmental abnormalities are for the most part consistent with the psychopharmacotherapy used for the general infant population. However, child neurologists must be aware that some psychotropic medications have different effects in children with mental retardation. For example, stimulants, the drugs of choice for treating attention-deficit hyperactivity disorder, show decreasing efficacy as the severity of mental retardation increases. In this chapter we will review some aspects of the child’s conditions that can be effectively treated by different categories of drugs.

30.2. Pharmacotherapy for seizures The medical treatment of seizures in children with developmental disorders of the CNS can often prove frustrating because of their limited response to conventional AEDs. In recent years the advent of new molecules has certainly had a positive impact on short-term seizure control. Difficulties are related to the limited understanding of epileptogenesis in children with brain malformations and the paucity of definite knowledge about the mode of action of most AEDs in humans (Meldrum, 1996). One major mode of action of AEDs at the cellular level is through the Na2þ channels, by preventing repetitive discharges and opposing the diffusion rather than the initiation of the epileptic discharge. Several AEDs are known to exert an activity on synaptic transmission. This is especially the case with vigabatrin and tiagabine, which increase the concentration of gamino-butyric-acid (GABA) in the CNS. Other agents

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Table 30.1 Mechanisms of action of antiepileptic drugs

AEDs Fenitoin Phenobarbital Carbamazepine Valproic acid Ethosuccimide Benzodiazepines Lamotrigine Vigabatrin Tiagabine Gabapentin Felbamate Topiramate Zonisamide

Inactivation of Na channels þþþ þþ þþþ þþ

Inhibition of Ca T channels

þ þþþ

þ þþþ þ þ þþ þþ

Other Ca channels

Increase of GABA activity

Antagonism to NMDA receptors

þ þ

þ þþ þ

þþ

þ þ

þ þþ

probably act on glutamatergic transmission. In most cases, however, mechanisms of action are multiple and remain largely unknown. Even for AEDs synthesized following a precise hypothesis, the actual mode of action does not always seem to be univocally related to their known properties. The supposed mechanisms of action of AEDs are summarized in Table 30.1. The exact role and clinical indications of new AEDs are not yet definitively established. In general, new AEDs should not be used as first-choice drugs for the treatment of most patients with epilepsy until more data on their safety profile are available. This is particularly relevant as far as cognitive and behavioral effects are concerned. Some (tiagabine, vigabatrin, lamotrigine) have been reported to aggravate certain generalized seizure types (Guerrini et al., 1998; Genton, 2000; Cerminara et al., 2004). Interactions between conventional and new AEDs have been summarized in Tables 30.2 and 30.3.

þ

þþþ þþþ þþþ þþ þ þþ þþ

þþ þþ

The optimal choice of the first drug in a child with newly diagnosed epilepsy secondary to a brain malformation will be based on the type of epileptic seizure and syndrome diagnosed. In addition, adverse effects will be taken into consideration. Drugs of first choice for different seizure types and syndromes usually associated with brain malformations are listed in Table 30.4. According to the drug’s specific titration schedule the dose is increased progressively as long as the seizure still occurs and as long as the drug is tolerated. However, the physician should bear in mind that in most cases the use of dosages larger than required may lead to adverse effects. Furthermore, there is little evidence that further dose increments in patients receiving dosages within the medium to high dose range results in seizure freedom in many cases (Perucca, 2005). A large proportion of patients will not become seizure free with the first medication. In these

Table 30.2 Interactions between conventional and new AEDs

Valproic acid Carbamazepine Fenitoin Phenobarbital Primidone

Felbamate

Gabapentin

Lamotrigine

Oxcarbazepine

Tingabine

Topiramate

Vigabatrin

None # # # #

None None None None None

" # # # #

# # # # #

None # # # #

# # # # #

None None None None None

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Table 30.3 Interactions between new and conventional AEDs

Phenobarbital Gabapentin Lamotrigine Oxcarbazepine Tiagabine Topiramate Vigabatrin

Valproic acid

Carbamazepine

Fenitoin

Phenobarbital

Primidonem

" 10–30% None # 25% None None #10% None

# None None None None None None

" 10–30% None None " None " 25% #

" None None " None None None

Unknown None None None None None None

Table 30.4 Drug choice by epileptic seizures and syndromes in children with brain malformations Partial seizures with or without secondary generalization First choice Carbamazepine/oxcarbazepine Second choice Lamotrigine, topiramate, valproic acid Consider Levetiracetam, tiagabine, zonisamide, phenytoin, phenobarbital, gabapentin, benzodiazepine, primidone Generalized tonic-clonic seizures First choice Valproic acid, carbamazepine Second choice Topiramate, lamotrigine Consider Phenobarbital, primidone, phenytoin Lennox–Gastaut syndrome First choice Valproic acid Second choice Topiramate, lamotrigine Consider Benzodiazepine, phenobarbital, felbamate, levetiracetam, zonisamide Infantile spasms First choice ACTH, vigabatrin Second choice Valproic acid, topiramate Consider Lamotrigine, tiagabine, benzodiazepine, pyridoxine, zonisamide

patients, the first monotherapy is usually replaced in an overlapping fashion by a second drug in monotherapy. There is now a much larger choice of drugs listed in Table 30.4 as second choice. However, randomized trials of the different AEDs have not been performed in patients with brain malformations and these choices are not supported by comparative trials of efficacy. Therefore, personal experience with the individual drugs and with side effects will often influence the choice. Theoretically, when a first AED has failed, it

makes sense to switch to a drug with a different mode of action rather than to a drug sharing the same primary mechanism (Beghi et al., 2003). The third therapeutic step in this population is usually a combination of the first two drugs. When combining two AEDs greater benefits could be expected by adding a drug with a mode of action that is different from and potentially complementary to that of the existing medication. As a rule, the use of more than two AEDs should not be encouraged. In most cases, seizure control will not significantly improve while the risk of cognitive adverse effects will dramatically increase. However, selected patients have shown to benefit from the use of polytherapy. It is good practice to use AEDs with different sites and mechanisms of action (e.g. drugs acting mainly on GABAergic synaptic transmission with drugs acting on Na2þ channels). Vigabatrin has been used in tuberous sclerosis associated with infantile spasms with excellent response (Curatolo et al., 2001). However, vigabatrin does not appear to be more effective than other AEDs in patients with other malformations, and caution with a prescription of vigabatrin might be indicated because of visual field restriction as an adverse effect. Adrenocorticotropic hormone and steroids have been the most popular treatment for infantile spasms. At high doses and when used for protracted time, side effects can be frequent and serious. Although data from appropriately controlled trials is still insufficient for a consensus statement, there is some evidence that steroids may not be the drug of choice when infantile spasms are associated with tuberous sclerosis complex, focal cortical dysplasia and neuronal migration disorders. AEDs, like all medications, pose possible risks (Table 30.5). Of major concern are catastrophic, idiosyncratic organ toxicities, such as chemical hepatitis and hepatic failure, pancreatitis, aplastic anemia and agranulocytosis, although these catastrophic adverse

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Table 30.5 Potential side effects of antiepileptic drugs GBP

LTG

TPM

Hepatotoxicity/pancreatitis Hematological Glaucoma Allergic dermatitis/rash Metabolic acidosis/renal stones Hyponatremia Weight gain Weight loss Ataxia Blurred vision

þ þ þ þ þ þ þ þ þ þ

þ þ þ* þ þ þ þ þ

TGB

OXC

þ*

þ*

þ þ

þ þ

ZNS

LEV

CBZ

VPA

þ

þ

þ

þ

BZP

* Stevens–Johnson syndrome also reported. BZP, benzodiazepines; CBZ, carbamazepine; LEV, levodopa; LTG, lamotrigine; OXC, oxcarbazepine; TGB tiagabine; TPM, topiramate; VPA, valproic acid; VGB, vigabatrin; ZNS, zonisamide.

events are less of a concern with the array of newergeneration AEDs. Additionally, most AEDs carry a risk of allergic reactions, which may progress to a Stevens–Johnson reaction. For women of childbearing age, traditional AEDs may pose a teratogenic risk, whereas there is limited pregnancy data for newer-generation AEDs. Although not necessarily life-threatening, impaired cognition, in some cases due to somnolence, is of concern with many AEDs. This is a side effect difficult to detect in children with mental retardation. Also difficult to detect, especially in nonverbal children, are ophthalmological signs of AED toxicity such as diplopia, eye pain or blurred vision. Other potential adverse effects of some AEDs include dizziness, ataxia, alopecia, weight gain/loss, gingival hyperplasia and gastrointestinal disturbances. In the last 10 years, several new AEDs have become available. However, little experience in the population of children with CNS malformations has been reported in the literature. Topiramate is emerging as an effective drug in partial seizures with or without secondary generalization and in the Lennox–Gastaut syndrome. The mechanisms of action of topiramate, apart from state-dependent blockade of sodium and calcium channels and inhibitory effect on carbon anhydrase, include the enhancement of GABA activity on GABAA receptors with elevation of cerebral GABA levels and antagonism of glutamate receptors. In children, the usual starting dose is 0.5–1 mg/kg per day; the steady-state dosage range varies from 2 mg/kg to 15 mg/kg daily. The most common side effects include weight loss, dizziness, sleepiness, headache, tremor and cognitive dysfunction (Uldall and Buchholt, 1999). Clinical

experience suggested that titration has to be slow, with increments every second week in order to permit the greatest possible number of children to benefit from its efficacy. Lamotrigine is more effective than topiramate on some generalized seizures. Improved alertness and behavior were apparent in many patients and the incidence of side effects was similar to that reported for other pediatric populations with symptomatic partial epilepsy. Although lamotrigine is well tolerated, a skin rash can occur in 5–10% of subjects and concern has been raised that the incidence may be higher in children. The rash usually begins within the first 8–10 weeks of exposure. To minimize the risk of rash, lamotrigine should be titrated slowly, with increments over a period of 6–8 weeks (Schmidt and Bourgeois, 2000). Levetiracetam is a novel AED shown to be effective in children against partial seizures and generalized seizures (Vigevano, 2005). Recently, the synaptic vesicle protein SV2A was identified as the binding site for levetiracetam. It suggests that levetiracetam has a mechanism of action that differs from that of other AEDs, probably linked to the exocytose modulation. Levetiracetam is well tolerated and its good safety profile makes it an attractive AED to use early in children with CNS malformations. Oxcarbazepine has a lower incidence of cognitive side effects than its parent compound carbamazepine. Its primary side effect relates to the idiosyncratic development of hyponatremia, which is increased in individuals with pre-existing renal disease. Felbamate has been effective in partial seizures in patients with tuberous sclerosis but has not been properly tested because of the high risk of causing

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aplastic anemia, leukopenia and thrombocytopenia, or hepatic damage. In patients with the Lennox–Gastaut syndrome its use is acceptable when all other AEDs have failed to control seizures. A blood count and liver function tests should be performed every 15 days in subjects treated with this drug. In most of the patients with early-onset seizures, total control may be an unreasonable aim. In these cases, treatment should be focused on the suppression of the more dangerous seizures (i.e. drop attacks) without producing unacceptable adverse effects and inability to participate in daily living activities. Despite therapeutic trials with first-line and new AEDs, some children and adolescents with brain malformations will continue to present seizures. In these cases alternative strategies should be explored. The ketogenic diet is an effective alternative therapy for intractable epilepsy (Kossoff et al., 2002). However, problems, difficulties and side effects limit its use in children with brain malformations. Vagal nerve stimulation has also been reported to be as effective in patients with previously intractable generalized or partial epilepsy due to tuberous sclerosis complex (Frost et al., 2001; Parain et al., 2001). When seizure activity proves intractable to medication the possibility of surgical treatment should be explored. This process involves the identification of converging electroclinical, morphological and functional imaging data and requires a common operational framework between epileptologists, neuroradiologists and clinical neurophysiologists. Whenever possible, the identification of a single epileptogenic area and its selective surgical removal could significantly improve the quality of life of patients with brain malformations. The success rate is high when the patients are screened carefully (Schmidt et al., 2004). In our experience early onset of seizures in hemimegalencephaly may indicate a very poor prognosis and surgery evaluation should be considered as soon as possible. Epilepsy surgery must be considered early even in patients with intractable partial epilepsy related to a focal dysplasia. In some patients, surgery is followed by improvement in cognitive and motor performance, suggesting a role for hemispherectomy in eliminating some deleterious effects due to the dysplastic epileptogenic hemisphere on the intact contralateral cerebral hemisphere (Leblanc et al., 1996; Maehara et al., 2002). In children with tuberous sclerosis the frequent presence in the same patient of multiple lesions on neuroimaging poses a serious challenge in defining surgical amenability (Curatolo et al., 2005). Isolated lesionectomy might not be sufficient to control current and feature seizure foci in patients with multiple tubers.

In fact, various pathological substrates are suitable for surgical treatment. However, the same pathological abnormality may be localized, diffuse or multifocal and therefore not always amenable to resection. In some patients with brain malformations, according to our experience, the continuation of drug treatment after resectomy is still required, and surgery may simply transform a refractory epilepsy into a drugresponsive form.

30.3. Pharmacotherapy for behavioral and psychiatric disorders Psychiatric disorders, including conduct disorders, depression and anxiety disorders, are common in the population of individuals with developmental disorders of CNS. Some seizure types and syndromes are particularly associated with psychopathology. For example, clinical experience suggests that children with frontal lobe abnormalities, such as cortical dysplasia and associated epilepsy, may have particular problems with hyperactive, disinhibited and disruptive behavior. 30.3.1. Mood stabilizers With time it has become apparent that AEDs have broader effects such as control of pain, migraines and mood stabilization, particularly in individuals with bipolar disorders. These effects are related to AEDs’ control of neuronal excitation and modulation of neurotransmitter activity. They exert their multiple effects via the inhibition or potentiation of ionic channels; disruption of neurotransmitter metabolism; impacting of neurotransmitter or other receptor functions; antagonism to excitatory neurotransmitters; potentiation of inhibitory neurotransmitters; and/or carbonic anhydrase inhibition. Additionally, AEDs may exert behavioral control through the suppression of clinical seizures or subclinical spike activity. This may be beneficial if the interictal, subclinical spike activity is causing behavioral change. In the extreme this may lead to the ‘forced normalization’ phenomena (i.e. better seizure control or spike suppression associated with worsening behaviors). It is unclear whether identifiable baseline EEG abnormalities can predict behavioral responsiveness to AED. Of the ‘older’ generation AEDs, valproic acid has yielded reductions in aggression and impulsivity but is limited by dose-related weight gain, long-term metabolic dysfunction and the potential for idiosyncratic hepatic and/or pancreatic toxicity, particularly in younger patients. Although carbamazepine has also

MEDICAL TREATMENT IN CHILDREN demonstrated efficacy in bipolar disorder and in controlling aberrant behaviors in patients with mental retardation, there are few data to support its use in children with associated autism and there is the concern of its apparent potential to induce mania. Analogous to the neuroleptics, ‘newer-generation’ AEDs have been touted to have comparable efficacy with more favorable side effect profiles and a broader spectrum of pharmacological mechanisms. Oxcarbazepine is very well tolerated in the pediatric population with reports of reductions in aggression and behavioral severity along with mood amelioration. Lamotrigine may be useful in individuals with self-injurious behaviors and may be very effective in dysregulated mood disorders, especially when depression is a major component. Lamotrigine is also well tolerated in children but requires a slow introductory titration rate to dampen potential allergic side effects. Topiramate is stated to reduce self-injurious and aggressive behaviors and may also provide an element of mood stabilization in those with bipolar phenomena, but weight loss can limit its use and induction of metabolic acidosis is a concern. 30.3.2. Neuroleptics Neuroleptics/antipsychotics, both traditional and the newer generation ‘atypicals’, derive their pharmacological properties primarily from their ability to modulate dopamine activity; the same properties that are likely to create undesirable side effects. However, there are antagonistic and agonistic effects on other neurotransmitters and their receptors that are likely to play a role in the therapeutic mechanisms and side effect profiles of these medications. In particular, a major difference between the two classes of agent is the relatively high ratio of 5-HT2A/dopamine D2 (5HT: serotonin) receptor antagonism displayed by most atypical neuroleptics, which may in part explain their more favorable side effect profile. There is evidence that these agents can ameliorate symptoms associated with mental retardation, particularly in targeting aberrant behaviors, including aggression, self-injurious behavior, ‘explosive’ outbursts, agitation, mood dysregulation (‘bipolar’ phenomena) and severe hyperactivity/impulsiveness (Palermo and Curatolo, 2004). Neuroleptics may also help manage sleep problems in selected patients with brain malformations. Although neuroleptics may be beneficial, they are limited by unfavorable side effect profiles and restricted pediatric experience. The induction of abnormal involuntary movements reduces the utility of neuroleptics and requires vigilant monitoring. These extrapyramidal side effects include dystonic reactions and

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involuntary dyskinesias, which can occur both during administration or following withdrawal, are often related to long-term use and can occasionally lead to a tardive dyskinesia syndrome (Malone et al., 1991; Campbell et al., 1997). Another ominous and dangerous side effect is the neuroleptic malignant syndrome, which can prove fatal. Other concerns include sedation, metabolic changes such as weight gain, alteration of lipid profiles, induction of a diabetic state and end-organ toxicity, particularly hepatotoxicity. Prolactinemia can lead to galactorrhea, and long-term neuroleptic use can cause reduction in bone density. Cardiac effects such as prolongation of the Q–Tc interval and induction of arrhythmias have been reported, and orthostatic hypotension can occur. Nevertheless, in some cases neuroleptics are the most effective medications available to date. Therefore, when using typical/ atypical neuroleptics in children, careful monitoring is necessary. Clinically, the older, ‘typical’ antipsychotics have documented evidence of utility in improving attention and vigilance in brain malformations. Haloperidol, a dopamine receptor antagonist, was found in several controlled clinical trials to have clinical efficacy in ameliorating the ‘core’ symptoms of social withdrawal and abnormal ‘object relations’, as well as irritability, hyperactivity, mood dysregulation and stereotypies (Anderson et al., 1989). Reports of improved performance on cognitive tasks of discrimination learning in highly structured settings while receiving haloperidol were not confirmed by later studies. Other traditional neuroleptics such as pimozide and thioridazine, although useful in controlling and reducing a number of maladaptive and problematic behaviors and involuntary movements, have fallen out of widespread use because of potential side effects, particularly prolongation of the Q–Tc interval and the increased risk of serious tachyarrhythmias (Goodnick et al., 2002). Because of more favorable side effect profiles (fewer extrapyramidal side-effects, less effect on prolactin levels, more weight neutrality for some agents) and reported efficacy, there has been considerable interest in the new ‘atypical’ antipsychotics (Aman and Madrid, 1999). Proposed mechanisms of action and neurobehavioral targets are summarized in Table 30.6. Risperidone has the most research data for atypical neuroleptics in autistic spectrum disorders. In an 8week, multisite, randomized, double-blind, placebocontrolled trial involving 101 autistic children and sponsored by the Research Units on Pediatric Psychopharmacology Autism Network, risperidone was found to be effective for tantrums, aggression, or self-injurious behaviors, and was relatively well tolerated (Research Units on Pediatric Psychopharmacology Autism

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Table 30.6 Atypical neuroleptics: proposed mechanisms of action and neurobehavioral targets

Receptor targets Dopamine-antagonist Dopamine-agonist Serotonin antagonist Serotonin agonist Adrenergic antagonist Cholinergic antagonist Histamine antagonist Uses Psychoses Bipolar disorder Aggressiveness Self-injurious behavior Mood dysregulation Hyperactivity

Risperidone

Olanzapine

Ziprasidone

Apriprazole

Quetiapine

Clozapine

D2 – 5HT2a – A-1,2 – H1

D1,2,4 – 5HT-2a,c – A-1 M1–5 H1

D2 – 5HT2 – A-1 – H1

– D2 5HT2a 5HT1a A-1 – –

D2 – 5HT2 – A-1 þþþ H1

D4 > D1,2,3 – 5HT – þþþ

þþþ þþ þþþ þþþ þþþ þþ

þþþ þþþ þþ þþ þþ þþ

þþþ þþ þ þ þ þ

þþþ þþþ

þþþ þþ

þþþ* þþ*

þþþ

þ, rare reports of efficacy or minimal clinical experience; þþ, modest reports of efficacy or moderate clinical experience; þþþ broad reports of efficacy or extensive clinical experience * Clozapine should be used for treatment-resistant cases.

Network, 2002; Arnold et al., 2003). There have been small, uncontrolled studies and case reports suggesting that aripiprazole, ziprasidone and olanzapine may be of benefit in controlling various maladaptive behaviors (Horrigan et al., 1997; Potenza et al., 1999). Clozapine is a very powerful and effective atypical neuroleptic but, because of the risk of agranulocytosis and/seizures and the need for weekly or bimonthly blood tests, this medication should be considered only for cases refractory to other neuroleptic options (Zuddas et al., 1996). 30.3.3. Stimulants/nonstimulants Drugs derived from methylphenidate or amfetamine salts have proven to be very useful for targeting the core symptoms of attention deficit hyperactivity disorder (ADHD): inattentiveness, hyperactivity and impulsivity (Aman, 1996; Handen et al., 2000). These medications are often used to target analogous behaviors in children with brain malformations as well. A potential limitation is an apparently high incidence of atypical responses (induction of ‘mania’/activation, irritability) and side effects (e.g. sleep disturbances, reduction of appetite, tics, increase in stereotypies, blunting of personality). The magnitude of the effect of stimulants appears to be less in children with mental retardation than in typical children. The caveat for their use in mental retardation is that atypical responses are prevalent, side effects may be dose or treatment limiting and a mood stabilizer may be more

efficacious in some cases. The relatively favorable safety of these medications, along with their long history of use in the pediatric population, suggests that they should be considered in selective cases of cognitive impairment involving children with debilitating hyperactivity and impulsiveness, and in the higherfunctioning child with mental retardation who manifests significant inattentiveness. A group of new generation ‘nonstimulants’ are built around the mechanism of selective inhibition of the norepinephrine reuptake transporter (SNRIs). The initially marketed SNRI atomoxetine, has been shown to be efficacious for ADHD, has an extended pharmacodynamic effect despite a relatively short serum halflife, does not exacerbate tics or disturb sleep and is not contraindicated if there is comorbid anxiety. Comparable to selective serotonin reuptake inhibitors (SSRIs), duration of therapy may be as important as dose because of a response lag following introduction of therapy or a change in dosage. However, there is no literature as yet describing the effects of atomoxetine in children with mental retardation and autistic spectrum disorders due to brain malformations. 30.3.4. Other drugs There is evidence of abnormal brain serotonin synthesis in autism, providing a theoretical justification for the use of serotonin reuptake inhibitors (SRIs) in children with mental impairment and autistic spectrum

MEDICAL TREATMENT IN CHILDREN disorders (Aman et al., 1999). Interest has been stirred by reports of high systemic serotonin levels, changes induced by tryptophan-free diets and serotonergic responsivity. The association of 5-HTTLPR, a putative marker for autism (serotonin transporter polymorphism), with anxiety and affective disorders also provides a rationale for the use of drugs affecting the serotonin system in clinical management. With the exception of fenfluramine, which has proved to be ineffective and with unacceptable side effects, the literature suggests that SRIs, both SSRIs (e.g., fluoxetine, sertraline, fluvoxamine and paroxetine) (Snead et al., 1994; Branford et al., 1998; Cook et al., 1992; Steingard et al., 1997; Yokoyama et al., 2002) and nonselective (e.g. the tricyclic antidepressants clomipramine, imipramine, desipramine), may have a role in the treatment of ‘interfering behaviors’, specifically those that are repetitive and aggressive, and may ameliorate, to a certain degree, social relatedness in autistic spectrum disorders (Alcami et al., 2000). This appears to be particularly true in cases where there is a family history of affective illness. A double-blind, placebo-controlled study of fluvoxamine in autistic adults found good tolerability and clinical superiority with respect to placebo in ameliorating repetitive behaviors and aggression, while reportedly improving social relatedness and language (Fukuda et al., 2001). However, the majority of published studies of SSRIs have been uncontrolled. DeLong et al., (2002) reported improvements in language function in an open label observation of fluoxetine, although such findings have not been replicated. Using placebo controlled designs, clomipramine and desipramine, serotonergic and relative norepinephrine uptake inhibitors, have both demonstrated efficacy in improving core symptoms, anger or uncooperativeness, hyperactivity and obsessive–compulsive behaviors. The SRIs appear to be generally safe and well tolerated in children, especially for the treatment of mood and obsessive–compulsive disorders. Induction of ‘manic’ behaviors, however, have been described in children with obsessive–compulsive disorder or bipolar-like syndromes, which could represent a major challenge in the treatment of autistic spectrum disorders. Other anxiolytics such as benzodiazepines can be useful when there is a need for immediate effect, particularly in combating overarousal, or when the anxiety is driving comorbid disorders such as obsessive– compulsive disorder. Because of sedation and potential for dependency, long-term use should be avoided, but these agents are relatively safe and well tolerated in short-term use. In general, sleep-related problems in children with brain malformations are part of a very complex clinical picture, and a comprehensive management strategy

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needs to take into account the wide comorbidity seen in these patients. Sedative hypnotics are used for enhancing and maintaining sleep. Antihistamines may be useful for sleep issues but may occasionally trigger an atypical response (e.g. hyperactivity, overstimulation). Alpha-adrenergic agonists, such as clonidine, are sometimes employed to reduce sleep latency, exerting their effect at least in part through sedation, but should be used cautiously in children because of cardiovascular side effects. The pineal hormone melatonin was initially reported to be useful in promoting sleep cycle regulation in pilots experiencing jet lag, with much anecdotal support as a sleep inducer for children with neurodevelopmental disabilities. In these subjects melatonin given 20 minutes before bedtime has shown some promising results (O’Callaghan et al., 1999). The mechanisms underlying this effect are not fully understood. Narcotics, such as chloral hydrate, must be used only as a last resort and should be used with extreme caution and monitoring, because of the risk of respiratory depression. Overall, sleep issues, such as insomnia or sleep disruption, should be evaluated for an underlying and treatable cause (e.g. sleep apnea) and various sleep hygiene and behavioral approaches should be tried prior to medication use. Chronic use of medication to induce or maintain sleep can lead to dependency.

30.4. Pharmacotherapy for spasticity A cortical malformation involving the sensory motor cortex can be characterized by spasticity with hypertonia of the pyramidal type, clonus, Babinski and tendency for permanent deformities. Spastic hemiplegia is the most common manifestation of involvement of the motor system and can be often associated with hemimegalencephaly and unilateral schizencephaly. Less frequently, hemiplegia can be associated with tuberous sclerosis when a large rolandic tuber is present. Several treatment options are available to manage spasticity, including physical, pharmacological and surgical interventions. It is not uncommon to use physical and occupational therapies in combination with botulinum toxin injection, intrathecal baclofen therapy and timed orthopedic interventions. A rational approach to therapy can be developed on the basis of the proposed pathophysiology and receptor–neurotransmitter interactions. Realistic and clearly defined goals should be established before initiating treatment. The aim of medication is to reduce the spastic symptoms. Baclofen is a structural analog of GABA. The exact mode of action is unclear, but it appears to block polysynaptic and monosynaptic afferents in the spinal cord by binding to GABAB receptors (Edgard,

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Table 30.7 Daily dosages of antispastic agents

Baclofen Diazepam Dantrolene Tizanidine Clonidine L-dopa

Initial dose

Maximum dose

Doses/d

2.5–5 mg/d 0.1–0.2 mg/kg/d 0.5–1 mg/kg/d 2–4 mg/d 5 mg/kg/d 2–5 mg/kg/d

3(–5) mg/kg/d 0.8 mg/kg/d 6(–12)mg/kg/d 9–12 mg/d 10 mg/kg/d 600–900 mg/d

3–4 1–3 2 1–3 3 3

2003). Its mechanism of action may be as a direct inhibitory neurotransmitter or through hyperpolarization of the afferent nerve terminals. Poor penetration of the blood–brain barrier and central side effects have also led to the intrathecal application of baclofen when patients do not respond sufficiently to oral medication (Butler and Campbell, 2000). Treatment is usually started at a low dose and gradually increased to a maximum of 2–3 mg/kg/day in 3–4 doses until efficacy is reached or side effects occur (Table 30.7). Benzodiazepines exert their antispasmodic action through GABAA receptors. In the spinal cord, diazepam appears to increase presynaptic afferent inhibition and depress mono- and polysynaptic transmission. There is evidence for enhanced postsynaptic inhibition in the reticular formation. Although diazepam has been demonstrated to have clinical efficacy against spasticity, central side effects, including drowsiness, often limit it in clinical practice (Engle, 1966). Dantrolene is a potentially underused treatment, particularly in nonambulatory patients. It is unique among antispasmodic agents in that it acts peripherally at the level of the muscle fiber rather than the spinal cord. Dantrolene uncouples electrical excitation from contraction by inhibiting the release of calcium from the sarcoplasmic reticulum. It is useful for symptomatic relief, especially clonus, in all types of upper motor neuron insult. Because it may significantly exacerbate weakness, it should be used with caution in ambulatory patients with cerebral palsy (Edgard, 2003; Joynt and Leonard, 1980).

30.5. Conclusions It is important that a treatment plan for a child with brain malformations be built upon a firm and justifiable foundation. A complete medical and neurological evaluation is necessary to identify any underlying and identifiable medical disorder that should be primarily

targeted for treatment. A thorough assessment of a child’s cognitive abilities and social–communication skills and a functional analysis of aberrant behaviors will allow for formulation of a comprehensive and effective therapy plan. Successful treatment approaches are multimodal and interdisciplinary, tapping into the expertise and utility of pediatric neurology and psychiatry, developmental pediatrics, behavioral and neuropsychology, speech and language therapy, physical and occupational therapy, and education. It is critical that clinicians utilize therapeutic interventions that are individualized and tailored to the needs of the child, rather than more global, nonspecific practices. Evidenced-based therapies should be favored. There is a tremendous need for methodologically sound research aimed at determining the relative efficacy of the various behavioral and educational treatment approaches, the efficacy and safety of pharmacological treatments, and appropriate and relevant outcome measures. Furthermore, evidence is mounting that earlier interventions result in more favorable longterm outcomes. It is therefore incumbent upon the medical and professional community to be knowledgable about the earliest signs and symptoms of brain malformations to ensure that evidence-based interventions are initiated as quickly as possible following identification.

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Perucca E (2005). Can drug resistance in epilepsy be minimized? Epileptic Disord 7 (suppl. 1): S14–S21. Potenza MN, Holmes JP, Kanes SJ, McDougle CJ (1999). Olanzapine treatment of children, adolescents, and adults with pervasive developmental disorders: an open-label pilot study. J Clin Psychopharmacol 19: 37–44. Research Units on Pediatric Psychopharmacology Autism Network (2002). A double blind, placebo-controlled trial of risperidone in children with autistic disorder. N Engl J Med 347: 314–321. Schmidt D, Bourgeois B (2000). A risk-benefit assessment of therapies for Lennox–Gastaut syndrome. Drug Saf 22: 467–477. Schmidt D, Lo¨scher W (2005). Drug resistance in epilepsy: putative neurobiological and clinical mechanisms. Epilepsia 46: 858–877. Schmidt D, Baumgartner C, Lo¨scher W (2004). The change of cure following surgery for drug-resistant temporal lobe epilepsy: what do we know and do we need to revise our expectations? Cognitive, psychosocial, and family function one year after pediatric epilepsy surgery. Epilepsy Res 60: 187–201. Semah F, Ryvlin P (2005). Can we predict refractory epilepsy at the time of diagnosis? Epileptic Disord 7 (suppl. 1): S10–S13. Siddiqui A, Kerb R, Weale ME, et al. (2003). Association of multidrug resistance in epilepsy with a polymorphism in

the drug-transporter gene ABCB1. N Engl J Med 348: 1442–1448. Sisodiya SM, Lin W-R, Harding BN, et al. (2002). Drug resistance in epilepsy: expression of drug resistance proteins in common causes of the refractory epilepsy. Brain 125: 22–31. Snead RW, Boon F, Presberg J (1994). Paroxetine for selfinjurious behavior. J Am Acad Child Adolesc Psychiatry 33: 909–910. Steingard RJ, Zimnitzky B, DeMaso DR, et al. (1997). Sertraline treatment of transition-associated anxiety and agitation in children with autistic disorder. J Child Adolesc Psychopharmacol 7: 9–15. Tishler DM, Weinberg KT, Hinton DR, et al. (1995). MDR1 gene expression in brain of patients with medically intractable epilepsy. Epilepsia 36: 1–6. Uldall P, Buchholt JM (1999). Clinical experiences with topiramate in children with intractable epilepsy. Eur J Paediatr Neurol 3: 105–111. Vigevano F (2005). Levetiracetam in pediatrics. J Child Neurol 20: 87–93. Yokoyama H, Hirose M, Haginoya K, et al. (2002). Treatment with fluvoxamine against self-injury and aggressive behavior in autistic children. No To Hattatsu 34: 249–253. Zuddas A, Ledda MG, Fratta A, et al. (1996). Clinical effects of clozapine on autistic disorder. Am J Psychiatry 153: 738.


Chapter 31

Surgical treatment of central nervous system malformations LORENZO GENITORI *,1, PIER ARTURO DONATI1, FLAVIO GIORDANO1, MASSIMILIANO SANZO1, FEDERICO MUSSA1, LUIGI SARDO1, BARBARA SPACCA1, GIOVANNI DI PIETRO1, AND GIUSEPPE OLIVERI2 1

Department of Neurosurgery, Ospedale Pediatrico Meyer, Firenze, Italy 2

Department of Neurosurgery, Ospedale Le Scotte, Siena, Italy

31.1. Introduction The surgical treatment of CNS malformations is a big challenge for neurosurgeons. The developing technologies of new radiological imaging techniques, as well as surgical tools, and improving knowledge of such diseases have dramatically changed the opportunity to treat different malformations with better results and better quality of life for patients and family. Neuroendoscopy, for example, has radically changed the surgical approach to many cerebrospinal fluid (CSF)related diseases, giving, in some cases, an opportunity to treat the hydrocephalus in a minimally invasive and more physiological way. Nonetheless, new biomaterials such as resorbable plates and screws have changed the possibilities for surgical management of craniofacial diseases. This chapter aims to describe the current state of the art in the surgical management of CNS malformations on the basis of our experience and reviews of the pertinent literature (Table 31.1).

31.2. Management of hydrocephalus and CSF-related disturbances 31.2.1. Neuroendoscopy Neuroendoscopy appeared at the beginning of the last century and has begun to modify general neurosurgery during the last 10 years thanks to technological progress in optical fibers. Besides radically changing the neurosurgical treatment of hydrocephalus, nowadays neuroendoscopy is becoming an alternative and effective treatment for other intracerebral and periventricular lesions located into third and lateral ventricles, such as

arachnoid and colloid cysts. Furthermore neuroendoscopy allows biopsy and sometimes removal of intraand paraventricular tumors, including vascular malformations. The high incidence of hydrocephalus during childhood, isolated or associated with almost all the cerebral pathologies, makes neuroendoscopy a valid and suitable tool for its multimodal treatment. First, endoscopic third ventriculostomy (ETV) is today recognized as the gold standard treatment for obstructive hydrocephalus, both in children and infants, with an overall success rate of 75% in many published series (Sainte-Rose et al., 2001; Santamarta et al., 2005). Obstructive hydrocephalus due to aqueductal stenosis in children aged more than 1 year is characterized by 98% of patients shunt-free after ETV (Hellwig et al., 2005). Posterior fossa tumors with hydrocephalus should be treated first by ETV, followed by a direct approach to the tumor a few days later (Sainte-Rose et al., 2001). In infants the number of CSF shunting procedures has been reduced by neuroendoscopy. Posthemorrhagic hydrocephalus in preterm newborns can also sometimes be treated by neuroendoscopy instead of traditional techniques (Gorayeb et al., 2004). Indeed, in all cases of obstructive hydrocephalus (obstruction of the outlets of fourth ventricle, cysts, Chiari malformation, complex craniosynostosis) ETV may be considered the first-choice treatment (Decq et al., 2001). In case of shunt failure too, ETV can be proposed instead of ventriculo-peritoneal shunt revision, achieving an 82% success rate in terms of shunt-free children (Boschert et al., 2003). In shunted children affected by slit ventricle syndrome, ETV may be considered an alternative choice (Chernov et al., 2005).

*Correspondence to: Lorenzo Genitori, Department of Neurosurgery, Ospedale Pediatrico ‘Meyer’, Via Luca Giordano 13, Florence, Italy. E-mail: [email protected], Tel: þ39-055-566-2934 or þ39-348-443-1370.

