Noonan Syndrome and Related Disorders - A Matter of Deregulated Ras Signaling (Monographs in Human Genetics Vol 17)

Noonan Syndrome and Related Disorders A Matter of Deregulated Ras Signaling

Monographs in Human Genetics Vol. 17

Series Editor

Michael Schmid

Würzburg

Noonan Syndrome and Related Disorders – A Matter of Deregulated Ras Signaling Volume Editor

Martin Zenker

Erlangen

25 figures, 17 in color, and 16 tables, 2009

Basel · Freiburg · Paris · London · New York · Bangalore · Bangkok · Shanghai · Singapore · Tokyo · Sydney

Martin Zenker Institute of Human Genetics University Hospital Erlangen University of Erlangen–Nuremberg Schwabachanlage 10 D–91054 Erlangen

Library of Congress Cataloging-in-Publication Data Noonan syndrome and related disorders : a matter of deregulated ras signalling / volume editor, Martin Zenker. p. ; cm. -- (Monographs in human genetics, ISSN 0077-0876 ; v. 17) Includes bibliographical references and indexes. ISBN 978-3-8055-8653-5 (alk. paper) 1. Genetic disorders. 2. Ras oncogenes. 3. Ras proteins. I. Zenker, Martin. II. Series. [DNLM: 1. Noonan Syndrome--genetics. 2. Noonan Syndrome--physiopathology. 3. Signal Transduction. 4. ras Proteins--genetics. W1 MO567P v.17 2009 / WD 375 N817 2009] RB155.5.N66 2009 616⬘ .042--dc22 2008035626

Bibliographic Indices. This publication is listed in bibliographic services, including Current Contents®. Disclaimer. The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publisher and the editor(s). The appearance of advertisements in the book is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements. Drug Dosage. The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. © Copyright 2009 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland) www.karger.com Printed in Switzerland on acid-free and non-aging paper (ISO 9706) by Reinhardt Druck, Basel ISSN 0077–0876 ISBN 978–3–8055–8653–5 e-ISBN 978–3–8055–8654–2

Contents

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1 9 20 40 55 66 73 83 94 104 109

Editorial Schmid, M. (Würzburg) Preface Zenker, M. (Erlangen) History of Noonan Syndrome and Related Disorders Noonan, J.A. (Lexington, Ky.) The Clinical Phenotype of Noonan Syndrome Allanson, J.E. (Ottawa) Molecular Genetics of Noonan Syndrome Tartaglia, M. (Rome); Gelb, B.D. (New York, N.Y.) Genotype-Phenotype Correlations in Noonan Syndrome Sarkozy, A.; Digilio, M.C.; Marino, B.; Dallapiccola, B. (Rome) LEOPARD Syndrome: Clinical Aspects and Molecular Pathogenesis Sarkozy, A.; Digilio, M.C.; Zampino, G.; Dallapiccola, B.; Tartaglia, M. (Rome); Gelb, B.D. (New York, N.Y.) The Clinical Phenotype of Cardiofaciocutaneous Syndrome (CFC) Roberts, A.E. (Boston, Mass.) Molecular Causes of the Cardio-Facio-Cutaneous Syndrome Tidyman, W.E.; Rauen, K.A. (San Francisco, Calif.) The Clinical Phenotype of Costello Syndrome Kerr, B. (Manchester) The Molecular Basis of Costello Syndrome Sol-Church, K.; Gripp, K.W. (Wilmington, Del.) Endocrine Regulation of Growth and Short Stature in Noonan Syndrome Binder, G. (Tübingen) The Heart in Ras-MAPK Pathway Disorders Digilio, M.C.; Marino, B.; Sarkozy, A.; Versacci, P.; Dallapiccola, B. (Rome)

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128 138 151

165 166

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Myeloproliferative Disease and Cancer in Persons with Noonan Syndrome and Related Disorders Kratz, C. (Freiburg) Neurofibromatosis Type 1-Noonan Syndrome: What’s the Link? Denayer, E.; Legius, E. (Leuven) Animal Models for Noonan Syndrome and Related Disorders Araki, T.; Neel, B.G. (Toronto, Ont.) Towards a Treatment for RAS-MAPK Pathway Disorders Joshi, V.A.; Roberts, A.E.; Kucherlapati, R. (Boston, Mass.) Author Index Subject Index

Contents

Editorial

This volume 17 of Monographs in Human Genetics is an in-depth discourse on the disorders of the Ras-MAPK pathway (Noonan-, cardio-facio-cutaneous-, Costello-, and LEOPARD syndromes). Like the two preceding volumes of this book series, it deals with important hereditary diseases with high clinical impact, and whose molecular causes have been unravelled in recent years. Noonan syndrome belongs to one of the most frequent monogenic disorders occurring in approximately one in 1,000 to 2,500 children and therefore has significant importance in public health genomics. Molecular analyses have led to the surprising result that all four syndromes can be traced back to specific mutations in genes coding for molecules that interact in the Ras-MAPK pathway. This exciting discovery does not only permit the precise diagnosis of the diseases, but also clears promising ways for potential therapies in the future. Martin Zenker, the Editor of the present volume, succeeded in bringing together the leading experts working on these diseases and received their contributions in a very short space of time. The articles treat both the clinical and molecular data exhaustively and give the reader a very timely update and outline of these related disorders. I thank Martin Zenker and all the authors for their time and effort to render possible the publication of this book. Furthermore, I gratefully acknowledge the constant promotion of this book series by Thomas Karger. Michael Schmid Würzburg, August 2008

Preface

Noonan syndrome (NS), which is recognized as one of the most common monogenic disorders, was defined as a separate entity by Jacqueline Noonan in 1968. Thirty-three years later, the first gene for NS was identified by Marco Tartaglia and colleagues. Their discovery represented the spark for a series of new gene discoveries eventually showing that mutations that alter the function of molecules interacting in a common signalling cascade, the Ras-MAPK pathway, are responsible for NS and the clinically related disorders cardio-facio-cutaneous syndrome (CFCS), LEOPARD syndrome (LS), and Costello syndrome (CS). Together, these findings unexpectedly related this group of disorders to a signalling pathway which was previously known for its involvement in tumorigenesis. Thereby, the association of certain types of malignancies and tumor-like lesions with NS, LS, CFCS, and particularly CS has been elucidated. Vice versa, studies on the significance of somatic mutations in the same genes in sporadic tumors have been stimulated and yielded exciting new findings. Notably, the genes mutated in Neurofibromatosis 1 and a newly defined Neurofibromatosis 1-like phenotype encode negative regulators of the same pathway. Thus, the known clinical relations between all these conditions have become intelligible through the achievements of molecular research. The Editor of this volume of Monographs in Human Genetics greatly acknowledges the contributions of excellent experts in the field. Their comprehensive reviews provide most updated data on the various clinical and molecular aspects of known disorders of the Ras-MAPK pathway. Jacqueline Noonan herself is giving an historical overview in the first chapter. The book ends with a chapter on current and possible future treatment options for this group of disorders. Together the contributions to this volume nicely show the close relationship between clinical issues and molecular research and the mutual benefit for people working in either of these fields. It is of note that the previously established clinical entities are strongly correlated with certain mutated genes or – in the case of LS – specific functional consequences of certain mutations. The proposed term neuro-cardio-facial-cutaneous syndromes for all disorders caused by germline mutations in components of the Ras-MAPK pathway may be useful as a superordinate, but currently there is no need to replace the established nosology, which is also used in this book. The content of this volume certainly does not represent a story that has been completed, but it is much more than a progress report. The chase for genes for NS and related disorders seems to have reached a

IX

plateau, although it is obvious that there are still patients who do not have a mutation in the known genes. Following strict diagnostic criteria, the underlying mutation may now be found in more than 80% of patients with NS, 90% of patients with CFCS, and virtually all cases with CS. Future research will reach out for new goals by focusing on the refinement of genotype-phenotype correlations by studying larger cohorts, as well as on the development of model systems to explore the precise molecular pathogenesis of dysregulated Ras-MAPK signaling. One of the most fascinating prospects may be the possibility to invent treatment options for NS and related disorders by pharmacological modulation of Ras-MAPK signaling. Concerted international efforts will be required to reach these goals. Martin Zenker Erlangen, August 2008

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Preface

Zenker M (ed): Noonan Syndrome and Related Disorders. Monogr Hum Genet. Basel, Karger, 2009, vol 17, pp 1–8

History of Noonan Syndrome and Related Disorders J.A. Noonan Department of Pediatrics, Division of Cardiology, College of Medicine, University of Kentucky, Lexington, Ky., USA

Abstract

Noonan Syndrome (NS)

Deregulation of the RAS pathway by some recently discovered germline mutations reveals that this pathway, known to play an impohrtant role in human oncogenesis, also plays an important role in fetal development, cognition and growth. In this volume, the clinical phenotype of Noonan syndrome and related disorders will be reviewed, the genes for these syndromes discussed and possible treatment options will be considered. This chapter will include a brief history of Noonan syndrome and related disorders, including LEOPARD, cardio-facio-cutaneous, Costello and Neurofibromatosis-Noonan syndrome. In addition, speculation as to the possible cause of the distinctive and similar facial phenotypes seen in infancy in these syndromes will be discussed. When Noonan syndrome (NS) was described, it was suspected that a genetic cause would be found. The exciting discovery that mutation of the PTPN11 gene was the cause of NS in about half the cases demonstrated that deregulation of the RAS pathway could cause a variety of congenital malformations. This important discovery showed that the RAS pathway plays a role not only in human oncogenesis but also in fetal development, cognition and growth. This chapter will briefly discuss the history of NS and related disorders including LEOPARD, cardio-facio-cutaneous, Costello and Neurofibromatosis-NS. In addition, speculation as to the possible cause of the distinctive and similar facial phenotypes seen in early infancy in these syndromes will be discussed. Copyright © 2009 S. Karger AG, Basel

In 1962, Noonan [1] presented at the Midwest Society for Pediatric Research a clinical study of associated noncardiac malformations in children with congenital heart disease and described nine patients who shared distinctive facial features which included hypertelorism, downslanting palpebral fissures, low set posteriorly rotated ears, ptosis and malar hypoplasia. In addition, most were short in stature, all had pulmonary stenosis and additional deformities included undescended testes and a chest deformity. In 1968 [2], she published these nine and an additional ten patients. Dr. John Opitz [3] proposed the eponym Noonan syndrome be given to this syndrome. He felt that she was the first to describe this condition to occur in both sexes, to be associated with normal chromosomes, to have a congenital heart defect and to be familial in some cases. Several authors have suggested that the first reported patient with what is now called NS was reported by Kobylinski [4] (1883). This was a 20-year-old male who had marked webbing of the neck. It was this feature that seemed to prompt most of the early reports. Funke [5] (1902) reported a patient with a webbed neck as well as short

stature, micrognathia, cubitus valgus and other minor abnormalities. This report was followed by Ullrich [6] (1930) who reported an 8-year-old girl with similar features. Turner [7] (1938) reported older females who had facies similar to Ullrich’s but, in addition to short stature, had sexual infantilism. Before Turner syndrome was shown to be a sex chromosome abnormality, Flavell [8] (1943) introduced the term ‘male Turner syndrome’. This term led to considerable confusion in the literature for a number of years. Ullrich [9] (1949) reported a series of patients whom he had noted for over two decades. In that study, there was a 4:1 predominance of females over males. He noted the similarity between his patients and mice that had been bred by Bonnevie. Bonnevie was a mouse geneticist who had bred a mutant strain of mice with a webbed neck who also had lymphedema. The term Bonnevie-Ullrich syndrome became popular particularly in Europe. This term was used to describe children, some of whom would now be recognized as having NS while others would be recognized as having Turner syndrome. In 1959, Turner syndrome was found to have a 45, X chromosome pattern. Reports of ‘male Turner Syndrome’ or Turner phenotype in males were reported throughout the 1960s and 70s. Heller [10] (1965) reviewed 43 cases from the literature and reported five additional cases of his own. These early reports were mainly by endocrinologists who used this term for patients with a variety of testicular problems with or without short stature. A vigorous attempt to find a chromosomal abnormality in the ‘male Turner syndrome’ was unsuccessful. When chromosome studies became more widely available, it became clear that not all girls previously diagnosed with Turner syndrome had Turner syndrome but, in reality, had NS. Some, but certainly not all of the males previously called ‘male Turner syndrome’, fit the clinical description of NS. NS is one of the most common nonchromosomal syndromes seen in children with congenital heart disease.

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It occurs worldwide. The estimated incidence of NS is reported to be 1:1,000 to 1:2,500. NS is an autosomal dominant disorder with complete penetrance but variable expressivity. Many cases however are sporadic. Some patients have such a mild phenotype that they are never recognized while individuals with severe manifestations can be recognized as abnormal in early infancy. Allanson et al. [11] made the important observation that the phenotype in NS changes significantly over time. Some cases previously felt to be sporadic were later recognized as familial when photographs of parents taken at a similar age to the affected child were compared. It is not uncommon for NS to be first recognized in a parent after an affected child is born. Noonan reported a high incidence of valvular pulmonary stenosis and noted that the valves were often dysplastic. Ehlers et al. [12], in 1972, reported the first case of hypertrophic cardiomyopathy and this report was followed by Hirsch et al. [13] in 1975. In 1992, Burch et al. [14] demonstrated that the microscopic findings are similar to those seen in nonsyndromic familial hypertrophic cardiomyopathy. In the 1970s, lymphatic problems were reported. Intestinal lymphangiectasia was reported by Vallet et al. [15] in 1972 and pulmonary lymphangiectasis by Baltaxe et al. [16] in 1975. In the 1970s and 1980s, there were several reports describing lymphangiograms showing lymphatic dysplasia. Lymphatic abnormalities are reported in less than 20% of patients but may present serious problems. Fetuses are commonly recognized to have cystic hygroma. Prolonged pleural effusions following heart surgery are common. Some infants are born with hydrops and chylous thorax. This may be difficult to manage and is a cause of death in some severely affected infants. Easy bruising is common in NS. Kitchens and Alexander [17] in 1983, described partial deficiency of Factor XI and others have described deficiencies of Factor VIII and XII as well as thrombocytopenia and platelet dysfunction. In the 1990s, the occurrence of myeloproliferative

Noonan

disorders, including juvenile myelomonocytic leukemia was reported in NS. In 1992 Sharland et al. [18] reported on a large clinical study of patients with NS and discussed the feeding problems which are so often a problem in infancy. He also called attention to the frequent eye findings. Growth hormone studies and the use of growth hormone began to be reported in the 1990s. In 1994 [19], the gene for NS was mapped to the long arm of chromosome 12. One family, however, did not link suggesting more than one gene was involved. The search for the mutant gene began but it was not found until 2001. Tartaglia et al. [20], found a mutation in the PTPN11 gene. This mutation is found in about half of the patients with NS. In the past three years, three additional genes hav e been identified, KRAS, SOS1 and RAF1. It is likely that additional genes will be found to represent the 25–30% still without a known mutation. Mutations in the PTPN11 gene have a very high incidence of congenital heart disease of at least 80%. Pulmonary stenosis is most commonly found and there is a low incidence of hypertrophic cardiomyopathy. The most recently identified gene, RAF1, has a high incidence of hypertrophic cardiomyopathy. Patients with SOS1 have some cutaneous findings similar to Cardio-facio-cutaneous syndrome. Genotypephenotype correlations are discussed in a separate chapter of this book.

LEOPARD Syndrome

LEOPARD syndrome is a rare autosomal dominant disorder that shares many phenotypic features with NS. This syndrome was described by Gorlin et al. [21] in 1969. The facial features are similar to NS but in addition there are multiple lentigines and café-au-lait spots as well as deafness. In 2002, two groups of investigators found PTPN11 mutations in LEOPARD syndrome and demonstrated that LEOPARD syndrome and NS are allelic disorders caused by different missense

History of Noonan Syndrome and Related Disorders

mutations in the PTPN11 gene. About 90% of LEOPARD syndrome patients have a PTPN11 mutation but more recently a mutation in RAF1 has been shown to account for the remaining 10%. While NS most frequently has pulmonary stenosis and less commonly hypertrophic cardiomyopathy, LEOPARD syndrome has a very high incidence of hypertrophic cardiomyopathy and a lower incidence of pulmonary stenosis. These syndromes are very difficult to distinguish in infancy since lentigines do not appear until later in childhood and hearing loss may not be apparent in early infancy. Digilio et al. [22] recently reported 10 infants with suspected LEOPARD syndrome. Eight of these were found to have a mutation of the PTPN11 gene confirming the diagnosis of LEOPARD syndrome. They all had facies similar to NS although in some the findings were quite mild. Hypertrophic cardiomyopathy was present in seven of the eight infants and pulmonary stenosis in two of the eight. A single patient without hypertrophic cardiomyopathy had a dysplastic mitral valve. Although only one patient had lentigines, six of the eight did have café-au-lait spots. This suggested to Digilio that café-au-lait spots in early infancy in a patient with hypertrophic cardiomyopathy should strongly suggest the possibility of LEOPARD syndrome. It is of interest that, of the remaining two patients, one patient did have neurofibromatosis but had a Noonan phenotype. The second patient did not have a mutation of the PTPN11 gene. It will be interesting to see if that patient has a RAF1 mutation. LEOPARD syndrome is another example where the overlap between neurofibromatosis and NS exists.