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Table 31.1 Surgical procedures for CNS malformation in children 1996–2005 Procedure

N

%

Neuroendoscopy Shunting procedures Craniofacial repair for craniofacial dysmorphism Excision of encephalocele Posterior fossa decompression for Chiari type I anomaly Surgery for dysraphic state Fetal surgery

669 1385 353

25 51.9 13.3

20 129

0.7 4.8

111 3

4.2 0.1

31.2.1.1. Third ventriculocisternostomy Opening the floor of the third ventricle is a standard technique to put it in communication with the basal cisterns in order to divert CSF circulation from the aqueduct and the fourth ventricle. It is utilized for obstructive hydrocephalus due to aqueductal stenosis because of malformative pathology (aqueductal atresia, arachnoid cysts of lamina quadrigemina), tumors (posterior fossa, pineal or brainstem tumor, tectal hamartoma) and also in presence of aqueductal flow disturbance due to hemorrhages and/or infections. The surgical technique is standardized in all cases, using a rigid neuroendoscope of 9.5 F in outer diameter (about 2 mm) with a 30 angulated optic. The camera is oriented with the operative sheet. Under general anaesthesia the patient is positioned supine with the head slightly flexed and a precoronal 5 mm burr hole is made. After opening of the dural and arachnoidal surface, the endoscope is inserted ‘freehand’ without

Fig. 31.1. Endoscopic view of the floor of the third ventricle. The tuber cinereum and mammillary bodies are visible.

a stilet under direct view control. The presence of mandrin passed inside the operative channel prevents the passage of small brain particles into the endoscope during its introduction. After the lateral ventricle is reached and the foramen of Monro has been identified, the endoscope is passed into the third ventricle. Ventriculocisternostomy is always performed in the midline between the mammillary bodies and the tuber cinereum, as close as possible to the dorsum sellae, to avoid injury to the basilar artery complex (Fig. 31.1). The opening in the floor of the third ventricle is made with a 1 mm coagulator fiber followed by the insertion of a 2 Fr Fogarty balloon catheter inflated with 0.2 ml of saline solution in the cistern and then withdrawn into the third ventricle (Fig. 31.2). No forceps or blunt technique is used. After the perforation of the floor of the third ventricle, the neuroendoscope is always introduced through the stoma into the cisternal space to open the two layers of the Liliequist membrane,

Fig. 31.2. Endoscopic view. (A) Opening of the floor by coagulator. (B) The balloon of the Fogarty catheter is inflated through the stoma.

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phalic cisterns (Fig. 31.5); a suprasellar cyst may be marsupialized into the interpeduncular cistern while an interhemispheric cyst may be opened into the ventricular cavities (Abbott, 2004). A posterior fossa cyst (retrocerebellar, pontocerebellar angle, supracerebellar) may be marsupialized into the cisterna magna or into the pontocerebellar cisterns and the perimedullary cisternal spaces (Hopf and Perneczky, 1998). In case of endoscopic surgery failure, redo-endoscopy and direct microsurgical opening of the cyst through craniotomy are to be considered in the first instance; cystoperitoneal shunting today represents last-choice surgery (Pierre-Kahn et al., 2002). Fig. 31.3. Endoscopic view: the stoma is open between the third ventricle and the interpeduncular cistern

reaching the prepontine cistern after identification of the basilar artery. The endoscope is then retracted (Fig. 31.3). Irrigation is carried out carefully and manually if necessary; no continuous irrigation is used. The whole procedure is always carried out in a ‘freehand’ fashion and takes an average time of 30 min. 31.2.1.2. Septostomy Septostomy consists in opening the septum pellucidum (Fig. 31.4) in the case of monoventricular or biventricular hydrocephalus, or in fenestrating pathological septa inside the ventricles in multicystic hydrocephalus (Oi and Abbott, 2004). 31.2.1.3. Arachnoid cyst marsupialization This technique aims to open the wall of the cyst inside the CSF cisterns and/or ventricular cavities: for example, a temporo-silvian cyst can be put in communication with optical cistern, internal carotid artery and perimesence-

31.2.1.4. Placement of catheters In the presence of virtual ventricles or ventricles with multiple septations, neuroendoscopy enables a catheter to be placed in the selected place, also allowing its connection with an Ommaya reservoir for CSF tapping and/or delivering drugs (Oi and Abbott, 2004). 31.2.2. Shunting procedures The evacuation of superficial intracranial fluid in hydrocephalic children was described in detail for the first time in the 10th century by Abu Al-Kassim Khalaf ibn Abbas Al Zaharawi (936–1013). In 1893 the first permanent ventriculo-subarachnoid–subgaleal shunt was described by Mikulicz, who proposed a simultaneous ventriculostomy and drainage into the extrathecal low-pressure compartment. Between 1898 and 1925, lumboperitoneal and ventriculoperitoneal, ventriculovenous, ventriculopleural and ventriculoureteral shunts were invented but, in most cases, these systems had a high failure rate due to insufficient implant materials. Artificial CSF valves were proposed in 1948 by Ingram, by Bush at MIT in 1949 in collaboration with

Fig. 31.4. Endoscopic view: septostomy. (A) The Fogarty balloon is pushed through the septum pellucidum. (B) Opening between the two lateral ventricles.

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Fig. 31.5. MRI axial view. (A) Huge arachnoid cyst in the temporo-sylvian region with compression of the midline structures . (B) Postoperative (endoscopic approach). Note the almost complete disappearance of the cyst.

Matson, and by Nulsen and Spitz in Philadelphia (Aschoff et al., 1999). During the 1950s the Spitz– Holter shunt was developed, leading to a tremendous impact on neurosurgical procedures for hydrocephalus (Nulsen and Spitz, 1952). After a first generation of simple differential pressure valves, which are unable to drain physiologically in all body positions, a second generation of adjustable, autoregulating antisiphoning and gravitational valves was developed (Gruber et al., 1984). Many shunt systems also have a flexible flushing chamber (reservoir), which may be housed within the same unit as the valve or may be a separate unit along the shunt, depending on the design of the shunt system. Assuming that ‘the best shunt is no shunt’, none of the innumerable multicentric trials have showed that any shunting system is more effective than another. At the moment at least 127 different designs are available, but most of these are only clones (Drake and Kestle, 1996). 31.2.2.1. CSF shunt valves

ventricular pressure rises above the precalibrated opening pressure, allowing CSF outflow, and close when the pressure falls below the closing pressure of the valve (Pudenz, 1981). The limitation of standard differential valves is that the flow increases when the differential pressure increases (i.e. orthostatic pressure in standing position), leading to overdrainage complications (Pudenz and Foltz, 1991). 31.2.2.1.2 Programmable valves These valves have an adjustable ball–spring mechanism and operate as a differential device with the advantage that it is possible to modify the operating pressure of the valve once it has been implanted by means of an external device with a magnet placed on the skin (Benesch et al., 1994). Some authors have not reported higher efficacy and safety rates for these devices compared to precalibrated valves (Pollack et al., 1999). Other authors believe that this type of shunt is superior because ‘one cannot know in advance which case will turn out to be complicated’ (Zemack and Ramner, 2000).

31.2.2.1.1 Differential pressure pre-settled valves These valves are subdivided in four broad categories: slit valves, miter valves, diaphragm valves and ballin-cone valves (Drake and Saint-Rose, 1995). These systems have predefined operating pressures with three or five performance levels that vary from very low to high; differential pressure valves open when the intra-

31.2.2.1.3 Flow-regulating valves In these valves CSF flow through the device in correlation with variation of CSF pressure. In attempt to keep the CSF flow rate constant, the mechanism resistance increases as the pressure gradient increases (Hanlo et al., 2003).

SURGICAL TREATMENT OF CENTRAL NERVOUS SYSTEM MALFORMATIONS In conclusion, none of the described types of valve appears to be best for the initial treatment of pediatric hydrocephalus (Kestle et al., 2000). 31.2.2.2. Shunt surgery techniques Especially in children, the ventriculo-peritoneal route is preferred to the ventriculo-atrial route because it is easier to place and is followed by less morbidity (Hoffmann, 1982). Many studies have been carried out to evaluate the possibility of prevention and reversibility of pathological changes in hydrocephalic brain after shunting. In experimental models it has been demonstrated that, after early shunting, much damage to the gray (reduction of neuron size, disorientation, dendritic deterioration) and white matter (periventricular edema, axonal damage, demyelinization, gliosis) and brain metabolism can partially recover (Hakim and Hakim, 1984). 31.2.2.2.1. External ventricular drainage A short-term CSF shunt device may be needed for hydrocephalus following intraventricular hemorrhage, bacterial infection or after brain tumor surgery with a high risk of postoperative hydrocephalus. 31.2.2.2.2. Ventriculo-peritoneal shunt This is the most popular shunt procedure. The ventricular catheter is placed through an occipital or frontal burr hole and connected to the valve. The distal catheter is tunneled in the subcutaneous space and placed in the peritoneum. The advantages and disadvantages are as follows (Drake and Saint-Rose, 1995): Advantages:

less morbidity from shunt infections the possibility of placing a length of distal tubing to accommodate the patient’s distal growth Disadvantage: peritoneal adhesions or infection. The risk of seizures, which appears higher with frontal positioning, has been reported to be 5.5% in the first year after placement of a ventricular catheter. The risk rate drops to 1.1% after 3 years (Dan and Wade, 1986). 31.2.2.2.3. Ventriculo-atrial shunt This is a less commonly used procedure because of the high risk of infection (sepsis, pulmonary embolus, nephritis, cor pulmonaris and death) (Lundar et al., 1991). The shunt procedure is more demanding because the distal catheter is introduced into the transverse facial or jugular vein and the amount of distal tubing is standard and cannot be adapted to the child’s growth.

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31.2.2.3. Shunt complications CSF shunting represent the neurosurgical procedure with the highest failure rate (Drake and Saint-Rose, 1995). Most complications that require revision of the shunt occur between 6 months and 1 year after surgery (Sainte-Rose et al., 1991). The main causes of shunt dysfunction are:

Obstruction Infections Mechanical problems (migration, disconnection, malpositioning)

Other complications. Obstruction can occur in each component of the shunt device. The ventricular catheter may be obstructed by choroid plexus tissue or by the ventricular wall (Sainte-Rose, 1993). Blood cells, bacteria, proteins and other tissue debris may also block the ventricular catheter and/or the valve. Moreover, the tip of the peritoneal catheter may be obstructed by bowel loops, fat abdominal tissue and other abdominal pathologies (Drake and Saint-Rose, 1995). Shunt infection is usually caused by the child’s own bacterial organisms. The most frequent organism is Staphylococcus epidermidis, which is normally present on the surface of the child’s skin, in sweat glands and in hair follicles deep in the skin. These infections are most likely to occur 1 month after surgery and sometimes up to 6 months after surgery (Choux et al., 1992). Mechanical and other complications are described too. Shunts are very long-lasting systems, although their hardware may become disengaged as a result of the child’s growth, with migration into the body cavities where they were originally placed. The valve itself rarely breaks down because of mechanical malfunction even if the shunting device may over- or hypodrain CSF (Sgouros et al., 1995). The overdrainage may result in slit ventricles syndrome and/or subdural hematoma (Pudenz and Foltz, 1991); in these patients a cranial vault expansion and/or subtemporal decompression may be needed to achieve ventricular re-expansion (Epstein et al., 1988).

31.3. Craniofacial repair for craniofacial dysmorphism Surgical treatment of craniosynostosis aims to correct the deviated calvarial shape, to stop the compensatory growth and to modify its effects by normalizing the physiological functions. This can be achieved, but not always completely, by the ‘dynamization’ of the restricted skull growth and the redirection of the abnormally oriented growth vectors (David et al., 1982).

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In some craniosynostosis only affecting the cranial vault (scaphocephaly), a simple and wide suturectomy allows passive reshaping, especially in the first 2 months of life, utilizing the released directional vectors of growth with the final result of a good cranial expansion and cosmetic correction (Collmann et al., 1999). In other craniosynostosis involving the vault and the skull base simultaneously (e.g. brachycephaly, trigonocephaly, oxicephaly and most plagiocephaly), active reshaping is required by bringing vault regions into the desired position and remodeling shape, orientation and angles of the orbital bar (Genitori et al. 1995). The best time for this kind of correction is between the fourth and sixth months of life (Di Rocco et al., 1980). On the other hand, the management of complex craniofacial malformations (e.g. Crouzon, Apert and Pfeiffer syndromes, cloverleaf skull syndrome) is characterized by multistep surgery (Marchac et al., 1994). Initial anterior skull and orbital ridge remodeling with expansion and volumetric increase of the anterior cranial fossa aims to resolve intracranial hypertension, manage breathing and feeding problems and safeguard brain growth and visual function. Posterior skull expansion is sometimes needed when the occipital regions appear extremely flat; if Chiari type I anomaly coexists, occipital foramen opening may be combined (Cinalli et al., 1998). The second step is to address midfacial advancement, which is performed later, after the fourth year of life (Marchac et al., 1994). In cases of severe midfacial retrusion, causing psychological problems in pre- and school age, early maxillary distraction can be performed by means of mechanical devices that provide a progressive advancement and correction of the facial dysmorphism and subsequent enlargement of the nasal airway (Meling et al., 2004). This procedure is sometimes definitive or can prepare the child for subsequent programmed traditional midfacial advancement using the Le Fort III technique (Meazzini et al., 2005). Successively, when complete growth is achieved, treatment can be completed with rhinoplasty and canthopexy procedures (Tessier, 2000). 31.3.1. Preoperative assessment Early surgical correction is extremely important to achieve best functional and cosmetic result: the chance of an optimal aesthetic result decreases with child age especially after 12 months (Posnick, 2000). Unfortunately, toddlers in first months of life are characterized by ‘triple precariousness’ (large needs – insufficient supplies – inadequate control mechanisms) making

necessary an accurate clinical examination to detect concomitant pathologies (e.g. cardiopulmonary system, coagulopathies, etc.) and reduce anesthesiological and surgical risks (Di Rocco and Velardi, 2001). 31.3.2. Craniectomy and suturectomy This technique is only applied in infants in the first months of life with cranial deformities restricted to the vault (scaphocephaly). Goal of surgery is releasing the directional growth vectors in correspondence of the prematurely fused suture in order to allow a harmonic expansion of the brain. Vertex craniectomies, associated to strip craniotomies along coronal and lambdoid sutures must be preferred to small suturectomies to avoid precocious reossification. The bony defects will close by the end of the first year when the infant learns to walk. The advantage of this technique is represented by the possibility of a more precocious correction and a smaller skin incision (linear or Sshaped vertex incision) with reduced blood loss, the disadvantages are represented by a delayed cosmetic result (Jane and Persing, 1986). 31.3.3. Cranial vault remodeling When an immediate cosmetic result is required for scaphocephaly, more invasive procedures are employed. Many authors recommend the Marchac and Renier multisegment technique, which allows good cranial reshaping and volume expansion (Marchac and Renier, 1981). In these cases, in scaphocephalies we prefer the ‘Pi procedure’ described by Jane in 1986 (Jane and Persing, 1986) which accomplishes a satisfactory and immediate active remodeling of the cranial vault (Fig. 31.6). 31.3.4. Fronto-orbital advancement and remodeling This technique is used in various fashions for trigonocephaly, plagiocephaly (Fig. 31.7), brachycephaly and oxicephaly, to expand anterior cranial fossa and remodel frontal bone and orbital bar (Genitori et al., 1991, 1994). The procedure is characterized by a bicoronal skin incision, anterior lift of the scalp flap until orbital rims, elevation of the pericranium, detachment of the temporalis muscle and of the periosteum until the upper part of the orbital cavities and the frontozygomatic processes are exposed. A bifrontal bone flap, included between the coronal sutures and an horizontal line about 2.5 cm above the orbital rims, is then outlined and removed. The fronto-orbital bandeau is removed en bloc, avoiding to open the periorbital capsule, performing multiple osteotomies carried out along the orbital roofs, the frontozygomatic sutures,


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Fig. 31.6. Scaphocephaly. (A) Preoperative view from above. (B) Postoperative appearance after cranial vault remodeling (from above).

Fig. 31.7. Right anterior plagiocephaly. (A) Preoperative view. Note the facial scoliosis. (B) Postoperative (at 3 years). Note the symmetry of the craniofacial skull.

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Fig. 31.8. Brachycephaly. (A) Preoperative view. (B) Postoperative view after fronto-orbital advancement with bioresorbable plates and screws,

the lesser sphenoid wing, medially above the frontonasal suture and along the temporal bone with a fine oscillating saw or a chisel. At this point care is taken to detect dural lacerations and eventually repair them. The orbital bar is then bent by grooving the inner table or with a bender instrument, reshaped and repositioned with the new orientation and angle. A good stability especially in brachycephaly (Fig. 31.8) may be achieved with the use of bioresorbable lactic acid polymer plates (Kurpad et al., 2000). After its recontouring, the frontal bone is repositioned and ensured to the orbital bar or leaving it free to ‘float’ on the frontal lobes (Marchac et al., 1988). The temporal bone defect is filled advancing and rotating anteriorly the temporalis muscle. The bone surface is then covered by pericranium and the scalp is closed in layers. Treatment of anterior plagiocephaly can also be performed by advancing and remodeling the orbital bar only on the affected side (Genitori et al., 1994).

31.4. Excision of cephaloceles 31.4.1. Intrateutoria cephaloceles First question is deciding whether to treat or not a new-born with encephalocele (Brown and SheridanPereira, 1992). As a matter of fact, all meningoceles should be closed because they do not usually contain brain structures. On the other side, in case of large meningoencephaloceles with large amount of cerebral structures (sometimes exceeding the entire volume of

normal brain) and associated malformations, the surgical indication must be discussed with parents because of their poor prognosis. Prognostic factors to be considered are size of the encephalocele, the amount of vital brain tissue, the microcephaly and hydrocephalus associated. In these forms, the neurological outcome is usually dismal because of higher incidence of hydrocephalus and other brain malformations (Date et al., 1993). Goals of surgery are removing the sac with dysplastic tissue, preserving functional nerve structures and closing the malformation with not-dysplastic skin (McComb, 1996). In the early post-operative period, seizures, CSF collections, hydrocephalus and infection may occur (McComb, 1996). Seizures are due to presence of dysplastic and epileptogenic brain structures (Matson, 1969). It is usual to observe a CSF accumulation into the site of surgery. This ‘dead-space’ is to be avoided by compressive dressing: this phenomenon creates a ‘fifth’ ventricle which raises the risk of postoperative hydrocephalus (McComb, 1996). The hydrocephalus is more common in meningoencephaloceles than in meningocele being due to the loss of supplementary space of CSF accumulation and to coexistent subclinical infections (McLaurin, 1987). 31.4.2. Cranial vault cephaloceles Goal of surgery is cosmetic treatment without trying to remove all the intracranial portion of the content (McLaurin, 1964). Skin incision is tailored to the site


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Fig. 31.9. Three-dimensional CT (axial view). (A) Basal encephalocele. Note the opening of the base of the skull in the posterior ethmoidal and sphenoidal region. (B) Postoperative (transcranial route). Note the disappearance of the hole in the base.

and extension of the sac; the cranial defect is repaired with autologous bone. 31.4.3. Fronto-ethmoidal or sincipital encephaloceles The encephalocele is to be removed with its whole content by a subfrontal extradural route via an anterior bifrontal bone flap (David et al., 1984). The craniotomy is made just above anterior cranial fossa floor sometimes including a fronto-orbital osteotomy to dissect better the sac in a single-step procedure (Sargent et al., 1988). After sac excision and watertight dural closure with not-adsorbable suture, a cranial base plasty with a peduncularized autologous periosteum flap is created to seal the bony defect. A CSF leakage (rhinorrhea and/or CSF ‘tears’) may occur with risk of meningitis avoidable by an external lumbar drainage (McComb, 1996). 31.4.4. Basal encephaloceles The surgical management of trans-sphenoidal, intrasphenoidal and transethmoidal cephaloceles is still controversial because of its high morbidity, permanent impairment and mortality, especially in neonatal period and infancy (Yokota et al., 1986). The goal of surgery is the reduction of the prolapsed sac to lessen the traction on the vital structures, preserving their function and obtaining a watertight dural closure with reparation of the bone defect (Lai et al., 2002). The most important question still remaining is the route of the surgery: transcranial versus extracranial. As described by many authors, the transcranial transbasal route via a bifrontal

bone flap (Fig. 31.9) is followed by higher mortality and morbidity, especially in younger patients (Kai et al., 1996). On the other hand, since these lesions progressively enlarge, it is best to operate early in order to prevent further damage to the herniated brain tissue, preserve vision and avoid progressive respiratory distress (Abiko et al., 1988). Sometimes, urgent repair may be needed in patients with CSF leaks or hemorrhage after inadvertent removal of a cephalocele mimicking a nasal polyp (Choudhury and Taylor, 1982). So nowadays the extracranial approach is preferred even in infancy, especially in case of progressive and life-threatening symptoms (Kennedy et al., 1997). Different approaches may be performed: transpalatal, transnasal-transmaxillary, transnasal–trans-sphenoidal or combined approaches (Fig. 31.10). In the transpalatal approach, the sac can be easily viewed and dissected by paramedian splitting of the uvula and soft palate and partial osteotomy of the hard palate. The transnasal–transsphenoidal approach uses the well-known techniques of pituitary surgery to gain access to the sphenoid bone. In all these techniques, the common principle is not trying to push the whole sac inside the cranium but only to reduce the extent to which it stretches into the nasal cavity and epipharynx to stop traction on vital structures. Closure and reinforcing of the sac is made by application of multiple layers of oxidized cellulose and fibrin glue; the bone defect can be closed by autologous bone powder, nasal septum cartilage, autologous bone of nasal turbinates and sometimes other heterologous ossification inducers (Genitori et al., 2001). Reparation may be made through an endoscopic nasal approach, as described in an increasing number of cases reported in the literature (Lanza, 1996).

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Fig. 31.10. MRI (sagittal view). (A) Pure trans-sphenoidal encephalocele with third ventricular structures protruding into the epipharynx. (B) Postoperative aspect with complete reconstruction of the defect by an extracranial combined route.

31.4.5. Other forms of cranial dysraphism: atresic encephaloceles A horizontal skin incision in a rhomboidal fashion is made around the sac. The dysplastic skin is removed, with the nonvital inner tissue. The intracranial portion, if present, must be left in place. The cranial defect is closed by tubularizing the periosteum, which is then covered by autologous bony powder. The skin is closed with nonabsorbable sutures (McComb, 1996). 31.4.6. Congenital defects of the scalp (aplasia cutis congenita) Smaller lesions can be treated conservatively, waiting for spontaneous healing and epithelialization. Larger lesions must be repaired using rotational skin flaps, sometimes prepared in advance by implanting skin expanders. In cases of massive agenesis of the scalp, dessication and injury of the brain must be avoided by keeping the lesion moist (McComb, 1996).

31.5. Posterior fossa decompression for chiari type I anomaly In 1988 the American Association of Neurological Surgeons declared that, in Chiari I patients, posterior fossa decompression was always mandatory in the presence of signs of brainstem dysfunction, debatable in cases of mild symptoms and headache, and not recommended in asymptomatic patients (Haines and Berger, 1991). Today there is a general agreement that Chiari I anomaly characterized by cerebellar tonsil prolapse of at least 5 mm down to the foramen magnum, with appropriate symptoms, should be treated. In borderline cases (prolapse of 0–5 mm), the surgical indications must be eval-

uated in each individual clinical case. In cases of syringomyelia, surgery is mandatory even in the presence of limited tonsil descent to avoid further enlargement and clinical deterioration (Tubbs et al., 2003). The goal of surgery is to restore normal CSF flow, thus re-establishing a pressure balance between the intracranial and intraspinal subarachnoidal spaces by decompressing the inferior cerebellum and cervico-medullary region at the level of foramen magnum (Batzdorff, 1988). Nowadays, first-choice surgery consists of suboccipital craniectomy plus posterior C1 laminectomy both in simple Chiari I patients and in complicated cases with syringomyelia. The purpose of this simplified technique, first described by Isu, is to enlarge the posterior fossa at the level of the foramen magnum without complete dural opening (Isu et al., 1993). A midline vertical incision is made from just inferior to the inion to the C3 level. Myofascial dissection is carried out along the median raphe. Special care must be given to avoiding muscle dissection from C2 level (semispinalis cervicis and multifidus muscle) to prevent cervical instability and to reduce postoperative neck pain. Then a suboccipital craniectomy is carried out. The occipital bone is opened by a 3 3 cm suboccipital craniectomy performed using a high-speed drill and rongeurs to enlarge the foramen magnum. A posterior C1 laminectomy of about 2.5 cm is made. It is not necessary to extend the craniectomy laterally to reduce the surgical risk. A dense fibrous and constrictive band covers the atlanto-occipital membrane, causing intradural compression and arachnoid adhesion. This strip is cut and removed with the atlanto-occipital membrane until the outer dura is identified. This is bluntly dissected and sectioned until a good CSF pulsatility appears from the cisternal and medullary spaces. One must take care to respect the arachnoid layer as soon as it appears during

SURGICAL TREATMENT OF CENTRAL NERVOUS SYSTEM MALFORMATIONS the dissection of the outer layer of the dura (Genitori et al., 2000). In the past, other authors described many intradural procedures: opening of the dura in a Y fashion followed by a wide duraplasty graft (Batzdorff, 1996), dissection of arachnoid overlying the tonsils (Dyste and Menezes, 1988), coagulation of herniated cerebellar tonsils respecting the integrity of pia and arachnoid (Oakes, 1985), resection of cerebellar tonsils with subpial approach in cases of very high gliotic tonsils not reduced by simple coagulation (Fisher, 1995), obex occlusion with a piece of muscle (Hoffmann et al., 1987), section of filum terminale (Filizzolo et al., 1988). Surgical morbidity may take the form of vertebral artery damage, acute hydrocephalus, cerebellar ptosis, pseudomeningocele, CSF leakage, subdural collections, cervical instability and acute life-threatening signs of brainstem dysfunction (Tubbs et al., 2003). More aggressive surgery is followed by a higher rate of complications, especially pseudomeningocele (4%), CSF leakage (2%), aseptic meningitis (22%), acute hydrocephalus (1%), fluid collection in the operative wound and late arachnoid adhesions (Zerah, 1999). In modern series, authors like Zerah (1999) and Genitori et al. (2000) have stressed the good results of this technique, which is not associated with frequent compli-

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cations and is characterized by shorter hospitalization compared with the morbidity after surgery characterized by dural opening; surgical outcomes are good both in reducing syringomyelia and in improving its secondary effects such as scoliosis (Fig. 31.11). In any case, where there is clinical and/or radiological Chiari I recurrence or enlargement of syringomyelic cavities, dural expansion should be considered (Genitori et al., 2000). Recently, Milhorat and Bolognese (2003) have proposed intraoperative control using color Doppler ultrasonography (CDU) to tailor the extension of posterior fossa bony decompression and C1 laminectomy, and the eventual need for additional steps such as duraplasty and shrinkage or resection of the cerebellar tonsils. During the first surgical steps, CDU makes it possible to distinguish better all the posterior fossa structures, including aberrant vascular anatomy, asymmetrical herniations and neural displacement; this reduces the risk of surgical error, especially in patients undergoing reoperation with a lot of meningo-cerebral scarring (Milhorat and Bolognese, 2003). At the end of posterior fossa decompression, CDU serves to monitor whether the CSF circulation and pulsatility are restored by measuring CSF flow velocity and viewing CSF flow direction; optimal CSF flow has a peak

Fig. 31.11. MRI ( sagittal view). (A) Chiari type I anomaly. Note the cervical syringomyelia. (B) Postoperative, after posterior fossa decompression. Note the complete disappearance of the syringomyelia.

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velocity of 3–5 cm/s, bidirectional movement and a waveform exhibiting arterial, venous and respiratory variations (Milhorat and Bolognese, 2003). In presence of syringomyelia–hydromyelia, some authors have proposed putting a shunt between the fourth ventricle and the subarachnoidal spaces (Menezes et al., 1980) while others prefer to perform a syringostomy by myelotomy (Rhoton, 1976). Syringo-subarachnoid shunting using a small catheter has been also suggested, even though spinal cord injury has been described after insertion of catheter in the spinal cavity (Isu et al., 1990). A syringo-peritoneal or pleural shunt has been advocated because of the higher differential pressure compared with the subarachnoidal space (Barbaro et al., 1982). The catheter used is K- or T-shaped and 2 mm wide. The surgical technique consists of anchoring the catheter to the dura and placing its end in the planned cavity. The insertion of a valve device to regulate CSF drainage must be evaluated even if it is not usually employed (Batzdorff, 1988). Some technical reports describe the possibility of treating Chiari I anomaly by tapping the syringomyelial cavity via percutaneous aspiration after failure of previous treatment (Batzdorff, 1996). As regards the associated hydrocephalus, endoscopic third ventriculostomy is the first-choice surgery if hydrocephalus symptoms predominate (Decq et al., 2001). Posterior fossa surgery should be considered in the case of onset of Chiari symptoms even if hydrocephalus has been eliminated.

31.6. Surgery for dysraphic state 31.6.1. Closure of myelomeningocele (spina bifida aperta) Delivery by cesarean section is suggested to diminish local trauma to the malformation (Fig. 31.12). The mal-

Fig. 31.12. Myelomeningocele. Appearance of the placode before closure.

formation must be protected and kept moist using tulle gras (Genitori et al., 1993). At birth a number of precautions have to be taken to avoid hypothermia, hypovolemia and hypoglycemia (Reigel, 2001). A complete diagnostic work-up must be performed to evaluate the neurological status of the newborn and the associated problems (brain malformation, hydrocephalus, urological and orthopedic impairments) (Genitori et al., 1993). Surgery must be performed within the first 48 hours to avoid septic meningitis, sepsis and secondary injury to the placode requiring repair. Any delay after 72 hours increases this risk to 37% compared to 7% in cases of early closure (Charney et al., 1985). Neonatal meningitis is a serious complication because it impairs intellectual development (McLone et al., 1980). The neonate is positioned prone with all pressure points on smooth pads in a Trendelenburg position to reduce CSF leaking; warming tables are utilized. Tracheal intubation should be carried out in a donut position if possible to reduce trauma to the sac. The usual antiseptic drugs should be employed (i.e. povidone iodine must be avoided). Goals of surgery are: 1) identification of all anatomical planes according to the well-known embryological physiopathology; 2) reconstruction of the placode; 3) closure of meningeal coverings; 4) closure of the fascia and skin. The first step is an incision at the meningo-epithelial junction and dissection of the neural placode under the control of the operative microscope. Arachnoidal adhesions between the placode and underlying dura are lysed. Any other associated abnormalities must be identified and eventually removed (e.g. dermoids, lipomas, neuroenteric cysts, etc.). All residual epidermal and dermal elements must be removed to avoid the future formation of a dermoid or lipoma (McLone, 1998). At this stage, the placode is tubulized with nonabsorbable suture (nylon 5/0) and the recurrent spinal roots must be respected. The meningeal layer is then dissected as far as possible to cover the new spinal cord, aiming to maintain it submerged in CSF in order to avoid secondary tethering. Sometimes a duraplasty is created with an autologous flap (periosteum, fascia lata) or more frequently with artificial biocompatible material like PTFE (polytetra fluoroethilene). In a case with a huge kyphosis, kyphectomy is necessary during the first surgery to enable easy closure of the defect (Reigel, 1979). At the end of the procedure, a myofascial layer is prepared to cover the dura and the excess and dysplastic skin is excised before final closure is made with nonabsorbable sutures (nylon 3/0). A releasing incision in the fascia laterally away from the defect may be useful to obtain a tension-free closure. In the case of a large defect, the lumbo-sacral musculature is useful to create flaps to cover the malformations even if rotational flaps do not often function because

SURGICAL TREATMENT OF CENTRAL NERVOUS SYSTEM MALFORMATIONS of ischemia. Deep dissection of latissimus dorsi may be dangerous because of the risk of damage to retroperitoneal and pulmonary structures (Ramasatry and Cohen, 1995). In the early postoperative period the newborn must be carefully monitored. The recommended position is Trendelenburg prone or lateral protecting the wound from urine and fecal contamination. Periodical measurement of the cranial circumference and ultrasound tomography are performed to rule out hydrocephalus. Ventriculo-peritoneal shunting is mandatory at the first sign of hydrocephalus and/or in case of CSF leakage from the wound and/or brainstem dysfunction related to Chiari II. Endoscopic third ventriculostomy (ETV) achieves good results in secondary treatment of hydrocephalus in such children with shunt dysfunction (Jones et al., 1994). Surgery for Chiari type II anomaly should be considered if at least one of the four Griebel’s criteria is present (Griebel et al., 1990): 1) continuous stridor with respiratory difficulty; 2) recurrent ab ingestis pneumonia; 3) bradycardia or apnea; 4) cyanosis. The surgical technique involves wide dissection of the foramen magnum along with posterior C1 laminectomy; a large duraplasty is then constructed using autologous or artificial material (Genitori et al., 1993). However, this procedure is justified only if the hydrocephalus is well treated; in fact Chiari II may decompensate because of shunt dysfunction (Isu et al., 1990). Surgical mortality is near zero, while postoperative complications may be serious. The most frequent complication is wound dehiscence with CSF leak, followed by local infection (1–1.5%), neonatal sepsis, and all the other complications connected with shunt and posterior fossa surgery (Pang, 1995).

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on the dura. The laminae are repositioned, taking care to set them in the normal position, and sutured with resorbable 2/0 sutures. The fascia is closed. Subcutaneous tissue and skin are closed with the same suture (Chapman and Davis, 1993). 31.6.2.2. Caudal lipoma The paravertebral muscles are dissected off the laminae, very carefully in the zone of the schisis to avoid penetrating the dura. A two-level laminotomy is performed, above the lesion entry zone in the dura, if present. The surgical microscope is necessary to perform the next surgical step. The dura is opened in a craniocaudal fashion and suspended. Untethering is achieved by dividing the lipoma below the transitional zone, which is identified between the conus and lipoma to avoid neural elements (Choux et al., 1994). After division of the lipoma the cord often shows a remarkable degree of retraction. The dura is closed with a biocompatible artificial patch using a 5/0 running suture to avoid retethering between the scar and the dural elements (Fig. 31.13). The laminae are repositioned, setting them carefully in the normal position.

31.6.2. Detethering the spinal cord (occult spinal dysraphism) 31.6.2.1. Lipoma of the filum terminale The surgical approach starts with a skin incision in the midline; the subcutaneous layer is incised with a monopolar scalpel until the fascia is exposed. Then the paravertebral muscles are dissected off the laminae and a two- or three-level laminotomy is performed. Use of the surgical microscope is necessary to perform the next surgical step. The dura is opened in a craniocaudal fashion and suspended. At this point Trendelenburg position prevents loss of CSF and keeps the surgical field clear of CSF. The filum lipomatosus is identified, coagulated and sectioned using microscissors. Normally after this step there is a remarkable retraction of the proximal end of the filum. The dura is closed with a 5/0 Prolene running suture. Fibrin glue and oxidized cellulose are positioned

Fig. 31.13. MRI ( sagittal view). Dysraphic state. (A) Caudal lipoma, preoperative. (B) Postoperative. Note the detethering of the spinal cord.

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31.6.2.3. Lipomyelomeningocele

The surgical approach is planned on the basis of radiological evaluation (Fig. 31.15). The aim of surgery is untethering the spinal cord, using two different techniques according to the two types of split cord malformation (Pang, 1992). In type I split cord malformation the

osteocartilaginous spur must be removed while dealing with tight adhesions between the cord and dura. The skin incision is performed in the midline extending above and below the lesion. The laminae are dissected off the paravertebral muscles, starting where the spinous processes are normal. A minimal laminectomy is begun in a normal area to avoid the risk of kyphoscoliosis. The bony spur is then progressively exposed and removed after a subperiosteal dissection of the septum, avoiding lateral movements, which can injure the adjacent hemicords. The two dural sacs are progressively exposed and the dura is opened by an incision encircling the dural cleft and extended towards each extremity. The adhesions between the medial aspect of the hemicord and the dural sleeves must be progressively severed. The closure of the dura is performed with a duraplasty. In type II split cord malformation the procedure is simpler. The dural tube is single and the two hemicords with the median septum may have three different positions: 1) a complete fibrous septum transfixes the hemicords and is fixed on the ventral and dorsal surfaces of the dura; 2) the septum is ventral only, fixing the ventromedial aspect of the hemicords

Fig. 31.14. MRI (sagittal view). Dysraphic state. (A) Lipomyelomeningocele, preoperative. (B) Postoperative. The lipoma has not been completely removed, but the spinal cord is detethered.

Fig. 31.15. MRI (coronal view). Dysraphic state: diastematomyelia. Note the split cord malformation.

The initial surgical approach is the same as described for the other types of operation. At the level of the dura, it is important to distinguish the ‘normal dura’ from the capsule of the lipoma. The lipoma can be dissected and debulked using a CO2 laser or ultrasonic aspirator. It is not necessary to attempt to debulk it in the intramedullary space since it does not increase in size (Fig. 31.14). Careful dissection must be employed at the interface between the lipoma and the spinal cord. The filum can be identified and divided. In these cases, the detethering starts from the superficial planes. The nerve roots lie horizontally and cannot be liberated from the lateral surface of the lipoma (McLone, 2001). 31.6.2.4. Diastematomyelia

SURGICAL TREATMENT OF CENTRAL NERVOUS SYSTEM MALFORMATIONS to the dura; 3) the septum fixes the dorsal aspect of the hemicords (Pang, 1992). 31.6.2.5. Dermal sinus The skin incision is made around the dermal sinus opening. The tract itself is dissected free of the underlying subcutaneous tissues, down to the point where it pierces and penetrates the underlying muscular fascia. Every attempt is made to preserve the tract until the laminotomy is made at one or two levels above and below the tract where it enters the dura. Then the dura is opened in the cranial and caudal direction and two incisions are made around the stalk where it penetrates the dura. At this point, the stalk is sectioned and removed (Fig. 31.16). Associated lesions such as dermoids and lipomas should be removed using magnification under the operative microscope (Choux et al.,1994). Detethering of the spinal cord in other, rarer, forms of dysraphic-state-like neuroenteric cyst and anterior meningoceles should be treated in a multidisciplinary fashion with an anterior transabdominal and posterior approach that allows wide exposure of the malformation while preserving the surrounding neural structures (Fig. 31.17).

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31.7. Fetal surgery Fetal surgery represents a multidisciplinary approach to some CNS malformations and tumors diagnosed in utero (Flake and Harrison, 1995). Nowadays, these new techniques deal essentially with prenatal hydrocephalus (Cavalheiro et al., 2003) and myelomeningocele (Sutton et al., 2003) and are feasible thanks to a full collaboration between the obstetric surgeon, anesthesiologist and neurosurgeon (Hendrick et al., 1998). Prenatal imaging by fetal magnetic resonance imagine (MRI) is mandatory to gain a complete and precise evaluation of malformations (Oi et al., 1998). 31.7.1. Prenatal hydrocephalus The incidence of true fetal hydrocephalus ranges from 1 to 4:1000 births (Cavalheiro et al., 2003). In 70% of cases other CNS anomalies are associated (holoprosencephaly, Dandy–Walker complex, spina bifida, corpus callosum agenesia) and in 7–15% of fetuses systemic malformations coexist (Chervenak et al., 1985). In 3– 10% of cases different chromosomopathies have been screened, involving chromosomes 1, 6, 9, 13, 18, 21,

Fig. 31.16. Dysraphic state. (A) MRI (sagittal view). Dermal sinus and tract. (B) Intraoperative view. Note the stalk fixing the spinal cord.

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Fig. 31.17. MRI (sagittal view). Dysraphic state. (A) Neuroenteric cyst. (B) Postoperative appearance with removal of the cyst by an anterior approach.

22 or X (Strain et al., 1994). Fetal hydrocephalus may be due to ventricular obstruction (congenital tumors, intraventricular hemorrhage), CNS maldevelopment or acquired intrauterine damage (infections, hemorrhages) (Von Koch et al., 2003). The most frequent cause of isolated fetal hydrocephalus is aqueductal stenosis (Cavalheiro et al., 2003). In his ‘perspective classification of congenital hydrocephalus’, Oi divided prenatal hydrocephalus into four phases according to the gestational age of diagnosis (Oi et al., 1998). Phase II (22–31 weeks) corresponds to the period of ‘intrauterine preservation’; during this phase, if the hydrocephalus becomes progressive, the damage to CNS may be irreversible after birth. Between 22 and 31 weeks of gestational age, the fetal lungs are not sufficiently developed, requiring preservation in the uterus, and it is obvious that the earlier the onset of hydrocephalus the greater the damage to the developing CNS; hence the earlier the treatment the better the results from both motor and cognitive points of view (Weller and Shulman, 1972). Thus Phase II hydrocephalus is amenable to prenatal surgical treatment (Oi, 2001). Surgery is suggested only in case of hydrocephalus not associated with other systemic and/or brain malfor-

mations because surgical and patient outcomes are better (Cavalheiro et al., 2003). According to the guidelines of the International Fetal Surgery Registry (Harrison et al., 1982), the ideal candidate to be submitted to fetal surgery should have an isolated hydrocephalus from non-X-linked aqueductal stenosis diagnosed before 28 weeks gestation and before the cortical mantle thickness is less than 1.5 cm; the hydrocephalus must be moderate to severe and not associated with other fetal brain anomalies on MRI; its progression has been documented by periodic ultrasound tomography; infections and genetic anomalies must be ruled out by amniocentesis. Indeed, fetuses harboring hydrocephalus linked to another CNS malformation (e.g. Dandy–Walker complex, X-linked hydrocephalus) do not show improved intellectual outcome after fetal surgery (Von Koch et al., 2003). In the case of polymalformation hydrocephalus, termination of pregnancy should be suggested to the family. If the hydrocephalus is stable or resolving, the child is delivered at term and then treated (Von Koch et al., 2003). In the past, repeated cephalocentesis was performed to obtain temporary control until delivery was possible (Birnholz and Frigoletto, 1981) even if this technique involved a higher risk of fetus infection,

SURGICAL TREATMENT OF CENTRAL NERVOUS SYSTEM MALFORMATIONS hemorrhage and porencephaly (Cavalheiro et al., 2003). Today, fetal CSF shunting surgery is preferred, aiming to obtain temporary relief from intracranial pressure while waiting for the earliest possible delivery for definitive treatment in better general conditions (Von Koch et al., 2003). Ventriculo-amniotic shunts were proposed Clewell and colleagues (1982). This shunting technique is hindered by a high incidence of complications such as catheter obstruction, migration of the device into the amniotic cavity, and infection (Cavalheiro et al., 2003). Furthermore, fetal hydrocephalus is high-pressure but surrounded by a higher intrauterine pressure, which impedes its correct functioning (Oi et al., 1990). On this pathological basis Oi has proposed the use of a fetal-ventricular–maternalperitoneal shunt (Oi et al., 1989). Bruner has introduced a new type of ventriculo-amniotic shunting to improve the fixation (Bruner et al., 2001). As a rule, requirements for such shunts include a safe and simple insertion technique, valid scalp fixation and a one-way valve to prevent intraventricular reflux of amniotic fluid (Von Koch et al., 2003). As suggested by Cavalheiro et al. (2003) endoscopic third ventriculostomy may be considered in case of hydrocephalus due to pure aqueductal stenosis. 31.7.2. Myelomeningocele Much clinical and experimental evidence shows neurological deterioration in the affected fetus during pregnancy according to the ‘two hit’ hypothesis, the first hit being embryological spinal cord malformation (Heffez et al., 1990). Some reports have documented normal movement of the lower extremities in fetuses with spina bifida aperta before 17–20 weeks, followed by fairly complete paralysis in late gestation (Korenromp et al., 1986). This deterioration seems to be due to the exposure of nervous tissue to meconium and amniotic fluid (Drewek et al., 1997) and to direct trauma to the placode from the uterine wall during fetal movements (Hutchins et al., 1996). The amniotic fluid becomes more hypotonic thus more toxic as fetal urine output increases after kidney maturation, which takes place after 22 weeks gestation (Lind et al., 1972). Furthermore, there is evidence that the Chiari type II anomaly is also acquired as a result of the continuous CSF leakage from the placode, which leads to progressive hindbrain prolapse (Paek et al., 2000). These findings constitute the physiopathological background for myelomeningocele repair in utero (Heffez et al., 1990). According to a schedule promoted by three different institutions (Children’s Hospital of Philadelphia, Vanderbilt Medical University, University of California at Los Angeles) (Sutton

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et al., 2003), the selection criteria are: prenatal diagnosis between 16 and 25 weeks of gestation; level of the defect at S1 or above with documented leg motility on ultrasound and absence of foot deformity; absence of other fetal malformations or chromosomal anomalies; proposed date for surgery 26 weeks. The first cases were treated by an endoscopic technique pioneered by Copeland and colleagues (1993). This was performed between 22 and 24 weeks of gestation using a 4 mm rigid endoscope. First, the mother underwent laparotomy under general and epidural anesthesia, with exposure of the gravid uterus. Then, three endoscopic ports were inserted into the uterus (one for the endoscope and two operative channels for instruments). Because of its turbidity, amniotic fluid was tapped until the fetus was completely exposed and the fluid was replaced by carbon dioxide to maintain ambient intrauterine pressure. After positioning of the fetus, the placode was covered with a maternal split-thickness skin graft because it was not possible to use a standard skin suture. All the reconstruction was sealed by oxidized cellulose and fibrin glue (Bruner et al., 1999). The surgical results were not satisfactory because fetal morbidity and mortality, and maternal morbidity, were high: in the four cases treated by Tulipan and Bruner (Bruner et al., 1999) amnionitis, amniotic leakage, uterine dehiscence, placental abruption, preterm delivery and one death were observed. Furthermore, this technique was only palliative and not curative, as the skin graft was short-lived (Copeland et al., 1993). Accordingly, the technique of open intrauterine repair was developed, on the basis of experimental models suggesting that most secondary damage takes place during the third trimester of pregnancy (Tulipan and Bruner, 1998). The mother underwent cesarean section under general plus epidural anesthesia at 28– 30 weeks of gestation; this anesthetic combination seems to reduce the incidence of unwanted uterine contractions and allows sedation of the fetus too (Tulipan and Bruner, 2001). After the uterus is exteriorized through a Pfannenstiel’s incision and the fetus and placenta are localized by ultrasound scan, the Tulipan– Bruner trocar is inserted into the uterus (Bruner et al., 1999) to tap most of the amniotic fluid, which is conserved in warm syringes. A 5 cm incision is made in the uterus and the fetus is positioned with the placode in the middle of the hysterotomy (Fig. 31.18). The myelomeningocele is then closed using the standard neurosurgical technique with nonresorbable nylon 7/0 sutures for the placode tubulization and nylon 5/0 for the skin (Tulipan and Bruner, 2001). During the whole procedure, the fetal heartbeat is monitored by ultrasound and continuous electronic

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Fig. 31.18. Fetal surgery. Intrauterine repair of myelomeningocele. (A) Uterine incision and exposition of the fetal lesion. (B) Appearance after closure of the malformation (personal case).

fetal monitoring. The uterus is closed in layers with adsorbable sutures and the amniotic fluid is replaced, sometimes with saline solution until its turgor becomes similar to the preoperative state, in order to reduce the risk of uterine contractions (Tulipan and Bruner, 2001). The wall of the abdomen is closed in a standard fashion and the fetus continues to be monitored. The mother is administered tocolytic agents (indomethacin, terbutaline). In the postoperative period, both the mother and the fetus are periodically monitored until delivery by cesarean section, which is usually planned at 34–35 weeks gestation; delivery is anticipated only in the case of uncontrolled amniotic leak or premature contractions, trying to balance, in all cases, the risk of dehiscence of the hysterotomy and iatrogenic fetal immaturity (Tulipan and Bruner, 2001). In the series of 50 cases operated on by Tulipan and coworkers, surgical morbidity was low and included uterine contractions, placental abruption, amniotic leakage; uterine dehiscence with prolapse of the fetus into the peritoneal cavity was the most serious. In only one case did premature delivery occur. Surgical mortality in utero involved only one fetus (Tulipan and Bruner, 2001) even if, in other series, there is a perinatal mortality of about 6% due to the extreme prematurity (Johnson et al., 2003). Unwanted side effects of tocolytic therapy are possible in the mother, such as tachycardia, fever, dyspnea and pulmonary edema (Tulipan and Bruner, 2001). The newborn may show local dehiscence at the site of placode repair, which is usually managed conservatively (Tulipan and Bruner, 2001). The most encouraging surgical results are the lower incidence of Chiari II and of hydrocephalus (Tulipan and Bruner, 2001). Chiari type II anomaly after fetal surgery accounts for only 16% rather than the

described incidence of 95% (Tulipan and Bruner, 1999). Other studies have shown that hindbrain prolapse is reversed rather than prevented by fetal surgery: postoperative fetal MRI (Fig 31.19) at 3 weeks has well documented the ascent of these structures (Sutton et al., 1999). Resolution of Chiari II anomaly reduces the incidence of hydrocephalus to 42.7% from 90% (Sutton et al., 2003) thanks to restoration of CSF pathway at the level of the fourth ventricle outlets (Babcook et al., 1994). Despite a number of experimental and clinical studies to the contrary (Heffez et al., 1990; Massobrio and Genitori, 2003), Tulipan’s series did not show any neurological improvement, as

Fig. 31.19. Fetal surgery. MRI (sagittal view). (A) Appearance of fetal myelomeningocele. (B) After closure of the lesion. Note the ‘normal’ position of the spinal cord (personal case).



the neonates showed neurological impairment exactly corresponding to the level of the defect (Tulipan and Bruner, 2001). In some cases secondary, late tethering of the spinal cord has been described because of epidermoid inclusion cysts, which required further treatment (Mazzola et al., 2002).

References Abbott R (2004). The endoscopic management of arachnoidal cysts. Neurosurg Clin North Am 15: 9–17. Abiko S, Aoki H, Fudaba H (1988). Intrasphenoidal encephalocele: report of a case. Neurosurgery 22: 933–936. Aschoff A, Kremer P, Hashemi B, Kunze S (1999). The scientific history of hydrocephalus and its treatment. Neurosurg Rev 22: 67–93. Babcook CJ, Goldstein RB, Barth RA, et al. (1994). Prevalence of ventriculomegaly in association with myelomeningocele: correlation with gestational age and severity of posterior fossa deformity. Radiology 190: 703–707. Barbaro NM, Wilson CB, Gutin PH, Edwards MSB (1982). Surgical treatment of syringomyelia. Favorable results with syringoperitoneal shunting. J Neurosurg 61: 531–538. Batzdorff U (1988). Chiari I malformation with syringomyelia: evaluation of surgical therapy by magnetic resonance imaging. J Neurosurg 68: 726–730.

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Genitori L, Giordano F, Peretta P, et al. (2001). Surgical management of pure transsphenoidal encephaloceles in children: a series of 5 cases. Technical considerations on intra- and/or extracranial approaches. Childs Nerv Syst 17: 440. Gorayeb RP, Cavalheiro S, Zymberg ST (2004). Endoscopic third ventriculostomy in children younger than 1 year of age. J Neurosurg 100 (suppl. Pediatrics): 427–429. Griebel ML, Oakes WJ, Worley G (1990). The Chiari malformation associated with myelomeningocele. In: H Rekate (Ed.), Comprehensive Management of Spina Bifida. CRC Press, New York, pp. 67–92. Gruber R, Jenny P, Herzog B (1984). Experiences with the antisiphon device (ASD) in shunt therapy of pediatric hydrocephalus. J Neurosurg 61: 156–162. Haines SJ, Berger M (1991). Current treatment of Chiari malformations types I and II: a survey of the Pediatric Section of the American Association of Neurological Surgeons. Neurosurgery 28: 353–357. Hakim S, Hakim C (1984). A biomechanical model of hydrocephalus and its relationship to treatment. In: K Shapiro, A Marmarou, HD Portnoy (Eds.), Hydrocephalus. Raven Press, New York. Hanlo PW, Cinalli G, Vandertop WP, et al. (2003). Treatment of hydrocephalus determined by the European Orbis Sigma Valve II survey: a multicenter prospective 5-year shunt survival study in children and adults in whom a flowregulating shunt was used. J Neurosurg 99: 52–57. Harrison MR, Filly RA, Golbus MS, et al. (1982). Fetal treatment N Engl J Med 307: 1651–1652. Heffez DS, Aryanpur J, Hutchins GM, Freeman JM (1990). The paralysis associated with myelomeningocele. Clinical and experimental data implicating a preventable spinal cord injury. Neurosurgery 26: 987–992. Hellwig D, Grotenhuis JA, Tirakotai W, et al. (2005). Endoscopic third ventriculostomy for obstructive hydrocephalus. Neurosurg Rev 28: 1–34. Hendrick MH, Longaker MT, Harrison MR (1998). A fetal surgery primer for plastic surgeons. Plast Reconstruct Surg 101: 1709–1729. Hoffmann HJ (1982). Technical problems in shunts. Monogr Neurol Sci 8: 158–169. Hoffmann HJ, Neill J, Crone KR, et al. (1987). Hydrosyringomyelia and its management in childhood. Neurosurgery 21: 347–351. Hopf NJ, Perneczky A (1998). Endoscopic neurosurgery and endoscope-assisted microneurosurgery for the treatment of intracranial cysts. Neurosurgery 43: 1330–1336. Hutchins G, Meuli M, Meuli-Simmen C, et al. (1996). Acquired spinal cord injury in human fetuses with myelomeningocele. Pediatr Pathol Lab Med 16: 701–702. Isu T, Iwasaki Y, Akino M, Abe H (1990). Syringosubarachnoid shunt for syringomyelia associated with Chiari malformation (type I). Acta Neurochir (Wien) 107: 152–160. Isu T, Sasaki H, Takamura H, Kobayashi N (1993). Foramen magnum decompression with removal of the outer layer of the dura as treatment for syringomyelia occurring with Chiari I malformation. Neurosurgery 33: 845–849.

SURGICAL TREATMENT OF CENTRAL NERVOUS SYSTEM MALFORMATIONS Jane JA, Persing JA (1986). Neurosurgical treatment of craniosynostosis. In: MM Cohen Jr (Ed.), Craniosynostosis. Diagnosis, Evaluation and Treatment. Raven Press, New York, ch. 18, pp. 209–227. Johnson MP, Sutton LN, Rintoul N, et al. (2003). Fetal myelomeningocele repair: short-term clinical outcomes. Am J Obstet Gynecol 189: 482–487. Jones RF, Kwok BC, Stening WA, Vonau M (1994). The current status of endoscopic third ventriculostomy in the management of non-communicating hydrocephalus. Minim Invasive Neurosurg 37: 28–36. Kai Y, Nagahiro S, Yoshioka S, Ushio Y (1996). Application of the skull base technique to the repair of transsphenoidal meningoencephaloceles. Pediatr Neurosurg 25: 54–56. Kennedy EM, Gruber DP, Billmire DA, Crone KR (1997). Transpalatal approach for the extracranial surgical repair of transsphenoidal cephaloceles in children. J Neurosurg 87: 677–681. Kestle J, Drake J, Milner R, et al. (2000). Long-term followup data from the Shunt Design Trial. Pediatr Neurosurg 33: 230–236. Korenromp M, Van Good J, Bruinese H, Driek R (1986). Early fetal movements in myelomeningocele. Lancet 1: 917–918. Kurpad SN, Goldstein JA, Cohen AR (2000). Bioresorbable fixation for congenital pediatric craniofacial surgery: a 2year follow-up. Pediatr Neurosurg 33: 306–310. Lai SY, Kennedy DW, Bolger WE (2002). Sphenoid encephaloceles: disease management and identification of lesions within the lateral recess of the sphenoid sinus. Laryngoscope 112: 1800–1805. Lanza DC, O’Brien DA, Kennedy DW (1996). Endoscopic repair of cerebrospinal fluid fistulae and encephaloceles. Laryngoscope 106: 1119–1125. Lind T, Kendall A, Hyten FE (1972). The role of the fetus in the formation of amniotic fluid. Br J Obstet Gynaecol 79: 289–298. Lundar T, Langmoen IA, Hovind KH (1991). Fatal cardiopulmonary complications in children treated with ventriculoatrial shunts. Childs Nerv Syst 7: 215–217. McComb JG (1996). Encephaloceles. In: JR Youmans (Ed.), Neurological Surgery.WB Saunders, Philadelphia, pp. 829–842. McLaurin RL (1964). Parietal cephaloceles. Neurology 14: 764–772. McLaurin RL (1987). Encephalocele and cranium bifidum. In: NC Myrianthopoulos (Ed.), Handbook of Clinical Neurology. Elsevier Science, Amsterdam, pp. 97–111. McLone D (1998). Care of the neonate with a myelomeningocele. Neurosurg Clin North Am 9: 111–120. McLone DG (2001). Lipomyelomeningocele repair. In: DG McLone (Ed.), Pediatric Neurosurgery.WB Saunders, Philadelphia, pp. 302–306. McLone D, Czyzewsky D, Raimondi A (1980). Central nervous system infections as a limiting factor in the intelligence of children with myelomeningocele. Pediatrics 70: 338–342.

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Chapter 32

Neurorehabilitation of children with cerebral palsy KAREN MARIA BARLOW* Alberta Children’s Hospital, Calgary, Alberta, Canada

32.1. Introduction Malformations of cerebral development account for 7–19% of cases of cerebral palsy (Kwong et al., 2004; Russman and Ashwal, 2004). Not all children with disorders of cerebral malformation will fulfill criteria for cerebral palsy; however, the management of a child with cerebral palsy and its comorbidities provides a good framework with which to consider the challenges encountered in the rehabilitation (or habilitation) of these children. It is not possible here to cover every aspect of multidisciplinary care of rehabilitation medicine, instead this chapter aims to discuss recent concepts and advances in pediatric neurorehabilitation and highlight those areas that are frequently encountered by the pediatrician, neurologist or rehabilitation specialist when caring for a child with the complex medical needs of cerebral palsy. Key topics will include the pathophysiology and management of hypertonia and motor impairments, oromotor, cognitive and behavioral problems. Cerebral palsy describes a group of disorders of the development of movement and posture, causing activity limitation, that are attributed to nonprogressive disturbances that occurred in the developing fetal or infant brain. The motor disorders of cerebral palsy are often accompanied by disturbances of sensation, cognition, communication, perception and/or behavior, and/or by a seizure disorder (Bax et al., 2005). Although the clinical manifestations evolve as the child matures the underlying lesion is nonprogressive. Cerebral palsy is one of the most common developmental disabilities with an estimated prevalence of 2.0–2.5/1000 children with very little change over the last few decades (Nelson, 2003). The etiology of cerebral palsy is varied: the most common causes include preterm encephalopa-

thy (including periventricular leukomalacia), term neonatal encephalopathy (including birth asphyxia), teratogen exposure and cerebral malformations. A new classification system of cerebral palsy has been proposed to allow accurate description of the individual, predictions of current and future service needs, comparisons between groups and evaluation of change (Table 32.1) (Bax et al., 2005).

32.2. International classification of function, health, and disability The way we think about health and disease determines to a considerable extent what we do and say in our clinical encounters with patients. Two children with the same neurological lesion, e.g. unilateral schizencephaly leading to a right hemiparesis, may experience very different disabilities because of the intrinsic characteristics of the individual and extrinsic environmental factors. In 2001 the WHO endorsed a new health classification scheme, the International Classification of Functioning, Disability and Health (ICF) (Rosenbaum and Stewart, 2004). This complements their classification of diseases (ICD-10), but the ICF model provides a broad perspective within which to appreciate the spectrum of functioning and disability across a lifespan (Table 32.2). It seeks to classify abnormalities at two levels: 1) body structures (anatomical) and body functions (physiological and psychological), and 2) limitations in everyday activities and restrictions on participation in life situations and roles that interfere with functioning and health. Consider the case of a 12-year-old girl with a cerebral malformation, such as schizencephaly (body structure), who has a resultant right hemiparesis and epilepsy (body functions). She has difficulty in climbing stairs at school

*Correspondence to: Dr Karen Maria Barlow, Assistant Professor of Pediatric Neurology, University of Calgary, Alberta Children’s Hospital, Calgary, Alberta T2T 5C7, Canada. E-mail: [email protected], Tel: þ001-403-9437728, Fax: þ001-403-943-7609.

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Table 32.1 Components of cerebral palsy classification 1. Motor abnormalities A. Nature and typology of the motor disorder: the observed tonal abnormalities assessed on examination (e.g. hypertonia or hypotonia) as well as the diagnosed movement disorders present, such as spasticity, ataxia, dystonia or athetosis B. Functional motor abilities: the extent to which the individual is limited in his or her motor function in all body areas, including oromotor and speech function 2. Associated impairments The presence or absence of associated nonmotor neurodevelopmental or sensory problems, such as seizures, hearing or vision impairments, or attentional, behavioural, communicative and/or cognitive deficits, and the extent to which impairments interact in individuals with cerebral palsy 3. Anatomic and radiological findings A. Anatomic distribution: the parts of the body (such as limbs, trunk, or bulbar region) affected by motor impairments or limitations B. Radiological findings: the neuroanatomical findings on computed tomography or magnetic resonance imaging, such as ventricular enlargement, white matter loss or brain anomaly 4. Causation and timing Whether there is a clearly identified cause, as is usually the case with postnatal cerebral palsy (e.g. meningitis or head injury) or when brain malformations are present, and the presumed time frame during which the injury occurred, if known

Table 32.2 International classification of functioning, disability and health (ICF)

functioning or disability. This conceptual model therefore heightens our understanding of the interaction between the individual and the environment and the dynamic nature of disability (King et al., 2004; Rosenbaum and Stewart, 2004) (Fig. 32.1). The ICF model reflects the growing understanding of the role of the family in a child’s life and the importance of the insights of the parents and caregivers into their child’s abilities and needs. Although not new, over the past 10–15 years there has been an increasing trend for hospital, clinics and community-based services to adopt the practices of family-centred care.

32.3. Family-centred care Children with cerebral palsy often have complex and long-term needs that are best addressed using the model of family-centred care. It is considered best-practice in early intervention and pediatric rehabilitation and reflects three basics premises: that 1) parents know their children best and want the best for their children, 2) families are unique and different and 3) optimal child functioning occurs within a supportive family and community context (King et al., 2004). Family-centered service has been defined as being: made up of a set of values, attitudes, and approaches to services for children with special needs and their families. Family-centered service recognizes that each family is unique; that the family is the constant in the child’s life; and that they are the experts on the child’s abilities and needs. The family works together with service providers to make informed decisions about the services and supports the child and family receives. In family-centered service, the strengths and needs of all family members are considered.

Part I: Functioning and disability

(King et al., 2004; Lammi and Law, 2003; Law et al., 2003.)

Body structures and body functions (organ system level) Activity (individual level) and participation (societal level)

32.3.1. Evidence for family-centred care approach

Part II: Contextual factors Personal factors (intrinsic) Environmental factors (extrinsic)

(activity), does not participate in sports and has few friends (societal). She is shy but enjoys reading (intrinsic personality traits), is from a single-parent family and her classroom is on the second floor (extrinsic environmental factors). The ICF recognizes the ‘contextual factors’ that may impede or facilitate her level of

Several randomized controlled trials have shown improvement in children’s motor and developmental progress through using family centred approaches in educational and treatment programs. These studies used approaches that included involving parents in identifying goals and needs, fitting the therapy to these needs and priorities, involving the whole family and having a strong educational component (MoxleyHaegert and Serbin, 1983; Stein and Jessop, 1984, 1991). Improved child psychological adjustments were also seen in comprehensive programs where families were encouraged to take responsibility for managing

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Child characteristics relating to primary impairment Child characteristics relating to secondary impairments

Inherent child personality characteristics

Changes in basic motor abilities

Activity

Family ecology

Participation Health care services

Fig. 32.1. Multivariate model of determinants of motor change for children with cerebral palsy. Solid lines and oval are part of the proposed model; dotted lines and oval represent parts of an expanded model.

their child’s care, making informed decisions in partnership with service providers, and were given education and support (Stein and Jessop, 1984, 1991; Pless et al., 1994). These gains were still evident at least 4 years later (Stein and Jessop, 1991). Mothers have been shown to have reduced levels of anxiety and depression and higher levels of wellbeing in randomized controlled trials of programs focusing on informational, emotional and affirmational support. These programs emphasized parent–professional collaboration and a responsiveness to family needs (Stein and Jessop, 1984; Marcenko and Smith, 1992; Van Riper, 1999; Ireys et al., 2001). Other studies have shown increased satisfaction with service delivery when the model was changed to a family-centred approach (DeChillo et al., 1994; Stallard and Hutchison, 1995). King et al. (2004) provide a comprehensive review of the literature regarding the evidence for family-centred services. It is recommended that professionals involved in the care of children with complex needs examine their own beliefs and practices and those of the recipients of care using one of several measures available for this (King et al., 2003, 2004). In order to evaluate disease, treatment plans, service provision and the many other aspects of the care of a child with complex needs, objective outcome measures are needed. Measures used in pediatric clinical settings must be relevant to client goals and intervention effect and must possess good psychometric properties (Dietz, 1989).

32.4. Assessment of outcome There are many factors of interest when determining holistic outcome, including function, disability, individual activities and the child’s participation in society. Many factors act as barriers or facilitators, including the biological characteristics of the disease in question and the child’s personality traits (intrinsic variables) as well as external variables such as school environment, family dynamics and peer relationships (Majnemer and Mazer, 2004). A multivariate model of determinants of outcome for children with cerebral palsy has been proposed by Bartlett and Palisano (2000) and builds on the dynamic nature of disability delineated by the ICF framework and important role of family ecology. Undoubtedly other physical, social and attitudinal factors within the environment will be evaluated in future as determinants of outcome with cerebral palsy. This is important when we consider the changes that occur when adolescents make the transition to adulthood and the emphasis on autonomy increases. For instance, some studies indicate an alarming trend toward deterioration in physical, social and emotional wellbeing with increasing age. This occurs at a time when coordinated, comprehensive health services diminish for adults with cerebral palsy (Andersson and Mattsson, 2001; Bottos et al., 2001; Stevenson et al., 1997). The last decade has seen the development of tools designed to quantify various domains of outcome,

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including the Gross Motor Function Measure (GMFM; Russell et al., 1989), the Pediatric Evaluation of Disability Inventory (PEDI; Haley et al., 1992, 2005), the Functional Independence Measure for Children (WeeFIMW) (UDSMR, 1993) and others (Pencharz et al., 2001). The use of standardized measures serves to increase the accuracy of the assessment results, providing outcomes that are more reliable or reproducible. 32.4.1. Gross motor function measure The Gross Motor Function Classification System (GMFCS) is based on differences in functional abilities and limitations in cerebral palsy. There are five levels, which vary from level I (walks without restrictions) to level V (self-mobility is severely restricted). The gross motor function measure GMFM-66 is a measure of gross motor function that was designed and validated to assess changes over time (Rosenbaum et al., 2002). The gross motor development curves in Fig. 32.2 represent the average pattern of development for each classification level. The vertical dotted lines represent

the age at which 90% of children at a given level will have developed 90% of their predicted gross motor abilities. It is hoped that these curves will assist health-care professionals and therapists to identify 1) outcomes that are consistent with a child’s potential and 2) the extent to which interventions improve gross motor function beyond expectations. Therapies to improve independence in walking are warranted for children with classification levels I and II; these are not useful for children in levels III, IV and V, where therapy should focus on powered mobility. These predictions are only valid where function has been assessed using the GMFM-66. The Pediatric Evaluation of Disability Inventory (PEDI) was developed to provide a comprehensive clinical assessment of key functional capabilities and performance in children between the ages of 6 months and 7 years (Haley et al., 1992). The PEDI was designed primarily for the functional evaluation of young children; however, it can also be used for the evaluation of older children if their functional abilities fall below that expected of 7-year-old children without

GMFCS Level I to V 100 90 Level I 80

D

70 GMFM-66

Level II 60 C Level III

50 40 30

Level IV B

20

Level V A

10 0 0

1

2

3

4

5

6

7 8 9 10 11 12 13 14 15 Age (yrs.)

Fig 32.2. Predicted average development of gross motor ability by GMFM classification system category (levels I–V). Vertical dashed lines indicate age 90, the age in years at which children are expected to achieve 90% of their potential for motor development. Diamonds on the vertical axis marked A–D indicate the GMFM-66 score at which children are expected to have 50% chance of successfully completing selected gross motor test items: A (item 21): Therapist holds child sitting upright; child lifts head for 3 seconds. B (item 24): Child maintains sitting, arms free, for 3 seconds. C (item 69): Child walks forward 10 steps. D (item 87): Child walks down four steps, alternating feet. (Reproduced from Rosenbaum et al., 2002, # 2002, American Medical Association, with permission.)

NEUROREHABILITATION OF CHILDREN WITH CEREBRAL PALSY disabilities. The assessment was designed to serve as a descriptive measure of the child’s current functional performance as well as a method for tracking change across time. The PEDI measures both capability and performance of functional activities in three content domains: 1) self-care, 2) mobility and 3) social function. A similar instrument is the WeeFIMW, which measures similar constructs (Ziviani et al., 2001) in children between the ages of 6 months and 7 years). The WeeFIMW instrument may be also used with children above the age of 7 years. The WeeFIMW instrument is a minimal data set of 18 items that measures functional performance in three domains: self-care; mobility and cognitive. The Child Health Questionnaire is a generic instrument for measuring health outcomes in children and assessing functional status and well-being (Langraf et al., 1996). It has shown reliability and validity (Waters et al., 2000) and has been used to evaluate health related quality of life (HRQOL) in many childhood diseases. The original, full-length CHQ includes 98 items and is designed for completion by the child’s parent. Various short-form versions have been developed; CHQ-PF50 is the most tested and most widely used version. It has been used to assess HRQOL in cerebral palsy, where children with cerebral palsy were demonstrated to have deficits in HRQOL that were related to the severity of cerebral palsy (assessed by the GMFCS) (Vargus-Adams, 2005). Psychosocial HRQOL was better than physical HRQOL and many of the issues related to physical disabilities and parental impact. Finally, another measure of children’s functional health status, Health Utilities Index – Mark 3 (HUI-3), has been used in cerebral palsy (Feeny et al., 1992; Furlong et al., 2001; Kennes et al., 2002). This is a brief but comprehensive system for describing the health status and function (capacity) of individuals and assesses eight domains of function (health status) believed to contribute to quality of life: ambulation, dexterity, speech, vision, hearing, cognition, emotion, and pain. Each domain has five or six levels representing functional classes within that domain. These functional categories are ordered from normal function (not impaired, or a ranking of 1) to severely impaired (a ranking of 5 or 6, depending on the domain). Using the HUI-3 in a population-based study, parents assessed their children with cerebral palsy as having difficulty with ambulation (70%), dexterity (37%), speech (40%), vision (32%), hearing (3%), cognition (57%), emotion (10%) and pain (14%) (Kennes et al., 2002). This study highlights the comorbidities present in cerebral palsy and will be discussed later.

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32.5. Motor impairment Cerebral palsy is an upper motor neuron syndrome characterized by a disorder in supraspinal regulation of intensity, timing and sequencing of motor unit activation (Bourbonnais et al., 1991; Leonard, 1994). Motor impairments are a result of the primary underlying neurological deficits but also secondary impairments due to combinations of spasticity, dystonia, muscle contractures, weakness, motor control abilities, physical growth, abnormal biomechanical forces, bony deformities and changes in the physical and social environment. Although the cerebral malformation resulting in cerebral palsy is nonprogressive, secondary impairments change over time (Leonard and Goldberger, 1987). 32.5.1. Pathophysiology Decreased central voluntary activation and resultant changes in spinal motor neuron activity lead (at least in part) to abnormal patterns of motor unit recruitment with resultant decreased mobility of the affected limb (Rose and McGill, 2005). The paretic muscles are frequently immobilized in a shortened position; for example, the upper limb flexors, pronators and wrist flexors are shortened in spastic hemiparesis. These muscles have decreased longitudinal tension, i.e. they are unloaded. This leads to secondary changes including atrophy (decreased muscle mass) and decreased sarcomeres (shortening) (Gracies, 2005a, 2005b). Varying degrees of fiber type I and II atrophy and hypertrophy are found in the muscles of children with spastic cerebral palsy, together with deposits of fat and connective tissue. The decrease in number of type II fibers has been correlated with decreased power, although it is important to appreciate that decreased power and increased fatigability is also secondary to abnormal supraspinal and spinal motor activation (Rose et al., 1994; Rose and McGill, 2005). The pathological changes are associated with soft tissue contraction, which further limits range of motion at a joint and is an important factor in deformity and resistance to passive movement. In addition to the decreased central voluntary ability to synchronize motor unit activation and generate force, some muscles also demonstrate overactivity. The spindles in stiff muscles show increased sensitivity to stretch stimulation and a resultant decrease in the muscle’s ability to relax limiting the range of motion at a joint and a further propensity for contracture (Gracies, 2005b). The onset of motor unit activation is slow and recruitment is prolonged, contributing to reduced speed of movement and low levels of force production. The slow onset and prolonged duration of motor unit

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recruitment may also increase muscle cocontraction and thus limits speed and stride length during walking. As a result children with cerebral palsy expend excessive energy to overcome body mass and inertial forces. High energy cost and muscular fatigue limit endurance. 32.5.1.1. Spasticity Spasticity can be defined as hypertonia in which one or both of the following signs are present: 1) resistance to passive lengthening increases with increasing speed of stretch and varies with the direction of joint movement; 2) resistance to externally imposed movement rises rapidly above a threshold speed or joint angle (Sanger et al., 2003). This symptom reflects abnormal spinal a-motor neuron activity, which may be due to excessive excitation or lack of inhibition at central and even spinal levels. In a child with cerebral palsy, it is often associated with other symptoms including spasms, dystonia and cocontraction of muscles associated with voluntary movements. Spasticity can vary depending on the child’s state of alertness, activity or posture. It is influenced by anxiety, pain and surface contact, or other non-noxious sensory input. Spasticity is usually evident by the first year of life but changes with time; interestingly, it may resolve or improve in half the children with spastic diplegic cerebral palsy (Nelson and Ellenberg, 1982). The natural history of children with cerebral palsy secondary to a cerebral malformation is not known. 32.5.1.2. Dystonia Dystonia is often seen in children with cerebral palsy, frequently in association with spasticity. When spasticity and dystonia are seen together they were previously referred to as spastic dystonia, although current classifications and definitions discourage this terminology (Sanger et al., 2003). Dystonia is an involuntary alteration in the pattern of muscle activation during voluntary movement or maintenance of posture. It is a movement disorder in which involuntary sustained or intermittent muscle contractions cause twisting and repetitive movements, abnormal postures or both and is often diagnosed by the observation of abnormal twisted postures (Sanger et al., 2003). There is resistance at the joint to movement at all speeds (even very slow ones), i.e. dystonia is not velocity-dependent and simultaneous cocontraction of agonists and antagonists may occur. Dystonia is commonly triggered or exacerbated by attempted voluntary movement and may fluctuate in presence and severity over time. The severity and quality of dystonic postures may vary with body position, specific tasks, emotional state or level of consciousness. The roles of spasticity, dystonia and ‘lead-pipe’ rigidity have a complex relationship in cerebral palsy,

are sometimes difficult to determine and frequently change with time. Like spasticity and dystonia, rigidity may depend on the state of the child. Unlike dystonia, rigidity is not specific to particular tasks or postures. Children with cerebral palsy have reduced or impaired anticipatory postural control. When we reach forward for an object our centre of gravity also moves. Our body makes synchronous postural adjustments for this (generated within the central nervous system) to prepare for the shift of centre of gravity. Children with cerebral palsy prepare well in advance and the change in postural control and the prime movement may not be synchronized, resulting in poor coordination. Postural adjustment can be practiced as part of goaldirected movement, learning strategies for weight shifting and varying task requirements and environmental conditions are strategies that may be effective in improving anticipatory postural adjustment. 32.5.1.3. Evaluation of motor impairment Thorough evaluation requires the input of the multidisciplinary management team, which includes physicians, surgeons, physical and occupational therapists, nurses and caregivers. Motor impairment in cerebral palsy can be classified by affected body region, such as spastic diplegia, hemiparesis and tetraparesis, but it is more important to document the specific pattern in the individual patient (e.g. scissoring thighs, adducted shoulder), the degree of severity and the patient’s function, using object measures such as the GMFM (Bax et al., 2005; Rosenbaum et al. 2002; Tilton, 2004). The treatment program begins with a careful evaluation to determine whether muscle overactivity is interfering with some aspect of function, comfort, or care. The presence of hypertonicity alone is not considered sufficient to warrant its treatment. Sometimes a patient’s spasticity is aiding function and altering tone could be counterproductive, e.g. where lower extremity stiffness improves transfer ability in the presence of leg muscle weakness. It is essential to have goals that are to be achieved through the reduction of hypertonicity. Also, one must be sure that there are no other factors increasing spasticity that can be eliminated, such as urinary tract infection, pressure sores, fracture, dislocation or nutritional status. For many children, however, reducing spasticity, especially when combined with increasing muscle strength and appropriate orthotics, can provide overall functional benefit. The modified Ashworth Scale is the most commonly used measurement of spasticity in clinical practice, where tone is graded 0 (normal tone) to 4 (rigid in extension or flexion) (Table 32.3). It has good inter-rater reliability. Although it is a subjective measure, more

NEUROREHABILITATION OF CHILDREN WITH CEREBRAL PALSY Table 32.3 The modified Ashworth scale 0 1

1þ

2 3 4

No increase in muscle tone Slight increase in muscle tone, manifested by a catch and release or minimal resistance at the end of the range of movement (ROM) when the affected part is moved in flexion or extension. Slight increase in muscle tone, manifested by a catch followed by minimal resistance throughout the remainder (less than half) of the ROM. More marked increase in muscle tone through most of the ROM, but affected part easily moved. Considerable increase in muscle tone, passive movement difficult. Affected parts rigid in flexion and extension.

objective measures are rarely used and have been systematically reviewed by Wood and colleagues (Engl et al., 2004; Wood et al., 2005). The modified Ashworth Scale can also be used in all forms of hypertonia and does not differentiate between spasticity, dystonia and rigidity. More specific measures of dystonia and rigidity include the Barry–Albright dystonia scale and the Burke–Fahn–Marsden dystonia rating scale (Barry et al., 1999; Comella et al., 2003). In addition to the evaluation of type and severity of hypertonia, range of motion at specific joints, presence of contractions and hip subluxation, it is equally important to measure the degree of function and disability.