Cardio-Facio-Cutaneous Syndrome (CFC)

CFC is a multiple anomaly syndrome with significant mental retardation. It occurs sporadically and is characterized by failure-to-thrive, macrocephaly, a distinctive face similar to NS with

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bitemporal constriction, hypertelorism, downward slanting palpebral fissures, depressed nasal root and low set ears. There is usually significant cutaneous involvement consisting of dry hyperkeratotic scaly skin, sparse or absent eyebrows and sparse or absent eyebrows and sparse slow growing curly hair. This syndrome was first described 20 years ago by Reynolds et al. [23] who described eight patients whom they felt represented a distinct syndrome. These reports were followed by considerable controversy in the literature. Many questioned whether CFC was a unique and separate condition or a variant of NS. Unlike NS, CFC is quite rare. About 100 cases have been confirmed so far. Although the facies are similar to NS in infancy, at older age, the face remains broad and coarse and does not develop the inverted triangular shape seen in NS. Cutaneous manifestations are prominent but may overlap with NS, especially NS with the SOS1 mutation. In 2002, Kavamura et al. [24] published a CFC index to aid in the diagnosis of this syndrome. Fortunately, in 2006, two groups of investigators found BRAF as well as MEK1, MEK2 and occasional KRAS mutations to be responsible for CFC. Earlier it has been determined that patients with CFC did not have a mutation in the PTPN11 gene so that by now it is clear that CFC is distinct from NS. The most common mutated gene appears to be BRAF followed by MEK1 and MEK2 and occasional patients with a KRAS mutation. Like NS, cardiovascular malformations are frequent. About 75% [25] have some kind of a cardiac malformation. Forty-five percent of those are pulmonary stenosis and 40% hypertrophic cardiomyopathy.

Costello Syndrome

Costello syndrome is a rare disorder. It was first described by Dr. Costello in 1971 [26] at a meeting. He described two patients with mental

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retardation, high birth weight, feeding problems, coarse facies, nasal papillomata and loose integument of the back of the hands. In 1977 [27], he published these two cases in more detail. Following that, a number of authors reported patients who displayed the phenotype described by Costello but they were unaware of Costello’s report. DerKaloustian et al. [28], first used the term Costello syndrome in 1991. In 1992, Johnson et al. [29] reported eight patients with Costello syndrome and reviewed 29 cases that had been previously reported under a variety of titles who undoubtedly also had Costello syndrome. These patients have a distinctive facial appearance which may be difficult to distinguish from NS and CFC in infancy. In 1994, Lurie [30] wrote that Costello was likely a sporadic autosomal dominant mutation. By 1991, malignancies were being reported in Costello patients, particularly bladder carcinoma and rhabdomyosarcoma. Costello syndrome is characterized by polyhydramnios, overgrowth and edema with postnatal feeding difficulties and failure-to-thrive. Characteristic facial features include macrocephaly, a high forehead, usually curly hair, hypertelorism, fleshy nasal tip, full lips, wide mouth, full cheeks and fleshy earlobes. In infancy, there is excessive skin wrinkling. The skin appears very loose. There is postnatal growth retardation and developmental delay. The characteristic skin disorder in Costello syndrome suggested that there might be an elastin fiber abnormality. Hinek et al. [31] in 2000 found that fibroblasts from Costello syndrome were able to produce normal levels of tropoelastin and to properly position the microfibrillar scaffold but they were unable to assemble elastin fibers because of a deficiency in the elastin binding protein. In addition, they found fibroblast cultures from Costello syndrome patients showed an increased rate of proliferation. They postulated that disturbed elastogenesis could explain the interesting skin findings and that increased proliferation of fibroblasts in tissue culture might explain

Noonan

the increased tumor rate in Costello syndrome. It now makes sense that deregulation of the RAS pathway could explain the functional deficiency of the 67-kDa elastin binding protein (EBP) proposed by Hinek. In 2005 [32], Aoki et al. reported that germline mutations in HRAS caused Costello syndrome. This finding was soon confirmed by a number of other investigators. Although this is a rare disease, as soon as the gene for Costello was discovered, 40 samples of DNA from Costello patients were available for confirmation. This DNA was available from patients who had been clinically diagnosed with Costello syndrome at the 2003 and 2005 International Costello Syndrome Meeting and through the Costello Syndrome Family network. Of the 40 patients with the clinical diagnosis of Costello syndrome, 33 were confirmed to have the HRAS mutation. Since it is often difficult to distinguish between CFC and Costello, it is not surprising that seven patients suspected of having Costello syndrome had mutations in either BRAF, KRAS, MEK1 or MEK2 which confirmed the phenotypic overlap between these disorders. It is now felt that the term Costello syndrome should be limited to those individuals who do have a mutation of the HRAS gene. Similar to NS and CFC, cardiovascular malformations are frequent, occurring in about 50% and include pulmonary stenosis in about 40% and hypertrophic cardiomyopathy in another 40%. Unlike NS or CFC, chaotic atrial arrhythmias are relatively frequent in Costello syndrome, particularly in infancy.

Neurofibromatosis-Noonan Syndrome (NF-NS)

Since the mid 1980s, a number of investigators have written about the presence of Noonan phenotype associated with some patients with neurofibromatosis. Neurofibromatosis Type 1 (NF1) is an autosomal dominant disorder characterized by hamartomas in multiple organs. Mutations or deletions in the neurofibromin-1 gene (NF1)

History of Noonan Syndrome and Related Disorders

have been recognized as the cause of neurofibromatosis Type 1. The NF1 gene product acts as a negative regulator to the RAS mediated signal transduction pathway. This finding provided the first direct evidence that the RAS pathway played an important role in human development. NF1 has a prevalence of about 1:3,000. Colley et al. [33] examined 94 patients with NF1 and found that 9.5% had findings that seemed similar to NS. It appears clear that there is a clinical overlap between both syndromes. So far, the etiology of NF-NS is unclear. There has been one report [34] of a patient showing features of both syndromes who was found to have two mutations, a PTPN11 mutation which was inherited from the father and a de novo NF1 mutation. This is the first and only report so far of molecular occurrence of both disorders in the same patient. Huffmeier et al. [35] recently reported seven patients from five unrelated families with variable phenotypes of the NF1-NS syndrome spectrum. Heterozygous mutations or deletions of NF1 were identified in all patients. No PTPN11 mutation was found. The NF1 mutation segregated with the phenotype in both familial cases. They felt this supported the hypothesis that variable phenotypes of the NF1-NS spectrum represent variants of NF1 mutation in the majority of cases. The NF1-NS facial phenotype is similar to NS but usually quite mild. Short stature is not common. Cardiac defects are less common but pulmonary stenosis is reported as well as a variety of other cardiac defects. Hypertrophic cardiomyopathy is also seen. Digilio et al. [22] as discussed under LEOPARD syndrome have reported the difficulty in distinguishing between LEOPARD syndrome and neurofibromatosis in infancy.

Discussion

Bentires-Alj et al. [36] suggest that the phenotype overlap between NS, NF1 and the other related syndromes reflects a similar underlying

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pathogenesis, namely deregulation of the RAS pathway and proposed that all these syndromes be called Neuro-cardio-facio-cutaneous (NCFC) syndrome. There are clearly many phenotypic similarities in infancy in these syndromes. The facies typically show low set ears, downward slanting eyes and a short neck. Polyhydramnios is common and cystic hygroma is sometimes noted by fetal ultrasound. It is tempting to blame the cystic hygroma as a likely cause of the facial phenotype. However, an interesting study by Achiron et al. [37], causes some doubt at this explanation. They propose that NS has an evolving phenotype during in utero and postnatal life. Among 46,224 live born infants only seven newborn and four fetuses were found to have NS while some 30–40 NS would be expected. Unlike Bekker et al. [38] who found cervical cystic hygroma in midtrimester to be a reliable sign for in utero diagnosis of NS Achiron noted none of his cases had evidence of septated cystic hygroma and only one of the fetuses had transient nuchal translucency. This observation indicates lymphatic abnormalities are not a sine qua non for a prenatal diagnosis of NS. Since the great majority of patients with these syndromes do not have nuchal translucency in utero it is necessary to propose another explanation for the typical facial phenotype. Some infants with NS and related disorders are clearly recognized as dysmorphic at birth. The four fetuses in Achiron’s report all developed bilateral hydrothorax and generalized edema. All had typical facies of NS. All were very ill and two died in the neonatal period. The seven infants diagnosed at birth or in early infancy had typical clinical findings of NS. This suggests that the other 30 to 40 NS expected among the 46,224 newborn delivered were mild enough to be unrecognized at least through the first year of life. It is not uncommon for a diagnosis of NS to be delayed past five to six years of age and sometimes into adulthood. Is it possible that the facial phenotype becomes more typical with time demonstrating that the RAS pathway continues to play a role in an evolving phenotype?

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The RAS pathway must play a role in this common facial phenotype but it is still unknown. On the other hand it is also clear that the RAS pathway must play a role in early lymphatic development. Lymphatic problems are well recognized in NS. Chylous thorax may be present at birth or appear spontaneously later on or be a complication following heart surgery. Pulmonary and intestinal lymphangiectasia have been reported. Some cases occur in infancy but may be delayed until adulthood. Lymphedema may present for the first time in adulthood. Bloomfield et al. [39] reported the first neonatal case with lymphangiography. The infant was born at 33 weeks with severe edema and bilateral pleural effusions which proved to be chylous. The fluid was drained and the baby improved. On day 14 a lymphangiogram was carried out from the right foot. The six lymphatics that filled were dilated and saccular. There was no filling of lymph nodes in the groin or extension into the pelvic or para-aortic lymphatics or thoracic duct. Clearly the lymphatic system was dysplastic suggesting very abnormal development. Unfortunately the infant had two episodes of viral pneumonia and died at 5 months of age. I have personal knowledge of another infant with persistent chylous thorax who underwent lymphangiography. This demonstrated absence of the thoracic duct with multiple lymphatics draining directly into the chest cavity. There is need for further study of the role of RAS in lymphatic development. While lymphatic abnormalities are clinically recognized in only 20% of NS patients, they may be unrecognized clinically in many more. Few studies of the lymphatics have been carried out but the lymphangiograms so far performed have shown significant lymphatic abnormalities.

Conclusion

It is very exciting for me to learn that NS and these related disorders disturb the RAS pathway, demonstrating that this pathway plays an important role in development. Once we understand exactly

Noonan

how these mutations alter the pathway, it may be possible to develop strategies to treat at least the postnatal effects such as short stature and hypertrophic cardiomyopathy, to name a few. Already basic scientists are making important progress in understanding the effect of specific mutations on human development. Krenz et al. [40] have shown that a mutation of Q79R-Shp2 in NS results in increased activity of the extra cellular signal-regulated (ERK)1/2 and this results in hyperproliferation in valve primordia. Nakamura et al. [41] then generated a transgenic Q79R-Shp2 mouse model which showed again the role of enhanced ERK1/2 in cardiac malformation. They were able to prevent cardiac abnormalities by ERK1/2 modulation. Gauthier et al. [42] have demonstrated the important role of Shp2 in brain development. The normal Shp2 instructs cell precursors to make neurons and not astrocytes during the neurogenic period of development. A mouse knockin mutant Shp2 model is a phenocopy of human NS. This model was shown to inhibit basal neurogenesis and caused enhanced astrocyte formation. It will be very important for these patients to have continued long-term follow-up as they age. Follow-up of adults with syndromes is very

difficult. Registries to follow these patients will be important. In countries with established registries, it will be essential that these patients be followed long-term. As we follow these adults, we may be able to identify what role the RAS pathway plays in aging. There is plenty of exciting knowledge awaiting investigators as we continue to learn more about the RAS pathway. In the United States, without national registries, three support groups founded by mothers of affected children could play a role. The NS Support Group, Costello Family Network and Cardio-facio-cutaneous International are the three support groups. They hold international meetings every one to two years and families attend with their affected children. At these meetings, information about the syndrome is shared with families and the physicians attending always learn much from the families. The children are able to interact with affected peers which provides a lot of support. These groups have or are in the process of establishing registries which could play a very important role in long-term followup of patients with all these syndromes. Little is known of the natural history of these syndromes. These mutations likely continue to exert an effect on the RAS pathway throughout life.

References 1 Noonan JA: Hypertelorism with Turner phenotype. Am J Dis Child 1968;116:373–380. 2 Noonan JA, Ehmke DA: Associated noncardiac malformations in children with congenital heart disease. J Pediatr 1963;31:150–153. 3 Opitz JM: Editorial comment: the Noonan syndrome. Am J Med Genet 1985;21:515–518. 4 Kobylinski O: Über eine flughautähnliche Ausbreitung am Halse. Arch Anthropol 1883;14:342–348. 5 Funke O: Pterygium colli. Dtsch Zeitschr Chir 1902;63:163–167.

6 Ullrich O: Über typische Kombinationsbilder multipler Abartungen. Z Kinderheilkd 1930;49:271–276. 7 Turner HH: A syndrome of infantilism, congenital webbed neck, and cubitus valgus. Endocrinology 1938;25:566–574. 8 Flavell G: Webbing of the neck with Turner’s syndrome in the male. Br J Surg 1943;31:150–153. 9 Ullrich O: Turner’s syndrome and status Bonnevie-Ullrich; synthesis of animal phenogenetics and clinical observations on a typical complex of developmental anomalies. Am J Hum Genet 1949;1:179–202.

History of Noonan Syndrome and Related Disorders

10 Heller RH: The Turner phenotype in the male. J Pediatr 1965;66:48–63. 11 Allanson JE, Hall JG, Hughes M: Noonan syndrome: the changing phenotype. Am J Med Genet 1985;21: 507–514. 12 Ehlers KH, Engle MA, Levin AR, Deely WJ: Eccentric ventricular hypertrophy in familial and sporadic instances of 46, XX, XY Turner phenotype. Circulation 1972;45:639–652. 13 Hirsch HD, Gelband H, Garcia O, Gottlieb S, Tamer DM: Rapidly progressive obstructive cardiomyopathy in infants with Noonan’s syndrome. Circulation 1975;52:1161–1165.

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14 Burch M, Mann JM, Sharland M, Shinebourne EA, Patton MA, McKenna WJ: Myocardial disarray in Noonan syndrome. Br Heart J 1992;68: 580–585. 15 Vallet HL, Holtzapple PG, Eberlein WR, Yakovac WC, Moshang T Jr, Bongiovanni AM: Noonan syndrome with intestinal lymphangiectasia. J Pediatr 1972;80:269–274. 16 Baltaxe HA, Lee JG, Ehlers KH, Engle MA: Pulmonary lymphangiectasia in 2 patients with Noonan syndrome. Radiology 1975;155:149–153. 17 Kitchens CS, Alexander JA: Partial deficiency of coagulation factor XI as a newly recognized feature of Noonan syndrome. J Pediatr 1983;102:224–227. 18 Sharland M, Burch M, McKenna WM, Patton MA: A clinical study of Noonan syndrome. Arch Dis Child 1992;67: 178–183. 19 Jamieson CR, van der Burgt I, Brady AF, van Reen M, Elsawi MM, et al: Mapping a gene for Noonan syndrome to the long arm of chromosome 12. Nat Genet 1994;8:357–360. 20 Tartaglia M, Kalidas K, Shaw A, Song X, Musat DL, et al: PTPN11 mutations in Noonan syndrome: molecular spectrum, genotype-phenotype correlation and phenotypic heterogeneity. Am J Hum Genet 2002;70:1555–1563. 21 Gorlin RJ, Anderson RC, Blaw M: Multiple lentigenes syndrome. Am J Dis Child 1969;17:652–662. 22 Digilio MC, Sarkozy A, de Zorzi A, Pacileo G, Limongelli G, et al: LEOPARD Syndrome: clinical diagnosis in the first year of life. Am J Med Genet A 2006;140:740–746. 23 Reynolds JF, Neri G, Herrmann JP, Blumberg B, Coldwell JG, Miles PV, Opitz JM: New multiple congenital anomalies/mental retardation syndrome with cardio-facio-cutaneous involvement – the CFC syndrome. Am J Med Genet 1986;25:413–427. 24 Kavamura MI, Peres CA, Alchorne MM, Brunoni D: CFC index for the diagnosis of cardiofaciocutaneous syndrome. Am J Med Genet 2002;112:12–16.

25 Roberts A, Allanson J, Jadico SK, Kavamura MI, Noonan J, et al: The cardiofaciocutaneous syndrome. J Med Genet 2006;43:833–842. 26 Costello JM: A new syndrome. NZ Med J 1971;74:397. 27 Costello JM: A new syndrome: mental subnormality and nasal papillomata. Aust Paediatr J 1977;13:114–118. 28 Der Kaloustian VM, Moroz B, McIntosh N, Watters AK, Blaichman S: Costello syndrome. Am J Med Genet 1991; 41:69–73. 29 Johnson JP, Fried MH, Norton ME, Rosenblatt R, Feldman G, Yang S: Prenatal overgrowth with postnatal growth failure, dysmorphic facies, cutaneous features, and cardiomyopathy: overlap of AMICABLE, facio-cutaneous-skeletal (fcs) and Costello (cs) syndromes. Proc Greenwood Genet Cent 1992;12:98. 30 Lurie JW: Genetics of the Costello syndrome. Am J Med Genet 1994;52:358–359. 31 Hinek A, Smith AC, Cutiongco EM, Callahan JW, Gripp KW, Weksberg R: Decreased elastin deposition and high proliferation of fibroblasts from Costello syndrome are related to functional deficiency in the 67-kD elastinbinding protein. Am J Hum Genet 2000;66:859–872. 32 Aoki Y, Niihori T, Kawame H, Kurosawa K, Ohashi H, et al: Germline mutations in HRAS proto-oncogene cause Costello syndrome. Nat Genet 2005;37:1038–1040. 33 Colley A, Donnai D, Evans DG: Neurofibromatosis/Noonan phenotype: a variable feature of type 1 neurofibromatosis. Clin Genet 1996;49:59–64. 34 Bertola DR, Pereira AC, Passetti F, de Oliveira PS, Messiaen L, et al: Neurofibromatosis-Noonan syndrome: molecular evidence of the concurrence of both disorders in a patient. Am J Med Genet A 2005;136:242–245.