32.6. Management of hypertonia In the broadest terms, the goals of any hypertonicity treatment plan are to maximize active function, ease care and prevent secondary problems such as pain, subluxation and contracture. Treatment plans should be drawn up with active participation of the caregivers, as described above, should be concise and explicit should and include short- and long-term goals that are tailored to each child’s unique needs. The treatment approaches are many and include stretching, physical therapy, orthotics, botulinum toxin, oral medications, injections, intrathecal baclofen and orthopedic surgery (Tilton and Maria, 2001). The choice of treatment is influenced by several factors:

The distribution of spasticity, which may be primarily focal or generalized

Comorbidity, e.g. seizures Age of the patient Which options have been tried in the past

597

Dosing frequency, formulation and delivery Side effect profile Prior reaction to a treatment Cost Compliance with the treatment and follow-up.

Several broad parameters can help to shape decision making. Orthopedic surgery should be delayed until the child’s gait is mature. In the interim period, stretching, physical therapy and orthotics are used to maintain range of motion. Botulinum toxin provides selective weakness of the muscle groups injected and can be used as a single modality for focal spasticity or in conjunction with management for generalized spasticity but to help with specific areas. Oral medication has a relatively nonselective action seen in all muscle groups and is frequently associated with cognitive side effects. It is helpful if the child has painful spasms, severe generalized hypertonia, decreased sleep, seizures or dystonia. When the gait is mature, gait analysis and clinical examination can be used to determine the need for surgery. To avoid multiple surgeries and periods of immobility, all necessary surgery should be done concurrently, if feasible, and the child should be remobilized as soon as possible. Continued stretching and medical treatment are applied as needed to maintain range of motion and mobility. 32.6.1. Physical therapy and orthotics The main aims of physiotherapy include avoidance of contractures, improvement of strength, improvement of body perception, avoidance of interference with involuntary movement processes, encouragement of sensible voluntary movement processes and furthering of movement transitions, individual activities and social participation. Physical therapy for the child with cerebral palsy may encompass a regular exercise program, hydrotherapy, constraint therapy, horseback riding and various modalities, including cold and heat application, biofeedback and electrical stimulation. It is beyond the scope of this chapter to review each of these approaches in detail. The importance of regular stretching to maintain full range of motion and preventing contracture cannot be overstated. Children want to move and much of the need for range of motion exercises can be met by the daily activities of an active child. If a child deals with impairments by using strategies that minimize movement of the affected joint, however, the potential for contracture is increased. It is for this reason that regular stretching of all affected limbs is generally prescribed. Stretching can reduce severity of tone for several hours, providing a short-term, but not long-term, antispasticity action.

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The intensity of physical therapy needed to maximize gains has been the subject of several studies, with varying results. Bower et al. (2001) compared the effects of the usual amount of physical therapy to intensive therapy (1 hour per day, 5 days per week) in children aged 3–12 and found no significant difference. Intermittent intensive physical therapy has recently been suggested as a promising alternative (Trahan and Malouin, 2002). Strength training programs have been shown to increase lower limb strength, which was associated with improvements in GMFM scores for standing, running and jumping, and faster stair climbing (Dodd et al., 2002). Children with cerebral palsy have impaired postural control due to multiple factors: musculoskeletal problems (including contractures and reduced range of motion) and shifts in initial alignment, which affect reactive balance control (Nashner et al., 1983). Other motor components include the disruption of the spatial and temporal aspects of postural muscle responses during the recovery of stability following an unexpected external perturbation (Roncesvalles et al., 2002). Balance training has been shown to improve stability in children with cerebral palsy (Dodd et al., 2002; Shumway-Cook et al., 2003). Orthoses are designed to provide joint stability, to hold a joint in a functional position and to keep tight muscles stretched. The commonest orthotic is the ankle–foot orthosis (AFO) which is used to treat dynamic equinus. AFOs reduce ankle excursion and increase dorsiflexion angle at foot strike and have other benefits, although neither stride length nor walking speed are improved (Carlson et al., 1997). AFOs can improve the sit-to-stand transition in preambulatory children whose standing is impaired by equinus (Wilson et al., 1997). It is now common to reduce local spasticity with botulinum toxin injection or orthopedic surgery and then fit an AFO. 32.6.2. Botulinum toxin Botulinum toxin is an exotoxin produced by Clostridium botulinum, the organism also responsible for botulism. There are seven naturally occurring serotypes of the toxin, A–G, all of which are zinc proteases that target the synaptic vesicle fusion machinery at the neuromuscular junction. Denervation occurs because vesicles cannot fuse with the synaptic membrane and thus acetylcholine cannot be released. The seven serotypes differ in the specific component of the fusion machinery that they target, in their duration of action, in their unit potency and in their immunogenic potential. Two serotypes (A and B) are commercially available. Although spasticity management is not a current

Food and Drug Administration (FDA)-labeled indication in the USA for botulinum toxin, it is a common off-label application. At effective antispasticity doses, both types have roughly the same duration of clinical action, approximately 3 months. Potency is expressed in terms of mouse units, the amount of toxin required to kill 50% of mice in a standardized assay. Because of differences in molecular formulation and other variables, the potency of a single unit varies greatly among the commercial types and it is critical to specify the commercial brand when discussing units for dosing recommendations. The onset of weakness related to botulinum toxin chemodenervation typically begins within 14 days of injection. The duration of effect is usually 1–6 months, with an average of 3 months. Because axonal innervation of the neuromuscular junction is eventually re-established, multiple injection sessions, usually separated by at least 12 weeks, may be needed. Earlier reinjection is discouraged because of the increased risk of developing neutralizing antibodies that would render further treatments ineffective. The incidence of adverse effects reported is low and the effects are usually transient. These include injection-related local findings (e.g. pain and swelling), local weakness with increased falling, generalized weakness or lethargy, and incontinence (Pidcock, 2004). In comparison with drug therapy and intrathecal baclofen, botulinum toxin is injected into specific muscles. The clinical goals when treating a child with botulinum toxin include: improvement of function, prevention or treatment of secondary musculoskeletal complications, increasing comfort and facilitating care. Hip adductors, gastrocnemius and biceps are the most frequent muscles to be injected. The effectiveness of botulinum toxin type A in the management of cerebral palsy has been verified by objective measurement in several studies. The main effects are in reducing local spasticity, improvements in dynamic gait and ankle kinematics and it helps to delay surgical intervention until the child is older (Koman et al., 1994). In the upper limb the effect is more modest; the decrease in spasticity is not necessarily correlated with functional improvement (Corry et al., 1997; Edgar, 2001). Practitioners and caregivers usually agree that the benefits they see from botulinum toxin injection are greater than those revealed in clinical trials. The injection technique for botulinum toxin type A is similar to that used for local anesthetics. The area to be injected is prepared with an alcohol or povidone– iodine solution. Sterile gloves and a ‘no-touch’ skin entry site technique are used. A 1 ml tuberculin syringe and a 27-gauge, 1.5 in (38 mm) needle is typically used (Edgar, 2001). Correct placement in muscles other

NEUROREHABILITATION OF CHILDREN WITH CEREBRAL PALSY than the gastrocnemius can be ensured by muscle activation, ultrasound or electromyographic control (Chin et al., 2005). The use of local anesthetic cream, ethylene chloride or conscious sedation may be employed with or without midazolam (0.5 mg/kg orally with a maximum dose of 15 mg) given 30 minutes prior to the procedure. 32.6.3. Drug therapy 32.6.3.1. Benzodiazepines g-amino butyric acid (GABA) and glycine are the main inhibitory neurotransmitters in the CNS, typically used by small interneurons to mediate presynaptic inhibition in the spinal cord. However, they are also ubiquitous in inhibitory synapses of the brain stem, cerebellum, basal ganglia and cerebral cortex. Benzodiazepines have an inhibitory effect at both the spinal cord and supraspinal levels. Their effect is mediated by GABA, specifically GABAA, and results in presynaptic inhibition and a reduction of mono- and polysynaptic reflexes. They bind in both the reticular formation and spinal polysynaptic pathways. Different benzodiazepines have different lengths of action due to both the rates of metabolism and metabolically active metabolites. Diazepam is commonly used in the treatment of spasticity. It has a long duration of action. It is rapidly absorbed, reaching peak drug levels an hour after oral administration. It is metabolized in the liver to compounds that are active, including N-desmethyldiazepam and oxazepam. Diazepam is highly protein-bound and both it and its metabolites have a half-life of 20–80 hours. Sedation is the commonest side effect and therapy should be initiated slowly. It can reduce motor coordination and impair attention and memory. Intoxication can be provoked if diazepam is combined with alcohol and care must be taken if using drugs that inhibit its metabolism, e.g. cimetidine. Parents should be warned about the danger of acute withdrawal symptoms if the drug is stopped abruptly. Pediatric doses range from 0.12–0.8 mg/kg per day in divided doses (Gracies et al., 1997). Diazepam as an antispasticity medication is often used at night to suppress spasms that disturb sleep (Mathew and Mathew, 2005). Clonazepam is more frequently used as an antiepileptic medication to control myoclonic, atonic and absence seizures. Where epilepsy and hypertonia coexist, clonazepam would be a reasonable choice. It has also been used for dystonia. Less frequently, clonazepam has been used for spasticity, particularly where dystonia is also present and where night-time spasms are frequent. Sedative and behavioral side effects limit its use and where it is used it is commonly prescribed at night.

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32.6.3.2. Baclofen Baclofen is a selective GABAB agonist. GABAB receptors are located in laminae I–IV of the spinal cord, where primary sensory fibers end. GABAB receptor activation results in a presynaptic inhibition of the monosynaptic extensor and polysynaptic flexor reflex activity. There is wide intersubject variation in baclofen absorption and elimination but on average it is rapidly and extensively absorbed after oral administration. Oral use is limited because of poor blood–brain barrier penetration and a short (3–4 h) half-life. Baclofen is excreted mainly by the kidney in unchanged form, although 15% is metabolized in the liver. Therefore, the dose should be reduced in patients with impaired renal function and liver function parameters should be monitored periodically during treatment. Unfortunately, there is little evidence that baclofen increases function in children with cerebral palsy. In one double-blind crossover trial it was demonstrated that baclofen reduced spasticity significantly better than placebo and improved passive and active movement (Milla and Jackson, 1977). A small uncontrolled study demonstrated that it was well tolerated in children but there was no objective evidence of improvement in motor function and a trend towards impairment of motor progression. However, parents did report that it was easier to care for their child (Vargus-Adams et al., 2004). The dose should be titrated slowly. Frequent and high doses often produce somnolence or sometimes other untoward side effects before efficacious CNS levels are achieved. A typical starting dose is 2.5 mg/day, which can be titrated up gradually to a maximum of 20–60 mg/day (Gracies et al., 1997). Baclofen must be weaned gradually to avoid a withdrawal syndrome. While oral baclofen is effective in some children, other delivery systems have been investigated. Intrathecal administration of baclofen is discussed below. 32.6.3.3. Dantrolene Unlike the previous medications, dantrolene sodium does not exert its antispasticity effects through the central nervous system. It acts on skeletal muscle to inhibit the release of calcium at the sarcoplasmic reticulum and so uncouples the electrical excitation from contraction (Gracies et al., 1997). Its effects thus produce muscle weakness that is greater in fast-contracting muscles. The majority of placebo-controlled trials of dantrolene have shown a reduction of muscle tone, tendon reflexes and clonus, and an increase in range of passive motion (Gracies et al., 1997). As in other treatments of hypertonicity, the proportion of patients who demonstrated improvement in activities of daily living was usually much smaller than the proportion who had reduced

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clonus or improved ‘overall clinical response’. In children with cerebral palsy, dantrolene sodium was superior to placebo in four trials (Haslam et al., 1974). The degree of improvement appeared greater in children than in adults (Nogen, 1976). Dantrolene sodium is well absorbed, with peak blood levels 3–6 hours after ingestion. It is metabolized in the liver and eliminated in the bile and urine. Its half-life after oral administration is 15 hours. Doses in children range up to 12 mg/kg/day (Whyte and Robinson, 1990). Treatment is generally started at a low dose and titrated upward every 5–7 days as tolerated. Dantrolene is mildly sedating and may cause malaise, nausea, vomiting, dizziness, diarrhea and paresthesia. In comparison to baclofen or diazepam, it is less likely to cause lethargy or cognitive disturbances. Although dantrolene can weaken muscles (including respiratory muscles), the effects on spasticity are not generally accompanied by significant impairment of motor performance, except in patients with marginal strength. Dantrolene has also been associated with hepatotoxicity. In a large group of patients receiving dantrolene sodium for more than 2 months, the overall incidence of hepatotoxicity was 1.8%, symptomatic hepatitis occurred in 0.6% and fatal hepatitis in 0.3%, with the greatest risk in females older than 30 years of age taking more than 300 mg per day for more than 60 days, and in patients taking other medications simultaneously, especially those metabolized in the liver (Ward et al., 1986). Reversible enzyme elevation has been reported in children taking dantrolene. Liver function studies should be done prior to instituting treatment with dantrolene and periodically while on it. I generally avoid using dantrolene concurrently with valproate or where the patient has a possibility of an inborn error of metabolism. 32.6.3.4. Tizanidine Tizanidine is an a2-adrenergic receptor agonist on sites both spinally and supraspinally. It prevents the release of excitatory amino acids from the presynaptic terminal of spinal interneurons and it may facilitate the action of glycine, an inhibitory neurotransmitter. One Russian study in 30 diplegic children receiving tizanidine 6 mg/day reported improved motor ability, which was confirmed with electroneuromyography (Maksimov et al., 1999). Side effects were reportedly well tolerated. To date, no clinical trials of this agent in children have been published in the English-language literature. Common side effects include dry mouth and sedation. Other side effects include visual hallucinations and elevated liver function tests.

32.6.3.5. Intrathecal baclofen In the 1980s, reliable drug delivery systems became available for intrathecal infusions of drugs such as morphine, methotrexate and baclofen. As noted previously, baclofen acts by binding with GABAB receptors in the spinal cord and therefore it can be given intrathecally, often with a greater reduction in tone than when given orally. Intrathecal baclofen therapy can be considered for children where oral antihypertonicity medications have failed to give satisfactory relief, and its use in cerebral palsy has recently been reviewed (Boop, 2001; Krach, 2001). The dose varies from 50–1000 mg/24 h, much lower than oral doses and with no detectable blood level (Vloeberghs et al., 2005). The concentration of drug in the cisternal area is one-quarter the concentration that is present in the lumbar area when the infusion is in the lumbar area. The half-life of intrathecal baclofen in the cerebrospinal fluid is 5 hours. The primary advantages of delivering baclofen intrathecally include: it is associated with a low incidence of cerebral side effects; its effects are completely reversible; and it can be titrated to effect using a programmable magnetic wand. For example, if a person needs a bit of hypertonicity to facilitate ambulation during the daytime, the morning dose can be decreased. If they need less tone in order to be able to sleep better at night, the physician can program in a bolus before bedtime to help the patient relax through the night. The primary disadvantage of intrathecal baclofen is that it requires implantation of the foreign body and catheter. Over time, the pump may become infected and have to be removed, and the catheter may slip out of place, break or come loose from the pump and have to be reimplanted. Furthermore, the patient must return for refills every 2–3 months, which requires a percutaneous injection into the reservoir. Around 20% of patients have required revision or removal of their pumps because of complications (Vloeberghs et al., 2005). Even those without complications require replacement of their pump every 7–10 years as the batteries run down, which is at least a $10 000 expense. There have also been reports of drug overdose leading to respiratory arrest and the requirement for ventilatory and intensive care support until the drug effects wear off. This is almost always a consequence of programming error rather than pump failure. At the moment, there are only two absolute contraindications to intrathecal baclofen therapy: patients who have had an adverse reaction to the drug and patients who have no reliable access to or financial support for refilling the pump. A meta-analysis of a number of studies has shown that intrathecal baclofen is effective in tone reduction in spasticity of both cerebral and spinal origin (Creedon

NEUROREHABILITATION OF CHILDREN WITH CEREBRAL PALSY et al., 1997). Its effect on reducing spasticity in cerebral palsy has been reported in several studies (Albright, 1996; Gilmartin et al. 2000; Krach et al., 2004). It may decrease the progression of hip subluxation (Krach et al., 2004). Krach et al. (2005) demonstrated improvement in GMFM after 1 year of intrathecal baclofen therapy in those less than 18 years of age. However, the functional benefits of this treatment need to be investigated further and is part of further ongoing research (Vloeberghs et al., 2005). 32.6.4. Selective dorsal rhizotomy Selective dorsal rhizotomy has been used to reduce spasticity and improve function in ambulant children with spastic diplegia and to ease the care of children with spastic quadriplegia. The rationale of the treatment is consistent with neurophysiological evidence that spasticity is the result of decreased inhibition from upper motor neuron corticospinal tracts and interneuron inputs. Between 25% and 50% of dorsal rootlets are usually cut and, despite fairly sophisticated detection and monitoring in experienced hands, some uncertainty still exists about the ideal number to sever. Postoperatively, intensive physiotherapy is needed, despite positive changes in spasticity. Steinbok (2001) has examined this topic in an evidence-based review. A meta-analysis of three prospective randomized trials that have attempted to determine whether selective dorsal rhizotomy offers sustained relief of spasticity has also shown significant benefit in a select subgroup of spastic diplegic children (McLaughlin et al., 2002). Still, controversy remains as to whether selective dorsal rhizotomy is better than other surgical options such as intrathecal baclofen or orthopedic procedures. This is particularly true for children without ambulatory potential. 32.6.5. Orthopedic surgery The selection, timing and procedural use of orthopedic surgery have changed significantly in recent years. In the lower limb there is a definite trend to avoid, or at least to delay, surgery until a child’s gait has matured in late childhood, and towards single-event multilevel surgery (SEMLS) (Hodgkinson et al., 1999; Flett, 2003). An aggressive use of botulinum toxin-A in conjunction with physiotherapy, casting and orthotics has shifted surgical intervention in many cases to mid to late childhood. At that stage, gait analysis can be employed to help develop the surgical prescription (Gage and Novacheck, 2001; Schwartz et al., 2004). There have always been good reasons to avoid surgery in some early childhood situations, such as calf-length-

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ening procedures in spastic diplegia (responsible for iatrogenic crouch gait) and in hemiparesis (overlengthening or underlengthening). It is important to appreciate that after SEMLS it can take some children several months to regain their former mobility and up to a year to make satisfactory gains, during which time they require intensive therapy resources (Hodgkinson et al., 1999). Orthopedic surgery can correct muscle contractures and associated imbalance, but also bony deformities. Abnormal muscle tone and abnormal postural support place considerable stresses on growing bones, typically leading to femoral anteversion, coxa valga and tibial torsional deformity as well as valgus or equinovarus foot deformities. If spasticity is managed more effectively, these bony deformities might very well be less significant, but this is yet to be proven conclusively. Orthopaedic surgical options in the lower limb that can influence spasticity more directly include lengthening of the musculotendinous unit and tendon transfers, some of which are relatively new (e.g. split tibialis posterior transfer) (Gage and Novacheck, 2001). Musculotendinous unit lengthening procedures are increasingly being performed at the musculotendinous junction and allow a more controlled slide with less weakening, such as gastrocnemius recession and psoas lengthening at the brim of the pelvis. Achilles tendon lengthening, which used to be common practice in spastic diplegia, is now known to cause iatrogenic crouch gait and should be avoided (Segal et al., 1989). Overall, the aims of surgery are to reduce established deformity, improve cosmesis, improve gait pattern and reduce the energy cost of walking. 32.6.6. Occupational therapy Occupational therapy focuses on the development of skills necessary for the performance of activities of daily living. These activities include play, self-care activities such as dressing, grooming and feeding, and fine motor tasks such as writing and drawing. It can address cognitive and perceptual disabilities, especially in the visual– motor area and is involved in the adaptation of equipment and seating to allow better upper extremity use and to promote functional independence (Wilson, 1996). Furthermore, parent counseling is an important aspect of occupational therapy, embracing the concept of family-centred care providing parental support for improving the functional abilities of the child with cerebral palsy. As a result, improvement of functional abilities, improvement of participation in society and an increased quality of life are important outcomes of occupational therapy treatment. Different approaches to treatment are taken within occupational therapy, such

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as neurodevelopmental treatment (Bobath, 1980) and sensory integration (Ayres, 1972). Interventions can include training of skills, training of sensory–motor functions, instruction on joint protection and/or energy conservation, provision of splints, training of cognitive functions, advice and instruction on the use of assistive devices, counseling, and education of the primary caregiver. The evidence for the efficacy of the various approaches of occupational therapy is inconclusive because of methodological flaws in the studies and is a topic for further research. This has been systematically reviewed by Steultjens et al. (2004).

Drugs can be used to manage drooling and act through an anticholinergic blockade of muscarinic receptor sites to decrease parasympathetic nervous system stimulation to the salivary glands, but these drugs affect muscarinic sites throughout the body and can cause significant side effects. The drugs most often used to treat drooling include atropine, glycopyrrolate, scopolamine, benztropine and benzhexol hydrochloride (Tscheng, 2002; Jongerius et al., 2003).

Atropine is the classic anticholinergic medication

32.7. Oromotor problems Oromotor problems, including drooling, swallowing dysfunction and feeding difficulties, may lead to potential serious impacts on nutrition and growth, oral health, respiration and self-esteem. 32.7.1. Drooling Drooling is normal in infants and young children, particularly when a child is learning a new motor skill or cutting a new tooth, but has typically resolved by 24 months (Brei, 2003). It has been estimated that drooling persists abnormally in 10–38% of individuals with cerebral palsy and is likely to be due to disordered oromotor functioning (Brei, 2003; Senner et al., 2004). Children who drool have increased difficulty forming a bolus, reduced lip closure, slightly less intraoral suction, difficulty chewing, less spontaneous swallowing and more oral residue after the swallow (Ekedahl et al., 1974; Lespargot et al., 1993). Consequences of drooling include irritated facial skin, unpleasant odor, increased oral and perioral infections, hygiene problems and dehydration (Senner et al., 2004). Severe drooling often necessitates frequent clothing changes and can cause damage to books, electronic equipment and other educational materials. The most unfortunate consequence of drooling may be social isolation. Treatment options to help decrease severe drooling include behavioral and oral motor therapies, pharmacological management and surgical interventions and has been reviewed by Brei (2003). Behavioral and oromotor therapies are time-intensive strategies. Behavioral therapies aim to increase awareness of drooling, increase swallowing and improve head control, body tone and posture. Specific oral motor therapy is often advocated to reduce abnormal oral motor patterns and to facilitate more appropriate mouth and lip closure, tongue mobility and swallowing. Some of these techniques are difficult concepts for the child with cognitive impairment to grasp and results are variable.

and has effects at both central and peripheral receptors and exerts relatively greater effects on the heart, intestinal smooth muscle and CNS, so is not used to control drooling. However, I have had success using atropine ophthalmic eye drops sublingually (1–2 drops 1% solution 3–4 times a day) without systemic side effects, and this has been piloted in a group of adults with parkinsonism (Hyson et al., 2002). Glycopyrrolate, a quaternary ammonium compound structurally similar to atropine with antimuscarinic effects, is currently one of the most frequently used oral medications, because it has a long half-life and does not cross the blood–brain barrier. Several study trials of glycopyrrolate (0.04–0.4 mg/d in 2–3 divided doses) indicate that it does decrease drooling. However, dose-dependent side effects occur in 32–69% of patients. The most common side effects included dry mouth with overly thick secretions, urinary retention, constipation and behavior changes. Benztropine (0.5–1.0 mg/d gradually increasing to a maximum of 6 mg/d) is a synthetic agent with antimuscarinic effects that is effective in drooling. Side effects include irritability, insomnia, vomiting and listlessness. Scopolamine patch (one patch every 72 h) also decreases drooling. Common side effects include itching and rash at the patch application site, pupillary dilation, increased mouthing behaviors and mild sedation. However, significant side effects have also been reported, including scopolamine-induced psychosis and unilateral mydriasis. Because the scopolamine patch is designed to deliver a certain dose and should not be cut to lower the dose, its use in young children is not recommended (Jongerius et al., 2003).

For children resistant to medical treatment or who have unacceptable side effects, surgery is a possible option. The surgical approaches include gland excision, duct ligation, duct rerouting and interruption of the parasympathetic nerve supply to the glands (Hockstein et al., 2004). The use of intraglandular botulinum

NEUROREHABILITATION OF CHILDREN WITH CEREBRAL PALSY toxin injections to reduce saliva production is also receiving attention, has variable results and needs to be investigated further (Hassin-Baer et al., 2005). 32.7.2. Swallowing Swallowing and feeding difficulties in children with cerebral palsy are more common in those with moderate/severe cerebral palsy, occurring in 37% of those with diplegia or hemiparesis and 97% of those with quadriparesis. In a study of 12 709 children age 0.5– 3.5 years with moderate to severe cerebral palsy, feeding and mobility skills were the most powerful prognostic factors for survival. The highest mortality rates were for those children who could not lift their chest or head in the prone position, especially children who were solely tube-fed or who were totally dependent on caregivers for oral feeding (Strauss et al., 1998). Feeding or swallowing problems may initially present as upper or lower respiratory tract signs or symptoms (Loughlin and Lefton-Greif, 1994). Common anatomical airway abnormalities observed in children with cerebral palsy include hypotonia of the hypopharynx, supraglottic edema associated with gastroesophageal reflux and tracheobronchomalacia. Common acute respiratory symptoms during oral feedings include apnea, hypopnea, tachypnea, coughing, choking and hypoxemia. Aspiration of food, either with swallowing or more commonly after, results in chronic lung disease (recurrent wheezing, bronchitis, atelectasis and need for supplemental oxygen) rather than acute aspiration pneumonia. Gastroesophageal reflux is believed to be quite common in children with cerebral palsy, although its true prevalence remains unknown. It is present in 70–90% of those who present with failure to thrive, food refusal, small-volume feeds and vomiting (Del Giudice et al., 1999). The videofluoroscopic swallow study is a valuable assessment tool and provides dynamic images of the oral, pharyngeal and esophageal phases of deglutition (Mirrett et al., 1994; Rogers et al., 1994). It is most helpful in assessing pharyngeal motility and airway protection during swallowing. Commonly observed pharyngeal phase abnormalities include swallow delay, food residue after swallows, pharyngeal dysmotility and aspiration. These abnormalities are usually restricted to specific food textures. Aspiration more commonly occurs with thin liquids and is usually silent and not necessarily associated with coughing or choking. Gastrostomy tube feeding remains an important alternative nutritional source for children with cerebral palsy. Indications for this include dysphagia resulting in failure to thrive, aspiration with associated respi-

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ratory disease, insufficient fluid intake and/or refusal of oral medications, and excessive effort or stress during oral feedings. These relative indications need to be viewed in the context of the cultural, social and religious belief systems of each child’s family. Mothers often express a feeling of culpability for their child’s poor growth, see the need for surgery as a failure and need considerable support before and after the surgery (Craig et al., 2003). Major and minor postoperative complication rates are 10–30%. Common minor complications include leakage from the stoma, cellulitis and excessive granulation tissue formation. Fundoplication procedures for gastroesophageal reflux are associated with higher rates of postoperative complications, including persistent retching. Discharge planning should include management of gastrostomy tube feedings to insure appropriate weight gain and promotion of oral sensorimotor development. Family surveys after gastrostomy tube placement generally reveal high rates (55–95%) of self-limited tube-related problems. Parents are usually highly satisfied after the procedure (80–90%), with children having 50–60% fewer chest infections, 90% less vomiting, and a majority with oral intake improved or unchanged (Smith et al., 1999).

32.8. Speech and language disorders There is bilateral corticobulbar dysfunction in many cerebral palsy syndromes and therefore anarthric or dysarthric speech and other impairments related to oromotor dysfunction are common. In one study, articulation disorders and impaired speech intelligibility were present in 38% of children with cerebral palsy (Kennes et al., 2002). Impairment of articulation and speech intelligibility were examined in subjects with cerebral palsy who were deemed to have adequate intelligence, hearing and ability to perform the required tasks. Results identified characteristic dysarthric phonemic features such as anterior lingual place inaccuracy, reduced precision of fricative and affricate manners, and inability to achieve extreme positions in vowel articulatory space. Language (as opposed to speech) deficits in cerebral palsy are associated with verbal intellectual limitations associated with mental retardation (Falkman et al., 2002; Kennes et al., 2002). As impaired mobility can lead to limited interaction with others, children with cerebral palsy may not have the same opportunities to further develop their linguistic skills and achieve more complex speech patterns (Fennell and Dikel, 2005). Speech and language therapy aims to maximize children’s ability to communicate through speech, gesture and/or supplementary means such as communication aids, and to enable them to become independent

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communicators. As the problems experienced by children with a diagnosis of cerebral palsy range widely, there is no single universally appropriate form of treatment (Pennington et al., 2005). Intervention can focus on speech, on expressive or receptive language development or on helping children to develop conversation skills such as asking questions and ‘repairing’ conversation when misunderstandings occur. It can be given to children directly on a one-to-one basis or in groups, or indirectly by training familiar conversation partners to alter communication environments and provide more opportunities for interaction. Pennington et al. (2005) have systematically reviewed the various methods of therapy and found that operant and microteaching methods were effective and that children with severe cognitive impairment found iconic symbols easier to acquire than noniconic Blissymbols. Observational studies have found augmentative and alternative communication effective but this and other methods of speech therapy are the subjects of ongoing research.

32.9. Cognitive function Given the heterogeneous nature of the clinical picture in cerebral palsy, it is difficult, if not impossible, to make satisfactory generalizations about the relationship of cerebral palsy or disorders of cerebral malformation and cognitive functioning. Individuals with spastic quadriparesis are, in most cases, severely intellectually impaired, whereas half of hemiparetic children have IQs in the average range and 18% achieve scores over 100 (Nelson and Ellenberg, 1982). In children with spastic diplegia, there tends to be a general correlation between severity of motor deficit and level of retardation, in contrast with those children with dyskinetic cerebral palsy, in whom this relationship is lacking (Fennell and Dikel, 2005). In cases of extrapyramidal cerebral palsy, delayed or deficient language skills due to dysarthric incoordination of muscles of language and speech and significant gross motor handicaps can lead to false underestimation of intelligence. In these cases, motor function tends to be more impaired than cognition. There is also a strong association between greater intellectual impairment in children with cerebral palsy and the presence of epilepsy, an abnormal electroencephalogram or an abnormal neuroimaging study (Zafeririou et al., 1999). It may be difficult to accurately assess cognitive functioning because of the presence of motor, speech, visual, and auditory difficulties that impair the child’s performance. Many neuropsychological tests demand adequate vision and visual discrimination, visual-motor coordination, gross and fine motor skills, the ability to work quickly and efficiently and the ability to commu-

nicate responses to the examiner. Children with cerebral palsy have difficulties in one or more of these areas. For example, upper extremity motor impairment may interfere with hand–eye coordination and fine and gross motor skills that become sequentially increasingly demanding within performance scale subtests of the Wechsler Intelligence Scales. Another major issue affecting consideration of cognition and cerebral palsy pertains to the split between verbal IQ and performance IQ on Wechsler intelligence tests that is often found and may increase with age as performance remains the same but the verbal IQ increases (Ito et al., 1997). Summary scores in the reported IQ therefore serve in a limited capacity, as these are averages of a diverse array of strengths and weaknesses and so may misrepresent the child’s level of functioning.

32.10. Ophthalmological impairments Visual impairments and disorders of ocular motility are common (39–71%) in children with cerebral palsy (Schenk-Rootlieb et al., 1992; Zafeririou et al., 1999). There is an increased presence of strabismus, amblyopia, nystagmus, optic atrophy, refractive errors and visuoperceptual difficulties (Schenk-Rootlieb et al. 1992; Stiers et al., 2002). There is some evidence that with time (over 10 years), visual function may improve, although abnormalities are still present (Porro et al., 1998).

32.11. Hearing impairment Hearing impairment occurs in approximately 3–12% of children with cerebral palsy (Kennes et al., 2002; Russman and Ashwal, 2004). Hearing impairment can go unrecognized and should be specifically tested in the neonatal period whenever possible (Joint Committee on Infant Hearing, 2000).

32.12. Epilepsy The frequency of epilepsy in children with cerebral palsy ranges from 35–62%. Compared to other children with epilepsy, children with cerebral palsy are more likely to start having seizures in the first year of life, have partial epilepsy or episodes of status epilepticus and require polytherapy, and are less likely to remain seizure-free (Kwong et al., 1998). Children with spastic quadriplegia or hemiplegia have a higher incidence of epilepsy than patients with diplegia or ataxic cerebral palsy (Zafeririou et al., 1999). This topic has been reviewed by Wallace (2001). It is worthwhile noting that it may occasionally be difficult to differentiate partial complex seizures from

NEUROREHABILITATION OF CHILDREN WITH CEREBRAL PALSY dyskinetic movements in patients with dyskinetic cerebral palsy where video EEG telemetry is useful. It is beyond the scope of this chapter to discuss the treatment of epilepsy in detail. When possible, antiepileptic drugs should be chosen to treat both the epilepsy and one or more of the comorbidities present. Drugs such as carbamazepine, oxcarbazepine and sodium valproate could be chosen both for partial epilepsy and as mood-stabilizing drugs (Hirschfeld and Kasper, 2004; Spina and Perugi, 2004). Clonazepam or nitrazepam are useful where spasticity, dystonia or nocturnal spasms are present. Lamotrigine is useful when one wishes to avoid further sedation or cognitive side effects, particularly when using other anti-hypertonicity medication (Aldenkamp et al., 2003). The ketogenic diet is a useful alternative when seizures are difficult to control and the child is fed using a G-tube (Hosain et al., 2005).

32.13. Behavior The prevalence of disturbed behaviors or emotional maladaptations has been reported in 30–80% of children with cerebral palsy (Hourcade and Parette, 1984; McDermott et al., 2002). McDermott et al. found that parents of children with cerebral palsy were five times more likely to report behavior problems in school-aged children than parents of those with no known health problem. The problems were reported mainly in the domains of dependency and stubbornness. Parents of children with mental retardation were almost eight times more likely to report behavior problems and these included antisocial behavior, anxiety, hyperactivity and peer conflict (McDermott et al., 1996). Parents also emphasized the chronic stress and exhaustion they experienced as caregivers of children with cerebral palsy and other medical conditions, particularly during the early developmental years. Therapists often report that, while they are aware of the multiple stresses and needs of families presenting for rehabilitation, they often feel ill-equipped to address parenting concerns or behavior problems in their treatment sessions. It has been demonstrated that intervention consisting of monthly meetings between rehabilitation therapists and a team consisting of a child psychiatrist, a developmental pediatrician, psychologists and a preventive medicine specialist, resulted in statistically significant improvements in the subscales of the Vineland adaptive skills assessment and measures of family stress associated with the parent’s attitude toward the child with a disability. The magnitude of the improvement was greatest for children with mental development indices below 50 (McDermott et al., 2002).

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32.14. Conclusion No two children with cerebral palsy are alike and care must be tailored to the needs of the individual in their familial, educational and societal setting. Goals of care must be identified in collaboration with the family and be frequently reassessed and modified as the child changes and develops. Objective evaluation measures will enable therapists and physicians to mark the child’s progress, facilitate communication between the many specialties involved and allow further research in neurorehabilitation therapies. In the early years of development, motor impairments are often highlighted but later the important issues of cognition, communication and behavior become equally, if not more, important. There is a growing understanding of the frequency of these comorbidities and with time we will understand the interplay between them and their role in optimal functioning. The resources of a multidisciplinary team and the family allow us to make careful assessments for their presence and intervene when necessary. The changes that take place from adolescence to adulthood and how they relate to changes in service provision are only just beginning to be explored. This will undoubtedly be a key area of health service research in the future as the survival of children increases.