35 Huffmeier U, Zenker M, Hoyer J, Fahsold R, Rauch A: A variable combination of features of Noonan syndrome and neurofibromatosis type I are caused by mutations in the NF1 gene. Am J Med Genet A 2006;140: 2749–2756. 36 Bentires-Alj M, Kontaridis MI, Neel BG: Stops along the RAS pathway in human genetic disease. Nat Med 2006;12:283–285. 37 Achiron R, Heggesh J, Grisaru D, Goldman B, Lipitz S, Yagel S, Frydman M: Noonan syndrome: a cryptic condition in early gestation. Am J Med Genet 2000;92:159–165. 38 Bekker MN, Go AT, van Vugt JM: Persistence of nuchal edema and distended jugular lymphatic sacs in Noonan syndrome. Fetal Diagn Ther 2007;22:245–248. 39 Bloomfield FH, Hadden W, Gunn TR: Lymphatic dysplasia in a neonate with Noonan’s syndrome. Pediatr Radiol 1997;27:321–323. 40 Krenz M, Yutzey KE, Robbins J: Noonan syndrome mutation Q79R in Shp2 increases proliferation of valve primordia mesenchymal cells via extracellular signal-regulated kinase 1/2 signaling. Circ Res 2005;97:813–820. 41 Nakamura T, Colbert M, Krenz M, Molkentin JD, Hahn HS, Dorn GW 2nd, Robbins J: Mediating ERK 1/2 signaling rescues congenital heart defects in a mouse model of Noonan syndrome. J Clin Invest 2007;117:2123–2132. 42 Gauthier AS, Furstoss O, Araki T, Chan R, Neel BG, Kaplan DR, Miller FD: Control of CNS cell-fate decisions by Shp2 and its dysregulation in Noonan syndrome. Neuron 2007;54:245–262.

Jacqueline A. Noonan Department of Pediatrics, Division of Cardiology, College of Medicine, University of Kentucky 800 Rose Street, MN470 Lexington, KY 40536 (USA) Tel. +1 859 323 5494, Fax +1 859 323 3499, E-Mail [email protected]

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Noonan

Zenker M (ed): Noonan Syndrome and Related Disorders. Monogr Hum Genet. Basel, Karger, 2009, vol 17, pp 9–19

The Clinical Phenotype of Noonan Syndrome J.E. Allanson Children’s Hospital of Eastern Ontario, Ottawa, Canada

Abstract Noonan syndrome is an autosomal dominant condition notable both for its common occurrence and phenotypic variability. It is characterized by short stature, congenital cardiac defects, unusual chest shape, broad or webbed neck, cryptorchidism, typical facial appearance and developmental delay of variable extent. It is frequently overlooked in the mildly affected individual and diagnosis in an adult often follows the birth of a child with more florid manifestations. Copyright © 2009 S. Karger AG, Basel

The cardinal features of Noonan syndrome are short stature, congenital heart defects, broad or webbed neck, characteristic pectus deformity, a particular facial appearance which changes with age and, in some cases, mild intellectual handicap. This pattern of features was recognized and reported more than 40 years ago by Noonan and Ehmke [1], however it is likely that Kobylinski, in 1883, was the first to publish on the condition [2]. Birth prevalence is estimated to be between 1/1,000 and 1/2,500 although mild expression is said to occur in 1 in 100 [3]. Average age at diagnosis is 9 years [4]. Life expectancy is likely to be normal in the absence of serious cardiac defects. In one natural history study, non-accidental mortality was 7%, with half of all deaths occurring in adulthood. Cause of adult death

included hypertrophic cardiomyopathy, ischemic heart disease, breast cancer, and cerebral hemorrhage [5]. There are several excellent reviews [1, 3–9].

Craniofacial Features

Facial appearance changes with age (fig. 1) [4, 10]. In the newborn, key features include tall forehead, widespaced and down-slanting palpebral fissures, ptosis or thickened eyelids, epicanthal folds, depressed nasal root with upturned nasal tip, deeply grooved philtrum with high, wide peaks of the vermilion border (so-called cupid’s bow shape), low-set and posteriorly angulated ears with thick helices, small chin, and excessive nuchal skin with a low posterior hairline. During infancy, the head is relatively large in comparison to face size, with a tall and prominent forehead. Hypertelorism, ptosis or thick hooded eyelids remain characteristic. The nose is short and wide with a depressed root. During later childhood, the face may appear coarse or even myopathic. With increasing age, the face lengthens and becomes more triangular in shape with a broad forehead tapering to a small and

a

b

c

Fig. 1. Female with Noonan syndrome at different ages, showing how facial features change with time. (a) Baby, (b) child and (c) young adult.

pointed chin. In adolescence and young adulthood, the nose has a thin, prominent bridge and a wide base. The neck is longer with accentuated webbing (pterygium colli) or a prominent trapezius. In older adults, nasolabial folds are exaggerated and the skin appears thin and transparent [6, 10, 11]. The hair may be wispy or sparse during infancy and curly or woolly in older childhood and adolescence. Despite this subjective impression of age-related facial change, detailed measurements demonstrate the opposite. There is a Noonan-specific pattern of craniofacial widths, lengths, depths and arcs that is maintained over time. This is superimposed on normal changes that occur in face shape/size with age and is perceived as a change in gestalt [12]. Features likely to be seen irrespective of age include blue-green irides, frequently out of keeping with family eye colour, arched or diamond-shaped eyebrows, and low-set posteriorly angulated ears with thickened helices [4, 6]. Malocclusion is common and likely is related to the small chin and oral cavity [6, 10]. Relative or absolute macrocephaly is usual. Mean adult head circumference in males is 56.4 cm and in females is 54.9 cm [5].

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Cardiovascular Anomalies

Congenital heart defects occur in between 50 and 90% of affected individuals [6, 13]. Since this feature may prompt diagnosis, and because many published reports come from tertiary and quaternary medical centres which place emphasis on serious structural manifestations, there may be bias of ascertainment. The most common anomaly, seen in up to half, is a dysplastic and/or stenotic pulmonary valve [5, 8, 13–15]. It may be isolated or associated with other defects. Other common structural cardiac anomalies include atrial or ventricular septal defects and tetralogy of Fallot. Many other cardiac defects have been reported less commonly, including atrioventricular septal defect, aortic stenosis or dysplasia, coarctation of the aorta [16–18], bicuspid aortic valve, double chambered right ventricle, mitral valve anomalies [19], Ebstein anomaly, total anomalous pulmonary venous return, supravalvular pulmonary stenosis, coronary artery dilatation, coronary artery fibromuscular dysplasia causing ischemia [20], and giant aneurysms of the sinuses of Valsalva caused by deficiency of medial elastin [21].

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Hypertrophic cardiomyopathy, both obstructive and non-obstructive, occurs in 20– 33% [5, 13, 14, 18, 22, 23]. Hypertrophy may be mild or severe, and may present before or at birth, in infancy or childhood. It is histologically, echocardiographically and clinically indistinguishable from non-syndromic hypertrophic cardiomyopathy, except that arrhythmia and sudden death appear to be less common. Nonetheless, mortality appears to be higher in the children with Noonan syndrome and hypertrophic cardiomyopathy, with progression to cardiac failure in 25% [5]. Restrictive cardiomyopathy and dilated cardiomyopathy are reported but uncommon [24– 26]. The electrocardiogram is abnormal in almost 90%. Extreme right axis deviation with superior counter-clockwise frontal QRS loop is likely related to asymmetric septal hypertrophy. Left axis deviation may occur secondary to a conduction abnormality; there may be left anterior hemiblock or an RSR’ pattern in lead V1. Abnormal findings may occur in a structurally normal heart. In older individuals, arrhythmia and congestive cardiac failure may be more common than previously suspected [27].

Growth and Feeding

Birth weight is usually normal but may be increased due to subcutaneous edema. In this situation there is rapid loss of weight in the neonatal period. Feeding difficulties occur in 77% of infants [4, 5, 28], and may be mild (15%), characterized by poor suck, or severe (38%), requiring tube feeding [5]. They are usually related to hypotonia and poor coordination of oral musculature, however immature gut motility and delayed gastrointestinal motor development are documented in some individuals [28]. Failure to thrive occurs in 40%. It is self-limited and usually resolves by 18 months of age.

The Clinical Phenotype of Noonan Syndrome

Average birth length is 47 cm. Childhood growth tends to follow the general population third centile, with normal growth velocity. The pubertal growth spurt is frequently reduced or absent. Delayed bone maturation is common and allows prolonged growth potential into the 20s. Average adult height in males is 162.5– 169.8 cm and in females is 152.7–153.3 cm [5, 29]. Noonan syndrome growth curves are published [29, 30]. Growth hormone production is usually normal but a variety of physiological abnormalities are described which may or may not have consequences for growth or responsiveness to growth hormone [31]. One study suggested that children with more f lorid facial, thoracic and cardiac features of Noonan syndrome had higher peak growth hormone levels [32]. These children did not seem to differ in pre- or post-growth hormone treatment height when compared to children with a milder Noonan syndrome phenotype. Genotype was not reported but may be germane, because higher spontaneous and stimulated growth hormone secretion has been noted in children with PTPN11 mutations [33]. There is a growing body of lite rature on the use of growth hormone therapy in Noonan syndrome [34–38]. Growth velocity is clearly enhanced in the first year of treatment, and, to a lesser extent, in year 2. Growth velocity gradually seems to fall after three years of treatment. The accelerating effect on bone maturation may compromise final height prognosis, although gain in height of 1 SD appears to be sustained. Several studies show improvement in intermediate and final adult height [36, 39, 40]. Use of growth hormone treatment also varies from country to country. Considerable enthusiasm for use remains in the United States. In Canada, growth hormone is only prescribed if growth hormone deficiency is proven. Response to growth hormone therapy may be better in those individuals without PTPN11 mutations [33, 41, 42]. The inferior response to growth hormone, greater likelihood of short stature, and

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higher (compensatory) growth hormone levels in individuals with a PTPN11 mutation may be explained by the fact that SHP2 normally downregulates growth hormone receptor signaling. Gain-of-function mutations in PTPN11 will enhance this effect [33, 43].

Development and Behaviour

Early developmental milestones are often delayed, with average age of sitting at 10 months, first unsupported walking at 21 months and simple two-word phrases at 31 months. Joint laxity and hypotonia clearly contribute to the motor delay. Most children will do well in a normal school setting but 10–40% will require additional help [4]. A large cohort of affected individuals, followed for many years, has demonstrated a strong association between significant feeding difficulties in infancy and intellectual handicap requiring special education [5]. Mild mental retardation occurs in up to 35%, however, IQ ranges from 64 to 127 [3, 4, 6, 44]. In one study of 48 affected British children, detailed psychometric testing demonstrated a mean fullscale IQ of 84 and 25% likelihood of learning disability [45]. Verbal IQ was slightly higher than performance IQ. Mild to moderate clumsiness and coordination problems were noted in about half the children. Other publications report learning disability with specific visual-constructional problems and verbalperformance discrepancy [44, 46, 47], language delay [4] and strengths in abstract reasoning and social awareness [48]. Studies of behaviour in Noonan syndrome have been somewhat contradictory. One study has suggested an increased likelihood of stubbornness and mood disorders [49]. Another found a majority of a group of 26 individuals to be impulsive, hyperactive and irritable [47]. A more recent study of 48 affected children has shown good self-esteem and has failed to identify a behavioral phenotype [45]. Notably few children are reported with autism,

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sleep difficulties, severe aggression or anxiety [50]. Few details of psychological health are reported. In a cohort of 51 adults, 23% had depression and there was occasional substance abuse and bipolar disease [27]. In males, short stature, hypotonia and reduced athleticism appeared to be predisposing factors. Similar findings were not reported by Shaw et al. [5] although this natural history study had few questions on self-esteem and mental health. Detailed psychological assessment of 10 young adults demonstrated variable levels of intelligence and suggests moderate impairment of social cognition in terms of emotion recognition and alexithymia. In some individuals there were mild signs of anxiety and lowered mood. Key elements of this behavioral phenotype are deficiencies in social and emotional recognition and expression [51].

Ocular Anomalies

Ocular anomalies are among the most common findings in Noonan syndrome and have been well studied in two large cohorts [5, 52]. Strabismus and refractive errors are present in a majority, amblyopia in about one third, and nystagmus in about 10%. Anterior segment changes (prominent corneal nerves, anterior stromal dystrophy, cataracts, and panuveitis) are frequently found, while coloboma [52, 53], retinitis pigmentosa [54], congenital fibrosis of extraocular muscles [55] and spontaneous corneal rupture [53, 56] are rare associations. Optic nerve hypoplasia is occasionally reported, in contrast to cardiofaciocutaneous syndrome, which shares many characteristics with Noonan syndrome, and in which optic nerve hypoplasia appears fairly common.

Hearing

The hearing loss described in Noonan syndrome is usually a mild conductive loss secondary to recurrent otitis media, however sensorineural and

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mixed hearing loss, though quite rare, do occur [5, 57]. Qui et al. [58] found progressive high tone loss in 50% of 20 affected individuals. Temporal bone anomalies are reported [59].

Musculoskeletal Findings

The thorax usually displays pectus carinatum superiorly and pectus excavatum inferiorly due to precocious closure of sternal sutures (fig. 2). The chest is also broad with wide-spaced nipples. Shoulders are rounded and the upper chest appears long; with low-set nipples and axillary webbing. This chest phenotype provides a good clue to diagnosis. Cubitus valgus, brachydactyly and blunt fingertips are frequently found. There are less common reports of talipes equinovarus, joint contractures, scoliosis, vertebral and rib anomalies, and radio-ulnar synostosis. Joint hyper-extensibility occurs in 30% [4, 5]. The association between Noonan syndrome and malignant hyperthermia is poorly understood. Malignant hyperthermia has been linked to a Noonan phenotype and designated as King syndrome [60–64]. The possibility of malignant hyperthermia is of greater concern in individuals with significant muscular pathology or elevated creatine kinase. Giant cell lesions of the jaws identical to those found in cherubism are described [65–71]. This combination has been called Noonan-like/multiple giant-cell lesion syndrome. Cherubism may occur as an isolated autosomal dominant disorder caused by mutations in SH3BP2 [72], or as part of neurofibromatosis. In Ramon syndrome cherubism is associated with juvenile rheumatoid arthritis (polyarticular pigmented villonodular synovitis). Giant cell granulomas, and bone and joint anomalies that include polyarticular pigmented villonodular synovitis, are now recognized to be part of the Noonan syndrome spectrum, and have been reported in individuals with PTPN11 and KRAS mutations [66, 73, 74]. Polyarticular pigmented villonodular synovitis

The Clinical Phenotype of Noonan Syndrome

Fig. 2. The chest phenotype showing wide-spaced and low-set nipples, pectus deformity with pectus carinatum superiorly and pectus excavatum inferiorly, and rounded shoulders.

is histologically identical to the consequences of peri-articular bleeding caused by hemophilia (K. Reinker, personal communication). This is intriguing given the bleeding diathesis that can accompany Noonan syndrome.

Central Nervous System

Seizures of varied types are found in 10% of individuals, with mean age of onset of 11 years [5]. Structural anomalies of the central nervous system are unusual. Hydrocephalus is reported in about 5% [75–78]. Communicating hydrocephalus is generally described and Clericuzio and colleagues hypothesize that this may be related to extra-cranial lymphatic dysplasia [75]. They refer to studies documenting the drainage of cerebrospinal fluid from the subarachnoid space along the olfactory nerves to the nasal lymphatics, and from there to cervical lymph nodes [79]. There are several reports of Chiari I malformation in Noonan syndrome and additional individuals are known to the author [80, 81]. Other less common structural brain anomalies include schwannoma, Dandy-Walker malformation, and lateral meningocele [82]. Cerebrovascular anomalies have been described in a few individuals [83–88].

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Genitourinary System

Renal anomalies are commonly reported (10%), generally mild, and include dilatation of renal pelvis (most common), duplex systems, minor rotational anomalies, distal ureteric stenosis, and renal hypoplasia/aplasia [89]. In males, pubertal development varies from normal virilization with subsequent fertility, to delayed but normal pubertal development, to inadequate sexual development associated with early cryptorchidism and deficient spermatogenesis [90]. Mean age of onset of puberty is 14.5 years in males and 14 years in females [5]. Most females are fertile [6, 90].

Gastrointestinal System

Both splenomegaly (50%) and hepatomegaly (25%) are said to be common, although in this author’s experience these figures seem high. The cause is unknown, but one might suspect an association with congestive heart failure or myelodysplasia on occasion. Rarely reported anomalies include choledochal cyst and midgut rotation [89].

but are more common in LEOPARD syndrome [95, 96]. Rare findings include xanthomas of the skin and oral mucous membranes, leukokeratosis of the lips and gingiva, molluscoid scalp skin, and vulvar angiokeratoma.

Lymphatics

Postnatally, a lymphatic abnormality is found in less than 20%; it may be localized or widespread; it is most commonly appreciated at birth but may not appear until adulthood [97]. Dorsal limb lymphedema is the most common finding. It may contribute to increased birth weight, and usually resolves in childhood. Less common abnormalities include generalized lymphedema, pulmonary lymphangiectasia, chylous effusions in pleural or peritoneal spaces, intestinal or testicular lymphangiectasia, and localized lymphedema of scrotum or vulva. Adolescent or adult onset does occur. Lymphangioma is a rare complication [98, 99]. The most common underlying pathology is lymph vessel hyperplasia with or without a thoracic duct abnormality. Lymphatic aplasia, hypoplasia and megalymphatics are also described.

Hematology – Oncology Skin

Various skin manifestations are seen in Noonan syndrome including café-au-lait spots, pigmented nevi, and lentigines [91]. Keratosis pilaris atrophicans has been noted in several instances, predominantly over extensor surfaces and the face [92]. On occasion facial keratosis is severe enough to cause absence of eyebrows and lashes, as seen in cardiofaciocutaneous syndrome. Ectodermal features seem to be more prevalent when Noonan syndrome is caused by mutations in SOS1 [93, 94]. Prominent fetal fingertip pads are often seen [4]. Multiple subcutaneous granular cell schwannomas are occasionally reported

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Several different coagulopathies may occur, either alone or in combination [100, 101]. They affect about one third of individuals, however, many more will have a history of abnormal bleeding or easy bruising. The range of manifestations is broad, from severe surgical hemorrhage to asymptomatic laboratory abnormalities. There is poor correlation between bleeding history and actual defect. Laboratory findings include factor XI deficiency, factor XII deficiency, factor VIII deficiency [100, 102–106], von Willebrand disease, and platelet dysfunction, which may be associated with trimethylaminuria or acyclooxygenase deficiency [101, 107]. Some factor deficiencies

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seem to improve with age. There is no evidence of hepatic dysfunction or vitamin K-dependent coagulation factor deficiency. Aspirin-containing medications should be avoided. Congenital bone marrow hypoplasia, congenital hypoplastic anemia, and pancytopenia have been reported rarely [108, 109]. A low frequency association with myeloproliferative disorders (MPD) exists. These include, in particular, juvenile myelomonocytic leukemia (JMML), but, more rarely, acute lymphoblastic leukemia [110–113], chronic myelomonocytic leukemia [114, 115] and proliferation of erythroid precursors [116]. JMML associated with Noonan syndrome tends to have an earlier onset and milder presentation than sporadic JMML and spontaneous remission may occur [117]. One particular PTPN11 mutation, The73Ile, is found in almost half the children with Noonan syndrome and MPD, but is uncommon in individuals with Noonan syndrome without MPD [117, 118]. Somatic mutations in PTPN11 are a common cause of MPD unassociated with Noonan syndrome [117, 119]. Solid tumours such as pheochromocytoma, malignant schwannoma, vaginal and orbital rhabdomyosarcoma, and neuroblastoma have been reported rarely [120–126].

Immunological Findings

Autoimmune thyroiditis occurs in 5% of individuals with Noonan syndrome [127–130]. Other autoimmune disorders, such as lupus, celiac disease, vitiligo, anterior uveitis and vasculitis are described infrequently [4, 130]. In addition, levels of anti-thyroglobulin and anti-microsomal thyroid antibodies seem to be higher than in the general population [130]. Antiphospholipid syndrome with Moyamoyalike vascular changes is reported [131, 132].

Prenatal Period

During pregnancy certain features may suggest the diagnosis of Noonan syndrome. The commonest of these are polyhydramnios, seen in 33%, and cystic hygroma [4, 133–136]. Lack of septation of the cystic hygroma and regression prior to mid second trimester are associated with more favorable prognosis than those with later regression [134, 135]. Other ultrasonographic markers include scalp edema, pleural or pericardial effusion, ascites and/or hydrops [133, 137]. Chorioangiomas are described and may contribute to formation of edema through decreased fetal oncotic pressure secondary to loss of alpha-fetoprotein into amniotic fluid.

References 1 Noonan JA, Ehmke DA: Associated noncardiac malformations in children with congenital heart disease. J Pediatr 1963;63:468–470. 2 Kobylinski O: Über eine flughautähnliche Ausbreitung am Halse. Arch Anthropol 1883;14:342–348. 3 Mendez HMM, Opitz JM: Noonan syndrome: A review. Am J Med Genet 1985;21:493–506. 4 Sharland M, Burch M, McKenna WM, Patton MA: A clinical study of Noonan syndrome. Arch Dis Child 1992;67: 178–183.

The Clinical Phenotype of Noonan Syndrome

5 Shaw AC, Kalidas K, Crosby AH, Jeffrey S, Patton MA: The natural history of Noonan syndrome: a long-term follow-up study. Arch Dis Child 2007;92;128–132. 6 Allanson JE: Noonan syndrome. J Med Genet 1987;24:9–13. 7 Char FC, Rodriguez-Fernandez HL, Scott CI, Borgankoar DS, Bell BB, Rowe RD: The Noonan-syndrome: A clinical study of forty-five cases. Birth Defects 1972;8:110–118. 8 Noonan JA: Noonan syndrome: Update and review for the primary pediatrician. Clin Pediatr 1994;33:548–555.

9 Yoshida R, Hasegawa T, Hasegawa Y, Nagai T, Kinoshita E, et al: Proteintyrosine phosphatase, non-receptor type 11 mutation analysis and clinical assessment in 45 patients with Noonan syndrome. J Clin Endocrinol Metab 2004;89:3359–3364. 10 Allanson JE: Time and natural history: the changing face. J Craniofac Genet Dev Biol 1989;9:21–28. 11 Allanson JE, Hall JG, Hughes HE, Preus M, Witt RD: Noonan syndrome: the changing phenotype. Am J Med Genet 1985;21:507–514. 12 Allanson JE: Noonan syndrome: the changing face. Proc Greenwood Genet Ctr 2001;20:78–79.

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24 Shimizu A, Oku Y, Matsuo K, Hashiba K: Hypertrophic cardiomyopathy progressing to a dilated cardiomyopathylike feature in Noonan’s syndrome. Am Heart J 1992;123:814–816. 25 Wilmshurst PT, Katritsis D: Restrictive and hypertrophic cardiomyopathies in Noonan syndrome: the overlap syndromes. Heart 1996;75:94–97. 26 Yu CM, Chow LT, Sanderson JE: Dilated cardiomyopathy in Noonan’s syndrome. Int J Cardiol 1996;56:83–85. 27 Noonan JA: Noonan syndrome; in Goldstein S, Reynolds CR (eds): Handbook of Neurodevelopmental and Genetic Disorders in Adults. New York, Guilford Press, 2005, pp 308–319. 28 Shah N, Rodriguez M, St Louis D, Lindley K, Milla PJ: Feeding difficulties and foregut dysmotility in Noonan syndrome. Arch Dis Child 1999; 81:28–31. 29 Ranke MB, Heidemann P, Knupfer C, Enders H, Schmaltz AA, Bierich JR: Noonan syndrome: growth and clinical manifestations in 144 cases. Eur J Pediatr 1988;148:220–227. 30 Witt DR, Keena B, Hall JG, Allanson JE: Growth curves for height in Noonan’s syndrome. Clin Genet 1986;30:150–153. 31 Noordam C, van der Burgt I, Sweep CG, Delemarre-van de Waal HA, Sengers RC, Otten BJ: Growth hormone (GH) secretion in children with Noonan syndrome: frequently abnormal without consequences for growth or GH treatment. Clin Endocrinol 2001; 54:53–59. 32 Noordam K, van der Burgt I, Brunner HG, Otten BJ: The relationship between clinical severity of Noonan’s syndrome and growth, growth hormone (GH) secretion and response to GH. J Pediatr Endocrinol Metab 2002;15:175–180. 33 Binder G, Neuer K, Ranke MB, Wittekindt NE: PTPN11 mutations are associated with mild GH resistance in individuals with Noonan syndrome. J Clin Endocrinol Metab 2005;90: 5377–5381. 34 Ahmed ML, Foot AB, Edge JA, Lamkin VA, Savage MO, Dunger DB: Noonan’s syndrome: Abnormalities of the growth hormone IGF-1 axis and the response to treatment with human biosynthetic growth hormone. Acta Paediatr Scand 1991;80:446–450.

35 MacFarlane CE, Brown DC, Johnston LB, Patton MA, Dunger DB, et al: Growth hormone therapy and growth in children with Noonan’s syndrome: Results of 3 years’ follow-up. J Clin Endocrinol Metab 2001;86:1953–1956. 36 Raaijmakers R, Noordam C, K’aragiannis G, Gregory JW, Hertel NT, Sipila I, Otten BJ: Response to growth hormone treatment and final height in Noonan syndrome in a large cohort of patients in the KIGS database. J Pediatr Endocrinol Metab 2008;21:267–273. 37 Ogawa M, Moriya N, Ikeda H, Tanae A, Tanaka T, et al: Clinical evaluation of recombinant human growth hormone in Noonan syndrome. Endocr J 2004;51:61–68. 38 Thomas BC, Stanhope R: Long-term treatment with growth hormone in Noonan’s syndrome. Acta Paediatr 1993;82:853–855. 39 Kelnar CJH: Growth hormone therapy in Noonan syndrome. Horm Res 2000;53(Suppl 1):77–81. 40 Osio D, Dahlgren J, Wikland KA, Westphal O: Improved final height with long-term growth hormone treatment in Noonan syndrome. Acta Pediatr 2005;94:1232–1237. 41 Ferreira LV, Souza SA, Arnhold IJ, Mendonca BB, Jorge AA: PTPN11 (protein tyrosine phosphatase nonreceptor type 11) mutations and response to growth hormone therapy in children with Noonan syndrome. J Clin Endocrinol Metab 2005;90: 5156–5160. 42 Limal J-M, Parfait B, Cabrol S, Bonnet D, Leheup B, et al: Noonan syndrome: Relationships between genotype, growth, and growth factors. J Clin Endocrinol Metab 2005;91:300–306. 43 Stofega MR, Herrington J, Billestrup N, Carter-Su C: Mutation of the SHP-2 binding site in growth hormone (GH) receptor prolongs GH-promoted tyrosyl phosphorylation of GH receptor, JAK2, and STAT5B. Mol Endocrinol 2000;14:1338–1350. 44 Money J, Kalus ME: Noonan’s syndrome: IQ and specific disabilities. Am J Dis Child 1979;133:846–850. 45 Lee DA, Portnoy S, Hill P, Gillberg C, Patton MA: Psychological profile of children with Noonan syndrome. Dev Med Child Neurol 2005;47:35–38. 46 Cornish KM: Verbal-performance discrepancies in a family with Noonan syndrome. Am J Med Genet 1996;66:235–236.

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47 Sarimski K: Developmental and behavioural phenotype in Noonan syndrome. Genet Couns 2000;11:383–390. 48 van der Burgt I, Thoone G, Roosenboom N, Assman-Hulsman C, Gabreels F, et al: Patterns of cognitive functioning in school-aged children with Noonan syndrome associated with variability of phenotypic expression. J Pediatr 1999;135:707–713. 49 Wood A, Massarano A, Super M, Harrington R: Behavioral aspects and psychiatric findings in Noonan’s syndrome. Arch Dis Child 1995;72:153–155. 50 Ghaziuddin M, Bolyard B, Alessi N: Autistic disorder in Noonan syndrome. J Intellect Disabil Res 1994;38:67–72. 51 Verhoeven W, Wingbermühle E, Egger J, Van der Burgt I, Tuinier S: Noonan syndrome: psychological and psychiatric aspects. Am J Med Genet 2008;146A:191–196. 52 Lee NB, Kelly L, Sharland M: Ocular manifestations of Noonan syndrome. Eye 1992;6:328–334. 53 Ascaso FJ, Del Buey MA, Huerva V, Latre B, Palomar A: Noonan’s syndrome with keratoconus and optic disc coloboma. Eur J Ophthalmol 1993;3:101–103. 54 Schollen E, Matthijs G, Fryns JP: PTPN11 mutation in a young man with Noonan syndrome and retinitis pigmentosa. Genet Couns 2003;14:259. 55 Elgohary MA, Bradshaw P, Ahmad N: Anterior uveitis and congenital fibrosis of the extraocular muscles in a patient with Noonan syndrome. J Postgrad Med 2005;51:319–321. 56 Au YK, Collins WP, Patel JS, Asamoah A: Spontaneous corneal rupture in Noonan syndrome. A case report. Ophthalmic Genet 1997;18:39–41. 57 Cremers CWRJ, van der Burgt CJAM: Hearing loss in Noonan syndrome. Int J Pediatr Otorhinol 1992;23:81–84. 58 Qui WW, Yin SS, Stucker FJ: Audiologic manifestations of Noonan syndrome. Otolaryngol Head Neck Surg 1998;118:319–323. 59 Naficy S, Shepard NT, Telian SA: Multiple temporal bone anomalies associated with Noonan syndrome. Otolaryngol Head Neck Surg 1997;116: 265–267. 60 Chitayat D, Hodgkinson KA, Ginsburg O, Dimmick J, Watters GV: King syndrome: a genetically heterogeneous phenotype due to congenital myopathies. Am J Med Genet 1992;43:954–956.

61 Hunter A, Pinsky L: An evaluation of the possible association of malignant hyperpyrexia with Noonan syndrome using serum creatine phosphokinase levels. J Pediatr 1975;96:412–415. 62 King JO, Denborough MA: Anestheticinduced malignant hyperpyrexia in children. J Pediatr 1973;83:37–40. 63 Rissam HS, Mittal SR, Wahi PL, Bidwai PS: Post-operative hyperpyrexia in a case of Noonan’s syndrome. Indian Heart J 1982;34:180–182. 64 Steenson AJ, Torkelson RD: King’s syndrome with malignant hyperthermia. Am J Dis Child 1987;141:271–273. 65 Addante RR, Breen GH: Cherubism in a patient with Noonan’s syndrome. J Oral Maxillofac Surg 1996;54:210–213. 66 Betts NJ, Stewart JC, Fonseca RJ, Scott RF: Multiple central giant cell lesions in a Noonan-like phenotype. Oral Surg Oral Med Oral Pathol 1993;76:601–607. 67 Chuong R, Kaban LB, Kozakewich H, Perez-Atayde A: Central giant cell lesions of the jaws: a clinicopathologic study. J Oral Maxillofac Surg 1986;44:708–713. 68 Dunlap C, Neville B, Vickers RA, O’Neil D, Barker B: The Noonan syndrome/cherubism association. Oral Surg Oral Med Oral Pathol 1989;67: 698–705. 69 Hoyer PF, Neukam FW: Cherubismus – eine osteofibröse Kiefererkrankung im Kindesalter. Klin Paediatr 1982;194:128–131. 70 Sugar AW, Ezsias A, Bloom AL, Morcos WE: Orthognathic surgery in a patient with Noonan’s syndrome. J Oral Maxillofac Surg 1994;52:421–425. 71 Weldon L, Cozzi G: Multiple giant cell lesions of the jaws. J Oral Maxillofac Surg 1982;40:520–522. 72 Mangion J, Rahman N, Edkins S, Barfoot R, Nguyen T, et al: The gene for cherubism maps to chromosome 4p16.3. Am J Hum Genet 1999;65:151–157. 73 Jafarov T, Ferimazova N, Reichenberger E: Noonan-like syndrome mutations in PTPN11 in patients diagnosed with cherubism. Clin Genet 2005;68: 190–191. 74 Wolvius EB, de Lange J, Smeets EEJ, van der Wal KGH, van den Akker HP: Noonan-like/multiple giant cell lesion syndrome: Report of a case and review of the literature. J Oral Maxillofac Surg 2006;64:1289–1292.

The Clinical Phenotype of Noonan Syndrome

75 Clericuzio CL, Roberts A, Kucherlapati RS, Tworog-Dube E, Allanson JE: Communicating hydrocephalus in Noonan syndrome: A consequence of lymphatic dysplasia? Proc Greenwood Gen Ctr 2008;27:81. 76 Fryns JP: Progressive hydrocephalus in Noonan syndrome. Clin Dysmorphol 1997;6:379. 77 Henn W, Reichert H, Nienhaus Z, Zankl M, Lindinger A, et al: Progressive hydrocephalus in two members of a family with autosomal dominant Noonan phenotype. Clin Dysmorphol 1997;6:153–156. 78 Heye N, Dunne JW: Noonan’s syndrome with hydrocephalus, hindbrain herniation, and upper cervical intracord cyst. J Neurol Neurosurg Psychiatry 1995;59:338–339. 79 Walter BA, Valera VA, Takahashi S, Ushiki T: The olfactory route for cerebrospinal fluid drainage into the peripheral nervous system. Neuropathol Appl Neurobiol 2006;32:388–396. 80 Ball MJ, Peiris A: Chiari (type I) malformation and syringomyelia in a patient with Noonan’s syndrome. J Neurol Neurosurg Psychiatry 1982; 45:753–754. 81 Holder-Espinasse M, Winter RM: Type 1 Arnold-Chiari malformation and Noonan syndrome. A new diagnostic feature. Clin Dysmorphol 2003;12:275. 82 Hughes HE, Hughes RM, Summers A, Hochhauser L: Noonan syndrome and lateral meningoceles: another link with neurofibromatosis. Proc Greenwood Genet Ctr 1987;6:159. 83 Hara T, Sasaki T, Miyauchi H, Takakura K: Noonan phenotype associated with intracerebral hemorrhage and cerebral vascular anomalies: Case report. Surg Neurol 1993;39:31–36. 84 Hinnant CA: Thromboembolic infarcts occurring after mild traumatic brain injury in a paediatric patient with Noonan’s syndrome. Brain Injury 1994;8:719–727. 85 Hinnant CA: Noonan syndrome associated with thromboembolic brain infarcts and posterior circulation abnormalities. Am J Med Genet 1995;56:241–244. 86 Robertson S, Tsang B, Aftimos S: Cerebral infarction in Noonan syndrome. Am J Med Genet 1997;71:111–114.