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Chapter 33

Educational, cognitive, behavioral and language development issues CARLA ARPINO*, ANNA VOLZONE, AND PAOLO CURATOLO Pediatric Neurology Unit, Department of Neuroscience, Tor Vergata University of Rome, Italy

33.1. Introduction Cerebral malformations are responsible for several developmental disabilities that are heterogeneous both in type and severity. Virtually any type of disorder – cognitive, motor, behavioral, speech and language – can be present. Recent progress in molecular genetics has improved our knowledge of the role played by several genes in causing malformations. Although animal models show that the behavioral deficits following insults to cortical neuronal differentiation are more pronounced and extended than those following insults during neurogenesis (BergerSweeney and Hohmann, 1997), these findings have not been confirmed in humans. Furthermore, as far as our understanding of the genetic complexity of cerebral malformations tends to improve, the spectrum of clinical phenotypes also increases. In fact, the same gene may determine, at the same maturational stage, different clinical and neuroradiological phenotypes depending on the type of defect (e.g. deletion, missense mutation, mosaic mutations) and gender (Table 33.1). Thus, predicting the clinical phenotype (i.e. the developmental disorder and its related disability) according to the genotype remains difficult, because of the possible effect of several confounding factors. Among these, we can mention interaction among genes, involved sequentially and serving different functions at different stages (Sarnat, 2000, 2004) and our incomplete knowledge of the function of specific molecules resulting from specific mutations. The demand for educational, cognitive/language and behavioral treatments for developmental disorders resulting from cerebral malformation or acquired disorders has been continuously increasing over the last few

decades, both because of earlier diagnosis of these disorders and because, in most cases, this approach (i.e. ‘rehabilitation’) represents the only available ‘therapeutic’ chance. The 77% of children referred to specialists for the first investigation of their ‘delay’ receive some rehabilitation treatment within the first 6 months following medical evaluation (Majnemer et al., 2002). Scarcity of resources, the lack of a clear definition of ‘treatment’, the paucity of standardized treatments, the limited availability of guidelines to inform medical decisions and the necessity for fulfilling parents’ requests are all aspects that physicians and therapists have to deal with in daily practice and make the job difficult.

33.2. The basic assumption of rehabilitation: brain plasticity Rehabilitation strategies, including educational, cognitive/language and behavioral treatment, should be based on the assumption of plasticity, which implies the brain’s capacity to be molded by experience, the capacity to learn and remember and the ability to reorganize and recover after injury. It is known that the CNS is able to modify its structure through 1) the creation of new neuronal circuits, 2) the improvement of the circuits already present but underused, 3) the creation of collateral synapses that may join different neural circuits and 4) the suppression of redundant circuits (Johnston et al., 2003; Johnston, 2004). Adverse events occurring during cerebral morphogenesis and neuronal migration (i.e., during the first and second trimester of pregnancy) typically result in brain malformations. Age is an important factor for

*Correspondence to: Dr Carla Arpino, Department of Neuroscience, Child Neurology, ‘Tor Vergata’ University of Rome, Rome, Italy. E-mail:[email protected].

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Table 33.1 Genotype-phenotype correlations in brain malformations. Gene

LIS1 gene (17p13.3)

Male

MRI

Genotype

Clinical features

Isolated Lissencepaly (LISS) more evident on posterior regions (Guerrini et al., 2005) rarely isolated subcortical band heterotopia (Cardoso et al., 2002)

40% exhibit a deletion (Pilz et al., 1998), 25% show an intragenic mutation (Cardoso et al., 2000, Kato and Dobyns, 2003)

Subcortical Band Heterotopia (SBH) in the parietal and occipital regions (Gleeson, 2000)

Point mutation and somatic mosaicism

Miller-Dieker phenotype (Kato et al., 2003)

Large deletion with inclusion the 14-3-3 (17p13.3) Mutation of the coding region and missense mutations

From milder (infrequent seizures, mild clumsiness and normal intelligence: missense mutations) to severe phenotype (frequent seizures, severe delay language, mental retardation: truncation mutations) (Sicca et al., 2003) Mild global developmental delay (Leventer et al., 1998; Sicca et al., 2003) Epilepsy is present in almost all patients and is intractable in about 65% (Guerrini et al., 2001) Miller-Dieker syndrome (Kato et al., 2003)

SBH in sporadic and in familiar females (Matsumoto et al., 2001) more evident on anterior regions (Gleeson, 2000)

Cognitive function ranges from normal to severe retardation and correlates with the thickness of the band (Guerrini et al., 2003)

MRI

Genotype

Clinical features

No gender-related differences

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DCX gene (Xq22.3q23)

Female

Sporadic male with SBH (Pilz et al., 1999) in the parietal and occipital regions (D’Agostino et al., 2002; Sicca et al., 2003)

Missense mutation or mosaicism

Severe phenotype with intractable epilepsy, spasticity and moderate-severe mental retardation (D’Agostino et al., 2002)

No gender-related differences SBH predominant on anterior regions (Pilz et al., 1999; D’Agostino et al., 2002; Guerrini et al., 2003)

Missense mutation Somatic mosaicism

Cognitive function ranges from normal to severe retardation and correlates with the thickness of the band (Guerrini et al., 2003)

RELN gene (7q22) ARX (Xp22.13)

FLN1 (Xq28)

ARFGEF2 (20Q13.13)

LISS more severe in anterior regions with cerebellar hypoplasia Isolated agenesis of the corpus callosum (ACC)

Bilateral/Monolateral Periventricular Heterotopia (B-M/PNH) Isolated or bilateral subependymal nodules of gray matter Bilateral Periventricular Heterotopia

Severe mental retardation (Guerrini et al., 2003)

Different splice site mutations

Mental retardation. Familiar cases are reported

Missense mutations, large deletions, frameshifts, nonsense mutations, and splice site mutations

Normal development

Heterozygous mutations

Normal to borderline IQ and epilepsy with different degree of disability

Large mutations in the gene ADPribosylation factor guanine nucleotideexchange factor-2 (ARFGEF2 gene)

Severe developmental delay, microcephaly, early onset severe epilepsy, including infantile spasms (Lu et al., 2005)

LISS (Dobyns et al., 1999; Pilz et al., 1999; Pilz et al., 2002)

Hemizygous mutation

Severe mental retardation (Pilz et al. 1999, 2002)

No gender-related differences LISS more evident in posterior regions, abnormal signal of white matter, ACC, cystic or fragmented basal ganglia (Bonneau et al., 2002) Periventricular nodular heterotopia

Missense mutations, deletions, frameshifts, nonsense mutations, splice site mutations

X-linked Lissencephaly with abnormal genitalia (Dobyns et al., 1999; Kato et al., 2005)

Missense mutations, mosaicism and deletion.

Prenatal lethality. Two males with milder phenotype in two males (Guerrini et al., 2004)

No gender-related differences

EDUCATIONAL, COGNITIVE, BEHAVIORAL AND LANGUAGE DEVELOPMENT ISSUES

LISS more severe in anterior than posterior regions

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cerebral reorganization, in addition to the size and location of the lesion. In fact, the immature brain seems to be more adept at reorganizing its synaptic connections than the adult brain (Johnston, 2003; Carr et al., 1993). Studies on the role of ipsilateral corticospinal projections in the efficacy of reorganization after an insult show a reduced reorganizational potential for lesions occurring late in the third trimester compared with lesions acquired during the first, second and early third trimester (Staudt et al., 2002, 2004; Vandermeeren et al., 2002, 2003). Furthermore, reorganizational potential differs according to specific functions: brain plasticity following left-sided lesions in the developing brain seems to be greater for language than for motor and visual functions (Krageloh-Mann, 2004). The type of lesion, cortical–subcortical or periventricular, and the timing of the insult, at term or preterm, have been suggested as the primary factors responsible for inter- versus intrahemispheric organization of language after congenital brain lesions (Brizzolara et al., 2002). With regard to visual functions, there is no clear evidence for better outcome in early lesions than in later lesions (Krageloh-Mann, 2004). There seems to be a critical period for plasticity in the visual cortex that starts a few days or weeks after vision is established and ends before adulthood (Feller and Scanziani, 2005).

33.3. How to plan a treatment for a child with a brain malformation The difficulty of predicting the type of disability and the heterogeneity of the cerebral malformations should be kept in mind when planning treatment and assessing its efficacy. The child is a competent individual who is in interactive relationship with caregivers and environment. According to the synactive theory of development promoted by Als (1982), child development in the early stage has a hierarchical organization based on both dependent and independent subsystems, such as autonomous, motor, behavioral, attention/interaction and autoregulation systems. In particular, the motor system provides stability for all the others. Early damage can therefore affect the subsystems by interfering with the sensorimotor abilities that the child uses for experience and knowledge of the surrounding environment. The resulting damage can then range from psychomotor disturbance to the typical presentation of cerebral palsy. Clinical experience shows that the child who can competently acquire posture or movement against gravity has more opportunity to engage in play activities with his/her caregiver, promoting interaction, socialization and cognitive learning. Furthermore, the ability to grasp and manipulate a toy and to move around enriches the

child’s learning opportunities and his/her superior cognitive functions. Motor system instability leads to reduced and undifferentiated experiences with objects and toys, which in turn result in an early disturbance in development, with age-related symptoms and presentation. Interrelations between motor and cognitive processing provide examples where systems are more intertwined than previously thought. Fine motor skills and visual–motor coordination mature together with higher cognitive functions, neither reaching full maturity until late adolescence (Diamond and Lee, 2000; Rosenbaum and Chaiken, 2001). Thus a developmental disorder can involve motor, sensorial, cognitive, neuropsychological and communicative–interactive competences or can globally fragment development. The clinical features and intervention strategies may be different according to severity, developmental stage, expression of disease and associated disorders. Diagnosis therefore has to be viewed from a developmental point of view, with reciprocal interactions between development and disorder: the disturbance is transformed by emerging competences, which in turn are affected by the disturbance. We should also consider that development in children with brain impairment is not a linear but an unpredictable process; it does not seem to happen spontaneously or as a natural process of maturation, being the result of complex interactions between the child and a stimulating environment (Lebeer and Rijke, 2003). Based on the abovementioned considerations, planning a treatment for CNS malformations should not underestimate the interdependence of systems and should involve a global approach. To ensure the success of the intervention, a team with an interdisciplinary approach is necessary. To set individualized treatment plans and goals, this team of rehabilitation specialists should work with the patient and their family. Team members should include:

Family: parents and siblings should receive per

sonal support, training in management strategies and practical assistance; Rehabilitation health workers, in particular: a physical therapist whose work is addressed toward maximizing functional abilities and mobility with the purpose of increasing independence a speech–language pathologist in order to maximize the child’s ability to communicate, through speech, gesture, and/or supplementary means, such as communication aids a neuropsychologist involved in the evaluation of temporary or permanent problems with thinking skills secondary to a brain malformation


an occupational therapist whose aim is to assist

the patient in carrying out activities of daily living at the highest possible level of independence. School: each child with disability should receive special education and related services and an individualized education program addressed to the student’s special needs. This represents an opportunity for teachers, parents, school administrators, related services personnel, and students (when appropriate) to work together to improve the educational results obtained by children with disabilities. The child’s special education teacher contributes important information and experience about how to educate children with disabilities. Because of his/her training in special education, this teacher can talk about such issues as how to modify the general curriculum to help the child learn and the supplementary aids and services that the child may need to be successful in the regular classroom.

The rehabilitation goals are broken down into shortterm objectives. Goals may be academic, address social or behavioral needs, relate to physical needs, or address other educational needs. The goals must be measurable, meaning that it must be possible to measure whether the student has achieved the goals. Finally, the leading figure for the ‘care’ strategy is the child neurologist. S/he evaluates the patient, prescribes a therapy program, supervises the medical care, re-evaluates the patient to determine if goals are being achieved and assists with discharge plans. S/he is a ‘key worker’ or ‘link person’ (Sloper, 1999) and is the reference person for parents’ requests about any problem related to the disabled child. 33.3.1. The use of the international classification of functioning, disability and health Children with CNS malformations as the background to their brain damage have additional neuroimpairments more frequently than those with perinatal CNS lesions or no identifiable antecedents (Beckung and Hagberg, 2002). Thus, planning a treatment requires a careful and exhaustive assessment, in particular concerning functioning and disability. The functional assessment, the process of describing a child’s strengths and challenges in the context of the essential activities that occur within a child’s everyday environment (Msall and Tremont, 1999), represents the core of a rehabilitative treatment. As it is known, rehabilitation is a multidisciplinary area that encompasses several components (medical, psychosocial, educational, and biotechnological); thus, it is a complex intervention the effectiveness

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of which is difficult to evaluate. Efforts have been made over the last 20 years to 1) reduce the barrier between medical and social models of rehabilitation through the identification of a common language for different specialists; 2) strengthen the idea that the effectiveness of rehabilitation (either with the meaning of a ‘cure’ or simple ‘managing’), like any other treatment, must be proved. The need for treatment evaluation is reinforced by the fact that we deal with chronic disorders entailing high financial costs. The consciousness of the necessity of this approach has inspired World Health Organization expert committees in the development of models of disablement and related classification systems. The recommendations of this experts’ panel have produced, since 1980, the International Classification of Impairment, Disability and Handicap (ICIDH) (World Health Organization, 1980), which has been later revised as the ICIDH-2 (World Health Organization, 1997); the last revision, the International Classification of Functioning, Disability and Health (ICF) (World Health Organization, 2001), has been accepted by 191 countries as the international standard for describing and measuring health and disability. The functional assessment, in the framework of the ICF, conceived as a response to the problem of evaluating the effectiveness of health care processes, implies the description of body functions and structures, activities and participation, and includes environmental factors as important contributors to understanding the complexity of sources of disability (Beckung and Hagberg, 2002).

33.4. What are the available educational, cognitive–language, behavioral approaches? There are several rehabilitation methods for treatment of language, cognitive and/or behavioral disorders following a brain malformation and often coexisting in the same child. There are, at least, two main theoretical approaches. For some disabilities, we can follow the development of children and provide the primary intervention in the family (counseling), encouraging the child’s general development. We can define this approach as ‘watching and waiting’. Otherwise, we can plan treatment to improve specific functions and/or to effect global cognitive stimulation. The main available therapeutic approach and methods for language, cognitive, behavioral and learning disabilities, in the context of brain malformations, are described below. 33.4.1. Speech therapy based on social interaction approach This is based on three key concepts: 1) language development is motivated by the child’s desire for,

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and attempts to acquire, a means of participation in social interaction with the important people in his or her life; 2) the interactions and relationships that the child has with other, competent, language users are critical for determining the rate and course of language development; 3) there is considerable variation among children regarding their environments, including the extent and nature of their interactions and relationships with care providers. This approach emphasizes that the social uses and contexts of language are important for development. 33.4.2. Augmentative and alternative communication Visual supports in the form of the picture exchange card system and other augmentative and alternative communication aids, including voice output communication aids, have been offered as viable tools for improving expressive and receptive language. 33.4.3. Functional communication training This has been used to reduce problem behavior exhibited by individuals with developmental disabilities. 33.4.4. Auditory integration training Auditory integration training is said to address the hearing distortions, hyperacute hearing, and sensory processing anomalies that cause discomfort and confusion in persons suffering from learning disabilities, including autism. Auditory training seeks to retrain the auditory system by correcting hearing distortions. Various tones are presented to the child in the hope of ‘desensitizing’ resistance to auditory input. 33.4.5. Treatment and education of autistic and related communication-handicapped children

them to achieve their individual potentials. The program is comprehensive, aimed at enhancing language and communication, social/play and the preacademic and independent living skills of young children so that they may take better advantage of opportunities in their communities and may require less professional attention as they grow older. 33.4.7. ‘Floor Time’ approach Floor Time approach is based upon Greenspan’s theories of the six functional milestones necessary for a child to succeed in further learning and development. Floor Time includes interactive experiences, which are childdirected, in a low-stimulus environment, ranging from 2–5 hours a day. During a preschool program, Floor Time includes integration with typically developing peers. 33.4.8. Sensory integration therapy Ayres describes sensory integration therapy as sensory stimulation and subsequent adaptive responses that evolve according to the child’s neurological needs. Therapy techniques include vestibular stimulation such as swinging in a hammock, and tactile stimulation achieved by brushing parts of the child’s body. This therapy is viewed as a direct intervention that can improve nervous system function. This is done by providing the child with enhanced levels of sensory information gleaned during physical activities that are meaningful to the child and that elicit adaptive behaviors. 33.4.9. Portage Portage is a home educational programme comprising 580 developmentally progressive skills for children from birth to 6 years, taught by a visiting teacher and practiced by parents with the child.

The Treatment and Education of Autistic and Related Communication Handicapped Children (TEACCH) approach includes a focus on the person with disability and the development of a program around this person’s skills, interests and needs. The major priorities include centering on the individual, adopting appropriate adaptations and a broadly based intervention strategy building on existing skills and interests.

33.4.10. Biofeedback

33.4.6. Lovaas

This is a multidisciplinary educational program focusing on the child’s psychological, cognitive and motor growth, an all-embracing day-long educational process in groups led by a ‘conductor’.

The focus is to serve children of all levels of functioning while providing an educational setting that allows

This uses visual and auditory stimuli to provide a subject with information about psychophysiological processes that are often outside their immediate awareness and control. 33.4.11. Conductive education

EDUCATIONAL, COGNITIVE, BEHAVIORAL AND LANGUAGE DEVELOPMENT ISSUES 33.4.12. The Orton–Gillingham techniques These techniques are taught in a small number of state school systems today, usually within special education classes. An intensive, sequential phonics-based system teaches the basics of word formation before whole meanings. The method accommodates and uses the three learning modalities, or pathways, through which people learn – visual, auditory and kinesthetic. 33.4.13. Feuerstein’s instrumental enrichment Instrumental Enrichment (IE) is a cognitive education program that was developed in the 1950s by Professor Reuven Feuerstein. The program has been successfully used in 70 countries as a tool for the enhancement of learning potential in challenged individuals and those in high-risk environments. IE materials are organized into instruments that comprise paper and pencil tasks aimed at such specific cognitive domains as analytic perception, orientation in space and time, comparative behavior, classification, and more. IE consists of 14 instruments that focus on specific cognitive functions. Learning how to learn takes place through repetition – not repetition of the IE tasks themselves but of the cognitive functions that enable individuals to think effectively. Tasks become increasingly complex and abstract, and the instruments reinforce cognitive functions in a cyclical manner. Deliberately free of specific subject matter, the IE tasks are intended to be more readily transferable to all life situations. Through IE, students develop the ability to apply their cognitive functions to any problem or thinking situation. Feuerstein’s method is based on an optimistic view of the learning process. Indeed, he believes that each individual is modifiable because the human organism is an open, flexible system. Therefore it is possible to make structural cognitive changes – within the constraints imposed by age, the severity of delay, the exogenous and endogenous limitations – through the assistance of another person who acts as a mediator. The mediator places him/herself between the individual and the source of stimulus and encourages active and intentional behavior. In the educational relationship, the mediator (parent, educator or teacher) selects sequences and includes stimuli in the teaching-learning process. 33.4.14. Bright Start Bright Start is a flexible cognitive curriculum for young children, designed for use with children functioning at developmental levels from 3–6 years, including those who are ‘normally developing’, those who are sociologically at risk of school failure

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(e.g. children from very poor families) and those who have a low IQ. The primary goal of Bright Start is that of ‘stretching the mind’, i.e. broadening children’s understanding and thinking processes, thereby increasing their educability. 33.4.15. Mixed models A combination of approaches can be used, depending on developmental stage, specific needs and feasibility.

33.5. When, how, and for how long to treat a child with brain malformation 33.5.1. When to start a treatment In our daily clinical practice, we are often asked to distinguish between normal and abnormal movements or between delay and disorder. Because of limited knowledge of outcome predictors, it is difficult to identify, among children affected by a specific malformation, those who will benefit from early treatment and those whose muscular tone will normalize without any treatment; this limits our ability to make clinical decisions. As an example, early hypotonia or hypertonia might represent a mild and transient symptom or an early indicator of cerebral palsy; a delay in an early precursor of communication might represent either a transient deviance from normal development or an early indicator of language disorder. Many children with brain impairment develop well, in spite of negative risk factors or prognosis (Lebeer and Rijke, 2003). In a large follow-up study, Nelson and Ellenberg (1982) found that half the children diagnosed with cerebral palsy at 1 year of age, including 16% of those with moderate to severe tetraparesis, had no more motor symptoms at the age of 7 years. Neither the length of birth anoxia, the type of chromosome damage, the extent of brain tissue loss or the degree of spasticity justify a prediction of the degree of functional deficit (Lebeer and Rijke, 2003). Taking into account the limited reliability of predictors of outcome, all at-risk infants should receive careful pediatric follow-up, including developmental screening and functional evaluation. In the presence of any ‘alarm signal’ and/or ‘dysfunction’, a more detailed assessment should be mandatory. In fact, an appropriate recognition of ‘delay’ is necessary for referral to early intervention services that may help these children to overcome or improve motor /cognitive/behavioral dysfunction and help families grow more confident in caring for children with special needs. There is no agreement on the effectiveness of early intervention. Early rehabilitation is helpful, since it

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may increase the adaptive skills of the patient and promote ‘family communication’ (Korkmaz, 2000; Kasari, 2002), but it is necessary to change the target and the intensity of therapy in time. In an evidencebased review of neurodevelopmental treatment methods, no studies were found on the efficacy of treatments under 6 months of age, and only four out of 21 studies evaluated treatment under 12 months of age (Butler and Darrah, 2001). Two controlled studies of the treatment of cerebral palsy evaluated the effects of physical therapy administered during the first year of life to infants deemed at risk for neurologic sequelae, and found no advantages for treated patients (Goodman et al., 1985; Piper et al., 1986). In conclusion, it might be prudent and reasonable to arrange a rehabilitation treatment for high-risk infants as early as possible in order to ‘maximize the chance’ afforded by plasticity and reduce the severity of possible handicap later in life, but we also need more research addressed at evaluating whether early treatment is really more effective than late treatment and to quantify the benefit. 33.5.2. How to treat In accordance with our practical experience, we present some examples of specific intervention profiles that may be adopted as educational, cognitive–language and behavioral treatment for selected brain malformations (Tables 33.2–33.6). 33.5.3. For how long to treat To our knowledge, there are no studies addressing the question of how long therapy needs to be given to achieve the optimal outcome (Kanda et al., 2004).

Furthermore, the majority of studies examining the effect of duration and intensity of a treatment on its efficacy concern motor disabilities. However, it is important to underline the fact that early damage, e.g. a brain malformation, can affect several domains by interfering with sensorimotor competences. It has been reported that randomized trials conducted over a period of about 1 year might be insufficient to prove treatment effects (Piper et al., 1986) and that more intensive and longer trials of early intervention are necessary to demonstrate the efficacy of physiotherapy. The longest duration of treatment in a recent review of neurodevelopmental treatment was only 21 months (Butler and Darrah, 2001). Five of the 21 studies examined in the report had treatment durations between 6 and 12 months and the remaining 15 had treatment durations of less than 6 months. Some studies have demonstrated that rehabilitation programs with a higher frequency of treatments result in better outcomes (Mayo, 1991; Bower et al., 1992), especially when combining intensive therapy periods with periods without therapy (Trahan and Malouin, 2002), while other studies have shown no difference (Wright and Nicholson, 1973; Scherzer et al., 1976; Bower et al., 2001). Most studies on the intensity of therapies have compared the number of treatments provided by rehabilitation centers per week or per month (Reddihough et al., 1991). In a controlled trial, Palmer et al. (1988) reported that the motor outcome of the group receiving 12 months of physical therapy starting earlier was no better than that of the group receiving 6 months of physical therapy, after 6 months of infant stimulation (a program that consists of 100 explicitly defined and illustrated cognitive, sensory, language and motor activities of increasing developmental complexity appropriate for children from birth to 3 years of age). The amount of therapy provided

Table 33.2 Educational, cognitive, language intervention in the context of frontal lobe cortical dysplasia (male, 8 years) Disability

Intervention

Clumsiness

Training to improve gross and fine motor function, maintain posture and move according to fixed coordinates around the child’s individual space Enhance cognitive skills using specific treatment method (according to learning profile and age) ex reading and writing, text comprehension and elaboration (use of maps, charts and simplified text) School program goals aiming at the student’s special needs (Individualized Education Program) Train teachers and parents to follow schedules and charts as stated in the program Remediation of executive function deficit: work on problem solving, reward/ punishment reinforcement techniques Progressively delay gratification, visualize time intervals, use tokens for activities Remediation of social problems (teach to react to frustration, bring child under adult control, make a contract and establish rules)

Borderline cognitive functioning and learning disabilities

Attention deficit/hyperactivity symptoms Impulsivity Troublesome behavior


619

Table 33.3 Educational, cognitive, language intervention in the context of tuberous sclerosis (male, 10 years) Disability

Intervention

Moderate mental retardation and learning disabilities

Choose a cognitive/educational method, ex TEACCH, aimed at developing personal and social autonomy, and functional/adaptive skills School program goals aiming at the student’s special needs (Individualized Education Program) Parent training

Autism Aggressive/self-injurious behavior Social problems

Inadequate communication ability Rigidity Stereotypes

Sleep disorders

Analyze situation and occurrence pattern, and evaluate extinction/prevention strategies Bring child under adult control Teach/practice social interactions Teach how to react to frustration Train joint attention Alternative communication Use visual/verbal cues to indicate daily schedules and minimize changes Analyze stereotypes: noninterfering with other activities, can be tolerated and must be ignored; interfering or disturbing ones should be substituted with more acceptable ones by extinction Structure regular sleep schedules

Table 33.4 Motor, cognitive and educational intervention in the context of left hemimegalencephaly (female, 4 years) Disability

Intervention

Right hemiplegia

Choose a treatment method according to the child’s performance and age, ex: constraint-induced movement, a treatment for substantially increasing the use of extremities affected by neurological injuries in adults and in young children with cerebral palsy. Prescription of specific devices when necessary Speech and language therapy based on cognitive program and social interaction: expand vocabulary, help correct construction of a complete sentence (syntaxis); potentiate semantic comprehension (categorization, memorization strategy) Train visual/motor pursuit (puzzle, labyrinths, computer-based training) Be prepared to adapt treatment strategy in the presence of surgical intervention

Mild mental retardationVerbal IQ < performance IQ Visual-spatial, visual-constructive difficulties

Table 33.5 Motor, cognitive and educational intervention in the context of right hemimegalencephaly (female, 7 years) Disability

Intervention

Left hemiplegia

Choose a treatment method, ex: neurodevelopmental treatment using inhibition of abnormal reflexes and facilitation of normal movement. Prescription of specific devices when necessary Choose a cognitive education program (e.g. Feuerstein method) aimed at developing personal and social autonomy and functional/adaptive skills. Reinforce communication and expression of needs and emotions Play activities (building a tower, following simple pathways, hand–eye coordination)

Moderate mental retardationVerbal IQ > performance IQ Visual-spatial, visual-constructive difficulties Learning disabilities

School program goals to specific needs of the student (Individualized Education Program), facilitate global identification of words Be prepared to adapt treatment strategy in the presence of surgical intervention

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Table 33.6 Habilitation/management in the context of holoprosencephaly (female, preterm, 2 months) Disability

Intervention

Motor and autonomous instability (respiratory disturbances, gastro-esophageal reflux, startles)

Adjustment of macro-environment (room temperature, noise, illumination), adjustment of micro-environment (boundaries, center of gravity), adjustment of cutaneous stimulation (facilitators to be inserted in daily routine: wrapping, handling) Postural stability, organization of motor-postural scheme, (axial, distal stabilization, holding and boundary, head–limb realignment) Infant massage

Abnormal general movements and motor derangement Behavioral derangement (hyperexcitability, fussiness, irritability, inconsolability) Poor visual and auditory orientation

Favor emotional interaction with caregiver through verbal cues during motor stabilization

at home by parents or other trained individuals is not usually reported because of the difficulty in assessing compliance with treatment regimens: the quality, amount and consistency of home treatment administered by trained caregivers is rarely documented or even mentioned in studies that evaluate outcomes. Methods for encouraging compliance with home-based treatments are needed in future studies of therapeutic effectiveness (Kanda et al., 2004). Among communicative disorders, Law et al. (2004) reported that speech and language therapy might be effective for children with phonological or expressive vocabulary difficulties. There was mixed evidence concerning the effectiveness of intervention for children with expressive syntax difficulties and little evidence available considering the effectiveness of intervention for children with receptive language difficulties. No significant difference was found between interventions administered by trained parents and those administered by clinicians. The review identified longer duration (> 8 weeks) of therapy as being a potential factor in good clinical outcome. Although the model of early treatment has been adopted by the scientific community, the neurological theories underpinning early intervention have been criticized (Ferry, 1981). Neither a quantitative nor a qualitative analysis has so far provided unequivocal evidence that any early intervention therapy is effective. It is, however, quite possible that failure to demonstrate the efficacy of early intervention is due to inadequate methods of assessment (Turnbull, 1993).

33.6. The evaluation of effectiveness: the problem of outcome measures Evaluating the effectiveness of intervention implies the development of outcome measures (Table 32.7).

Outcomes should include measures of the effect of a treatment, across different dimensions: impairment, functional limitations, and disability, i.e. changes at home and at school (Lollar et al., 2000). Despite all the progress, measurement tools are still limited for the pediatric age, especially for children under 2 years of age.

33.7. Specific factors conditioning treatment procedures Among cerebral malformations, those involving cortical development are often highly epileptogenic, with the onset of epilepsy in the first year of life and drug-resistant seizures. Epilepsy surgery is a viable option for patients for whom adequate trials of medical therapy have failed. Surgical intervention at an appropriate time, whenever possible, offers the best chance for seizure freedom and improved cognitive outcome (Gupta et al., 2004). Cognition may be improved by the reduction of seizures and the decreased use of antiepileptic drugs (Loring and Meador, 2001). The results of postoperative disorders, e.g. cognitive deterioration and/or behavioral disorders, are controversial. Although several studies show surgery to be effective (Williams et al., 1998), about 10% of children undergoing surgical treatment display a significant decline in cognitive skills, while Devlin found neither postoperative significant cognitive deterioration nor loss of language, and behavioral improvement in 92% of those who had behavior problems preoperatively. The etiology of the lesion seems to play a major role in determining the outcome. With longer-term followup, only 47% of children with developmental abnormalities are reported to be still seizure-free in contrast to 77% of children with acquired abnormalities (Doring et al., 1999).

Table 33.7 Tools for measuring the functional outcomes Age of subjects

Target

Type of measure

Reference

Health Status Measure for Children (HSMC) Functional Status II-R (FS IIR) WeeFIM

4 domains

0–4 years 5–13 years

Parent questionnaire

Eisen et al., 1979

50 items

0–16 years


18 items/6 domains

Stein and Jessop, 1990 Msall et al., 1994

237 items/3 domains

6 months–8 years (nondisabled children)6 months–12 years (disabled children)All ages (with mental age less than 7 years) 6 months–7.5 years

To assess children’s heath related outcomes in core domains To assess parents’ perception of illness on their children To track development of functional independence in children with disabilities

Caregiver questionnaire

Haley et al., 1991

163 items/ 6 domains

11–17 years

To assess child’s functional skills and behaviors Specifically deals with health assessment in adolescents

Self-administered questionnaire

Starfield et al., 1993

50 items/14 domains

2 months–5 years 5–15 years


Landgraf et al., 1996

Questionnaire for Identifying Children with Chronic Conditions (QUICCC)

39 items/3 domains

1–18 years

Parent interview

Stein et al., 1997

POSNA Pediatric Musculoskeletal Functional Health Questionnaire ABILITIES Index

3 domains

2–18 years

Measures and compares health of specific groups of children and estimates benefits of specific treatments To identify children with chronic health conditions within the consequences of an underlying biological, psychological or cognitive disorder Measure outcomes of orthopedic interventions for children with musculoskeletal disorders Document the nature and extent of childhood disability across audition, behavior, intelligence, limbs, intentional communication, tonicity, integrity of health, eyes and structure domains To asses the functional motor skills of disabled children To assess QOL among healthy adolescents or with disabilities

Parent and adolescent self-report

Daltroy et al., 1998

Parent/caregivers questionnaire

Simeonsson et al., 1995

Therapist scoring

Russell et al., 2002

Self-report

Edwards et al., 2003

Pediatric Evaluation of Disability Inventory (PEDI) Child Health and Illness Profile – Adolescent Edition (CHIP-AE) Child Health Questionnaire (CHQ)

Gross Motor Function Measures Youth Quality of Life Instrument – Research version (YQOL-S)

9 domains

88 items/5 domains

5 months–16 years

76 items/4 domains

12–18 years

Observation/interview of/with caregiver

621

Items/domains


Tool

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As a consequence, children with cerebral malformations and drug-resistant epilepsy may have special needs in terms of rehabilitation treatment. In fact, their rehabilitation planning must be frequently re-evaluated and eventually modified taking into account the clinical evolution of epilepsy, the outcome of surgical intervention and the degree of modifiability of the clinical picture in response to treatment, in specific periods of time. There may be a time when rehabilitation treatment can only be addressed to stabilize the clinical status and to contrast cognitive regression related to frequent and long-lasting seizures (e.g. hemimegalencephaly, cortical dysplasia, neurocutaneous syndromes associated with epileptic spasms). At other times, particularly after obtaining seizure control, treatment may be directed at maintaining acquired abilities and favoring emerging abilities. In any case, it is necessary to provide parents with a specific definition of the goals of treatment, and to distinguish between ‘treatment’ and ‘management’ (Bax, 2003). Moreover, cerebral malformations are biological risk factors that put the children at increased risk for developmental disability, such as mental retardation, which is in turn associated with increased risk of psychiatric disorder. The analysis of the 1999 UK Office for National Statistics survey of the Mental Health of Children and Adolescents in Great Britain (Meltzer et al., 2000) suggests that rates for conduct disorders, anxiety disorders, attention deficit hyperactivity disorder/hyperkinesis and pervasive developmental disorders are higher among children with mental retardation than among their non-mentallyretarded peers. Among cerebral malformations, tuberous sclerosis shows the highest risk for autism

compared with other malformations (Curatolo et al., 2002, 2004). High rates of comorbidity have also been observed for cerebral malformations and hyperactivity/ inattention. These features have serious implications for the child’s relationships with parents, siblings and peers. A successful intervention should be aimed at educating the child and family about the disorder and to adopting psychosocial strategies that may help the family and school cope with the child (Shah et al., 2005).

33.8. Educational, cognitive/language, behavioral treatment: the need for guidelines 33.8.1. The evidence-based medicine approach The lack of consistency among studies on the definition of interventions and outcome measures, the involvement of different specialists speaking ‘different languages’, the largely heterogeneous training of therapists, makes the comparison of treatment difficult and limits the production of guidelines in support of specific interventions. To deal with these problems, the scientific community has produced and largely supported the ‘evidence-based’ approach, with the objective of helping clinicians in their daily clinical practice, through the development of a critical lecture of the scientific literature. Based on the assumption that the higher the appropriateness of the study design, the higher the quality of the results, Sackett (2002) proposed five levels of evidence (Table 33.8). The first two levels relate to the results of randomized clinical trials, which are considered the goal-standard design for the evaluation of treatment. Guidelines

Table 33.8 Sackett’s criteria: level of evidence Level

Description

Level I

Large randomized trials, producing results with high probability of certainty. These include studies with positive effects that show statistical significance and studies demonstrating no effect that are large enough to avoid missing a clinically significant effect Small randomized trials, producing uncertain results. These are studies which have a positive trend that is not statistically significant to demonstrate efficacy or studies showing a negative effect that are not sufficiently large to rule out the possibility of a clinically significant effect Nonrandomized prospective studies of concurrent treatment and control groups, e.g. cohort comparisons between contemporaneous subjects who did and did not receive the intervention Nonrandomized historical cohort comparisons between subjects who received the intervention and earlier subjects who did not Case series without controls. The clinical course of a group of clients is described, but no control is undertaken of confounding variables. This is a descriptive study that can generate hypotheses for future research but does not demonstrate efficacy

Level II

Level III Level IV Level V

EDUCATIONAL, COGNITIVE, BEHAVIORAL AND LANGUAGE DEVELOPMENT ISSUES should be based on levels of evidence in order to allow clinicians to assess to what extent they can trust the results of a study and thus choose the best treatment.

33.9. Conclusions The cognitive, language and educational treatment for children affected by brain malformations is complex and requires a multidisciplinary approach. Treatment can range from therapy to management according the severity of the clinical picture and type of disorder. The current knowledge on treatment efficacy is still limited, although a lot of progress has been made. A better integration of a functional approach with up-to-date information on genetics and natural history may lead to the identification of undetected deficits that are known to be associated with a specific genetic disorder or malformation, possibly leading to earlier and better treatment. A correct analysis of the child’s environment and the appropriate involvement of the family in sharing the therapeutic objectives are essential since the quality of life of both family and child represents the final goal of every rehabilitative process. In the near future, basic and applied research on treatment of brain damage, either maldevelopment or acquired lesions, should focus on neuroplasticity, neuroprotection and neurorehabilitation. In fact, rationale treatment of brain damage implies the protection of developing neural networks against insults and the enhancement of brain plasticity. Rehabilitative intervention may contribute to maximize recovery by modulating neuroplastic mechanism. To conclude, there is an urgent need of studying in detail biological mechanisms underpinning the processes of neuroplasticity, neuroprotection and neurorehabilitation, in order to provide the best treatment to the child with brain malformation.