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87 Schon F, Bowler J, Baraitser M: Cerebral arteriovenous malformation in Noonan’s syndrome. Postgrad Med J 1992;68:37–40. 88 Tanaka Y, Masuno M, Iwamoto H, Aida N, Ijiri R, et al: Noonan syndrome and cavernous hemangioma of the brain. Am J Med Genet 1999;82:212–214. 89 George CD, Patton MA, El Sawi M, Sharland M, Adam EJ: Abdominal ultrasound in Noonan syndrome: A study of 44 patients. Pediatr Radiol 1993;23:316–318. 90 Elsawi MM, Pryor JP, Klufio G, Barnes C, Patton MA: Genital tract function in men with Noonan syndrome. J Med Genet 1994;31:468–470. 91 Daoud MS, Dahl PR, Su WP: Noonan syndrome. Semin Dermatol 1995;14:140–144. 92 Pierini DO, Pierini AM: Keratosis pilaris atrophicans faciei (ulerythema ophryogenes): A cutaneous marker in the Noonan’s syndrome. Br J Dermatol 1979;100:409–416. 93 Tartaglia M, Pennacchio LA, Zhao C, Yadav KK, Fodale V, et al: Gain-offunction SOS1 mutations cause a distinctive form of Noonan syndrome. Nat Genet 2007;39:75–79. 94 Zenker M, Horn M, Wieczorek D, Allanson J, Pauli S, et al: SOS1 is the second most common Noonan gene but plays no major role in cardio-facio-cutaneous (CFC) syndrome. J Med Genet 2007;44:651–656. 95 Lohmann DR, Gillessen-Kaesbach G: Multiple cutaneous granular cell tumours in a patient with Noonan syndrome. Clin Dysmorphol 2001;19:301–302. 96 Sahn EE, Dunlavey ES, Parsons JL: Multiple cutaneous granular cell tumors in a child with possible neurofibromatosis. J Am Acad Dermatol 1997;36:327–330. 97 Witt DR, Hoyme HE, Zonana J, Manchester DK, Fryns JP, et al: Lymphedema in Noonan syndrome: Clues to pathogenesis and premature diagnosis and review of the literature. Am J Med Genet 1987;27:841–856. 98 Bloomfield FH, Hadden W, Gunn TR: Lymphatic dysplasia in a neonate with Noonan’s syndrome. Pediatr Radiol 1997;27:321–323.

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99 Evans DG, Lonsdale RN, Patton MA: Cutaneous lymphangioma and amegakaryocytic thrombocytopenia in Noonan syndrome. Clin Genet 1991;39:228–232. 100 Sharland M, Patton MA, Talbot S, Chitolie A, Bevan DH: Coagulation-factor deficiencies and abnormal bleeding in Noonan’s syndrome. Lancet 1992;339:19–21. 101 Witt DR, McGillivray BC, Allanson JE, Hughes HE, Hathaway WE, et al: Bleeding diathesis in Noonan syndrome: a common association. Am J Med Genet 1988;31:305–317. 102 de Haan M, van der Kamp JJP, Briet E, Dubbeldam J: Noonan syndrome: partial factor XI deficiency. Am J Med Genet 1988;29:277–282. 103 Emmerich J, Aiach M, Capron L, Fiessinger JN: Noonan’s syndrome and coagulation-factor deficiencies. Lancet 1992;339:431. 104 Kitchens CS, Alexander JA: Partial deficiency of coagulation factor XI as a newly recognized feature of Noonan syndrome. J Pediatr 1983;102:224–227. 105 Massarano A, Wood A, Tait RC, Stevens R, Super M: Noonan syndrome: Coagulation and clinical aspects. Acta Paediatr 1996;85:1181–1185. 106 Singer ST, Hurst D, Addiego JE Jr: Bleeding disorders in Noonan syndrome: three case reports and review of the literature. J Pediatr Hematol Oncol 1997;19:130–134. 107 Humbert JR, Hammond KB, Hathaway WE: Trimethylaminuria: the fishodour syndrome. Lancet 1970;2:770–771. 108 Feldman KW, Ochs HD, Price TH, Wedgwood RJ: Congenital stem cell dysfunction associated with Turnerlike phenotype. J Pediatr 1976;88:979–998. 109 Sackey K, Sakati N, Aur RJA, Shebib S, Sabbah RS, Rifai S: Multiple dysmorphic features and pancytopenia: a new syndrome? Clin Genet 1985;27:606–610. 110 Attard-Montalto SP, Kingston JE, Eden T: Noonan’s syndrome and acute lymphoblastic leukaemia. Med Pediatr Oncol 1994;23:391–392. 111 Johannes JM, Garcia ER, De Vaan GA, Weening RS: Noonan’s syndrome in association with acute leukemia. Pediatr Hematol Oncol 1995;12:571–575.

112 Piombo M, Rosana C, Pasino M, Marasini M, Cerruti P, Comelli A: Acute lymphoblastic leukemia in Noonan syndrome: report of two cases. Med Pediatr Oncol 1993;21:454–455. 113 Roti G, La Starza R, Ballanti S, Crescenzi B, Romoli S, et al: Acute lymphoblastic leukaemia in Noonan syndrome. Br J Haematol 2006;133:448–450. 114 Bader-Meunier B, Tchernia G, Mielot F, Fontaine JL, Thomas C, et al: Occurrence of myeloproliferative disorder in patients with Noonan syndrome. J Pediatr 1997;130:885–889. 115 Fukuda M, Horibe K, Miyajima Y, Matsumoto K, Nagashima M: Spontaneous remission of juvenile chronic myelomonocytic leukemia in an infant with Noonan syndrome. J Pediatr Hematol Oncol 1997;19:177–178. 116 Kratz CP, Nathrath M, Freisinger P, Dressel P, Assmuss H-P, et al: Lethal proliferation of erythroid precursors in a neonate with a germline PTPN11 mutation. Eur J Pediatr 2006;165:182–185. 117 Kratz CP, Niemeyer CM, Castleberry RP, Cetin M, Bergstrasser E, et al: The mutational spectrum of PTPN11 in juvenile myelomonocytic leukemia and Noonan syndrome. Blood 2005;15:2183–2185. 118 Jongmans M, Sistemans EA, Rikken A, Nillesen WM, Tamminga R, et al: Genotypic and phenotypic characterization of Noonan syndrome: New data and review of the literature. Am J Med Genet A 2005;134:165–170. 119 Tartaglia M, Niemeyer CM, Fragale A, Song X, Buechner J, et al: Somatic mutations in PTPN11 in juvenile myelomonocytic leukemia, myelodysplastic syndromes and acute myeloid leukemia. Nat Genet 2003;34:148–150. 120 Becker CE, Rosen SW, Engelman K: Pheochromocytoma and hyporesponsiveness to thyrotropin in a 46,XY male with features of Turner phenotype. Ann Intern Med 1969;70: 325–333. 121 Cotton JL, Williams RG: Noonan syndrome and neuroblastoma. Arch Pediatr Adolesc Med 1995;149:1280– 1281. 122 Jung A, Bechthold S, Pfluger T, Renner C, Ehrt O: Orbital rhabdomyosarcoma in Noonan syndrome. J Pediatr Hematol Oncol 2003;25:330–332.

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123 Kaplan MS, Opitz JM, Gosset FR: Noonan’s syndrome: A case with elevated serum alkaline phosphatase levels and malignant schwannoma of the left forearm. Am J Dis Child 1968;116:359–366. 124 Khan S, McDowell H, Upadhyaya M, Fryer A: Vaginal rhabdomyosarcoma in a patient with Noonan syndrome. J Med Genet 1995;32:743–745. 125 Lopez-Miranda B, Westra SJ, Yazdani S, Boechar MI: Noonan syndrome associated with neuroblastoma: a case report. Pediatr Radiol 1997;27:324–326. 126 Ijiri R, Tanaka Y, Keisuke K, Masuno M, Imaizumi K: A case of Noonan’s syndrome with possible associated neuroblastoma. Pediatr Radiol 2000;30:432–433. 127 Chaves-Carballo E, Hayles AB: UllrichTurner syndrome in the male: review of the literature and report of a case with lymphocytic (Hashimoto’s) thyroiditis. Mayo Clin Proc 1966;41:843–854.

128 Vesterhus P, Aarskog D: Noonan’s syndrome and autoimmune thyroiditis. J Pediatr 1973;83:237–240. 129 Amoroso A, Garzia P, Vadacca M, Galluzzo S, Del Porto F, et al: The unusual association of three autoimmune diseases in a patient with Noonan syndrome. J Adol Health 2003;32:94–97. 130 Lopez-Rangel E, Malleson PN, Lirenman DS, Roa B, Wiszniewska J, Lewis ME: Systemic lupus erythematosus and other autoimmune disorders in children with Noonan syndrome. Am J Med Genet A 2006;139:239–242. 131 Ganesan V, Kirkham FJ: Noonan syndrome and Moyamoya. Pediatr Neurol 1997;16:256–258. 132 Yamashita Y, Kusaga A, Koga Y, Nagamitsu S-I, Matsuishi T: Noonan syndrome, Moyamoya-like vascular changes, and antiphospholipid antibodies. Pediatr Neurol 2004;31: 364–366.

133 Achiron R, Heggesh J, Grisaru D, Goldman B, Lipitz S, et al: Noonan syndrome: A cryptic condition in early gestation. Am J Med Genet 2000;92:159–165. 134 Benacerraf BR, Greene MF, Holmes LB: The prenatal sonographic features of Noonan’s syndrome. J Ultrasound Med 1989;8:59–64. 135 Donnenfeld A, Nazir MA, Sindoni F, Librizzi RJ: Prenatal sonographic documentation of cystic hygroma regression in Noonan syndrome. Am J Med Genet 1991;39:461–465. 136 Zarabi M, Mieckowski GC, Mazer J: Cystic hygroma associated with Noonan’s syndrome. J Clin Ultrasound 1983;11:398–400. 137 Bawle EV, Black V: Nonimmune hydrops fetalis in Noonan’s syndrome. Am J Dis Child 1986;140:758–760.

Judith E. Allanson Department of Genetics, Children’s Hospital of Eastern Ontario 401 Smyth Road Ottawa, ON K1H 8L1 (Canada) Tel. +1 613 737 2233, Fax +1 613 738 4822, E-Mail [email protected]

The Clinical Phenotype of Noonan Syndrome

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Zenker M (ed): Noonan Syndrome and Related Disorders. Monogr Hum Genet. Basel, Karger, 2009, vol 17, pp 20–39

Molecular Genetics of Noonan Syndrome M. Tartagliaa  B.D. Gelbb aDipartimento

di Biologia Cellulare e Neuroscienze, Istituto Superiore di Sanità, Rome, Italy; for Molecular Cardiology, Departments of Pediatrics and Genetics & Genomic Sciences, Mount Sinai School of Medicine, New York, N.Y., USA bCenter

Abstract Noonan syndrome (NS) is a genetically heterogeneous disorder that can result from mutations in the PTPN11, SOS1, KRAS, RAF1 and MEK1 genes, which encode transducers participating in the RAS-MAP kinase (MAPK) signaling pathway. The disorder is generally transmitted as an autosomal dominant trait, although many cases result from de novo mutations. Defects in the PTPN11 gene, which encodes the Src homology 2 (SH2) containing protein tyrosine phosphatase SHP-2, account for approximately 50% of cases. The more than 60 mutations that have been reported are almost all missense changes, and promote upregulation of protein function. Two additional distinct classes of missense PTPN11 mutations have been identified as somatic lesions in hematological malignancies and germline defects in LEOPARD syndrome (LS), which is clinically related to NS. While the former are generally more activating compared to the NS-causing mutations, the latter cause loss of catalytic activity of the phosphatase. Defects in the KRAS proto-oncogene account for roughly 2% of NS cases and engender gain of function in RAS signaling through reduced KRAS GTPase activity or increased GDP/GTP dissociation rate. As documented for PTPN11, the distributions of affected residues and amino acid substitutions in NS and cancer appear to be largely mutually exclusive. Missense mutations in SOS1 occur in approximately 10% of affected individuals. SOS1 is a RAS-specific guanine nucleotide exchange factor that catalyzes the release of GDP from RAS, facilitating the conversion of its inactive GDP-bound form to active GTP-bound RAS. NS-causing SOS1 mutations are activating and affect residues placed

in domains that stabilize the catalytically autoinhibited conformation of the protein. Finally, a small percentage of NS results from missense mutations in the RAF1 and MEK1 genes. RAF1 is a member of a small family of serine-threonine kinases, which are effectors of RAS that activate the dual specificity kinases MEK1 and MEK2. Activated MEK proteins, in turn, activate the MAPKs, ERK1 and ERK2. RAF1 gene mutations are observed in about 5% of NS cases and affect residues clustered in three regions of the protein with amino acid substitutions within the consensus 14–3– 3 recognition sequence around Ser259 accounting for 75% of the mutations. Since 14–3–3 binding at residue Ser259 stabilizes RAF1’s catalytically inactive conformation and impairs its translocation to the plasma membrane, mutations affecting this motif promote increased RAF1 activity. Additional studies are required to fully understand the functional consequences of mutations affecting residues placed within the other two mutational hot spots within the activation segment region of the kinase domain and at the C-terminus. RAF1 gene mutations also account for approximately 3% of subjects with LS, and possibly a relevant fraction of pediatric cases with isolated hypertrophic cardiomyopathy. A single missense MEK1 mutation has been reported in two unrelated subjects with sporadic NS. MEK1 gene mutations are estimated to account for less than 2% of affected individuals. No data on the effect of the predicted amino acid change on MEK1 function and MAPK signaling is currently available. Copyright © 2009 S. Karger AG, Basel

Identification of the Noonan Syndrome Disease Genes: A Brief History

From a genetic point of view, Noonan syndrome (NS; OMIM 163950) was a poorly understood condition until recently. Autosomal dominant inheritance was apparent for the majority of families with the disorder, although evidence suggestive of an autosomal recessive form had been reported [1]. Genetic mapping studies for this disorder were performed with small kindreds with the first report appearing in 1992. Since NS shares some features with neurofibromatosis, markers flanking the NF1 and NF2 genes were tested and excluded allelism of NS to those traits [2, 3]. Next, Jamieson and co-workers studied a large Dutch kindred transmitting the trait to perform a genome-wide scan and observed linkage with several markers at chromosome 12q22-qter, which they named NS1 [4]. They also documented that NS was genetically heterogeneous, based on linkage exclusion to NS1 in some kindreds. The NS1 locus was refined to a region of approximately 7.5 cm using novel STRs [5]. Legius and co-workers studied a fourgeneration Belgian family transmitting the trait, achieving independent linkage to NS1, and refining the critical interval further to approximately 5 cm [6]. A positional candidacy approach was taken to identify the NS disease gene residing at NS1 [7], and Tartaglia and co-workers established PTPN11 as the NS1 disease gene a few years later [8]. PTPN11 was considered an excellent candidate because it mapped to the NS1 critical region and because its protein product, SHP-2, occupied a critical role in several intracellular signal transduction pathways controlling diverse developmental processes, including cardiac semilunar valvulogenesis [9]. Subsequent studies performed with large, clinically well-characterized cohorts provided an estimate of the relative importance of PTPN11 mutations in the epidemiology of NS, defined the spectrum of molecular defects in the disorder, and established genotype-phenotype correlations [10–13]. Based on those efforts, it has

Molecular Genetics of Noonan Syndrome

now been established that PTPN11 mutations account for approximately 50% of individuals with NS, are almost always missense changes that affect specific regions of the protein, and are more prevalent among subjects with pulmonary valve stenosis and short stature, and less common in individuals with hypertrophic cardiomyopathy (HCM) and/or severe cognitive deficits. Following the identification of PTPN11 as a NS disease gene, PTPN11 mutations have been identified in individuals with Noonan-like syndrome and multiple giant cell lesions in bone (NL/MGCLS, which is also known as NS with cherubism; OMIM 163955) [10, 14] and LEOPARD syndrome (LS; OMIM 151100) [15, 16], two developmental disorders known to be closely related to NS. Based on the higher prevalence of pediatric myeloproliferative disorders and leukemias in NS, Tartaglia and co-workers discovered that a different class of missense mutations in PTPN11 occurs as somatic events in myeloid and lymphoid malignancies [17–19], and the identity of PTPN11 mutations conferring susceptibility to these hematological disorders was characterized [17, 20]. While the spectrum and distribution of NS-causing and leukemia-associated mutations provided the first hint about their possible consequences on SHP-2 function, biochemical characterization of a relatively large panel of germline or somatic mutations identified multiple mechanisms promoting SHP-2 gain of function [17, 21–27]. Since SHP-2 has a critical positive role in RAS signaling (fig. 1), and the NS-causing PTPN11 mutations increased RAS-mediated signal flow, researchers hypothesized that mutations in other genes encoding proteins participating in this transduction pathway might underlie the half of NS cases without a mutation in PTPN11. This candidate gene approach represented the best available gene hunting strategy since no sufficiently informative PTPN11 mutation-negative family transmitting the trait had been identified to support a linkage study. Mutation analysis of candidate genes has allowed the identification of

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SOS1: NS SOS1 SHC

HRAS: CS KRAS: NS, CFCS GDP-RAS

GRB2 Neurofibromin

SHP2 PTPN11: NS, LS

Fig. 1. Schematic diagram showing the RAS-MAPK signal transduction pathway. The syndromes and their mutated proteins are as indicated. The double ovals in dark grey and the light grey ovals represent generic dimerized cell-surface receptors binding to their ligand.

three additional NS disease genes, KRAS, SOS1 and RAF1, in the last two years [28–33]. KRAS codes for one of the three members of the RAS family, while SOS1 and RAF1 are, respectively, a RAS-specific guanine nucleotide exchange factor (GEF) and an effector of RAS with serine/ threonine kinase activity functioning as the upstream component of the RAS-associated MAPK cascade. The initial structural and biochemical characterization of mutations in these genes has provided evidence for their activating effects on protein function as well as on the hyperactivation of the RAS-MAPK transduction pathway [29–34]. Genotype-phenotype correlation analyses have also documented that mutations in these genes are associated with distinct phenotypes. Specifically, KRAS defects were frequently found in children with a severe phenotype approaching cardiofaciocutaneous syndrome (CFCS; OMIM 115150) or Costello syndrome (CS; OMIM 218040) [28, 29, 35], two disorders clinically related to NS,

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GDP-RAS

NF1: NF1, NFNS

RAF

RAF1: NS, LS BRAF: CFC

MEK

MEK1: CFCS, NS MEK2: CFCS

ERK

Gene expression

while the phenotypes associated with SOS1 and RAF1 mutations included ectodermal abnormalities, normal growth and absence of cognitive deficits [33, 36], and HCM and hyperpigmented cutaneous lesions [30, 31], respectively. Mutations in KRAS, SOS1 and RAF1 have been estimated to account for approximately 15% of affected individuals, indicating that other disease genes responsible for a relatively large portion of Noonan syndrome remain to be identified. These genes are likely to encode proteins with role in the RASMAPK signaling pathway. While other genes are expected to be identified in the next following years, mutational screening efforts focused on genes that encode transducers participating in the RAS-MAPK signaling pathway and are mutated in disorders clinically related to NS have allowed the identification of additional molecular lesions involved in NS pathogenesis. Indeed, a single missense mutation in MEK1, which encodes a dual specificity kinase

Tartaglia  Gelb

ATG 1

2

3

N-SH2

a

3

4

5 6 7 8

C-SH2

104 112

9

TGA 10 11 12 13 14 15 16

PTP

216 221

Germline transmitted

524 Somatically acquired

b Fig. 2. PTPN11 gene organization, SHP-2 domain structure and location of affected residues in human disease. (a) The PTPN11 gene and its encoded protein. The numbered, filled boxes at the top indicate the coding exons; the positions of the ATG and TGA codons are shown. The functional domains of the SHP-2 protein, consisting of two tandemly arranged SH2 domains at the N-terminus (N-SH2 and C-SH2) followed by a protein tyrosine phosphatase (PTP) domain, are shown below. The numbers below that cartoon indicate the amino acid boundaries of those domains. (b) Location of mutated residues in the three dimensional structure of SHP-2 in its catalytically inactive conformation (green, N-SH2 domain; cyan, C-SH2 domain; pink, PTP domain). Residues affected by germline (left) or somatically acquired (right) mutations are shown with their lateral chains colored according to the classification proposed by Tartaglia et al. (2006) (red, group I; yellow, group II; green, group III; cyan, group IV; orange, group V; violet, group VI; blue, unclassified).

that activates ERK proteins, has been identified [37]. According to this study, MEK1 gene mutations are estimated to account for approximately 3% of PTPN11- and SOS1-mutation negative NS cases. No data on the effect of this mutation on MEK1 function and MAPK signaling is currently available. Next, we will briefly review current knowledge on the molecular genetics of NS. Specifically, we discuss the function of the identified disease

Molecular Genetics of Noonan Syndrome

genes, the diversity of disease-causing mutations and their consequences on protein function and intracellular signaling.

PTPN11

The PTPN11 gene (OMIM 176876) spans more than 90 kb, comprising 16 exons with an open reading frame of 1,779 bases. It encodes SHP-2,

23

Table 1. Classification and relative distribution of germline and somatic PTPN11 mutations Mutation group

Predicted effect on SHP-2 functiona

Germline origin (n = 573) n (%)

Somatic origin (n = 256) n (%)

I

A/I switching

243 (42.4)

217 (84.8)

II

A/I switching and catalysis

66 (11.5)

3 (1.2)

III

A/I switching and specificity

27 (4.7)

27 (10.5)

IV

A/I switching and/or catalysis

195 (34.0)

4 (1.6)

V

SH2 pY-binding

28 (4.9)

5 (1.9)

VI

SH2 orientation or mobility

12 (2.1)

–

others

–

2 (0.4)

–

a

A/I = Active/Inactive conformation; SH2 = Scr homology 2 domain; pY = phosphotyrosyl-containing peptide.

a widely expressed cytoplasmic Src homology 2 (SH2) domain-containing, non-membranous protein tyrosine phosphatase functioning as an intracellular signal transducer that is required during development [38–40]. SHP-2’s structure is composed of two tandemly arranged aminoterminal SH2 domains (N-SH2 and C-SH2), a single catalytic domain (PTP) and a carboxyterminal tail containing two tyrosyl phosphorylation sites and a proline-rich stretch (fig. 2). Both the N-SH2 and C-SH2 domains selectively bind to short amino acid motifs containing a phosphotyrosyl residue and promote SHP2’s association with cell surface receptors, cell adhesion molecules and scaffolding adapters. Crystallographic data indicate that the N-SH2 domain also interacts with the PTP domain using a separate site [41]. As these subdomains show negative cooperativity, the N-SH2 domain functions as an intramolecular switch controlling SHP-2 catalytic activation. Specifically, the N-SH2 domain interacts with the PTP domain basally, blocking the catalytic site. Binding of the N-SH2 phosphopeptide-binding site to a phosphotyrosyl ligand promotes a conformational change of the domain that weakens the auto-

24

inhibiting intramolecular interaction, making the catalytic site available to substrate, thereby activating the phosphatase. Although it has been demonstrated that SHP2 can either positively or negatively modulate signal flow depending upon its binding partner and interactions with downstream signaling networks, it is now established that SHP-2 positively controls the activation of the RAS-MAPK cascade induced by a number of growth factors and cytokines [38–40]. In most cases, SHP-2’s function in intracellular signaling appears to be distal to activated receptors and upstream to RAS. While the mechanisms of SHP-2’s action and its physiological substrates are still poorly defined, accumulated evidence supports the view that both membrane translocation and PTPase activity are required for SHP-2 function. Available records based on more than 500 germline defects indicate that NS-causing PTPN11 mutations are almost always missense changes and are not randomly distributed throughout the gene [27]. Mutations have been classified into six major groups on the basis of their predicted effect on protein function (table 1 and fig. 2). Most of the mutations affect

Tartaglia  Gelb

residues involved in the N-SH2/PTP interdomain binding network that stabilizes SHP-2 in its catalytically inactive conformation or are in close spatial proximity to them. These mutations are predicted to up-regulate SHP-2 physiological activation by impairing the switch between the active and inactive conformation, favoring a shift in the equilibrium toward the latter, without altering SHP-2’s catalytic capability. Recent biochemical and molecular modeling data consistently support this view [24, 27, 42]. A number of mutations, however, affect residues contributing to the stability of the catalytically inactive conformation but also participating in catalysis or controlling substrate specificity. For a number of these defects it can be speculated that the individual substitution does not markedly perturb substrate affinity and/or catalysis, and that protein activation by N-SH2 dissociation might prevail. Finally, a few missense mutations affect residues located in the phosphopeptide binding cleft of each SH2 domain. Experimental evidence supports the idea that these amino acid substitutions promote SHP-2 gain of function by increasing the affinity of the protein for the phosphorylated signaling partners [24, 27] (our unpublished observations). Like many autosomal dominant disorders, a significant (but not precisely determined) percentage of cases results from de novo mutations. To investigate the parental origin of de novo mutations in NS, Tartaglia and co-workers studied 46 families, each consisting of an affected individual heterozygous for a PTPN11 mutation and unaffected parents [43]. Among the fourteen informative families identified in the study, the mutation was of paternal origin in all cases. Moreover, advanced paternal age was noted among fathers of sporadic NS cases with or without PTPN11 mutations, consistent with many, but not all, other autosomal dominant disorders with paternal origin of spontaneous mutations. Notably, a sex-ratio bias in transmission of the PTPN11 mutations was also observed within families transmitting

Molecular Genetics of Noonan Syndrome

NS as well as for individuals with sporadic NS. This bias favored males by a factor of 2:1. The available data point to this bias being attributable to sex-specific developmental effects of PTPN11 mutations that favor survival of affected male embryos compared to female ones. Among families transmitting the trait, there were more transmitting mothers than fathers, a significant difference that can be ascribed to reduced fertility of male individuals with NS [44]. PTPN11 mutations have been identified in two phenotypes closely related to classic NS. An A-to-G transition at position 923 (Asn308Ser) was documented in a family with NL/MGCLS [10]. In this family, two siblings had lesions in the mandible while their mother only had typical features of NS [45]. The same mutation has been observed in individuals with sporadic NS and families segregating the condition without any bony involvement. More recently, mutational analysis of three unrelated families inheriting this disorder revealed PTPN11 mutations in two [14]. Both of the mutations, Asp106Ala and Phe285Leu, have also been observed in patients with NS. Thus, NL/MGCLS, which was introduced as a distinct nosologic entity characterized by the association of some cardinal features of NS with giant cell lesions of bone and soft tissue [46], should be considered as part of the NS phenotypic spectrum. Consistent with this view, this trait is genetically heterogeneous. Missense PTPN11 mutations have also been identified in LS [15, 16], a developmental disorder closely related to NS, with major features including multiple lentigines, short stature, distinctive face, cardiac defects and electrocardiographic conduction abnormalities, abnormal genitalia and sensorineural deafness [47, 48]. Analysis of several unrelated individuals with a phenotype fitting or suggestive of LS has confirmed the presence of a heterozygous PTPN11 mutation in the vast majority of cases. Tyr279Cys and Thr468Met represent the most common defects, even though additional mutations have been documented (see

25

‘LEOPARD syndrome: Clinical aspects and molecular pathogenesis’ in this volume). The elucidation of the pathogenesis of NS, particularly with respect to the developmental perturbations, depends upon studies of animal models. Araki and co-workers generated and characterized a knock-in mouse bearing the Asp61Gly mutation in the Ptpn11 gene [49]. Consistent with biochemical data on human SHP-2 mutants expressed transiently in cell culture, embryo fibroblasts derived from Ptpn11D61G/+ mice exhibited enhanced Shp-2 activity and increased association of Shp-2 with Gab1 after stimulation with EGF. Cell culture and whole embryo studies revealed that increased Ras/Mapk signaling was variably present, appearing to be cell-context specific. Both homozygous and heterozygous mice had a conspicuous phenotype. The former genotype was an embryonic lethal. At day E13.5, these embryos were grossly edematous and hemorrhagic, had diffuse liver necrosis and severe cardiac defects. Among the Ptpn11D61G/+ embryos, approximately one half had ventricular septal defects, double-outlet right ventricle and increased valve primordia size. Myocardial development was grossly normal. The other half of these embryos had mitral valve enlargement. Other aspects of the NS phenotype were also observed in the heterozygotes, including proportional growth failure, cardiofacial dysmorphism, and a mild leukocytosis with increased neutrophils and lymphocytes in adult mice. Splenomegaly was present due to extramedullary hematopoiesis. There was a myeloid expansion in the bone marrow and spleen. Factor-independent myeloid colonies grew from the marrow and had increased sensitivity to IL-3 and GM-CSF. Hence, this genetic defect engendered a mild myeloproliferative disease similar to that observed in some NS patients. New information concerning gain-of-function Shp-2 and development has emerged through work with transgenic flies [50]. The Drosophila homolog of PTPN11, corkscrew (csw), acts downstream of several receptor tyrosine kinases

26

controlling developmental processes [51]. While ubiquitous expression of leukemia-associated csw transgenic alleles engendered embryonic or larval lethality, expression of an NS-causing allele, N308D, resulted in ectopic wing vein formation. Activation of Ras was necessary but not sufficient for the expression of these phenotypes. Since the ectopic wing vein phenotype closely resembled that observed with Egfr gain of function, epistatic studies with genes relevant for Egfr-Ras-Mapk signaling showed that the N308D allele interacted genetically with nearly all genes in the pathway, documenting dependence on the activation of the receptor by its ligand for ectopic wing vein formation [50]. Children with NS are predisposed to a spectrum of hematologic abnormalities, including juvenile myelomonocytic leukemia (JMML), a clonal myeloproliferative disorder of childhood characterized by excessive proliferation of immature myelomonocytic cells that infiltrate hematopoietic and non-hematopoietic tissues [52, 53]. The hallmark of JMML cells is the hypersensitive pattern of myeloid progenitor colony growth in response to GM-CSF, which is due to a selective inability to down-regulate RAS. Indeed, approximately 50% of children with JMML exhibit either oncogenic RAS mutations or neurofibromin loss of function, the latter is a GTPase activating protein (GAP) for RAS encoded by the NF1 tumor suppressor gene. PTPN11 mutation analysis on a relatively large number of children with NS and JMML has demonstrated the presence of germline mutations in the majority of cases, as well as the occurrence of genotype-phenotype correlations [17, 20, 22]. In particular, one mutation, a C-to-T transition at position 218 (Thr73Ile), was observed to occur in a large percentage of children, a striking finding since that lesion has a very low prevalence among NS-causing mutations. The association between this specific amino acid change and JMML in NS and the key-role of SHP-2 in RAS signaling and hematopoiesis raised the possibility that a distinct class of lesions in PTPN11, possibly

Tartaglia  Gelb

acquired as a somatic event, might play a role in leukemogenesis. Indeed, somatic missense mutations in PTPN11 have been demonstrated to occur in approximately one-third of isolated JMML as well as variable proportions of other myeloid and lymphoid malignancies of childhood [17–19, 22, 27, 54, 55]. The prevalence of PTPN11 mutations among adult patients with myeloid or lymphoid disorders appears to be considerably lower than observed among pediatric cases [27, 56–59] (our unpublished data), even though SHP-2 overexpression has been documented in adult human leukemia [60]. Similarly, PTPN11 is only rarely mutated in non-hematologic cancers [59, 61]. As observed in NS, the vast majority of PTPN11 lesions identified in this heterogeneous group of hematologic malignancies are missense changes that alter residues located at the interface between the N-SH2 and PTP domains. Remarkably, the available molecular data indicate that specificity in the amino acid substitution is relevant to the functional deregulation of SHP-2 and disease pathogenesis (table 1 and fig. 2). Indeed, comparison of the molecular spectra observed with the NS and leukemias indicate a clear-cut genotypephenotype correlation, strongly supporting the idea that the germline transmitted PTPN11 mutations have different effects on development and hematopoiesis than those acquired somatically. Consistent with this, the biochemical behavior of SHP-2 mutants associated with malignancies tend to be more activating than observed with the NSassociated mutant proteins [24, 27, 42]. Moreover, the leukemia-associated PTPN11 mutations upregulate RAS signaling and induce cell hypersensitivity to growth factors and cytokines more than the NS defects do [17, 22, 23, 25]. Overall, the available genetic, modeling, biochemical and functional data support a model in which distinct gain-of-function thresholds for SHP-2 activity are required to induce cell-, tissue- or developmental-specific phenotypes, each depending on the transduction network context involved in the phenotype. According to this model, SHP-2

Molecular Genetics of Noonan Syndrome

mutants associated with NS have relatively milder gain-of-function effects, which are sufficient to perturb development processes but inadequate to deregulate hematopoietic precursor cell proliferation. The PTPN11 mutations observed in isolated JMML and other hematologic malignancies produce mutant SHP-2 proteins with higher gains in function. Since these molecular lesions are observed almost exclusively as somatic defects, it is likely that they affect embryonic development and/or fetal survival. The PTPN11 mutations observed in NS with JMML produce SHP-2 with intermediate activity, which would explain the relatively benign clinical course of the leukemia compared to that observed in isolated JMML.

KRAS

Genetic linkage exclusion studies and PTPN11 genotyping established that one or more additional NS genes existed. Based on the link between the functions of SHP-2 and RAS, two groups independently used a candidate gene approach to discover that KRAS mutations can cause NS [28, 29]. Four missense heterozygous mutations in the KRAS gene were identified in seven individuals among 212 PTPN11 mutation-negative subjects with NS. Of note, a generally more severe NS phenotype was associated with KRAS mutations including one subject with JMML and craniosynostosis and two exhibiting a phenotype at the interface with CFCS and CS. Consistent with this, KRAS mutations were also identified in a small percentage of individuals diagnosed as having CFCS [29, 62]. The diversity of mutations associated with these developmental disorders as well as their phenotypic spectrum have been investigated further, refining the picture of a clustered distribution of germline disease-associated KRAS defects, and confirming the high clinical variability [35, 37]. On the whole, available data indicate that NS-causing KRAS mutations are missense and account for less than 3% of

27

PM1 PM2 PM3 G1

G2

G3

Switch I Switch II G domain

AUG

1

2

Isoform A

3 4

AUG

UAA

5

6

UAA Isoform B

a

b

Fig. 3. KRAS gene organization and protein domain structure. (a) Schematic diagram (above) and three dimensional representation (below) of the structural and functional domains defined within RAS proteins. The conserved domain (G domain) is indicated, together with the motifs required for signaling function (PM1 to PM3 indicate residues involved in binding to the phosphate groups, while G1 to G3 are those involved in binding to the guanine base). The hypervariable region is shown in grey, together with the C-terminal motifs that direct post-translational processing and plasma membrane anchoring (dark grey). The GTP/GDP binding pocket is shown in cyan (guanine ring binding surface) and yellow (triphosphate group binding surface) together with the Switch I (green) and Switch II (magenta) domains, according to the GTP-bound RAS conformation. (b) KRAS gene organization and transcript processing to produce the alternative KRAS isoforms A and B. The numbered black and grey boxes indicate the invariant coding exons and exons undergoing alternative splicing, respectively. KRASB mRNA results from exon 5 skipping. In KRASA mRNA, exon 6 encodes the 3′-UTR.

affected individuals. As previously documented for PTPN11, the distributions of affected residues and amino acid substitutions in NS and cancer appear to be largely mutually exclusive (table 2 and fig. 3). The KRAS gene (OMIM 190070) spans more than 45 kb, is divided into 6 exons, and produces two transcripts through alternative splicing, resulting in two proteins called KRASA and KRASB (fig. 3) [63]. Exon 1 contains most of the 5′ untranslated region, with the last few bases of it residing in exon 2 along with the translation initiation ATG shared by the two mRNAs. For the KRASA transcript, exon 5 contains the stop codon and a portion of the 3′ untranslated region,

28

of which the remainder resides in exon 6. For the KRASB transcript, exon 5 is skipped so exon 6 comprises a portion of the coding region, the stop codon and the entire 3′ untranslated region. As with the other members of the RAS family, KRAS isoforms use GDP/GTP-regulated molecular switching to control intracellular signal flow [64, 65]. They exhibit high affinity for both GDP and GTP, low GTPase activity, and cycle from a GDP-bound inactive state to a GTP-bound active state, the latter allowing signal flow by protein interaction with multiple downstream transducers (fig. 1). GDP/GTP cycling is controlled by GAPs, which accelerate the intrinsic GTPase activity, and GEFs, which promote release of GDP.