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Index Pgae numbers in italics, e.g. 198, refer to figures. Page numbers in bold, e.g. 343, denote tables. n denotes a note. Abelson helper integration-1 (AHI1), 120–1, 521 mutations on chromosome 6 (JBTS-3), 523 abnormal neuronal and glial apoptosis, 392–4, 395 abortion, 79, 196, 249, 451, 453 acetyl-coenzyme A (acetyl CoA), 462, 470 acetylcholine, 543, 544, 598 acetylcholine receptor (AChR), 409, 413 acetylcholinesterase, 414, 542, 544, 545 acridine orange fluorochrome, 158, 542–3 adenohypophyseal (pituitary) placode, 259, 260, 262, 287, 288–9 adenohypophysis, 276, 288, 434 adenosine triphosphate (ATP), 462, 470 adenosine triphosphatase (ATPase), 422 aggression/aggressiveness, 141, 563, 564, 619 agranulocytosis, 560, 564 agrin, 220, 222, 227, 230, 414, 416 agyria, 167, 205, 206, 209, 210, 210–1, 219, 221, 223, 225, 227, 418 abnormal rhythmic scalp EEG, 511 anterior, 209 bilateral diffuse, 511 ‘classic agyria syndrome’, 205 D-2-hydroxyglutaric aciduria, 469 epilepsy due to cortical malformations, 402 imaging, 495 occipital, 221, 410, 415, 464t, 469, 495 posterior dominant, 200 typical diffuse, 167 AHI-1 (Abelson helper integration1), 120–1, 521, 523 Aicardi syndrome, 18, 76, 80–1, 121, 155, 392 associated with HME, 165 medical treatment of children, 557 X-linked, 495, 520 Aicardi-Goutires syndrome, 81 AIDS (congenital), 380–1 alisphenoid bone, 248, 281, 294, 295, 299, 329 alleles, 137, 201, 441 allelic loss, 136, 137 alopecia, 54–5, 61, 62, 62, 63, 162, 163, 165, 343, 345 altered cell cycle, 463, 467 alveolar arch, 250, 291 alveolar bone, 250 295, 310 alveolar process, 291, 308

alveolus, 291, 296, 305 Alzheimer’s disease, 181, 452 ambulation, 224, 413, 415, 416, 418, 595 amfetamine/amphetamine, 43, 45, 564 amino acid metabolism disorders, 468–9 amino acid proteins, 137, 197 amino acids, 264, 464t, 468, 470, 541–2, 544, 600 7 amino-acid duplication of threonine 44 to lysine 50 (residues 6–12 of homeodomain), 441 21–27 and 110–167 amino acids, 440 42–129 and 178–253 amino acids, 197 42–140 amino acid (R1 domain), 199 craniofacial clefts, 257, 314 Amish lethal microcephaly, 518, 522 amniocentesis, 18, 518, 584 amygdala, 6, 390, 433, 452, 453, 462, 491 amyoplasia, 7, 9, 411, 424 Andermann syndrome, 76, 80, 413, 518, 520, 521 anencephaly, 7, 9, 260, 286, 287, 298, 338 aneuploidy cerebral dysgeneses, 451 sex chromosome, 453–4 somatic, 452–3 Angelman syndrome, 455 angiomyolipomas (AMLs), 139, 142, 143, 145, 146, 148 anophthalmia, 28, 46, 75, 439, 440 anosmia (Kallman’s syndrome), 33, 260, 520 antibodies, 180, 180, 413, 546 antidiuretic hormone (vasopressin), 41, 49, 433 antiepileptic drugs (AEDs), 79, 533, 605, 620 clonazepam, 599 combination, 560 conventional versus new, 559–60 dosages, 559 effectiveness, 402–3 first-choice, 559, 560 mechanisms of action, 559 medical treatment of children, 557, 558 mood stabilizers, 562–3 new, 559–60, 561, 562 pharmacotherapy for seizures, 558–61 polytherapy, 560 pregnant mothers, 381 risks, 560–1 second-choice, 560 seizure freedom/control, 559, 560

antiepileptic drugs (AEDs) (Continued ) side effects, 561, 563 teratogenic drugs and toxins, 81, 381 titration schedule, 559 antioxidants, 422, 462 aorta, 354 dorsal, 317–8, 323, 354 ipsilateral dorsal, 255, 282, 283 aortic arches, 255, 282, 283, 307, 317, 323, 353 aortic stenosis, 343, 345, 454 Apert syndrome, 81, 574 apnea, 41, 97, 108, 119, 347, 348, 378, 581 apneic spells, 27, 97 ApoER2, 212, 215 apoptosis, 11, 26, 67, 68, 69, 70, 286, 301, 304, 494 apraxia, 78, 120 aqueduct of Sylvius, 22, 57, 98, 344t, 446 arachnoid, 238, 283, 446, 570, 578, 579 arginine, 49, 50, 434, 436, 440 arhinencephaly/arrhinencephaly, 9, 13, 44, 346 Aristaless-related homeobox protein (ARX gene), 75, 207, 209, 210, 211, 212, 215, 494, 522, 525, 613 arterial walls: congenital malformations, 359–61 fibromuscular dysplasia, 360–1 moyamoya disease, 359–60 arteries angular, 260, 270 anterior cerebral, 24, 45, 70, 71, 76, 77, 282, 298, 354, 359, 360 aortic arch, 255, 282 collicular and posterior choroidal, 366 congenital vascular malformations, 353–9, 360 extracranial, 354, 355, 355 hypoglossal, 355, 358 internal maxillary, 290, 293 intracranial, 354, 355, 355, 364 middle cerebral, 24, 70, 359, 360, 366, 379, 380 middle meningeal, 283, 357 occlusion, 71, 360 ophthalmic, 359, 360 otic/stapedial, 355, 357, 358 pericallosal, 71, 77, 366 posterior cerebral, 70, 71, 110, 354 renal, 360, 361 sphenopalatine, 261, 270, 290, 299, 307

628 arteries (Continued ) trigeminal, 353, 358 vertebral, 97, 356, 358, 359, 360 arteriography, 76–7 conventional, 355–6, 362, 365 conventional carotid, 357, 359 arteriovenous malformations (AVMs), 355, 363–4, 366, 367 arthrogryposis, 223, 424, 494 arthrogryposis multiplex, 229, 242, 413 Asn125Ser, 440, 440 ASPM gene, 522 astrocytes, 68, 540, 543, 544, 545 binucleated, 157 dysplastic, 131 fibrillary, 534 neuromere agenesis, 109 protoplasmic, 377 pyruvate carboxylase, 470 sex chromosome aneuploidy, 454 astrocytic markers, 158, 159, 546, 547 astrocytomas, 143, 147–8 ataxia, 4, 62, 97, 120, 121, 214, 347, 418, 423, 454, 455, 523, 561 cerebellar, 115, 116, 119, 122, 123, 424 congenital, 115, 116 nonprogressive congenital, 116, 122 truncal, 61, 121, 122 ATR gene, 522, 527 ATR-X gene, 527 attention-deficit hyperactivity disorder (ADHD), 141, 558, 564, 618, 622 autism, 109, 122, 123, 141, 146, 226, 399, 403, 468, 454, 527, 563, 564–5, 616, 619, 622 vermis hypoplasia and, 121 autistic spectrum disorders, 459, 563 autopsy, 53, 54–5, 57, 110, 153, 161, 164, 170, 180, 182, 191, 196, 235, 342, 347, 423, 534, 535, 550 autosomal dominant (AD) disorders, 495, 517 Apert syndrome, 81 cephalopolysyndactyly syndrome of Greig, 81 recurrence risks, 522n Rieger syndrome, 441 autosomal dominant inheritance, 347 521–2 autosomal dominant syndromes, 96, 495, 524, 527 autosomal dominant systemic disease tuberous sclerosis, 164–5 autosomal recessive (AR) disorders, 518, 519 acrocallosal syndrome, 81 cerebellar hypoplasia, 117, 122 congenital disorders of glycosylation, 123 hereditary motor and sensory neuropathy, 412–3 inborn errors of metabolism, 519 Joubert syndrome, 347

INDEX autosomal recessive (AR) disorders (Continued ) lissencephaly, 117, 209 Marden-Walker syndrome, 423 Nijmegen breakage syndrome, 494 autosomal recessive inheritance, 76, 119, 124, 341, 344, 521–2 autosomal recessive syndromes, 412, 526 autosomal recessive traits, 80, 81 axonal growth cones, 44, 69, 544 axonal pathfinding, 67, 68–9, 109 axonal terminals, 159, 534 axonal transport, 460, 534, 541, 543 axons, 47, 105, 135, 157, 180, 214, 220, 411, 433, 435–6, 437, 534, 535, 538, 546 callosal, 68–71, 76, 77–9, 413 callosal (aberrant projections), 74–5 collateral, 69–70 commissural, 46, 67 corticospinal tract, 121 motor or sensory, 409 myelinated, 415, 541 spinocerebellar, 106 wallerian degeneration, 83 baclofen, 565–6, 566, 599, 600 balloon cells, 396, 488–9 basal ganglia, 7, 15, 22, 23, 25, 48, 60, 71, 80, 81, 110, 111, 220, 346, 412, 461, 469, 539, 599, 612 basal plate, 93, 440 basement membranes, 220, 222, 230, 416 basioccipital bone, 90, 94, 95, 96, 252, 278, 279–80, 281, 283, 284, 298, 327–30 basipostsphenoid bone, 255, 283, 284, 297, 330f Bayley Scales of Infant Development (BSID-II), 30 Beckwith-Wiedeman syndrome, 242 Beemer-Langer syndrome, 342, 347 behavior aberrant/troublesome, 566, 618 atypical neuroleptics: mechanism of action, 564t automutilating/self-injurious, 62, 141, 564t, 563, 619 children with cerebral palsy, 605 obsessive-compulsive, 455 behavioral abnormalities/problems, 30, 62, 78, 140–1, 146, 192, 462, 615, 620 behavioral and psychiatric disorders, 399, 557, 558, 562–5 benzodiazepines, 170, 391, 559–60, 565–6, 599 Bergmann glial cells, 95, 222, 535–6, 543, 547 bioamines/biogenic amines, 435, 436 biopsy, 62, 413, 544, 569 muscle, 229, 412, 414, 415, 416, 419, 422, 423

biopsy (Continued ) skin, 62, 166, 171 sural nerves, 413 bipolar disorders, 141, 562–3, 564t birth weight, 455, 461 blastema, 290, 306 Blechschmidt model, 258, 272 blindness, 41, 43, 62, 119, 227, 418, 467 blood flow, 186, 363, 366, 437, 463, 492 blood oxygen level dependent (BOLD) signal, 186 blood supply, 250, 254, 261, 270, 272, 307, 434, 436 anatomy of aortic arches, 283 Carnegie staging system, 304 congenital vascular malformations, 353, 354, 359–60 blood vessels, 111, 142, 361, 379, 535, 536 cerebral, 220, 379, 410 cerebral (tortuosity), 466 embryonic, 353 intracranial or abdominal, 361 meningeal and choroid, 220 net-like, 359–60 rhombomeres, 290, 291, 292, 295 blood-brain barrier, 140, 220, 221, 463, 468, 565–6, 599, 602 bones, 249, 255, 259, 264, 269, 279, 280, 283, 288, 289, 301, 305, 306, 307, 310, 311, 330, 342, 445 bifid or abnormally ossified, 347 chondral, 290 craniofacial, 256, 282 lacrimal, 286 membranous, 263, 299 nonaxial, 254 occipital, 62 paraxial, 297 bone marrow failure, 123 bone morphogenic protein (BMP) family of genes, 7, 18, 20 BMP-2 gene, 410, 439, 441 BMP-4, 309, 310, 438, 439 BMP-7, 264, 439 botulinum toxin, 565, 597, 598–9, 601 brachycephaly, 61, 62, 452, 574, 576, 576 brain atrophy, 400, 464t, 469 brain development, 461, 462, 470 brain growth, prenatal/fetal, 461, 469 brain metabolism in fetal life, 459–68 critical periods of vulnerability, 459–60 effects of micronutrients deficiency, 461–2 brain plasticity, 611, 614 brain size, 452, 453 brain tissue, 240, 470, 493, 549, 544, 577 brainstem, 4, 23, 71, 89, 90, 92, 98, 107, 109, 110, 111, 123, 157, 209, 219, 227, 355, 368, 380, 411, 412, 420, 421, 433, 434, 437, 452, 455, 490, 535, 539, 540, 599

INDEX brainstem (Continued ) asymmetrical, hypoplastic, dysplastic, 346 fetal, 96, 410 flat, 223, 224, 226 hypoplastic, 228, 419 intrinsic dysgeneses (Chiari malformations), 95–6 brainstem dysfunction, 33, 578, 579 brainstem dysplasia, 80, 423 brainstem hypoplasia, 224, 225, 228, 380, 423, 471 bromodeoxyuridine (thymidine analog), 182 Brun: subpial glial layer, 27, 536 bundle of Probst, 68, 72–3, 76, 76, 77, 79

Cajal-Retzius cells, 215, 545 Cajal-Retzius neurons, 69, 539, 540, 542 calcification, 130, 131, 133, 143, 165, 166–8, 237, 363, 367, 484 cerebral, 482 cerebral cortical gyri, 380 dystrophic, 158, 379 intracerebral, 379, 469 intracranial, 162, 526 right occipital periventricular, 495 calcium antagonists, 359, 362 callosal dysgenesis, 39, 44t, 46, 47, 81 callosal plate, 9, 69, 70 calmodulin, 538, 541 calretinin, 196, 538, 541, 542, 543 calvaria, 7, 14, 94, 260, 283, 285, 297 capillary hemangiomas, 55, 362, 363 carbamazepine, 170, 402, 559–61, 562–3, 605 cardiac defects/dysfunction, 28, 141, 144, 183, 345, 366, 367 cardiac rhabdomyoma, 129, 142, 144, 146 cardiomyopathy, 416, 418, 419, 421, 422, 470 Carey-Fineman-Ziter (CFZ) syndrome, 411, 421, 423–4 Carnegie staging system (embryology), 302–5, 309 carnitine palmitoyl transferase type II (CPT2), 463, 465, 470–1 carotid artery, 355–8, 358, 360, 361, 366, 444 cartilage, 111, 249, 264, 280, 281, 285, 289, 297, 301, 307, 438 cranial base formation, 298, 299 nasal, 260, 263, 287, 288, 344t, 348, 577 quadrate, 281, 295 rhombomeres, 292, 295, 296 thyroid, 296, 326 catabolism, 459, 469 cataracts, 47, 119, 223, 224, 227, 345, 424, 495 catecholamines, 379, 463, 543 caudate nuclei, 6, 16, 17, 22, 25, 68, 110, 122, 424, 472, 484, 539, 544

cavum septum pellucidum, 42, 206, 454 cell cycle, 467, 468, 536, 538 cell differentiation, 137, 138, 439 cell membranes, 493 formation, 461 phospholipid synthesis, 470 properties, 471 cell migration, 45, 105, 132, 133 cell proliferation, 134, 136, 137, 138, 213, 254, 468, 479 cellular lineage, 6, 10, 171, 536, 537, 538–9, 546 cellular maturation, 280 markers in nervous system, 536–47 cellular pleomorphism, 131–2, 165 cephaloceles, 576–8 occipital, 219, 576 posterior, 98 cerebellar agenesis, 116, 117, 118 subtotal, 124 usage, 118 cerebellar aplasia, 9, 106, 118 cerebellar atrophy, 122, 123, 226, 229, 412, 413, 455, 464, 519, 527 cerebellar cortex, 4, 157, 467, 535, 538, 539, 540, 542, 544, 547 Chiari malformations, 95 craniofacial clefts, 250, 251 imperfect lamination, 109 neuromere agenesis, 109 cerebellar cortical abnormalities, 59, 123, 541 cerebellar dysgenesis, 344, 346, 347, 521 cerebellar dysplasia (CB-DYS), 179, 464–6 cerebellar hemispheres, 7, 8, 63, 95, 116, 118, 122, 124, 369 cerebellar hypoplasias, 115–27 cerebellar agenesis, 118 clinical manifestations, 115–6 development of cerebellum, 116–7 facial hemangioma, 122–3 familial, 116, 121, 122, 124 global cerebellar hypoplasia, 122–3 pontocerebellar hypoplasias, 123–4 prominent midline (vermis) involvement, 118–21 terminological confusion, 118–9 cerebellar tissue, 91, 107 cerebellar tonsils, 89, 90, 95, 98, 439, 443, 446, 578, 579 cerebellar vermis, 7, 8, 43, 57, 58, 90, 98, 119, 228, 346, 347, 453, 454 hypoplasia, 116, 117, 118, 121, 122, 124, 211, 229, 423, 472, 521, 523 cerebello-ocular-renal syndromes (CORS), 115, 119–21, 523 cerebello-trigeminal-dermal dysplasia (Go´mez-Lo´pez-Hernańdez syndrome), 54–5, 61–3, 64 cerebellum, 4, 10, 27, 57–8, 63–4, 89, 90, 91, 97, 107, 109, 130, 157, 197, 206, 220, 222, 223, 226, 230, 367,

629 380, 411, 412, 446, 452, 455, 470, 471, 481, 524, 534, 535, 539, 540, 542, 546, 599 development, 116–7 intrinsic dysgeneses (Chiari malformations), 95–6 cerebral angiography, 76–7 cerebral aqueduct, 92, 93, 94, 95, 380, 381 cerebral atrophy, 411, 412, 413, 465, 455, 468 cerebral cavernous malformations (CCMs), 367–9 cerebral cortex, 4, 6, 8, 26, 71, 77, 78, 154, 205, 377, 433, 463, 467, 540, 542, 543, 545, 546, 599 Chiari malformations, 96, 97 craniofacial clefts, 259 fetal hydrocephalus, 381 lissencephalies, 205, 525 schizencephaly, 238, 239 cerebral cortical architecture: disorders, 177–246 cerebral dysgeneses, 377, 402, 467–8, 472, 533, 548 genetic disorders, 423–4 cerebral dysgeneses associated with chromosomal disorders, 451–8 cerebral hemispheres, 44, 45, 129, 228, 235, 236, 304 abnormalities, 443 normal, 68 posterior, 77 cerebral malformations, 219, 410–1, 425, 496, 591 cerebral mantle (brain tissue), 43, 132, 235, 236, 378, 380, 381 cerebral palsy, 27, 243, 377, 389, 520, 591, 592, 604, 614, 617, 618 determinants of motor change, 593 neurorehabilitation of children, 591–609 pharmacotherapy for spasticity 565–6 cerebral vasculature, 353, 357 cerebrospinal fluid (CSF), 445, 518, 540–1, 547–8, 586 Chiari malformations, 90, 92, 96 hypothalamic-pituitary unit, 434, 436 cerebrovascular anomalies, 363–70 arteriovenous malformations, 363–4 cerebral cavernous malformations, 367–9 vascular malformations of meninges, 369–70 cerebrum, 83, 130, 260, 346, 411, 443, 534 cesarean section, 90, 161, 580, 586 Chiari malformation, 89–103 clinical correlates, 96–8 diagnosis, 96–7 microscopic findings, 95–6 pathogenesis: mechanical and hydrodynamic theories 89–90, 98

630 Chiari malformation (Continued ) pathogenesis: molecular genetic theory, 89, 90–6, 98 treatment, 97–8 Chiari I malformations, 89, 94, 96, 97, 98, 164, 168, 444, 446, 523, 570, 574, 578–80 Chiari II malformations, 9, 55, 60, 79, 89, 90, 92, 92–6, 97, 98, 548, 581, 585, 586 cholesterol biosynthesis, 18, 21 defects, 465, 471, 527 cholesterol hypothesis, 21–2 choline acetyltransferase, 542, 544 chondrocranium, 90, 91, 94, 326 choreoathetosis, 27, 32, 469 choroid plexus (‘butterfly sign’), 26, 95, 446 chromaffin cells, 534, 540, 543 chromatography-electronspray ionization mass spectroscopy, 472 chromosomal aberrations/disorders, 18, 75, 78, 96, 115, 494, 520, 527, 585 cerebral dysgeneses, 451–8 CNS malformation syndromes, 518 macrocephalies, 527 ring arrangements, 18 chromosome 1p36 microdeletion syndrome, 518, 526 chromosome 22q11 microdeletion syndrome, 518, 523, 526 chromosome 22q13 deletion syndrome, 518, 527 chromosome X, 96, 154, 181, 197, 214, 345, 453, 454, 493, 495, 584 chromosome deletions, 81, 119, 611 1p36, 518, 526 3q24–3q25.33, 119 7q36, 518, 520 9q22.3, 520 13q32, 520 15q11-q13, 455 18p11.3, 520 22q11, 518, 523, 526 22q13, 518, 527 hemizygous (incomplete), 107, 612 chromosome duplications, 18, 81, 119, 494, 518, 520 chromosomes: disorders of number 452–4 sex chromosome aneuploidy, 453–4 somatic aneuploidy, 452–3 chromosomes: disorders of structure, 454–6 circle of Willis, 354, 354, 359, 361, 362 cleft formation four-D theory, 248 integrative theory (Carstens), 301 pathological anatomy, 309–10, 334–5 cleft lip, 81 cranial base formation (frontoorbitosphenoid model), 299 craniofacial clefts, 247, 248, 249, 251, 264, 270

INDEX cleft lip (Continued ) malformation associated with HPE, 28, 29, 33 rhombomere 20 : premaxilla and vomer, 290, 291 cleft palate, 81, 247, 248, 251, 288, 290, 299 chromosome structure (disorders), 454 cranial base formation, 299 pathological anatomy, 309, 310 clinical syndromes: cortical malformations, 391–8 abnormal neuroblast migration, 394–6 abnormal neuronal and glial proliferation or apoptosis, 392–4, 395 holoprosencephaly, 397–8 clonazepam, 56, 61, 605 CNS (central nervous system) craniofacial clefts, 251, 252, 255, 271, 278, 281, 305 fetal hydrocephalus, 381 inborn errors of metabolism, 519 LIS type II, 220, 224, 226, 227, 229 multiple expression of genetic defect, 410 rostral rhomboencephalic derivatives, 292 serine-deficiency syndrome, 468 sex chromosome aneuploidy, 454 vascular malformations, 363 CNS: acquired induced, and secondary malformations, 377–85 congenital infections, 378–81 ischemic/hypoxic infarcts in fetal brain, 377, 378 teratogenic drugs and toxins, 381, 382 CNS abnormalities/anomalies, 347, 413, 424, 445, 451, 527 CNS defects/disorders, 154, 424, 425 CNS malformations, 78, 420, 469, 518, 614 embryology and neuropathological examination, 533–54 teratogenic drugs and toxins, 381 CNS malformations: comparative manifestations, 387–476 epilepsy in patients with cerebral malformations, 389–407 neuromuscular disorders associated with cerebral malformations, 409–31 CNS malformations: management, 555–625 educational, cognitive, behavioural, and language development issues, 611–25 medical treatment: children, 557–68 neurorehabilitation of children with cerebral palsy, 591–609 CNS malformations: neuroendocrine complications, 433–50 anterior pituitary gland: development, 437–9

CNS malformations: neuroendocrine complications (Continued ) auxological and endocrinological abnormalities in children with hydrocephalus and spina bifida, 444–5, 446 brain abnormalities: link with endocrine diseases, 439–43 endocrine function: CNS control, 436–7 endocrinopathies associated with midline cerebral and cranial malformations, 443–4 CNS malformations: neuromuscular disorders, 412–20 hereditary motor and sensory neuropathy, 412–3 peripheral neuropathy with agenesis of corpus callosum, 412–3 CNS malformations: other myopathies, 421–4 genetic disorders, 423–4 metabolic disorders, 421–3 CNS malformations: surgical treatment, 569–90 cephaloceles: excision, 576–8 hydrocephalus and CSF-related disturbances, 569–73 surgery for dysraphic state, 580–3, 584 COACH syndrome, 121, 347 Coffin-Siris syndrome, 520 cognitive defects/disorders, 8, 115, 119, 120, 122, 123, 140–1, 192, 381, 400, 403, 423, 468, 493, 564, 615 Cohen syndrome, 523 colliculi, 7, 8, 53, 59, 67, 78, 95, 107, 109, 250 colobomas, 28, 80, 119, 121, 224, 227, 310, 344, 520 COMA (congenital oculomotor apraxia), 121, 121 combined pituitary hormone deficiency (CPHD), 439 condyles, 257, 274, 284, 295–6 congenital arterial anomalies, 354–5, 356 congenital disorders of glycosylation (CDGs), 411, 422–3, 472, 519 congenital fibrosis of extraocular muscles (CFEOM), 411, 423, 424 congenital infiltrating lipomatosis of face (CILF), 155, 163 congenital muscle fiber-type disproportion (CMFTD), 421 congenital muscular dystrophies (CMDs/ MDCs), 115, 124, 411, 414–20, 535 Fukuyama-type, 414, 416, 417–8 LIS type II, 219, 222, 227, 229 MEB disease, 414, 416–8 muscular dystrophy, 410, 411, 424 Walker-Warburg syndrome, 414, 418, 420–1 congenital muscular dystrophy: due to dystroglycanopathies, 411, 416–9, 420–1

INDEX congenital muscular dystrophy: type 1A (MDC1A), 219, 220–2, 410, 411, 414–5, 416, 418 congenital muscular dystrophy: type 1B, 414, 420 congenital muscular dystrophy: type 1C, 222, 230, 411, 414, 416 brain involvement, 220, 226–7 congenital muscular dystrophy: type 1D, 220, 222, 228, 411, 414, 416, 419 congenital oculomotor apraxia (Cogan) (COMA), 121, 121 congenital vascular malformations in childhood, 353–75 cerebral arteries: absence, 358–9 congenital arterial anomalies, 354–5, 356 embryological development, 353–4 contractures, 123, 412, 415, 416, 418, 421, 423, 424, 597 corneal clouding, 54–5, 61, 62, 62 coronoid process, 295, 307 corpus callosotomy, 80, 83, 200–1, 403 corpus callosum, 4, 6, 41, 43, 59, 130, 132, 156, 162, 165, 167, 168, 170, 183, 206, 207, 208n, 209, 211, 220, 225, 225, 228, 237, 239, 344, 380, 412, 417, 419, 422, 424, 480, 535 absence, 14, 14–5, 418, 440 body, 15, 16, 80, 82 chromosome structure (disorders), 454 dysgenesis, 42, 60, 95, 119, 419, 469, 470, 471, 518 embryology, 68–70, 71–3 genu, 15, 15, 25, 69, 70, 453 hypoplasia, 75, 79, 96, 99, 225, 381, 423, 468, 471 rostrum, 69, 73, 77–80, 82 splenium, 15, 15–6, 25, 26, 69, 70, 71, 74, 77, 79, 82 vascular supply, 70–1, 74 corpus callosum: agenesis, 10, 45, 47, 68, 72, 156, 162, 211, 346, 420, 439, 453, 465–6, 470, 518 accompanying SOPD, 39 brain malformations: genotypephenotype correlations, 612 clinical presentation, 78–81 genetic testing and counseling, 520, 521, 523 hereditary motor and sensory neuropathy, 413 imaging, 480, 481, 494, 495 inborn errors of metabolism, 519, 527 neuroanatomical appearance of brain, 76–8 partial, 73, 79, 81, 82, 132, 412, 412, 422 teratogenic causes, 520 total, 74, 78, 79, 81, 83 cortex, 3, 27, 60, 78, 130, 135, 136, 139, 157, 166, 179, 183, 195, 205, 207, 214, 222, 228, 237, 244, 377, 399, 433, 485, 496, 508, 535

cortex (Continued ) cortical malformations, 392 dysgenetic, 240, 243 dysplastic, 24, 166, 237, 243 frontal, 392, 493 imaging, 480, 482, 488, 490, 491, 492, 493, 494, 496 nutritional deficit hypothesis, 462 visual, 77, 614 cortical abnormalities, 179, 183, 511 cortical cytoarchitectural disorganization, 130, 132 cortical development: malformations, 182, 403 cortical dysgenesis, 165, 237, 240 cortical dysplasia, 23, 25, 27, 43, 45, 132, 137, 157, 167, 168, 169, 227, 229, 389–92, 394, 402, 424, 491, 506, 507, 527 antiepileptic drugs (effectiveness), 402, 403 behavioral and psychiatric disorders, 562 factors conditions treatment procedures, 622 hemispheric or diffuse epileptiform discharges, 510 without balloon cells, 396, 398 cortical heterotopia (C-HT), 465–6 cortical malformations, 31, 181, 205, 210, 215, 238, 244, 403 clinical neurophysiology, 503–16 effect of uncontrolled seizures, 399–401 imaging, 479–502 intractable epilepsy, 389–90 cortical mantle, 235, 243, 396, 421, 488, 584 cortical plate, 10, 69, 133, 181, 196, 197, 213, 378–9, 534, 535, 539, 542, 544 cortical thickness, 206, 485 corticospinal tract, 67, 69, 121, 240, 411, 417 corticotroph cells, 434–5, 442 corticotropin-releasing hormone (CRH), 49, 435, 436 Couly-Le Douarin model, 300–1 Cowden syndrome, 155, 521, 524 cranial base, 94–5, 297–301 cranial malformations, 443–4 cranial vault, 96, 523, 573 cephaloceles, 576–7 remodeling, 574, 575 cranial volume: disorders, 526–7 craniectomy, 574, 578 cranio-cerebello-cardiac (3C) syndrome, 123 craniocaudal pattern formation, 258–9 craniofacial clefts neural tube programming and pathogenesis, 247–76, 277–339 Tessier’s classification, 331

631 craniofacial development, 18, 110, 257, 291, 535 craniofacial developmental fields: assembly, 301–10, 331–2, 334–7 connecting cranium with face, 307–8 cleft formation: pathological anatomy, 309–10, 334–5 craniofacial development: major themes, 305–7 oronasal soft tissues: assembly, 308–9 craniofacial malformations/anomalies, 26, 27, 28–30 craniofacial mesoderm: anatomy, 277–84 paraxial mesoderm: non-pharyngealarch, 283–4 paraxial mesoderm: somitomeres and somites, 277–81, 317–22, 324, 326–30 pharyngeal arches: vascularization, 282–3, 323, 325 craniofacial neural crest: anatomy, 284–96, 314–6, 337–9 craniofacial repair for craniofacial dysmorphism, 570, 573–6 fronto-orbital advancement and remodeling, 574–6 craniofacial structures, 11, 78, 94, 247–8 craniopharyngeal canal, 439, 443 craniosynostosis, 54–5, 62, 229, 260, 573–4 cranium, 167, 271, 307–8 creatine kinase, 229, 414, 422, 423 curvilinear multiplanar reformatting, 482, 485 cyclopia, 13, 21, 28, 29, 77, 285, 288, 298, 339 cytoarchitectural dysplasia, 488, 506 cytochrome P450 enzymes, 460 cytogenetics, 18, 27, 213, 454, 518, 520, 523, 526 cytomegalovirus (CMV), 378–9 cytomegaly, 137, 390 cytoplasm, 19, 106, 159, 538, 541, 543, 545, 547, 548, 549

Dandy-Walker malformation (DWM), 7, 24, 43, 60, 63, 80, 109, 118–9, 122, 123, 344, 346, 381, 423, 465, 480, 481, 518, 523, 583, 584 dantrolene, 565–6, 566, 599–600 Dekaban-Arima syndrome, 121, 347 dendrites, 26, 69, 83, 135, 157, 158, 159, 180, 214, 391, 534, 537, 538, 545, 546, 573 dendritic arborization, 26, 78, 130, 455 dendritic spine morphology, 452, 454 dentate nuclei, 53, 57, 58, 109, 117, 228, 464, 470, 539, 540 dermatome, 105, 255, 256, 281, 300, 317, 320 dorsal, 252 lateral, 280 ventral, 253

632 dermis, 155, 249, 259, 260, 264, 280, 285, 286, 287, 306, 309 epaxial, 300 neuromeric origin, 300 somite-transformation into, 252 dermomyotome (cervical neuromere c2 and below), 318, 320 developmental anatomy, 247, 249, 251 developmental delays abnormal neuroblast migration, 396 brain malformations: genotypephenotype correlations, 612 cerebellar hypoplasias, 115, 116, 117, 119, 122, 123, 124 cortical dysplasia without balloon cells, 396 environmental toxin hypotheses, 467 global, 160, 494 imaging cortical malformations, 479 imaging of genetically determined malformations, 494 developmental disorders, 30, 391, 472, 614 diabetes, 21, 424, 461, 467, 520 diabetes insipidus (DI), 31, 33, 41, 46, 50, 444 diabetes mellitus, 45, 54–5, 60, 61, 467 molecular genetic testing and genetic counseling, 517–31 diencephalon/thalamus, 6, 9, 76, 108, 109, 153, 287, 288, 315, 438, 442 basal, 289 caudal, 259 dorsal, 107 floor of, 438 thalamo-cortical radiations, 239 thalamus, 7, 13, 16, 17, 22, 25, 60, 67, 80, 197, 220, 259, 380, 434, 452, 454, 472, 484, 539, 540 diet, 460, 462, 468, 471, 472, 565 diffusion tensor imaging (DTI), 23, 485, 493 DNA (deoxyribonucleic acid), 145, 157, 199, 200, 201, 214, 237, 256, 264, 345, 378n, 379, 441, 443, 494, 518, 549 DNA-binding, 46, 109, 257, 440–1 dopamine, 435, 436, 461, 466, 563 dopamine-antagonist, 563, 564 doublecortin (DCX) gene, 10, 76, 191, 196–9, 214, 422 Down syndrome (trisomy-21), 46, 328, 451, 452, 454, 494 drop attacks, 392, 396, 403, 562 Duchenne muscular dystrophy, 223, 410, 411, 416 duraplasty, 579, 580, 581, 582 dysembryoplastic neuroepithelial tumors, 10, 395, 392, 394 dysequilibrium syndrome (Hutterite type), 522 dysmorphism (craniofacial), 5, 9, 111, 413, 468, 570, 573–6

INDEX dysmyelination, 157, 225, 416, 418, 465, 471, 490 dysphagia, 97, 378, 603 dysraphism occult spinal, 581–3, 584 dystonia, 27, 32–3, 122, 469, 596, 597, 599, 605 dystroglycan (glycoprotein), 220, 230, 414, 416 dystrophin gene (chromosome Xq21.2), 411

echocardiography, 144, 146, 348 ECoG (electrocorticography), 399, 503, 504, 509 ectoderm, 9, 63, 155, 249–51, 255, 258–9, 264, 272, 275, 280, 285, 287, 300, 304, 306, 307 ectopic expression, 9, 91, 92, 93, 98, 107, 109, 110, 111 Edwards syndrome (trisomy 18), 18, 81, 96, 119, 452–3, 494, 520 Ehlers-Danlos syndrome, 179, 183, 360 electrocerebral maturation, 399, 400–1 embryos, 7, 11, 105 assembly of face, 302 bilaminar, 278 Carnegie staging system, 302–3 congenital vascular malformations, 353, 354 craniofacial clefts, 250, 252, 254, 255, 258, 261, 262, 264, 269, 271, 273, 277, 278, 279, 311, 316, 323 craniofacial development, 305 crown-rump length, 302, 303–5 dorsal and ventral sectors, 279 generalized vertebrate, 316 neural axis, 250 neuromere agenesis, 105 neuromeric organization, 271 trilaminar, 278, 302, 305 embryogenesis, 136, 183, 248, 249, 257, 290, 302, 308, 328, 338, 409 embryology and neuropathological examination of CNS malformations, 533–54 encephaloceles, 24, 89, 97, 98, 225, 227, 228, 286, 418, 576 encephalocraniocutaneous lipomatosis, 155, 165 encephalopathy, 81, 356, 359, 389, 422, 425, 463, 468 endocrine abnormalities/disorders, 42, 49, 453, 520 endocrinopathies, 27, 31–2, 443–4 endoderm, 254, 255, 269, 272, 278, 304, 306, 315, 317 endomeres (endoderm developmental zones), 278 endomysial connective tissue 414, 424 endoscopic third ventriculostomy (ETV), 569, 580, 581, 585

endothelial cells, 93, 137, 158, 353, 362, 363, 378–9, 381, 536 environmental toxin hypotheses, 467–8 ependyma, 7, 94, 98, 108, 158, 378, 470, 535, 545, 546, 548 ependymal abnormalities, 93–4, 95, 98, 471 ependymal cells, 32, 92, 92–3, 111, 158, 159, 380, 537, 538, 543, 544, 547, 549, 550 epiblasts, 254, 258–9, 269, 272, 278, 301, 409 epidermal nevus syndrome (ENS), 10, 490 HME, 155, 160, 161, 164, 167, 170, 171 neurocutaneous syndrome associated with HME, 162–3 epilepsia partialis continua, 161, 169, 394, 509 epilepsy, 3, 4, 8, 79, 377, 496, 542, 591, 604 abnormal neuroblast migration, 395, 396 absence seizures, 599 age on onset, 192, 193, 241, 509 antiepileptic drugs, 403 atonic, 200, 241, 396–7, 599 brain malformations: genotypephenotype correlations, 612 cerebellar hypoplasias, 122, 123, 124 Chiari malformations, 97 cortical malformations, 392, 402 drop attacks, 83, 193, 200–1 early-onset severe, 612 effect of uncontrolled seizures, 400 environmental toxin hypotheses, 467 extratemporal, 389, 390, 486 focal seizures/focal epilepsy, 177, 183, 241, 243, 392, 393–4, 395, 396, 480, 486, 490, 491 generalized, 83, 193, 400, 402, 560, 561 imaging, 479, 491, 493, 496 infantile spasms (drug choice), 560 medical treatment of children, 557, 558 multifocal, 184, 392 myoclonic, 80, 122, 170, 193, 241, 469, 510, 599 neurorehabilitation, 604–5 partial, 170, 193, 400, 402, 480, 487, 507, 561, 604–5 polymicrogyria, 396–7 schizencephaly, 239, 240–2, 243, 244 seizure freedom, 487, 505, 559, 620 seizure frequency/type, 557 semeiology, 183–4 simple/complex partial, 193, 395 symptomatic generalized, 193, 399 temporal, 389, 490 tonic/tonic-clonic, 83, 183, 193, 241, 392, 395, 396–7 epilepsy: refractory/intractable, 24, 31, 83, 140, 161, 163, 167, 170, 177,