Tartaglia  Gelb

Table 2. KRAS affected residues and amino acid changes germinally transmitted or somatically acquired [28, 34, 35, 37, and 62] (germline mutations); catalogue of somatic mutations in cancer (COSMIC), http://www.sanger.ac.uk/perl/ genetics/CGP/cosmic?action=gene&ln=KRAS (November 30, 2007) (somatic mutations). Amino acid

Amino acid change

Germline origin (n = 30) n (%)

Lys5

Asn

1 (3.3)

Glu

1 (3.3)

Ala

–

566 (5.2)

Cys

–

1319 (12.3)

Asp

–

3861 (35.9)

Phe

–

16 (A)

2 family members, CALM, neurofibromas, axillary freckling, Lisch nodules in 1, learning difficulties, scoliosis, macrocephaly, hypertelorism, downslanting palpebral fissures, ptosis, low-set ears

1-bp deletion exon 45 (c.7877del G)

7-year-old boy, CALM, axillary freckling, Lisch nodules, optic glioma, MR, hypertelorism, downslanting palpebral fissures, ptosis, low-set ears, thoracic abnormality, cubitus valgus

Partial NF1 gene deletion

4-year-old, 5 CALM, delayed psychomotor development, PS, relative macrocephaly, hypertelorism, downslanting palpebral fissures, ptosis, low posterior hairline, webbed neck, thoracic abnormality

Missense mutation exon 21 (c.3587T>G; p.L1196R)

2 family members, CALM, neurofibromas in 1, aortic insufficiency in 1, hypertelorism, downslanting palpebral fissures, low posterior hairline, webbed neck, low-set ears, thoracic abnormality

4-bp deletion exon 21 (c.1756_1759 del 4)

2 family members, CALM, neurofibromas, axillary freckling, hypertelorism, downslanting palpebral fissures, low posterior hairline, webbed neck, thoracic abnormality

1-bp deletion exon 18 (c.3060 del A)

42-year-old, CALM, neurofibromas, axillary freckling, Lisch nodules, short stature, macrocephaly, hypertelorism, downslanting palpebral fissures, ptosis, low posterior hairline, webbed neck, lowset ears, thoracic abnormality, scoliosis

8-bp deletion exon 6 (c.796 del GTTTGGCC)

Denayer  Legius

Table 1. (continued) Reference

Clinical findings

Hüffmeier et al. [26]

20-year-old, CALM, neurofibromas, axillary Whole gene deletion freckling, Lisch nodules, learning difficulties, short stature, macrocephaly, hypertelorism, downslanting palpebral fissures, low posterior hairline, webbed neck, low-set ears, thoracic abnormality, scoliosis

– and Noonan syndrome – with a prevalence of 1/1,000 to 1/2,500 – are rather common disorders chance association is a possibility. There has been one report of a female patient with typical findings of NFNS and missense mutations in both the PTPN11 gene on chromosome 12q and the NF1 gene on chromosome 17q [20]. In the study by Colley et al. [19] one of the families showed independent segregation of NF1 and Noonan syndrome whereas in other families half of those affected with NF1 had manifestations of the Noonan syndrome. Bahuau et al. identified a heterozygous truncating mutation in exon 16 of the NF1 gene in a family in which 8 members had NF1/Noonan syndrome, 2 had NF1 only, and 2 had NS only. All 10 patients with features of NF1 carried the mutation, whereas the 2 patients with ‘Noonan syndrome only’ did not [21, 22]. Carey et al. identified a 3-bp deletion in exon 17 of the NF1 gene in one family with NFNS [23]. More recently several reports confirmed NFNS to be a variant of NF1 caused by mutations in NF1 (table 1). Baralle et al. found 2 mutations in 2 individuals with NFNS, a 3-bp deletion in exon 25 and a 2-bp insertion in exon 23–2. In four other individuals no mutations were found in NF1 nor in PTPN11 [24]. De Luca et al. identified heterozygous NF1 mutations in 16 of 17 unrelated subjects with NFNS, including nonsense mutations, out-of-frame deletions, missense changes, small in-frame deletions and one large multi-exon deletion. They noted a high prevalence of in-

Neurofibromatosis Type 1-Noonan Syndrome: What’s the Link?

Molecular findings

frame defects affecting exons 24 and 25, which encode a portion of the GAP-related domain of the protein. No defect in PTPN11 was observed. They provided further evidence that NFNS and Noonan syndrome are genetically distinct disorders by excluding mutations in exons 11–27 of NF1 in 100 PTPN11-negative Noonan syndrome patients [25]. Hüffmeier et al. found heterozygous mutations or deletions of NF1 in seven patients from 5 unrelated families who presented with a variable combination of features of Noonan syndrome and neurofibromatosis type 1 [26].

Some Tumour Types and Leukaemias Occur in Both NF1 and Noonan Syndrome

Haematological malignancies occur with increased frequency in both NF1 and Noonan syndrome. Children with NF1 are predisposed to juvenile myelomonocytic leukaemia (JMML) and other haematological malignancies (ALL, non-Hodgkin lymphoma) [27, 28]. In Noonan syndrome a spectrum of haematological abnormalities has been described including isolated monocytosis, a CMML-like condition that remits spontaneously [29] and JMML [30]. JMML is a rare (annual incidence of 1–2 per million) myeloproliferative disorder characterized by leukocytosis with tissue infiltration and it has a severe and often lethal course. The incidence of JMML is increased 200- to 500-fold in children with NF1. Loss

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of the normal NF1 allele (LOH, loss of heterozygosity) is common in JMML cells from children with NF1 [31]. Sporadic cases of JMML can be caused by somatic mutations of NRAS or KRAS [32], or PTPN11 [33]. Somatic PTPN11 mutations found in JMML (most frequent mutation: E76K) differ from the PTPN11 mutations in Noonan syndrome. They exhibit stronger biochemical and biological effects than germline PTPN11 mutations. Moreover patients with Noonan syndrome who develop JMML have specific germline PTPN11 mutations, most frequently T73I. Apart from the leukaemias there is also overlap in the type of solid tumours observed in NF1 and Noonan syndrome. Rhabdomyosarcoma [34, 35] and neuroblastoma [36–38] have been reported in association with both syndromes. Giant cell tumours of the bone are usually benign, but locally aggressive tumours characterized by the presence of multinucleated giant cells. They have been associated with NF1 [39–41], Noonan syndrome [42–44] and NFNS [45, 46]. Another example are granular cell tumours which are small, cutaneous or subcutaneous nodules. Immunohistochemistry studies have shown that they stain positive for S100, a protein that is a marker of Schwann cells and other cells of neuro-ectodermal origin. They are now generally accepted to have a Schwann cell origin, comparable to neurofibromas. They have been reported in individuals with NF1 [47] and Noonan syndrome [48].

generation of intracellular cyclic-AMP. However most important is its function as a RAS GTPase Activating Protein (GAP). By means of its GAPrelated domain (exons 20–27a) neurofibromin functions as a negative regulator of the RASMAPKinase cascade. It stimulates the hydrolysis of the GTP bound to RAS and thus converts active GTP-bound RAS to inactive GDP-bound RAS. Inactivating mutations in the NF1 gene disturb the GAP activity of the protein resulting in more active RAS and increased signalling through the RAS-MAPK pathway. In 2001 Costa et al. linked the learning difficulties in NF1 individuals to the GAP-activity of neurofibromin by showing that Nf1 mice lacking the alternatively spliced exon 23a exhibited learning difficulties but no apparent developmental abnormalities or tumour predisposition [51]. They proposed that hyperactive RAS resulting from Nf1 haploinsufficiency leads to overactivity of inhibitory GABAergic neurons. The increased inhibition by GABAergic neurons would then lead to the learning disorder phenotype in these animals. To prove this hypothesis they showed that genetically (by crossing Nf1+/– mice with Kras or Nras+/– mice) as well as pharmacologically (by use of the farnesyltransferase inhibitor lovastatin) diminished RAS function rescued the learning difficulties observed in Nf1 heterozygous animals [52]. It can be hypothesized that the learning problems observed in the other NCFC syndromes are also the result of an increased RAS activation in certain brain cells.

The NF1 Gene

The NF1 gene located on chromosome 17q11.2 is a large gene (~350 kilobases) containing 60 exons. It acts as a tumour suppressor gene and NF1 related tumours originate as a result of a somatic inactivation of the normal NF1 copy in an NF1 patient (Knudsons second hit) [10, 49, 50]. The NF1 protein product, neurofibromin, is a large 327 kDa protein which has different functions such as regulation of adenylyl-cyclase activity and

134

The Neuro-Cardio-Facial-Cutaneous (NCFC) Syndromes: An Expanding Group of Phenotypically Overlapping Disorders

Apart from phenotypic overlap with NF1 Noonan syndrome also shares phenotypical features with Costello, LEOPARD and cardio-facio-cutaneous (CFC) syndrome. Recently the term ‘neurocardio-facial-cutaneous (NCFC) syndromes’ has

Denayer  Legius

been coined to group these syndromes. These conditions all share a variable degree of learning disabilities or mental retardation, congenital heart defects, facial dysmorphy and skin abnormalities. In addition they all predispose in some way to malignancy (except for CFC syndrome). During recent years a common genetic and pathophysiologic basis has become obvious. In 2001 gain-of-function mutations in the PTPN11 gene, located on chromosome 12q24.1, have been found to cause about 50% of cases of Noonan syndrome. The PTPN11 gene encodes the non-receptor protein tyrosine phosphatase SHP-2 which relays signals from activated receptor complexes to downstream signalling molecules, like RAS. Noonan-associated PTPN11 mutations result in an enhanced phosphatase activity and activation of the RAS-MAPK pathway [53]. Shortly thereafter specific PTPN11 mutations have also been found in LEOPARD syndrome [54, 55]. Subsequently germline mutations in other components of the RAS-MAPK cascade have been identified in Costello syndrome (HRAS) [56], CFC syndrome (KRAS, BRAF, MEK1/2) [57–59] and also in nonPTPN11 associated Noonan syndrome (KRAS mutations in less than 2% [59], SOS1 in 10% [60, 61] and RAF1 in 3–17% [62, 63]). Functional studies have revealed that most of these mutants result in hyperactivation of the RAS-MAPK cascade. This hyperactive MAPK-signalling is now held responsible as a mechanism for several of the overlapping symptoms in the different NCFC syndromes such as specific facial features, learning difficulties and heart defects. Therefore the presence in NF1 individuals of a pulmonary valve stenosis

and/or facial features typically seen in Noonan syndrome is not surprising. This also explains why differentiation between these syndromes on clinical grounds is not always simple. As an illustration in 1996 a young woman with a prior diagnosis of LEOPARD syndrome and hypertrophic cardiomyopathy who had a de novo missense mutation in exon 18 of the NF1 gene was described [64]. Later on PTPN11 analysis in this individual proved to be negative. Recently a new member of the group of NCFC syndromes has been identified, an autosomal dominant condition caused by germline mutations in the SPRED1 gene [65]. Affected individuals presented with café-au-lait spots, skinfold freckling and macrocephaly. Most of the individuals fulfilled the NIH diagnostic criteria for NF1. Some typical features of NF1 were systematically absent in the reported patients such as Lisch nodules, neurofibromas and central nervous system tumours. In adults multiple lipomas were observed. A Noonan-like facial morphology has been observed in some patients. One individual had a pulmonary valve stenosis. The SPRED1 gene is, like NF1, a negative regulator of the RASMAPK pathway and acts between RAS and RAF to inhibit the activation of RAF by active RAS. A second hit was found in melanocytes from a café-au-lait spot of an affected individual, supporting the hypothesis that biallelic inactivation of SPRED1 is responsible for some of the observed features. This new syndrome again confirms that dysregulated RAS-MAPK signalling can be responsible for symptoms seen in either NF1 or Noonan syndrome.

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Neurofibromatosis Type 1-Noonan Syndrome: What’s the Link?

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18 Kaplan P, Rosenblatt B: A distinctive facial appearance in neurofibromatosis von Recklinghausen. Am J Med Genet 1985;21:463–470. 19 Colley A, Donnai D, Evans DG: Neurofibromatosis/Noonan phenotype: a variable feature of type 1 neurofibromatosis. Clin Genet 1996;49:59–64. 20 Bertola DR, Pereira AC, Passetti F, de Oliveira PS, Messiaen L, et al: Neurofibromatosis-Noonan syndrome: molecular evidence of the concurrence of both disorders in a patient. Am J Med Genet A 2005;136:242–245. 21 Bahuau M, Flintoff W, Assouline B, Lyonnet S, Le Merrer M, et al: Exclusion of allelism of Noonan syndrome and neurofibromatosis-type 1 in a large family with Noonan syndromeneurofibromatosis association. Am J Med Genet 1996;66:347–355. 22 Bahuau M, Houdayer C, Assouline B, Blanchet-Bardon C, Le Merrer M, et al: Novel recurrent nonsense mutation causing neurofibromatosis type 1 (NF1) in a family segregating both NF1 and Noonan syndrome. Am J Med Genet 1998;75:265–272. 23 Carey JC, Stevenson DA, Ota M, Neil S, Viskochil DH: Is there a Noonan syndrome: Part 2:Documentation of the clinical and molecular aspects of an important family. Proc Greenwood Genet Center 2007;17:52–53. 24 Baralle D, Mattocks C, Kalidas K, Elmslie F, Whittaker J, et al: Different mutations in the NF1 gene are associated with Neurofibromatosis-Noonan syndrome (NFNS). Am J Med Genet A 2003;119:1–8. 25 De Luca A, Bottillo I, Sarkozy A, Carta C, Neri C, et al: NF1 gene mutations represent the major molecular event underlying neurofibromatosis-Noonan syndrome. Am J Hum Genet 2005;77:1092–1101. 26 Huffmeier U, Zenker M, Hoyer J, Fahsold R, Rauch A: A variable combination of features of Noonan syndrome and neurofibromatosis type I are caused by mutations in the NF1 gene. Am J Med Genet A 2006;140: 2749–2756. 27 Stiller CA, Chessells JM, Fitchett M: Neurofibromatosis and childhood leukaemia/lymphoma: a populationbased UKCCSG study. Br J Cancer 1994;70:969–972.

28 Bader JL, Miller RW: Neurofibromatosis and childhood leukemia. J Pediatr 1978;92:925–929. 29 Bader-Meunier B, Tchernia G, Mielot F, Fontaine JL, Thomas C, et al: Occurrence of myeloproliferative disorder in patients with Noonan syndrome. J Pediatr 1997;130:885–889. 30 Choong K, Freedman MH, Chitayat D, Kelly EN, Taylor G, Zipursky A: Juvenile myelomonocytic leukemia and Noonan syndrome. J Pediatr Hematol Oncol 1999;21:523–527. 31 Shannon KM, O’Connell P, Martin GA, Paderanga D, Olson K, Dinndorf P, McCormick F: Loss of the normal NF1 allele from the bone marrow of children with type 1 neurofibromatosis and malignant myeloid disorders. N Engl J Med 1994;330:597–601. 32 Flotho C, Valcamonica S, Mach-Pascual S, Schmahl G, Corral L, et al: RAS mutations and clonality analysis in children with juvenile myelomonocytic leukemia (JMML). Leukemia 1999;13:32–37. 33 Kratz CP, Niemeyer CM, Castleberry RP, Cetin M, Bergstrasser E, et al: The mutational spectrum of PTPN11 in juvenile myelomonocytic leukemia and Noonan syndrome/myeloproliferative disease. Blood 2005;106: 2183–2185. 34 Matsui I, Tanimura M, Kobayashi N, Sawada T, Nagahara N, Akatsuka J: Neurofibromatosis type 1 and childhood cancer. Cancer 1993;72: 2746–2754. 35 Moschovi M, Vassiliki T, Anna P, Maria-Alexandra M, Polyxeni NK, Kitsiou-Tzeli S: Rhabdomyosarcoma in a patient with Noonan syndrome phenotype and review of the literature. J Pediatr Hematol Oncol 2007;29:341–344. 36 Ijiri R, Tanaka Y, Keisuke K, Masuno M, Imaizumi K: A case of Noonan’s syndrome with possible associated neuroblastoma. Pediatr Radiol 2000;30:432–433. 37 Lopez-Miranda B, Westra SJ, Yazdani S, Boechat MI: Noonan syndrome associated with neuroblastoma: a case report. Pediatr Radiol 1997;27: 324–326. 38 Origone P, Defferrari R, Mazzocco K, Lo CC, De Bernardi B, Tonini GP: Homozygous inactivation of NF1 gene in a patient with familial NF1 and disseminated neuroblastoma. Am J Med Genet A 2003;118:309–313.