INDEX 182–3, 183–4, 186, 193, 207, 241, 243, 244, 367, 389, 390, 395–8, 403, 485, 486, 507, 508, 534, 612, 620, 622 epilepsy in patients with cerebral malformations, 389–407 epilepsy surgery, 184, 186, 620 epileptiform discharges, 391, 503, 506, 509–11 cortical malformations, 393–4 EEG features associated with cortical malformations, 399, 401 effect of uncontrolled seizures, 399, 400 imaging epileptogenic networks, 492 interictal, 492, 505 multifocal and generalized, 399 epileptogenesis, 74, 137, 181, 196, 391, 494 epithelia, 249, 253, 255, 257, 260, 277, 306 Carnegie staging system, 304 cleft formation: pathological anatomy, 310 connecting cranium with face, 308 cranial base formation (frontoorbitosphenoid model), 299 developmental fields: biological basis, 301 nasal, 286, 287, 288 oral, 300, 440 ethmocephaly, 28, 28 ethmoid, 249, 279, 293, 301 lateral structure, 289 medial structure, 289 orbital lamina of, 286 perpendicular plate, 248, 250, 260, 262 posterior, 577 prosomere zone p5, 286 ethmoid bone, 261, 285, 329, 344 ethmoid complex, 263, 332, 338 ethmoid labyrinth, 260, 286, 287 ethmoid sinuses, 286, 289 exoccipital bone, 94, 252, 278, 279–80, 281, 284, 298, 327–8 exons, 137, 138, 197, 199, 213, 214, 215, 440, 442, 443 eyelids, 55, 300, 310, 338, 369 eyes, 5, 17, 41, 205, 285, 299, 343, 379, 411, 416, 523 abnormalities/malformations, 61, 96, 97, 122, 219, 418 craniofacial clefts, 276, 323, 325 malformations associated with HPE, 28, 29 Rieger syndrome, 441

face asymmetry, 160 connection with cranium, 307–8 embryology, 249 neuromeric origin of facial soft tissues, 300

facial angiofibromas, 141, 143, 144, 147 facial dysmorphism, 54–6, 81, 183, 207, 526, 533, 574 facial hemangioma, 119, 354, 355, 356, 359, 369 and cerebellar hypoplasia, 122–3 fatty acid oxidation disorders, 463, 469, 470–1 feeding difficulties, 81, 79, 97, 124, 207, 414, 603 Feingold syndrome, 527 fetal alcohol syndrome, 462, 520 fetal surgery, 570, 586 fetuses, 26, 69, 71, 78, 535, 549, 550 anencephalic, 338 brain metabolism, 459–68 cerebellar hypoplasias, 117 cerebral dysgeneses secondary to metabolic disorders, 459–76 Chiari malformations, 93 congenital vascular malformations, 356 Joubert-like syndromes, 121 metabolic disturbances (effect on maternal health), 460 neuromere agenesis, 107, 111 toxoplasmosis, 380 fiber necrosis, 414, 415, 424 fibroblast growth factor-8, 106, 107, 110, 117, 259, 261, 264, 316, 438, 439 fibroblasts, 93, 97, 540 filamin-1 (or filamin-A) protein, 10, 181, 182, 183, 521 floor plate, 8, 22, 92, 93, 215, 409, 544 fluid-attenuated inversion recovery (FLAIR), 131, 480, 482, 483–4, 495, 504 fluorescence in situ hybridization (FISH), 209, 213, 454, 455, 525, 526, 527 focal cortical dysplasia (FCD) abnormal rhythmic scalp EEG, 512 advanced MRI, 485 associated with SOPD, 42 cortical malformations, 392, 402, 403 epilepsy, 391, 402 epilepsy due to cortical malformations hemispheric or diffuse epileptiform discharges, 509, 510 imaging, 488–9, 491, 492 intracranial EEG, 506–7, 507–8 Taylor-type, 488, 492, 548 focal dysplasia, 4, 55, 133, 402, 411, 562 focal hypoplasia, 79, 81 folic acid, 93, 96, 383, 462 follicle-stimulating hormone (FSH), 49, 435, 443 foramen magnum, 89, 90, 91, 95, 97, 98, 252, 257, 279, 283, 327, 329, 356, 358, 446, 578, 581 forebrain commissures: embryology and malformations, 67–87 foregut endoderm (FGE), 258, 263, 264, 301, 307

633 fornices, 48, 74, 79, 434 fossae, 108, 120, 121, 347, 523 anterior, 307 cranial, 281, 283, 291, 574 fragile X syndrome, 453, 454, 527 frenula, 21, 343, 344, 347, 348 bifid, 308 hypertrophic, 341 labial, 28 oral, 342 follicle-stimulating hormone (FSH), 49, 435, 443 fukutin gene (on chromosome 9q31), 10, 220, 222, 223, 227, 410, 416, 522, 525 fukutin-related protein (FKRP), 220, 222, 226, 227–8, 230, 418 Fukuyama congenital muscular dystrophy (FCMD), 10, 222–3, 411, 414, 416, 417–8, 495, 548 functional MRI (fMRI), 186, 196, 240, 480, 483, 492

GABA (g-amino butyric acid), 147, 543, 544, 554, 558, 565–6, 599 gabapentin (GBP), 403, 559–60 gadolinium, 131, 134, 186, 364, 369, 482 gangliocytomas, 392, 394, 524 gangliogliomas, 10, 186, 390, 392, 394, 490 ganglia, 316, 318, 321–2, 424, 544 autonomic, 249 dorsal root, 409, 539 ganglion V, 315 ganglion cells, 44, 46, 47, 409, 443, 540, 545 ganglionic eminences, 110, 197, 215 gene mutations, 196, 465, 497 cerebellar hypoplasias, 117 heterozygous, 443, 612 Joubert syndrome, 121 genes, 9–10, 10n, 117 associated with CNS malformation syndromes, 521–2 brain malformations: genotypephenotype correlations, 612–3 cerebellar hypoplasias, 117 cerebral dysgeneses, 451 Chiari malformations, 92 craniofacial clefts, 250, 255, 256, 279, 311 defective, 4, 111 developmental, 64, 155 etiological, 171 expression and gradients, 6 extracellular signaling molecules, 256 homozygous deletions, 523 imaging of genetically determined malformations, 493 intracellular transcription factors, 256 missense mutations, 21 mitogenic, 154 mutation (identifiable), 17

634 genes (Continued ) neuromere agenesis, 111 non-Hox, 255 overexpression, 4 overlapping expression, 10 pituitary development, 438 posterior fossa: malformations, 523 re-expression, 10 transcription factors, 64 tumor-suppressor, 171 genetic counseling, 4, 348, 472, 480, 493, 517–8, 519, 602 identifying nongenetic causes, 383 imaging of genetically determined malformations, 493 genetic mechanisms, 45–7, 353 genetic programming, 3, 98, 105, 249, 290 genetic syndromes, 18, 80–1, 411 genetic testing, 145, 200, 425, 517–8, 519 genotype-phenotype correlation, 20–1, 520, 612–3 germinal matrix, 177, 377, 538, 539, 542, 545 germline mosaicism, 144, 145, 517 germline mutations, 518, 524 giant cell tumors, 133, 146 giant cells, 131, 132, 135, 136, 137, 139, 391 glaucoma, 224, 227, 369, 561 glia limitans, 220, 222, 223, 228, 230, 415 glial cells, 70, 95, 158, 177, 379, 381, 468, 490, 537, 538, 538, 540–3, 545, 546, 548, 550 energy metabolism, 470 subependymal primitive, 68 glial fibrillary acidic protein (GFAP), 92, 94, 135, 157, 170, 196, 535, 537, 538, 546, 547, 548 astrocytic marker, 159 ependymal distribution, 93 glial nodules, 470, 524 glioblasts, 10, 68, 534, 536, 547 gliomas, 480, 487, 491, 505 gliosis, 8, 58, 59, 109, 157, 165, 239, 380, 412, 465, 470, 482, 483 globus pallidus, 17, 110, 472, 539, 540 glutamate, 169, 391, 470, 490, 544, 554, 561 glutaric acidemia type 1 (GA-1), 464, 469, 471 glycine, 471, 544, 554, 600 glycogen, 158, 422, 459, 466n, 549 glycoproteins (LIS type II), 220, 222 glycosylation, 115, 123, 124, 219, 223, 229, 416, 465, 472 disorders, 423, 527 N-glycosylation disorders, 423 N-glycosylation pathway, 123 N-linked, 472 glycosyltransferases, 222, 223, 226, 228, 410, 419 Goldberg-Schprintzen syndrome, 522, 526 Golgi cells/zones, 135, 138, 422–3, 542

INDEX Golgi impregnations, 26, 78, 157, 534 Go´mez-Lo´pez-Hernańdez syndrome, 54–5, 61–3, 64 gonadotropin-releasing hormone (GnRH), 49, 260, 288, 435, 443, 444 gonadotropins, 443, 444, 445 Gorlin syndrome, 165, 527 gray matter, 15, 16, 58, 70, 76, 131, 157, 158, 167, 179, 191, 196, 222, 453, 454, 511, 612 cortical, 156, 177, 238, 243 heterotopic, 25, 39, 42, 45, 47, 48, 156, 180, 205, 235, 236, 243, 346, 471, 494 imaging, 482, 488, 490, 494 nodules, 156, 482 subcortical, 25, 236, 452 Gross Motor Function Measure (GMFM), 594–5, 596, 598, 601, 621 growth hormone (GH), 49, 435, 439, 441 deficiency, 31–2, 39, 41, 44, 79, 123 growth hormone deficiency (GHD), 439, 440, 442–5, 447 treatment, 443 growth retardation/failure, 41, 42, 48, 81, 381, 445, 454, 468 guanosine triphosphatase (GTPase), 137, 138, 521 gyral abnormalities, 23, 167, 179, 346, 490, 494, 495, 519 gyral patterns, 185, 346, 455, 471, 480, 482, 488 gyri, 23, 48, 56, 72–3, 130, 222, 237, 379, 380, 418, 452, 454, 526, 535 imaging, 488, 493, 494 parahippocampal, 69, 70, 483, 493 precentral and postcentral, 494

hamartin (TSC1 protein), 134, 136, 137, 138, 521, 524 hamartomas, 138, 142, 143, 147, 362, 490 hypothalamic, 346, 347, 348 lingual, 347 retinal, 143, 346 hamartomatous disorders, 129–76, 523–4 headache, 91n, 97, 363, 369, 578 hearing, 347, 348, 378, 595, 603, 616 impairment, 123, 380, 604 heart, 242, 282, 283, 317, 324, 421, 422, 438, 441, 461 defects/failure, 118, 119, 207, 348, 365, 453, 454, 526 hemangioma, 165, 365 part of PHACE syndrome, 122 cutaneous, 354, 355, 358, 359, 362 facial, 119, 354, 355, 356, 359 intracranial, 355, 362 leptomeningeal, 369 neck, 354, 356, 359 hemimegalencephaly (HME), 6, 10, 77, 80, 153–76, 389, 402, 489, 495,

509, 512, 524, 533, 535, 538, 557, 562, 565, 622 associated hemimegalencephaly, 161–5 clinical features of isolated and associated HME, 160–1 histopathological comparison with TSC, 165 immunocytochemical reactivities, 158–9 isolated, 155, 156, 157, 160, 161, 169, 171 macroscopic (gross) findings in published cases, 156–7 management, 170–1 neuropathology, 156–60 severe, 160, 167, 168, 172 hemiparesis, 42, 47, 160, 161, 162, 170, 171, 601, 603 cortical dysplasia without balloon cells, 396 right, 591 hemispherectomy, 170, 171, 390, 562 hemispherotomy, 170, 171, 489 Hensen’s node, 5, 19, 68, 107, 110, 154, 258, 272, 303, 318 hepatocytes, 422, 472 hepatotoxicity, 561, 600 heterotopias, 27, 94, 119, 166, 195, 196, 391, 411, 422, 453, 454, 471, 490 cerebral, 10, 177, 182, 423, 470 disorder of migration and organization, 524–5 glioneuronal, 26, 27, 157, 223, 396 imaging, 492, 494, 495, 496 leptomeningeal, 157, 222, 223, 219 neuronal, 219, 381 subcortical, 195, 196, 396, 535 subependymal, 396, 481, 494 heterozygosity, 136–7, 138, 440 hippocampal abnormalities, 60, 207, 214, 237 hippocampal atrophy, 390, 483, 490, 491 hippocampus/hippocampi, 27, 54, 69, 70, 182, 197, 411, 433, 452, 454, 462, 508, 534, 542, 546 abnormal orientation, 80 dysplastic, 55 gray and white matter pathology, 400–1 imaging, 491, 493 murine, 75 primordial, 69 structural changes, 467 Hirschsprung’s disease (aganglionic megacolon), 9, 10, 54, 155, 165, 526 holoprosencephaly (HPE), 4, 6–11, 13–37, 46, 60, 61, 75, 111, 156, 243, 260, 263, 288, 291, 299, 338, 346, 444, 452, 453, 471, 511, 517, 518, 535, 583 holoprosencephaly: alobar, 13–5, 26–33, 398, 443, 533

INDEX holoprosencephaly: alobar (Continued ) MRI, 23–4, 26–8, 29, 31, 32 holoprosencephaly: lobar, 13, 15, 16, 28, 29, 30, 32, 33, 76f, 76, 79–80, 398, 443, 533 holoprosencephaly: semilobar, 13, 15, 16, 26–33, 398, 443, 533 homeobox (HOX) genes, 46, 91–5, 98, 105, 110, 215, 255, 264, 272, 278 ‘Hox’ versus ‘non-Hox’, 264 molecular basis of (embryonic) segmentation, 256–7 mutation or deletion, 93 neural tube segmentation, 6 two classes, 264 homeobox proteins, 521–2 homeodomains, 441, 442, 443 homeodomain proteins, 69, 256, 314 homeodomain transcription factors, 20, 439 homeotic genes (Hox-negative), 237, 273 hormone-secreting cell types, 434–5 hormones, 49, 96, 433, 434, 435, 437, 439, 445, 459, 463 Hoyeraal-Hreidarsson syndrome, 123 hydranencephaly, 45, 124, 219, 235, 243, 346 hydrocephalus, 23, 25, 26, 28, 29, 32, 41, 54–6, 57–9, 74, 75, 81, 89, 90, 98, 118, 119, 123, 146, 166, 179, 219, 223, 225, 227, 239, 244, 344, 346, 363, 365, 366, 380, 417, 418, 420–1, 422, 423, 445, 527, 570, 576, 580, 581 acute, 579 congenital, 94 familial, 520 fetal, 381 gross, 61 monoventricular or biventricular, 571 multicystic, 571 obstructive, 95, 96, 97 polymalformation, 584 posthemorrhagic, 94 prenatal, 583 X-linked with aqueductal stenosis, 10 hydromyelia, 111, 535, 580 Chiari malformations, 94, 96, 97, 98 hyoid bone, 263, 281, 301, 326 greater cornu, 263 neural crest (r4/r5), 296 hyperactivity, 563, 564, 605 hypertelorism, 9, 10, 28, 29, 78, 83, 260, 286, 298, 455 hypertension, 142, 143, 161, 366, 461 hypertonia, 596, 597–602 hypertonicity, 32, 596, 597, 599, 600 hypertrophy, 153, 162, 163, 170, 422 hemicorporal, 164, 165, 171 hypoblast, 258, 269, 272, 278 hypoglossal canal, 356, 358 hypoglycemia, 41, 44, 48, 49, 444, 463, 580 hypogonadism, 453, 455

hypogonadotrophic hypogonadism (HH), 260, 424, 444 hypomelanosis of Ito, 155, 162, 164, 355, 490 hypomelanotic macules (ash leaf), 141–2 hypomyelination, 465, 468, 471, 488 hypopituitarism, 40, 242, 440, 443, 440 diagnosis and management (SOPD), 48–50 hypoproliferation hypothesis, 463, 467 hypotelorism, 9, 10, 21, 28, 29, 29, 78, 263, 520 hypothalamic hamartomas, 342, 403, 508 hypothalamic-pituitary axis, 40, 44, 46 hypothalamic-pituitary-adrenocortical (HPA) axis, 463 hypothalamic-pituitary-thyroid (HPTh) axis, 463 hypothalamic-pituitary unit, 433–6 hypothalamic-releasing factors, 48, 49, 443 hypothalamus, 6, 22, 25, 31, 49, 259, 346, 437, 444, 445, 480 hypothyroidism, 31, 41, 48, 463 hypotonia, 42, 32, 183, 224, 226, 229, 239, 412, 415, 417, 421, 422, 423, 425, 451, 452, 455, 469, 472, 603, 617

immunocytochemical markers, 78, 535, 536, 537, 550 technical notes, 548–9 immunocytochemical reactivity, 93, 541 immunocytochemical reactions, 536, 542 immunocytochemistry, 3, 26, 158, 170, 180, 363, 534, 535, 546, 547 terminology, 549 ultrastructural, 543 immunohistochemistry, 139, 196, 197, 223, 549 immunoreactivity, 536, 538, 539, 544, 547, 549 absent, 415 infantile epileptic encephalopathy (Ohtahara syndrome), 509–10, 510 infantile febrile convulsions, 183 infantile hypercalcemia, 454 infantile spasms, 80, 396, 402 abnormal neuroblast migration, 395 antiepileptic drugs (effectiveness), 402 brain malformations: genotypephenotype correlations, 612 cortical malformations, 392, 393–4 drug choice, 560, 560 hemispheric or diffuse epileptiform discharges, 510 schizencephaly, 241 infants, 8, 389, 518, 535, 537, 547–8, 617, 618 cerebellar hypoplasias, 115, 124 cerebral dysgeneses, 456 cerebral dysgeneses (fetal life), 472 Chiari malformations, 94, 95, 97

635 infants (Continued ) craniofacial clefts, 326 effect of uncontrolled seizures, 399 extratemporal epilepsy, 390 neonatal myasthenia gravis, 413 neuromere agenesis, 107, 110, 111 neuromuscular disorders, 425 nutritional deficit hypothesis, 461 premature, 94, 115, 124, 377, 379 routine MRI, 480, 482 secondary amyoplasia, 424 toxoplasmosis, 380 ventral horn cell disease, 412 infarcts/infarction, 70, 71, 74, 96, 109, 110, 380 infundibulum, 44, 108, 293, 438, 446 inheritance autosomal dominant, 18, 521–2 autosomal recessive, 18, 76, 119, 124, 341, 344, 521–2 dominant, 439 recessive, 96, 182, 439 X-linked, 181, 345, 347, 439, 521–2 intelligence, 61, 192, 193, 194, 220, 414, 603, 612 nonverbal, 194 performance, 604, 619 scores, 183, 453 verbal, 194, 399, 604, 619 interhemispheric fissure, 25, 26, 68, 72–3, 75 lipomas and arachnoidal cysts, 81–3 posterior, 15, 15 interictal epileptiform discharges, 509–11 imaging epileptogenic networks, 491 interictal zone, 504, 506 intermandibular muscle, 330 interneurons, 109, 214, 600 GABAergic, 180, 181 ischemia, 547–8, 581 cerebral tissue, 378 cortical necrosis, 237 delayed cerebral, 362 fetal cerebral, 77

Joubert syndrome (juvenile nephronophthisis), 4, 7, 63, 116, 119–21 autosomal recessive disorder, 347 gene implicated, 521 Joubert-syndrome-related disorders (JSRDs or JBTSs), 517, 523 Kallman’s syndrome, 18, 260 keratitis, 54, 61, 62–3 kidneys, 55, 119, 129, 138, 142, 143, 146, 242, 343, 471, 523, 599 cystic dysplastic, 121 fetal, 585 polycystic, 345 kinases, 136, 199, 413, 522

636 Klippel–Trenaunay syndrome (KTS), 155, 161, 162, 164, 170, 171 Klippel–Trenaunay–Weber syndrome, 155, 163, 490 Kreisler (patterns of gene expression), 321

labiomaxillary clefts, 301, 302, 309, 311, 334–5 lacrimal bone, 261, 286, 294, 301, 308, 316 lacrimal duct, 286, 293, 301, 337 lacrimal system, 301, 305 Lambotte syndrome, 18 lamina terminalis, 9, 44, 68, 70, 78, 162, 260 lamination, 17, 205, 308, 536, 542 abnormal, 95 cortical, 94, 169, 180, 222, 238, 381, 471, 488, 506 laminectomy, 578, 579, 581, 582 lamotrigine (AED), 170, 200, 402, 403, 559–60, 563, 605 language disorders/disabilities, 140, 452, 453, 467, 603–4, 615, 617 lateral ventricles, 6, 15, 24, 26, 43, 48, 59, 60, 62, 73, 74–6, 94, 131, 132, 164, 166, 168, 169, 177, 179, 180, 206, 222, 228, 235, 238, 243, 380, 446, 454, 533, 569, 570 learning disorder/disability, 42, 140, 141, 146, 160, 161, 239, 453, 462, 467, 557, 618–9 Leigh encephalopathy, 81, 469, 536, 537 Lennox–Gastaut syndrome, 139, 193, 194, 392, 394, 396, 511, 561, 562 leukodystrophy, 220, 414, 421, 471 Lhermitte–Duclos disease, 10, 155, 521, 524 LIM homeobox genes, 439 lin-11, 441 isl-1, 441 mec-3, 441 limb-girdle muscular dystrophy, adultonset, 226, 230, 418 lipomas, 155, 161–6, 168–71, 580, 583 callosal, 480 caudal, 581–2 hemifacial, 155, 161, 162, 165–6, 170, 171 interhemispheric, 76–9, 81–3 intracranial, 165 resection, 83 subcutaneous, 162, 165 supracollicular, 55, 62 lipomatosis, 165, 166 encephalocraniocutaneous, 155, 163, 164 facial, 171 hemifacial, 160, 168–9 infiltrating, 162 LIS-1 gene (lissencephaly-1 or PAFAH1B1), 10, 76, 199–200, 206–10, 213–4

INDEX lissencephalies, 8, 31, 80, 156, 166–9, 199, 201, 346, 379, 389, 391, 403, 417, 422, 452, 464–5, 466n, 490, 493, 510, 517, 518, 522, 548 lissencephaly type I (‘classic lissencephaly’; ‘LIS’), 10, 94, 199, 200, 205–18, 395, 456, 493, 511, 512, 525 lissencephaly type II (cobblestone lissencephaly), 94, 205, 219–34, 416, 418, 420–1, 496, 522, 517, 525 lissencephaly and cerebellar hypoplasia (LCH), 208, 209, 210, 211, 212 lungs, 54–5, 129, 138, 146–7, 345, 584, 603 buds, 257, 269, 297, 323 pulmonary edema, 586 pulmonary hypoplasia, 342, 412, 423 transplantations, 143 luteinizing hormone (LH), 49, 435, 443

macrencephaly, 81, 119, 122 macrocephaly, 32, 160, 164, 165, 166, 170, 366, 425, 469, 470, 518, 527 macrocephaly cutis marmorata congenita, 527 Majewski short-rib polydactyly syndrome, 18, 342, 347 malnutrition, 383, 460, 461, 462 mammillary body (MB), 45, 55, 434–6, 446, 570 mandible bone (r3 structure), 96, 263, 279, 280, 282, 295, 301, 303, 306, 307, 323, 326, 329 mandibular arch, 281, 326 mandibular fields, 280, 292, 332 Marden–Walker syndrome, 411, 421, 423 Marinesco–Sjo¨gren syndrome (MSS), 411, 421, 423, 424 markers of cellular maturation in nervous system, 536–47 masticatory muscles, 96, 155, 279, 280, 295 mastoid process, 253, 256, 278 mastoid temporal bone, 284, 296 maturational markers, 547–8 medial fibroplasia, 360 mega cisterna magna, 119, 177, 237 megalencephaly, 10, 133, 164, 392, 494, 522, 527 Melnick-Needles syndrome, 182, 525 memory, 399, 400, 452, 461, 467, 599 meninges, 306, 346, 363, 535 vascular malformations, 369–70 meningitis, 94, 579, 580 meningoceles, 165, 346, 576, 583 Menkes disease, 354, 355, 466, 472 mental retardation/mental deficits, 8, 42, 43, 61, 78, 80, 81, 397, 389, 453, 455, 557, 603, 619 abnormal neuroblast migration, 395 Angelman syndrome, 455

mental retardation/mental deficits (Continued ) cerebellar hypoplasias, 122, 124 cerebral dysgeneses, 451 cerebral malformations, 420 chromosome structure (disorders), 454 congenital vascular malformations, 359 effect of uncontrolled seizures, 399 environmental toxin hypotheses, 467 genetic cause, 452 genotype-phenotype correlations, 612 hereditary motor and sensory neuropathy, 413 Marden–Walker syndrome, 423 microcephalies, 527 mood stabilizers, 563 neuroleptics, 563 neuromuscular disorders, 425 oligophrenin-related X-linked, 518, 521 Rieger syndrome, 441 schizencephaly, 235, 239 sex chromosome aneuploidy, 454 somatic aneuploidy, 452, 453 teratogenic drugs and toxins, 381 mesencephalic neural crest (MNC), 10, 78, 110, 111 anatomy of aortic arches, 283 cranial base formation (frontoorbitosphenoid model), 299 craniofacial clefts, 249–50, 261, 262, 264, 271, 285, 307 craniofacial clefts: Tessier’s classification, 332 craniofacial development, 302, 306, 307 neuromeric origin of facial soft tissues, 300 mesencephalon (midbrain), 5–6, 8, 9, 14, 17, 29, 78 absence, 108 agenesis, 9, 106–10 atresia of cerebral aqueduct, 92 cerebellar hypoplasias, 117 Chiari malformations, 94 craniofacial clefts, 249, 253, 261, 276, 315–6, 323 neuromere agenesis, 105–9, 111 neuromeres, 253 pituitary development, 438 posterior, 107 reticular formation, 434 rostral, 67, 535 rostral rhomboencephalic derivatives, 292 mesenchymal cells, 278, 287, 409 mesenchyme anterior and posterior tongues, 286 cleft formation, 310 connecting cranium with face, 308 cranial base formation, 298, 299 craniofacial clefts, 251, 257, 264, 272, 279, 280, 281, 306, 317, 324

INDEX mesenchyme (Continued ) deficiency of p5, 288 developmental fields: biological basis, 301 formation of cranial base, 297 formation of lip and prolabium, 309 frontonasal, 306 premaxillary, 308 prosencephalic derivatives (sixteensomite stage), 285 residual, 286 rhombomeres, 290, 292, 295, 299 sphenoethmoid, 308 mesoderm, 90, 249–54, 257, 272, 279, 280, 283, 296, 297, 299, 300, 305, 325, 438 messenger RNA (mRNA), 135, 182, 223, 345, 416, 438, 440, 472, 540 metabolic acidosis, 470, 471, 561, 563 metabolic categories, 464–6 metabolic disorders brain metabolism in fetal life, 459 cerebral dysgeneses in fetal life 464–6 neuromuscular disease, 411 secondary cerebral dysgeneses in fetal life, 459–76 metencephalon (cerebellum/upper pons), 6, 106, 107, 108, 111, 117, 250, 259, 315–6, 330 microcephaly/microcephalies, 21, 25, 54, 78, 81, 207, 209, 210, 211, 229, 346, 380, 412, 425, 451, 452, 453, 455, 462, 517, 523, 576, 612 microdysgenesis, 396–7, 485, 505, 510 micrognathia, 96, 207, 343–4 microlissencephaly (MLIS), 207, 209, 210, 211, 392 microphthalmia, 28, 46, 227, 229, 418, 439 microtubules, 159, 197, 199, 212, 214, 345 migration and organization disorders, 524–6 Miller–Dieker syndrome (MDS), 206, 207, 212, 214, 456, 511, 518, 525 clinical, imaging, and genetic features, 208 facial features, 209 gene implicated, 521 genotype-phenotype correlations, 612 imaging, 493 lissencephaly type I, 10, 494 mitochondria, 159, 278, 462, 470 mitochondrial cytopathies, 411, 421–2 mitochondrial disorders, 81, 123, 124, 472, 495, 527 mitosis, 20, 25, 93 mitotic activity, 106, 157 mitotic cycle, 93, 158, 547 mitotic spindles, 158, 165, 547 Mohr syndrome (OFDS type II), 341, 344 Mohr–Majewski syndrome (OFDS type IV), 341, 344

molar tooth sign, 119, 120–1, 121, 347, 523 molecular genetic theory (Chiari malformations), 90–6, 98–9 mosaic mutations, 181–2, 191, 611 mosaicism, 197, 200, 201, 524, 612 motor deficits/motor delay, 62, 124, 170, 183, 192, 226, 235, 239, 415, 423 motor disabilities/dysfunction, 32–3, 381, 618 motor function, 243, 594–5 motor impairment, 97, 123–4, 183, 240, 359, 595–7, 605 motor neuron disorders, 410, 412, 421, 423, 425 motor skills, 115, 244, 461 Mowat-Wilson syndrome, 76, 523, 527 mumps, 94, 380, 381 muscle cells, 142, 143, 416 muscle fibers, 409, 410, 411, 422 muscle tone, 229, 240, 599, 601, 617 muscle-eye-brain (MEB) disease, 124, 219, 220, 222, 223–6, 227, 230, 411, 414, 416–8, 495–6, 522, 525 myasthenia gravis, neonatal, 413–4 myelencephalon, 6, 106, 109, 250, 315, 330 myelination, 25, 70, 157, 158, 167, 180, 196, 222, 223, 377, 461, 464–6, 482, 484, 489, 496, 538 aberrant, 471 delayed, 23, 412, 416, 464, 470 impaired, 466, 469 marker, 547 myelomeningocele, 344, 445, 580, 580–1, 583, 585–7 myoblasts, 282, 409, 410 cleft formation: pathological anatomy, 310 craniofacial clefts, 253, 256, 257, 325 formation of lip and prolabium, 309 non-PA PAM, 283 oronasal soft tissues: assembly, 309 r6 neural crest, 296 rostral rhomboencephalic derivatives, 292 vascularization of PAs, 283 myopathies, 164, 411, 421–4, 535 myopia, 223, 224, 227, 229 myotome, 106, 255, 256, 280, 281, 303, 317, 320, 409 of amphioxus, 153 dorsal, 105, 252, 253 occipital somite, 328 ventral, 253 Na2+ channels, 558, 559, 560 neocerebellum, 117, 412 neonates, 3, 8, 71, 74, 248, 370, 377, 378, 389, 391, 399, 463, 535, 539, 544, 549, 550, 580, 581, 586, 587 basal encephaloceles, 577 cerebellar hypoplasias, 117, 122

637 neonates (Continued ) Chiari malformations, 93, 94 cortical malformations, 392 epilepsy, 391 epileptiform discharges, 510 hearing impairment, 604 hepatic steatosis, 472 hypoxia/ischemia, 548 intracranial aneurysms, 361 Miller–Dieker syndrome, 511 neuroendoscopy, 569 posthemorrhagic hydrocephalus, 569 pyruvate dehydrogenase complex deficiency, 470 toxoplasmosis, 380 varicella-zoster virus, 380 neoplasms, 367, 390, 394, 505 nerves, 315 alveolar, 293 cervical, 95–6, 252 cranial, 92, 95, 97, 107, 252, 254, 259, 261, 282, 322, 355, 539 cranial sensory, 321 glossopharyngeal, 283 infraorbital, 293 medial, 57 medial nasal, 263 peripheral, 54, 220, 380, 421 sensorimotor, 252 sensory, 279, 280, 282, 283, 295 sphenopalatine, 261, 263, 299 spinal, 252–3, 303, 320 trochlear, 53, 57, 97 trigeminal, 63, 111, 249, 283, 369 vagus, 147, 296, 403, 437, 562 neural crest, 4, 63, 80, 161, 162, 535, 540 assembly of face, 302 bone and cartilage derivatives, 301 Carnegie staging system, 304 caudal, 248 cephalic, 263 cranial, 63 disorders, 10, 246–351 ectodermal lineage, 269 formation of cranial base, 297–8 formation of larynx and trachea, 296 gradients of genetic expression, 9 induction of non-neural tissues, 246–351 initial migration, 9 interhemispheric lipoma, 83 neuromere agenesis, 111 nonneural derivatives, 9 pharyngeal arches: spatial relationships, 297 pituitary development, 438 prosencephalic, 9, 10, 78, 162 prosomere-6, 287, 288 rhombencephalic, 10 role in development of head and neck, 250 terminal differentiation, 78 vomerine, 291

638 neural crest cells, 111, 155, 172, 249, 253, 254, 255, 259, 263, 264, 281, 282, 284, 285, 300, 316, 325, 409 neural folds, 5, 111, 248, 249, 254, 259, 263, 284, 291, 297–8, 299, 301–3, 305–7, 316, 440 neural plate, 5, 105, 250, 253, 254, 259, 276, 278, 282, 288, 303, 305, 438 neural tube, 5, 17, 67, 68, 116, 250, 251, 252, 275, 279, 280, 292, 295, 315–7, 320, 535, 546 agenesis, 9 axes and gradients, 5–6, 10–1 before closure of neuropores, 5 Chiari malformations, 89, 90, 92–5, 97–9 compartmental boundaries, 106 dorsal part, 9 dorsal structures, 5, 110 duplication, 9 early fetal life, 6 ectopic, 110 floor plate, 7, 8–9, 18, 315 formation of cranial base, 297 midline, 109 murine, 92 neuromere agenesis, 105, 106, 107, 109, 110, 111 rostral, 107 segmentation, 6, 9, 91–3, 94, 99, 106, 109, 254–6 skeletal muscle embryology, 409 ventral structures (motor neurons), 5 neuraxis, 4, 109, 252, 253, 259, 264, 287, 289, 297, 301, 306 neuroblast migration, 4, 6, 76, 81, 377, 378, 535, 542, 547 neuroblast migration: abnormal, 394–6 neuroblast migratory anomalies/ disorders, 4, 75, 166, 167, 170 neuroblasts, 133, 237, 287, 536, 538, 540, 547 neurocranium, 263, 279, 290, 306 neurocristopathies (Bolande), 4, 9, 10, 11, 263, 287 neurocutaneous disorders, 354, 480, 490 pathogenesis, 155–6 neurocutaneous syndromes, 4, 5, 9, 107, 122, 153, 160, 622 neurodevelopmental disorders, 144, 461, 467, 468, 469 neuroembryology, 3, 247, 249, 252, 284 neuroendocrine cells, 540, 543 neuroendocrine complications, 433–50 neuroepithelial cells, 133, 536, 540, 549 apoptosis disorders, 10 precursors, 136, 546 primitive, 538 proliferation, 11 proliferation disorders, 10 skeletal muscle embryology, 409 undifferentiated, 93, 541, 545, 547 neuroepithelium, 93, 106, 107, 111, 171, 182, 537, 538, 543

INDEX neurofibromatosis, 9, 490 type I (NF-1), 10, 96, 123, 155, 161–3, 164, 354, 523 type II, 389 neurofilament proteins (NFP), 78, 538, 541 neurohypophysis, 287, 288, 436 neuroimaging, 548, 549 cerebral dysgeneses, 451 cortical malformations, 403 role in identifying malformations, 479–88 neuromere formation, 93, 105–6 neuromeres, 6, 91, 92, 106, 250–3, 261, 264, 278, 284, 298, 300, 301, 311, 315–6 agenesis, 110–1 basal prosencephalic, 110 basal telencephalic, 110 diencephalic, 111 medulla, 253 mesencephalic, 8, 11, 91, 110, 111 neural crest tissue, 110–1 number and types, 106 selective deletion, 111 neuromeric levels, 279, 289 neuromeric mapping, 250–2, 282, 300–1 neuromeric model, 250, 251, 307 neuromeric system, 251, 302, 310, 311 neuromuscular defects/disorders, 122, 219, 495 neuromuscular junction, 410 transmission defect, 411, 413–4 neuron-specific enolase (NSE), 78, 538, 540–1, 547–8 neurons, 8, 31, 63, 92, 95, 106, 110, 121, 129, 135, 153, 158, 180, 206, 222, 228, 230, 238, 260, 287, 288, 325, 379, 380, 381, 397, 434, 454, 463, 468, 492, 505, 538, 550 abnormal/atypical, 129, 130–1 abnormality (cause of epilepsy), 391 adrenergic, 543 autonomic, 433, 437 binucleated, 157, 165 cholinergic, 543 chromatolytic, 158 cortical, 158, 215, 223, 472, 506 cortico-cortical pyramidal, 26 cranial, 64 dysmorphic, 135 dysplastic, 130, 135, 137, 140, 391, 487, 541 early postmigratory, 215 ectopic, 506 enlarged with Nissl bodies, 157 excitatory and inhibitory, 196 fusiform marginal, 180 GABAergic, 196, 212, 215, 542 giant, 157, 488 glutamate-containing pyramidal, 542 granule cell, 472 heterotopic, 3, 130, 170, 177, 195, 210, 377, 486

neurons (Continued ) hypertrophic, 157 hypothalamic, 433 hypothalamic magnocellular, 436 immature, 540, 545 laminar fusiform, 180 large, 170 layer-3, 69, 79 magnocellular, 435 molecular, 472 monodendritic, 543 multinucleated, 157 neurosecretory, 433 normotopic, 196 origins, 321 paraventricular magnocellular, 436 parvicellular, 435 peptidergic, 436 pheromonal, 304 postganglionic, 437 postmitotic, 197 postmitotic young, 181 preganglionic, 433, 437 pyramidal, 69, 83, 180, 391, 540 skeletal muscle embryology, 409 subcortical heterotopic, 159 neuropathological examination, 80, 74, 533–54 neuropathology, 3, 170, 220, 235, 534, 544, 549, 550 cerebral dysgeneses, 451, 459 hereditary motor and sensory, 412–3 motor demyelinating, 415 schizencephaly, 238–9 sex chromosome aneuploidy, 454 neuropeptides, 437, 536, 543, 546 neuropil, 159, 180, 543, 549 neurorehabilitation: children with cerebral palsy, 592–604 neurotransmitters, 139, 437, 459, 460, 463, 468, 534, 538, 540, 542–6, 562, 563 enzymes, 544–5 Nijmegen breakage syndrome, 494, 522, 527 NMDA receptors, 136, 139, 181, 391, 399 nonsense mutations, 213, 224, 226, 612 Norman–Roberts syndrome, 209 notochord, 5, 5, 7, 8, 18, 19, 92, 254, 257, 258, 278, 279, 287, 303, 305, 305, 315, 317–8, 323, 326–7, 409, 424, 455, 540, 545 rostral, 284, 289 nutritional deficit hypothesis, 461–2 nystagmus, 40, 61, 97, 347, 604

ocular anomalies, 21, 381, 494 ocular hypertelorism/telecanthus, 341, 343–4, 455 oculomotor apraxia, 119, 121, 347 Ohtahara syndrome (infantile epileptic encephalopathy), 392, 509–10

INDEX olfactory bulbs, 15, 42, 55, 108, 197, 238, 288, 494 olfactory nerves, 13, 305 olfactory system, 250, 288, 304, 316 olfactory tracts, 15, 42, 44, 55 oligodendrocytes, 158, 540, 543, 544, 545, 547 ontogenesis, 3, 68, 534, 536, 538, 541, 545 opercularization, 209, 464, 469 optic atrophy, 44, 379, 495, 604 optic chiasm (OC), 43, 45, 48, 60, 80, 108, 434, 434–6, 438, 446 optic cup, 286, 289, 304, 315 optic disc, 40, 80, 443 optic nerves, 60, 80, 108, 143, 227, 242, 261, 298, 439 abnormalities, 46 atrophy, 40 colobomata, 344 head, 40, 40, 44 optic nerve hypoplasia (ONH), 39, 40, 41–4, 44, 46 associated with hypopituitarism, 48 diagnosis, 47 endocrinopathies, 443, 444 midline cerebral malformations, 444 oral-facial-digital syndromes (OFDS), 341–51 orthodenticle (OTX/otx) family of genes, 92, 106, 107, 110, 117, 315 orthotics, 596, 597–8, 601 ossification, 297–8, 299, 307 osteogenesis, 269, 285, 286 Otahara syndrome, 161, 169

pachygyria (broad gyri), 23, 75, 80, 94, 130, 156, 166, 168, 191, 193, 205, 206, 210–1, 219, 223–6, 228, 230, 346, 379, 402, 414, 419, 421, 422, 464–5, 466n, 470, 481, 490, 548 abnormal neuroblast migration, 396 changes, 226 cortical, 192, 471, 488 diffuse, 510 EEG, 400, 511, 512 hemispheric or diffuse epileptiform discharges, 510 imaging, 494, 495 perirolandic, 495 posterior dominant, 200 typical diffuse, 167 unilateral familial, 155 X-linked with aqueductal stenosis, 10 Pallister Hall syndrome, 18, 342, 343, 345, 346, 347, 520 panhypopituitarism, 41, 43, 44, 49, 79, 440 paraventricular nucleus (PVN), 45, 434, 436 paraxial mesoderm (PAM), 90, 94, 250, 251, 252, 254–7, 260–2, 285, 287, 291, 305, 306, 310, 323

paraxial mesoderm (PAM) (Continued ) assembly of face, 302 cranial base formation, 297, 298, 299 defective segmentation, 105 formation of larynx and trachea, 297 hypaxial, 280 mesodermal ‘first response’ to brain formation, 297 neuromere agenesis, 105 neuromeric origin of facial soft tissues, 300 non-pharyngeal-arch, 283–4 rostral rhomboencephalic derivatives, 292 second somitomere, 262 somitomeres and somites, 277–81, 317–22, 324, 326–30 parenchyma, 58, 109, 142, 158, 166, 170, 364, 367, 377, 558 parental consanguinity, 44, 55, 61, 63, 79, 344, 496, 517, 526 ‘family consanguinity’, 81, 209, 228, 341 parietal bone, 279, 290, 292, 295, 326, 329 Parkes Weber syndrome (PWS), 161, 162, 164 Parry-Romberg syndrome, 155 Pascual-Castroviejo type II syndrome, 355, 369 same as cutaneous hemangiomas: vascular anomaly complex, 362 Patau syndrome (trisomy 13), 18, 76, 81, 119, 453, 494, 520 Patched (PTCH) gene, 8, 18, 19–20, 22, 22, 368 Pena-Shokeir syndrome, 411, 412, 423 peripheral nervous system, 4, 9, 197, 214, 220, 278, 303, 381, 410, 415, 540, 544 periventricular heterotopia, 39, 80, 155, 164–5, 167–8, 524 periventricular leukomalacia (PVL), 42, 45, 77, 379, 380, 465, 466n, 591 periventricular nodular heterotopia, 177–89 bilateral (‘variant’ syndromes), 179, 183, 391 bilateral, asymmetrical type, 178, 179, 180, 182, 183, 184 PEX genes, 422, 471 Pfannenstiel’s incision, 585 Pfeiffer syndrome, 574 PGP-9.5, 78, 538, 541 PHACE syndrome, 122 pharyngeal arches (PAs), 250–2, 261, 264, 271, 275, 280, 301, 306, 317–25, 329 anatomy, 281–2 assembly of face, 302 Carnegie staging system, 303, 304 cranial base formation (frontoorbitosphenoid model), 299 derivatives, 309

639 pharyngeal arches (PAs) (Continued ) formation, 253, 284, 297 formed from Sm2-Sm11, 253 four in mammals, 279 mesenchyme, 281, 307 neural crest bones and cartilages, 263 neuromeric origin of facial soft tissues, 300 non-pharyngeal-arch paraxial mesoderm: anatomy, 283 proximal/dorsal, distal/ventral, 255 rostral rhomboencephalic derivatives, 291, 292 vascularization: nutritional basis of derivative formation, 282–3, 323, 325 pharyngeal pouches, 106, 263, 275, 323 pharynx, 263, 282, 287, 289, 296, 323, 326 phenobarbital (AED), 170, 402, 559–60 philtrum, 260, 269, 286, 300, 309, 338 phosphorylation, 134, 136, 147, 199, 462, 541, 545, 546, 548 pial surface, 48, 135, 235, 369 Pierre Robin complex/sequence, 96, 423 piriform fossa, 249, 250, 269, 309, 310 pituitary disorders, 21, 44, 440, 447, 520, 521 pituitary dysfunction, 31, 49, 348, 520 pituitary dysgenesis/aplasia, 346, 439 pituitary gland, 17, 21, 25, 31, 41, 45, 60, 259, 279, 289, 433, 438, 577 ectopic/absent (heterozygous) posterior, 44 intermediate lobe, 437, 440, 442 morphological anomalies, 445 routine MRI, 480 sagittal view, 436 pituitary hormones, 41, 48, 49, 447 pituitary hypoplasia, 439, 440, 443 pituitary insufficiency, 39, 40–1, 44, 285 placodes, 61, 308, 321, 438, 580, 585, 586 adenohypophyseal, 259, 260, 262, 287, 288–9 adenophyseal, 285 appearance before closure, 580 cellular, 287 cranial, 316 epibranchial, 316 medial, 304 nasal, 260, 287, 288, 339 neural, 68 olfactory, 276, 285, 287, 316 optic, 260, 263, 276, 285, 286, 287, 304 petrosal epibranchial, 316 trigeminal, 63, 64, 276, 316 platelet-activating factor (PAF), 213, 521 pneumoencephalography, 76–7, 534 polydactyly, 119, 54, 341, 343, 347, 523 polyhydramnios, 123, 412, 413, 423 polymicrogyria (PMG) bilateral sylvian or parietooccipital, 402

640 polymicrogyria (PMG) (Continued ) cerebellar, 119, 219 cortical, 471 diffuse, 24, 225 frontoparietal, 223, 494, 496, 511 generalized, 522 hemispheric, 512 imaging, 480, 481, 492, 494, 495, 496 left temporal, 511 mitochondrial cytopathies, 421 nonketotic hyperglycinemia, 469 nonlaminated, 80 nonsyndromic, 526 occipital, 221, 414, 415, 419, 495 miscellaneous, 45, 54, 60, 95, 121, 130, 156, 166, 167, 179, 182, 205, 210, 213, 225, 237, 238, 239, 243, 346, 379, 396, 396–7, 401, 412, 464–5, 466n, 470, 487, 495, 509, 518–9, 523 opercular, 480 perisylvian, 392, 403, 481, 511, 526 region-specific bilateral symmetrical, 494 unilateral, 494 Pompe’s disease, 411, 421, 422 pontocerebellar hypoplasias (PCH), 8, 9, 123–4, 227, 410, 414, 418, 419 porencephaly, 42–5, 47, 48, 77, 344, 346, 379, 490, 585 posterior C1 laminectomy, 578, 581 posterior fossa, 4, 89, 90, 95–8, 116, 228, 237, 254, 355, 454, 455, 569, 570 anomalies, 53 cyst, 118, 346, 418, 571 decompression, 578–80 dura, 283 enlargement, 118 imaging, 115, 480, 481 malformations, 523 neural defects, 80 structures, 77 surgery, 581 Prader–Willi syndrome, 455 prechordal plate, 18, 315, 318 prechordal plate mesoderm (PCM), 252, 255, 258, 289 pregnancy, 161, 186, 238, 360, 364, 383, 445, 459, 461, 462, 468, 534 prenatal dysgenesis, 464–6 presphenoid bone, 248, 251, 261, 287, 289, 290, 291, 295, 297, 298, 301, 308, 328 progressive hemifacial atrophy (ParryRomberg syndrome), 155 prolactin (PRL), 49, 435, 439, 443, 563 prolactin-releasing hormone (PrRP), 435 proliferating cell nuclear antigen (PCNA), 547 PROP-1 gene, 439, 439, 440 prosencephalic derivatives (sixteen-somite stage), 284–9, 316, 337–9 prosomere-5, 285–7, 337–8 prosomere-6, 287–9, 339

INDEX prosencephalic neural crest (PNC) 260–1, 284 anatomy of aortic arches, 283 cranial base formation, 299 craniofacial clefts, 249, 250, 262, 264, 271, 275, 302, 306, 332 final migration, 287 neuromeric origin of facial soft tissues, 300 PNC migration, 260, 263, 285 prosencephalic derivatives, 285 rostral rhomboencephalic derivatives, 292 prosencephalon (‘forebrain’), 5, 6, 7, 9, 17, 18, 64, 83, 98, 105, 106, 154, 215, 237, 282, 283, 306, 346, 397, 434, 438, 443, 471, 535, 539 basal, 23, 25, 44, 45, 46, 286, 287, 289, 304 caudal, 264, 284, 302 cranial base formation, 299 craniofacial clefts, 249, 251, 257, 259–62, 264, 276, 279, 315–6 craniofacial development, 302, 305, 306 dorsal (alar) zone, 259, 287 p6 basal, 287–8 posterior, 259, 260, 285 prosencephalic derivatives, 285 prosomere zone p5, 287 rhombomeres (r0-r3), 289, 291, 292, 295 rostral, 264, 276, 284–5, 286 rostral zone (p6 and p5), 292 ventral, 19, 20, 259 protein O-mannosyltransferase-1 gene (POMT-1), 220, 222, 227, 230 protein O-mannosyltransferase-2 gene (POMT-2), 220, 222, 227 protein phosphatase 1 (PP-1), 191, 199 Proteus syndrome, 154, 155, 161, 162, 163–4, 170, 171, 490 psychomotor disorder/retardation, 42, 123, 191, 346–7, 360, 370, 423, 523, 614 PTEN (gene), 520, 521, 524, 527 PTEN hamartoma tumor syndrome (PHTS), 524, 527 pterygopalatine fossa, 290, 292–3, 295 puberty, 41, 48–9, 131, 141, 142, 347, 348, 443–5, 447, 461 Purkinje cells, 57, 109, 117, 455, 470, 472, 534, 535, 539–42, 544, 545, 546 pyruvate dehydrogenase (PDH) deficiency, 123, 465, 470, 519, 520, 527 pyruvate metabolism disorders, 464–5, 470 radial glia, 8, 159, 377, 540 radial glial cells, 6, 154, 378, 494, 534, 535, 536, 547, 549

radial glial fibers, 5, 17, 182, 196, 237, 381, 539, 542 radial neuroblast migration: disorders, radiation exposure, fetal, 381 Rathke’s pouch, 44, 46, 260, 262, 304, 305, 437–43 Rathke’s pouch homeobox (RPX) gene, 439–40 reelin localized on chromosome 7q22, 117 protein implicated in CNS malformation syndromes, 522 RLN, 10, 76 signaling pathway, 122 Reichert’s membrane, 222, 227 RELN gene, 208, 210, 212, 214–5, 522, 525, 613 mutations, 207, 209, 211 protein, 214 renal abnormalities, 141, 142, 144, 345 respiration/breathing, 121, 412, 433, 470, 523 respiratory difficulties/failure, 41, 97, 413, 414, 416, 418, 422, 600, 603, 620 reticulum endoplasmic, 422, 548 protein mutations, 414 sarcoplasmic, 599 retina, 9, 40, 111, 119, 129, 143, 197, 227, 379, 542 retinal dystrophy, 119, 121, 412 retinal ganglion cells, 44, 46, 47 retinoic acid (vitamin A), 20, 92, 93, 96, 110, 154, 257, 274, 381, 520 retinoic acid receptor (RAR), 92, 321 Rett syndrome, 495, 518, 522 rhombencephalic neural crest (RNC), 249, 250, 261, 271, 275, 282, 300, 302, 307, 332 rhombencephalon/rhomboencephalon (hindbrain), 5, 9, 64, 93, 117, 249, 252, 253, 259, 261, 279, 283, 302, 306, 315, 322, 346, 438, 585, 586 rhombic lip of His, 4, 95, 110, 117, 540 RNA (ribonucleic acid), 157, 158, 309, 345, 542, 543, 547, 549 rolandic fissure, 206, 206, 509 roof plate, 8, 25, 63, 93, 117, 325, 544 rostral rhomboencephalic derivatives (eleven-somite stage), 291–6 Rubinstein-Taybi syndrome, 18, 527 sacral agenesis, 7, 8, 9, 411, 424 sacrum, 7, 303, 424 sagittal dimension (4D theory), 248 Saguenay-Lac-St Jean, 413 scalp, 142, 163, 283, 300, 306, 359, 506, 508, 578 scanning electron microscopy (SEM), 252, 254, 303, 308, 324 schizencephaly, 6, 7, 8, 9–10, 24, 42, 43, 44, 45, 110, 130, 164, 166, 167,

INDEX 168, 179, 235–46, 379, 396, 397, 495, 520, 522, 557, 565 schizophrenia, 3, 461 Schwann cells, 249, 282, 409, 410, 415 sclera, 264, 286, 289, 291 sclerosis, 129, 390, 483, 487, 488, 507 sclerotome, 253, 255, 256, 279, 281, 284, 292, 295, 297, 298, 320, 546 scoliosis, 28, 56, 97, 413, 415, 416, 421, 575 Seckel syndrome, 522, 527 segmentation assembly of face, 302 craniofacial clefts, 275 craniofacial developmental fields, 302 craniofacial development: major themes, 305 formation of cranial base, 298 formation of larynx and trachea, 297 mesoderm/neural tube match, 254–6 mesodermal, 305 molecular basis: homeobox genes, 256–7 neural tube (disorders), 89–127 neuromere agenesis, 111 skeletal muscle embryology, 409 staging of embryos (general principles), 303 selective serotonin reuptake inhibitors (SSRIs), 565, 567 sella turcica, 41, 45, 346, 356, 434, 436, 443 empty, 445–7 septo-optic dysplasia (SOD), 15, 25, 40, 42, 46, 47, 60, 64, 242, 243, 421, 434, 439, 440, 443, 444 diagnosis, 520 gene implicated, 521 genetic testing and counseling, 520, 521 nongenetic causes, 520 septo-optic-pituitary dysplasia (SOPD), 39–52 septostomy, 571 septum/septa (r1 structure/s), 93, 248, 249, 250, 261, 263, 308, 309, 332, 582, 571, 583 bifid, 288 murine, 69 prosomere-6, 287, 288 septum pellucidum, 15, 26, 45, 60, 80, 183, 242, 243, 439, 440, 520, 571 absence (ASP), 40, 48, 54–6, 95, 225, 440, 443, 444, 444, 520 diagnostic radiological criteria, 48 rpx gene (mice), 440 septum pellucidum: agenesis, 39, 43, 44, 47, 236, 237, 239 Ser170Leu, 440, 440 serine-deficiency syndromes, 464, 468–9 serotonin, 135, 436, 461, 486, 491, 544, 564, 565 serotonin reuptake inhibitors (SRIs) 564, 565

sex chromosome aneuploidy, 451, 453–4 sexual infantilism, 41, 48, 49 Shapiro syndrome, 76, 79 short stature, 62, 119, 123, 347, 424, 445, 447, 453, 494, 527 shunting/surgical shunting complications, 573 CSF, 32, 569, 572–3, 585 cystoperitoneal, 571 hydrocephalus, 97, 571–3 LIS type II, 223, 225 macrocephaly, 32 pleural, 580 syringo-peritoneal, 580 ventricular, 59 ventriculo-atrial, 573 ventriculo-peritoneal, 22, 244, 573, 581 ventriculomegaly or colpocephaly, 171 signaling, 463, 299, 301, 303, 315 signaling molecules, 316, 438, 441 Simonart’s band, 309, 310 Simpson–Golabi–Behmel syndrome, 527 ‘single gene’ disorder models: metabolic cerebral dysgeneses amino acid metabolism disorders, 468–9 associated with cerebral dysgeneses in fetal life, 464–6 cholesterol biosynthesis disorders, 471 fatty acid oxidation disorders, 470–1 further research required, 470 glycoprotein metabolism disorders, 471–2 organic acid metabolism disorders, 469–70 peroxisome biogenesis (Zellweger) disorders, 471 pyruvate metabolism and tricarboxylic acid cycle disorders, 470 trace metal metabolism disorders, 472 single-event multilevel surgery (SEMLS), 601 single-photon emission computed tomography (SPECT) ictal, 169, 486, 491 imaging cortical malformations, 496 imaging epileptogenic networks, 491 interictal, 169, 195, 485 PNH, 186 quantitative, 491 role of imaging in identifying malformations, 479, 480 skeletal dysplasia, 342, 343–4, 345, 347 skeletal muscle embryology, 409–10 skin, 49, 129, 138, 147, 155, 160, 163, 196, 207, 249, 252, 280, 286, 306, 309, 338, 345, 524, 561 dysplastic, 580–1 facial, 300 forehead, nose, philtrum, 300 hyperextensible, 183

641 skin (Continued ) internal, 308 nasal, 288 neuromeric origin of facial soft tissues, 300 outer, 308 parietal and occipital, 61 prolabial, 310 retroauricular, 253 shunt complications, 573 upper lip, 260 skull, 252, 279, 281, 283, 290, 295, 302, 306, 326, 358, 577 fetal, 335 formation of cranial base, 298 prosencephalic derivatives (sixteensomite stage), 285 vertebrate, 254, 325 SLC12A6 gene (chromosome 15q), 413, 520, 521 SLC25A19, 522 SMAS (superficial musculo-aponeurotic fascia), 261, 296, 297 Smith-Lemli-Opitz (SLO) syndrome, 18, 21, 121, 123, 471, 520 imaging, 495 inborn errors, 519, 527 OFDS, 347 type II, 347 SMN gene, 10 social interaction, 30, 74, 461, 563, 565, 566, 602, 614, 616 solute carrier families, 521–2 soma, 26, 157, 214, 540, 541, 545 somatic aneuploidy, 452–3 somatic malformations, 182, 183 somatostatin, 435, 437, 536, 546 somatotrophs, 439, 440 somite pairs, 105, 302–3 somite stage-11, 284 Carnegie staging system, 303 completion of r2-r7 neural crest migration, 262 cranial base formation, 299 rostral rhomboencephalic derivatives, 291–6 somite stage-14, 262, 263, 289–91, 292, 296, 303, 338 somites, 5, 9, 105, 252–4, 317, 409 assembly of face, 302 caudal and rostral, 285 epithelialized (avian), 278 paraxial mesoderm, 277–81 relationship with somitomeres, 318 Sm8 onwards, 250 somites: cervical first (C1), 253, 271, 279 first (formed from Sm12), 250 second (C2), 253, 279 eight pairs, 252 somites: lumbar (five pairs), 252 somites: medial, 284 somites: occipital

642 somites: occipital (Continued ) first (O1), 252, 253, 256, 257, 274, 278, 279, 302, 303 first and second (Sm8 and Sm9), 255 first four, 253, 328 third and fourth (Sm10 and Sm11), 255 fourth (O4), 279, 288 fourth (murine), 257 formed from Sm8-Sm11, 250, 281, 284 human (four pairs), 252, 253–4, 279–80, 298, 303 miscellaneous, 271, 278, 281, 306 murine, 257, 273 somites: sacral (five pairs), 252 somites: thoracic, 252 second (T2), 279 third (T3), 279 somitogenesis, 252, 253, 254, 299, 318 somitomeres aortic arches, 283 assembly of face, 302 caudal, 281, 307 caudal to Sm7, 318 cranial, 307, 318 dorsal, 281 dorsal aspect, 283 epaxial, 280, 281 first eleven (humans), 255 formation of cranial base, 297 Hox gene code, 281 human head, 253 hypaxial, 280, 281 matching rhombomeres, 255 mesoderm, 255 moment when a somitomere becomes a somite, 253 numbering system ‘differs from existing textbooks’, 253 paired, 282 paraxial mesoderm, 277–81 relationship with somites, 318 rostral rhomboencephalic derivatives, 291 segmentation, 254 six head segments, 255 skeletal muscle embryology, 409 somitomeres, 250, 252–4, 271, 283, 306, 325 Sonic hedgehog (SHH) gene, 8, 310, 409 HPE, 17–21, 22, 520 implicated in CNS malformation syndromes, 521 mutations (HPE), 18–21, 22, 520 secondary amyoplasia, 424 Sonic hedgehog pathway, 19–20 Sonic hedgehog protein (Shh), 257, 471, 521 Sonic hedgehog signaling (Shh), 19, 465 Sotos syndrome, 10, 522, 527 spasticity, 27, 32, 207, 209, 224, 244, 418, 558, 565–6, 596, 597, 599, 605, 612, 617

INDEX speech disorders/impairment, 30, 347, 603–4 sphenethmoid (complex), 301, 307, 308 sphenoid/s, 261, 279, 283, 286, 288, 291, 292, 302, 304, 308, 332, 338 sphenoid bone (r1 structure), 260, 264, 285, 294, 329, 577 spina bifida, 28, 90, 94, 96, 98, 413, 444–5, 446, 583 spina bifida aperta, 580–1, 585 spinal central canal, 7, 8, 9, 67, 75, 89, 90, 92, 93, 94, 95, 98, 111 spinal cord, 5–6, 18, 46, 76, 89–98, 156, 197, 249, 252, 259, 315–6, 318, 323, 362, 365, 380, 433, 442, 443, 538, 586, 587, 599, 600 spinal cord: detethering, 581–3, 584 spinal muscular atrophy, 535 spinal trigeminal nucleus and tract, 63 spine, 5, 55, 274, 303, 424, 444 Sprintzen syndrome, 454 squamous temporal bone, 279, 295, 307, 329 formed from Sm3, 284 Stanford-Binet Intelligence Scale (SB-IV), 30 status epilepticus, 161, 400, 504, 557, 604 stem cells, 214, 377, 471, 487, 494, 534 Stevens-Johnson syndrome, 561, 561 STK9 gene, 345 stomodeum, 44, 262, 304 strabismus, 47, 61, 122, 223, 423, 472, 604 strokes, 186, 359, 360 Sturge-Weber syndrome, 10, 155, 354, 369, 380, 389 styloid process, 281, 326, 329 subacute necrotizing encephalomyelitis (Leigh syndrome), 81, 469, 536, 537 subarachnoid space, 7, 167, 222, 223, 230, 235, 416, 445, 446, 488, 490, 578, 580 subcortical areas, 129, 362, 492 subcortical band (laminar) heterotopia, 191–204 subcortical heterotopias, 23, 236, 399, 470, 481 subcortical transmantle hyperintensity, 483 subependymal giant cell astrocytomas, 130, 131, 133, 135, 136, 137, 146 subependymal nodules, 393, 482, 612 calcific, 163 subiculum (transitional cortex), 70, 75 substance P, 546 subventricular zone, 136, 377, 535, 540 sulcus/sulci, 23, 57, 58, 59, 115, 130, 191, 206, 237, 452, 453, 488 superficial musculo-aponeurotic fascia (SMAS), 261, 296, 297 suppression-burst pattern, 169, 394, 506, 510

suprachiasmatic nucleus (SCN), 434, 436 supraoccipital bone, 94, 95, 252, 278, 279–80, 281, 298, 328 supraoptic (SO) nuclei, 45, 433, 434, 436, 437 supraorbital neurovascular of V1, 285–6 sutures, 161, 580, 581, 586 absent, 62 coronal, 574 cranial, 257 craniofacial clefts, 260, 271, 279 frontonasal, 576 oronasal soft tissues, 309 prosencephalic derivatives (sixteen-somite stage), 285 spheno-occipital, 298 synaptic vesicle (SV) protein, 543, 544, 561 sylvian fissures, 23, 25, 43, 48, 54, 60, 70, 206, 238, 454, 469, 481, 509 synapses, 63, 78, 157, 391, 399, 437, 537, 545, 611 synaptic vesicle protein, 543, 544, 561 synaptogenesis, 377, 399, 459, 487, 544, 546, 554 synaptophysin, 40, 76, 78, 159, 170, 537, 538, 543, 544 synaptophysin immunoreactivity, 135–6, 536, 544 syndactyly, 28, 179, 341, 343, 347 syringomyelia, 94, 96, 578, 579, 580 tau proteins, 538, 546, 548 Taylor-type dysplasias, 391, 392, 403 TDGF-1/cripto gene, 18, 20, 22 tegmentum, 71, 157, 433 telencephalic internal structural heterotopia (TISH), 391 telencephalon, 20, 25, 26, 109, 250, 276, 287, 315 temporal bone, 281, 287, 290, 296, 326, 576 temporal lobe epilepsy, 390, 400, 486, 491 tentorium, 77, 118, 124, 283 tentorium cerebelli, 71, 89, 283 teratogens, 21, 45, 46, 92, 93, 96, 115, 381, 382, 520, 561, 591 Tessier clefting system, 250, 286, 287, 289, 292–5, 298, 306, 307, 308, 310, 332–4, 336–7 tetralogy of Fallot, 28, 56, 81, 343, 345 TGIF gene, 18, 22, 46, 520, 521 third ventricle, 15, 22, 48, 62, 76, 77, 92, 94, 95, 131, 180, 380, 433, 437, 438, 445, 446, 569, 571, 578 Thurston syndrome (OFDS type V), 341, 344 thyroid, 49, 283, 323, 437, 445, 463, 524 thyroid-stimulating hormone (TSH), 32, 48, 439, 443 thyrotrophs, 435, 439, 440 thyrotropin-releasing hormone (TRH), 49, 435, 436

INDEX thyroxine, 32, 48, 49, 463 tiagabine, 558, 559–61 tongue musculature, 253, 256, 328 topiramate, 170, 403, 559–61, 563 torcula, 89, 90, 94–5, 369–70 toxicity, 237, 238, 361, 560, 562 toxins, 21, 81, 92, 381, 382, 459, 460 toxoplasmosis, 379–80, 381 transcription factors, 106, 256, 438, 462, 520 transcription factors: mutation, 439–43 HESX-1 gene, 439–40 Lhx-3/Lhx-4, 441–3 pitx-2 gene, 440–1 transmantle cortical dysplasia, 130, 132, 133 trapezius muscle, 253, 256, 278, 296 tricarboxylic acid (TCA) cycle disorders, 464–5, 470 triploidy, 119, 518, 520 trisomies, 81, 96, 119, 494, 520 trisomy-13 (Patau syndrome), 18, 76, 81, 119, 453, 494, 518, 520 trisomy-18 (Edwards syndrome), 18, 81, 119, 452–3, 494, 520 trisomy-21 (Down syndrome), 46, 328, 451, 452, 454, 494 TSC-1 gene (chromosome 9q34), 133, 136, 138 characterization, 138 gene implicated in CNS malformation syndromes, 521 mutations, 129, 137, 143, 524 TSC-1/TSC-2, 130, 134, 135 TSC-2 gene (chromosome 16p13.3), 133, 137, 138, 141, 142, 521 encodes tuberin, 524 mutations, 129, 134, 136, 138, 143 tuberin (protein), 134, 136, 137, 138, 141, 521, 524 tuberin-hamartin complex (THC), 134, 135–6, 147 tuberous sclerosis complex (TSC), 6, 10, 129–51, 391, 486, 490, 517, 519, 524, 538, 540, 548 age of onset, 139, 140 cardiac manifestations, 142 clinical features, 129–30, 138–44 clinical management, 146–7 cutaneous manifestations, 141–2 diagnostic criteria, 144–6 familial, 129, 141, 144–6 genotype/phenotype correlations, 143–4 manifestations: neurological, 138–41 manifestations: nonneurological, 141–3 molecular genetics, 130, 137–8 neuropathology, 130–7 ophthalmic manifestations, 143 pathogenesis, 132–3 pathogenesis: developmental, 136–7 pathogenesis: molecular, 133–6 pulmonary manifestations, 143

tuberous sclerosis complex (TSC) (Continued ) renal manifestations, 142 treatment, 147–8 tubulins, 135, 199, 213, 545, 546 tumors, 162, 259, 288, 289, 482, 485, 487, 569, 403, 488 brainstem, 570 cerebellar, 116 developmental, 390, 403 diagnosed in utero, 583 dysembryoplastic neuroepithelial, 490, 491, 505 intra- and paraventricular, 569 low-grade, 390 mixed neural, 10 neuroepithelial, 540 pineal, 570 primitive, 289 Turner syndrome (45, X), 81, 453 turricephaly, 54, 62, 62, 63 twins, 9, 44, 54, 96, 345, 472 ultrasound, 27, 47, 54–5, 59–60, 90, 117, 142, 153, 165, 166, 171, 210, 243, 347–8, 425, 453, 455, 471, 472, 524, 585, 599 ultrasound transcranial sonography, 365 Unplugged (gbx-2) gene, 107, 110, 117, 315 upper beak epithelium (UBE), 260, 262, 285, 286 upregulation genes, 7, 8, 92, 98, 107, 110, 111 organizer genes, 9 urine, 49, 260, 345, 425, 460, 469, 585, 600 uterus, 461, 524, 585, 586 uvula, 53, 57, 89, 95, 305, 343, 577 valproate, 42, 56, 61, 402, 520, 600, 605 valproic acid, 81, 170, 381, 559–60 side effects, 561 Varadi syndrome (OFDS type VI), 18, 341, 347 VAs (venous angiomas), 363, 365, 482 vascular anomalies, 24, 45, 109, 160 vascular endothelial growth factor (VEGF), 282, 363 vascular malformations (VMs), 122, 162, 164, 362, 389 vascularization (biological system), 249, 260, 359 vasculature, 25, 142, 158, 251, 289 vasopressin (antidiuretic hormone), 31, 49, 50, 433–6 vein of Galen, 354, 369–70 aneurysmal malformations (VGAMs), 365, 366 malformations (VGMs), 365–7 velocardiofacial syndrome, 18, 454, 518 venous angiomas (VAs), 363, 365, 482

643 ventral horn cells, 422, 424 disease, 411, 412 ventricles, 54–5, 62, 167, 219, 535 abnormal, 170 absence, 60 cerebral, 463 diverticulum atrium right, 56 frontal horn, 166, 166, 464 hypoproliferation hypothesis, 463 ipsilateral, 495 normal, 71 ventricular dilatation, 59–60, 122, 166, 168, 225, 226, 243, 419 ventricular wall, 177, 178, 179, 180, 243, 573 ventricular zone, 136, 196, 197 ventriculo-peritoneal shunt, 22, 244, 569, 573 ventriculomegaly (VM), 59, 94, 96, 178, 179n, 191, 192, 243, 380, 410, 412, 464, 466, 470 vermis, 53, 57, 58, 61, 63, 95, 97, 98 agenesis, 95 aplasia, 346 clefting, 121 dysgenesis, 122 dysplastic upper, 120 inferior cerebellar, 80 neuromere agenesis, 108 posterior, 95 reduced size, 124 vermis hypoplasia, 121, 122–3, 206, 224, 227, 219 vertebrae, 106, 252, 254, 259, 280, 285, 297, 320, 326 first thoracic, 257 lumbar, 7 murine, 257, 274, 319 primitive, 328 seventh cervical, 257 third thoracic, 279 thoracic, 7 thoracic and lumbar (mouse), 319 true cervical, 284 vertebrobasilar system, 71, 354, 355, 357 vesicles, 182, 287, 317, 598 auditory, 315 brain, 116 cerebral, 90 eye, 5 mesencephalic, 106 optic, 261, 285, 286, 287, 289, 303, 315 otic, 304, 322–3 peripheral synaptic, 543 synaptic, 78, 534, 543, 544 telencephalic, 6 terminal synaptic, 543 Vici syndrome, 242, 520 vigabatrin (VGB), 135, 141, 147, 170, 558, 559, 560 side effects, 561 vimentin, 92, 98, 536, 537, 538, 546–550

644 vimentin (Continued ) maturational marker, 93 overexpression, 94 upregulation, 94 vimentin immunoreactivity, 94, 535, 536 vision, 47, 74, 196, 595 blurred (side effect of AEDs), 561 impairment, 40, 147, 412 SOPD, 40, 42 visuospatial difficulties, 453, 454, 455 vitamins, 383 vitamin A, 92, 110, 381 vitamin B, 422, 462 vitamin C, 422, 462 vitamin D, 541 vitamin E, 422, 462 VLDLR gene, 522, 525 vomer bone, 250, 251, 263, 264, 288, 290–1, 299, 302, 307, 310 Carnegie staging system, 304, 305 duplicated, 344 hypoplastic, 291 r1 structure, 248 voxels, 479, 485, 486, 487, 493 Waardenburg syndrome, 9, 10 Waldeyer’s ring, 260, 300 Walker-Warburg syndrome (WWS), 4, 18, 219, 220, 222, 223, 224, 226–30, 411, 414, 416, 420–1, 495, 525 cerebellar hypoplasias, 124 characteristic features, 227 familial, 228–9 gene implicated, 522 POMT2 mutations, 228–9

INDEX Walker-Warburg syndrome (WWS) (Continued ) prognosis, 418 Wechsler Adult Intelligence Scale (WAIS-III), 30, 61 West syndrome, 138, 161, 193, 392, 399, 510 Whelan syndrome (OFDS type VII), 344 Williams syndrome, 454–5 chromosome 7 at 7q11.23 (deletion), 454 posterior fossa: malformations, 523 wingless (WNT) family of genes, 7, 20 Wnt genes, 92, 105 Wingless-1 (Wnt-1), 107 Wnt-1, 316, 321 Wnt-1 produced at the isthmus, 261 wnt-1 gene, 107, 117, 259, 315 Wnt-2, 321 wnt-7B (murine gene), 63 Wolf–Hirschhorn syndrome (4p-), 455, 518 Wolff–Parkinson–White syndrome, 144

X-linked (XL) disorders/anomalies, 121, 208, 519, 525, 527 X-linked dominance, 80, 341, 518 X-linked lissencephaly with ambiguous genitalia (XLAG), 207, 209, 212, 215, 494 clinical, imaging, and genetic features, 208 gene implicated, 522 genotype-phenotype correlations, 612 X-linked recessive disorders, 75, 470, 518, 520

XLIS gene, 493 XO Turner syndrome, 81, 453 XXY Klinefelter syndrome, 81, 453

Y116C mutant, 443 yolk sac, 258, 269, 272, 278, 306, 317

Zellweger syndrome (ZS) (cerebrohepato-renal disease), 18, 46, 94, 411, 421, 465, 471, 519, 520, 526 features, 422 imaging, 495 prognosis, 422 ZFHX1B gene, 523, 527 zinc, 461, 462 zinc-finger gene (ZIC-1), 119, 521, 523 zinc-finger gene (ZIC-2), 7 dorsalizing gene, 8 mutations, 18, 20, 22, 25, 520, 521 zinc-finger gene (ZIC-3), 154 zinc-finger gene (ZIC-4), 119, 521, 523 zinc-finger homeobox gene (ZFHX1B) 76 zinc-finger proteins, 20, 256, 521 zinc-finger transcription factors, 20 ziprasidone, 564, 564 zonisamide, 559–61 zygoma, 248, 279, 292, 294, 295, 299, 305, 307 zygomaticofacial nerve, 307 zygomaticofacial neurovascular bundle, 295

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