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39 Ardekian L, Manor R, Peled M, Laufer D: Bilateral central giant cell granulomas in a patient with neurofibromatosis: report of a case and review of the literature. J Oral Maxillofac Surg 1999;57:869–872. 40 Krammer U, Wimmer K, Wiesbauer P, Rasse M, Lang S, Mullner-Eidenbock A, Frisch H: Neurofibromatosis 1: a novel NF1 mutation in an 11-year-old girl with a giant cell granuloma. J Child Neurol 2003;18:371–373. 41 Ruggieri M, Pavone V, Polizzi A, Albanese S, Magro G, Merino M, Duray PH: Unusual form of recurrent giant cell granuloma of the mandible and lower extremities in a patient with neurofibromatosis type 1. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1999;87:67–72. 42 Bertola DR, Kim CA, Pereira AC, Mota GF, Krieger JE, et al: Are Noonan syndrome and Noonan-like/multiple giant cell lesion syndrome distinct entities? Am J Med Genet 2001;98:230–234. 43 Cohen MM Jr, Gorlin RJ: Noonan-like/ multiple giant cell lesion syndrome. Am J Med Genet 1991;40:159–166. 44 Ucar B, Okten A, Mocan H, Ercin C: Noonan syndrome associated with central giant cell granuloma. Clin Genet 1998;53:411–414. 45 Posligua L, McDonald DJ, Dehner LP: Diffuse-type tenosynovial giant cell tumor in association with neurofibromatosis type 1-Noonan syndrome: possibly more than a chance relationship. Am J Surg Pathol 2006;30: 734–738. 46 Yazdizadeh M, Tapia JL, Baharvand M, Radfar L: A case of neurofibromatosisNoonan syndrome with a central giant cell granuloma. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;98:316–320. 47 Sahn EE, Dunlavey ES, Parsons JL: Multiple cutaneous granular cell tumors in a child with possible neurofibromatosis. J Am Acad Dermatol 1997;36:327–330.

48 Lohmann DR, Gillessen-Kaesbach G: Multiple subcutaneous granular-cell tumours in a patient with Noonan syndrome. Clin Dysmorphol 2000;9:301–302. 49 Legius E, Marchuk DA, Collins FS, Glover TW: Somatic deletion of the neurofibromatosis type 1 gene in a neurofibrosarcoma supports a tumour suppressor gene hypothesis. Nat Genet 1993;3:122–126. 50 Maertens O, Brems H, Vandesompele J, De Raedt T, Heyns I, et al: Comprehensive NF1 screening on cultured Schwann cells from neurofibromas. Hum Mutat 2006;27:1030–1040. 51 Costa RM, Yang T, Huynh DP, Pulst SM, Viskochil DH, Silva AJ, Brannan CI: Learning deficits, but normal development and tumor predisposition, in mice lacking exon 23a of Nf1. Nat Genet 2001;27:399–405. 52 Costa RM, Federov NB, Kogan JH, Murphy GG, Stern J, et al: Mechanism for the learning deficits in a mouse model of neurofibromatosis type 1. Nature 2002;415:526–530. 53 Tartaglia M, Mehler EL, Goldberg R, Zampino G, Brunner HG, et al: Mutations in PTPN11, encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome. Nat Genet 2001;29:465–468. 54 Digilio MC, Conti E, Sarkozy A, Mingarelli R, Dottorini T, et al: Grouping of multiple-lentigines/LEOPARD and Noonan syndromes on the PTPN11 gene. Am J Hum Genet 2002;71: 389–394. 55 Legius E, Schrander-Stumpel C, Schollen E, Pulles-Heintzberger C, Gewillig M, Fryns JP: PTPN11 mutations in LEOPARD syndrome. J Med Genet 2002;39:571–574. 56 Aoki Y, Niihori T, Kawame H, Kurosawa K, Ohashi H, et al: Germline mutations in HRAS proto-oncogene cause Costello syndrome. Nat Genet 2005;37:1038–1040.

57 Niihori T, Aoki Y, Narumi Y, Neri G, Cave H, et al: Germline KRAS and BRAF mutations in cardio-facio-cutaneous syndrome. Nat Genet 2006;38:294–296. 58 Rodriguez-Viciana P, Tetsu O, Tidyman WE, Estep AL, Conger BA, et al: Germline mutations in genes within the MAPK pathway cause cardio-faciocutaneous syndrome. Science 2006;311:1287–1290. 59 Schubbert S, Zenker M, Rowe SL, Boll S, Klein C, et al: Germline KRAS mutations cause Noonan syndrome. Nat Genet 2006;38:331–336. 60 Roberts AE, Araki T, Swanson KD, Montgomery KT, Schiripo TA, et al: Germline gain-of-function mutations in SOS1 cause Noonan syndrome. Nat Genet 2007;39:70–74. 61 Tartaglia M, Pennacchio LA, Zhao C, Yadav KK, Fodale V, et al: Gain-offunction SOS1 mutations cause a distinctive form of Noonan syndrome. Nat Genet 2007;39:75–79. 62 Pandit B, Sarkozy A, Pennacchio LA, Carta C, Oishi K, et al: Gain-of-function RAF1 mutations cause Noonan and LEOPARD syndromes with hypertrophic cardiomyopathy. Nat Genet 2007;39:1007–1012. 63 Razzaque MA, Nishizawa T, Komoike Y, Yagi H, Furutani M, et al: Germline gain-of-function mutations in RAF1 cause Noonan syndrome. Nat Genet 2007;39:1013–1017. 64 Wu R, Legius E, Robberecht W, Dumoulin M, Cassiman JJ, Fryns JP: Neurofibromatosis type I gene mutation in a patient with features of LEOPARD syndrome. Hum Mutat 1996;8:51–56. 65 Brems H, Chmara M, Sahbatou M, Denayer E, Taniguchi K, et al: Germline loss-of-function mutations in SPRED1 cause a neurofibromatosis 1-like phenotype. Nat Genet 2007;39:1120–1126.

Eric Legius Department of Human Genetics, Catholic University of Leuven Herestraat 49 BE–3000 Leuven (Belgium) Tel. +32 16 345903, Fax +32 16 346051, E-Mail [email protected]

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Zenker M (ed): Noonan Syndrome and Related Disorders. Monogr Hum Genet. Basel, Karger, 2009, vol 17, pp 138–150

Animal Models for Noonan Syndrome and Related Disorders T. Araki  B.G. Neel Ontario Cancer Institute, University Health Network, TMDT8–355, Toronto, Ont., Canada

Abstract Noonan syndrome (NS) and related disorders, including LEOPARD syndrome (LS), cardio-facial-cutaneous (CFC) syndrome, Costello syndrome (CS) and neurofibromatosis Type-I (NF1) can be grouped together as the ‘neuro-cardio-facio-cutaneous (NCFC) syndromes’ by virtue of their shared clinical features and molecular pathogenesis. Recent studies have shown that these diseases are caused by germline mutations in key components of the RAS-RAF-MEK-ERK kinase (hereafter, ‘RAS/ERK’) cascade. Presumably, the common features of these syndromes reflect abnormal function of this pathway during development and postnatally. Nevertheless, the detailed mechanism by which abnormal ERK activation results in shared syndromic phenotypes remains unclear. Moreover, although clearly related, specific NCFC syndromes are clinically distinguishable. How mutations within the same signaling pathway have such distinct phenotypic consequences is unknown. NCFC syndromes predominantly affect events that occur during embryogenesis and affect complex developmental/morphogenetic pathways. Consequently, it is difficult, if not impossible, to delineate their molecular pathogenesis using cell culture systems or human samples. Also, because the NCFC syndromes are fairly rare, multiple different alleles exist for each disorder, and the human population is extensively outbred, it is difficult to determine whether allele-specific phenotypic differences exist and/or whether there are key genetic modifiers. Animal models provide tools to address these issues, as well as to devise and evaluate potential therapeutic approaches. This review focuses on models of NS and related disorders, with a particular focus on mouse models. Copyright © 2009 S. Karger AG, Basel

It is now clear that germline mutations in members of the RAS/ERK cascade cause NCFC syndromes [1]. Individuals with these syndromes typically display some combination of facial abnormalities, cardiac defects and proportional short stature, although skin, lymphatic and genital abnormalities, as well as cognitive difficulties, ranging from mild to severe mental retardation, also are common. The phenotypic similarities between NCFC syndromes can be explained by effects of this common signaling pathway. Nevertheless, these syndromes are clinically distinguishable. For example, multiple neurofibromas and café au lait spots are observed in NF1 patients, and severe mental retardation is most often found in patients with CFC syndrome. The NCFC syndromes also differ markedly in their relative predisposition to malignancy. Predisposition to cancer is not a known feature of CFC syndrome, even though somatic mutations in BRAF, the gene mutated in most cases of CFC syndrome [2, 3], occur in ~70% of melanomas [4]. Other NCFC syndromes carry a high risk of cancer development. Brain tumors and hematological malignancies, particularly the rare disorder juvenile myelomonocytic leukemia (JMML), are associated with NF1. JMML, possibly other

hematological (e.g., acute lymphoblastic leukemia (ALL)) malignancies, and potentially neuroblastoma, are associated with NS (caused by PTPN11, KRAS, SOS1 or RAF1 mutations) [5–10]. Somatic PTPN11 mutations are also common in sporadic JMML [11, 12], and occur less frequently in a variety of other leukemias and myeloproliferative disorders (MPD). CS, caused by HRAS mutations [13], is associated with a high risk of rhabdomyosarcoma and bladder cancer; notably somatic HRAS mutations are also found in sporadic versions of these malignancies [14]. Although much progress has been made in defining the genetic basis for the NCFC syndromes, several fundamental issues remain to be addressed. First, the molecular and cellular mechanisms responsible for the defects seen in these syndromes remain poorly understood. Also unclear is how mutations in components of the same signaling pathway can cause the different features that allow individual NCFC syndromes to be distinguished clinically. Even within the same syndrome, genotype/phenotype relationships (e.g., whether individual mutant alleles contribute differentially to phenotype – and if so, how) are poorly defined. For example, biochemical data suggest that different NS mutations in PTPN11 can have substantially different effects on the catalytic activity of its gene product, SHP2 [15], making it possible, if not likely, that such alleles could have distinct phenotypic consequences. But studies of patients with PTPN11 mutations have failed to reveal clear differences, potentially because of the large number of alleles that exist and the relatively small numbers of patients surveyed. Alternatively (or in addition), unknown modifier loci might play critical roles in phenotype determination. Furthermore, some NCFC syndromes can result from mutations in different genes (e.g., mutations in KRAS, SOS1 or RAF1 can also cause NS, whereas mutations in MEK1 or MEK2, instead of BRAF, can cause CFC) [2, 3]. Certain RAF1 mutations may predispose to hypertrophic cardiomyopathy in NS [9, 10]. NS caused by

Animal Models for Noonan Syndrome and Related Disorders

PTPN11 and SOS1 mutations may also differ in some phenotypes [7, 8], including relative risk of malignancy [16, 17]. In general, though, whether the pathogenic impact of different disease-associated mutations is similar remains largely unknown. Even more confusing, although most mutant alleles associated with NCFC syndromes act as hypermorphic (gain-of-function) mutants in biochemical and/or transfection assays, others either have no effect compared to wild type (WT) or even act as hypomorphic (loss-of-function) or dominant negative mutants. Conceivably, gainand loss-of-function might have similar effects in some developmental pathways. Alternatively, the cellular pathogenesis of these disorders may be quite distinct. Ultimately, of course, one would like to reverse or remediate the defects in the NCFC syndromes (at least the postnatal defects, which are most likely to be treatable). However, the relative rarity of these syndromes limits the patient population likely to be available for any future therapeutic trials. These and other issues can be effectively, if not best, addressed by animal models. Here, we review the studies of the physiological functions of mutants associated with NCFC syndrome, with a particular focus on mouse models of NS.

Properties of Mutant Alleles Associated with NCFC Syndromes

Approximately 50% of NS cases are caused by mutations in PTPN11, which encodes the SH2 domain-containing protein tyrosine phosphatase SHP2. A key component of the RAS/ERK pathway, SHP2 and, in particular, SHP2 catalytic activity, is required for normal RAS activation by most, if not all, growth factors and cytokines [reviewed in 18; 19]. Multiple studies, culminating in the SHP2 crystal structure [20], established that SHP2 is regulated by an elegant ‘molecular switch’ mechanism that ensures that its catalytic activity is suppressed until it is needed at the

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right time and place. In the basal (closed) state, the amino terminal SH2 (N-SH2) domain interacts with the PTP domain to suppress catalytic activity [21]. Binding of a phosphotyrosyl peptide (e.g., from an appropriate tyrosyl phosphorylated receptor or adapter protein) to the N-SH2 domain disrupts this inhibitory interaction, resulting in ‘opening’ of the enzyme and potent activation. The physiological relevance of this regulatory mechanism was first demonstrated by the generation of ‘activated mutants’, which show enhanced enzymatic and biological activity ([22]; and see below). The discovery of NS- and leukemia-associated PTPN11 mutants provided even more dramatic validation. Almost all such mutants map to the N-SH2 or PTP domain and affect residues that participate in basal inhibition. Not surprisingly, these mutants almost invariably show enhanced catalytic (PTP) activity in vitro [15], indicating a shift towards the ‘open’ state of the enzyme. Consistent with this interpretation, NS-associated SHP2 mutants show increased binding to the adapter Gab1 and, when co-expressed with Gab1, enhance Erk activation in transfection assays [23]. SOS1, which encodes a major guanine-nucleotide exchange factor (GEF) for RAS proteins, is the second common gene mutated in NS. SOS1, like SHP2, is regulated by an auto-inhibitory module [24, 25]. Remarkably, similar to the effects of NS-associated PTPN11 mutants, SOS1 mutants affect key auto-inhibitory residues and RAS/ERK activation in transfection assays [7, 8]. SOS1 also has RAC-GEF activity [26], but whether NS-associated mutants affect RAC activation remains unclear. Most NS-associated RAF1 mutants also evoke enhanced MEK and ERK activity in 293T and COS7 cells [9, 10]. However, other RAF1 alleles have either the same or even lower activity than WT RAF1 in such assays. Some cancer-associated, somatic BRAF mutants also exhibit decreased activity under similar assay conditions, but when co-expressed with RAF1, show enhanced MEK/

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ERK activation. Such findings led to the conclusion that RAF1/BRAF heterodimers comprise the bona fide kinase that activates MEK in vivo [27, 28]. Conceivably, NS RAF1 mutants with ‘decreased’ activity might show increased activity upon co-expression of BRAF. Alternatively, NS RAF1 mutants may have decreased susceptibility to negative regulators (e.g., SPRED, SPROUTY proteins), whose effects could be obscured by the high levels of expression in transient transfection studies. Intriguingly, in this regard, it was reported recently that loss-of-function SPRED1 mutants cause a variant NF1-like syndrome [29]; conceivably, some NS-associated RAF1 proteins could be resistant to the inhibitory effects of SPRED1. Such complexities and potential complications in interpretation of heterologous expression experiments emphasize the need to assess the effects (both biochemically and biologically) or diseaseassociated mutants expressed at more physiological levels. LS, which shares multiple phenotypic features with NS, also is caused by PTPN11 mutations [5]. Yet surprisingly, LS alleles exhibit markedly decreased catalytic activity and act as dominant negative mutants to inhibit growth factor-evoked ERK activation [30, 31]. Some mutations reportedly cause both NS and LS [5]. It is unclear whether this represents misdiagnosis, similar phenotypes caused by SHP2 gain-of-function and partial deficiency, artifacts of the in vitro and ex vivo SHP2 assays, or as yet unknown PTP activity-independent effects of SHP2. Most CFC syndrome-associated mutants of BRAF and all MEK1/2 alleles cause increased ERK activation of downstream pathway in transient transfection experiments. Again, however, there are some exceptions [2, 3]. These cell line studies have provided a relatively easy way in which to test the effects of human disease-associated mutations on selected signaling pathways, and to provide initial insights into potential biochemical mechanisms of pathogenesis. Yet, as detailed above, some of the

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results obtained have been confusing, as they do not quite fit with the simple model that NCFC syndromes are diseases of RAS/ERK pathway hypermorphism. Even more importantly, heterologous cell lines provide limited insight into the cell biological basis of the type of cell- and tissue-specific phenotypes seen in these disorders.

Primary Cells/ex vivo Models

Primary cells from relevant tissues are potential improvements over heterologous cell systems for evaluating the effects of NCFC mutants. Several groups have studied the effects of disease-associated PTPN11 mutants on primary hematopoietic cells to gain insights into their role in leukemogenesis. Retroviral transduction of somatic leukemia-associated mutants into primary murine bone marrow (BM) cells recapitulates key cellular features of JMML, including the production of monocytic colonies in the absence of exogenous cytokines (factor-independent colony formation) and hypersensitivity of myeloid progenitors to the cytokine granulocyte-macrophage colonystimulating factor (GM-CSF) [32–34]. Unlike in human JMML, mouse myeloid progenitors expressing PTPN11 mutants also show increased sensitivity to IL3; such differences likely reflect intrinsic differences between murine and human hematopoiesis. Mutations associated solely with NS are less potent than those found in both NS and leukemia or in leukemia in this myeloid transformation assay [32]. Second site mutations (introduced into the most potent leukemia-associated mutant) indicate that PTP activity, one of the two C-terminal tyrosyl phosphorylation sites, and both SH2 domains are required for maximal transforming activity [32]. The latter finding suggests that even constitutively activated SHP2 must be targeted correctly via its SH2 domains to promote malignant transformation. Finally, transplantation of BM transduced with leukemia-associated mutants causes a fatal

Animal Models for Noonan Syndrome and Related Disorders

myeloproliferative disorder (MPD) characterized by overproduction of tissue-invading myeloid lineage cells in ~60% of recipients [32]. The remaining mice succumb to T cell leukemia/lymphoma, a type of neoplasm not associated with PTPN11 mutations in humans. Hematological disease may be strain-dependent however, as MPD (or lymphoid malignancy) is not observed following transduction of C57BL6 BM with potent leukemogenic mutants [33]. Such experiments have also provided some insight into the biochemical effects of leukemogenic mutants. Macrophages derived from BM transduced with such mutants display enhanced GM-CSF-evoked ERK activation [33, 34], whereas bone marrow mast cells from mice with SHP2-evoked MPD show increased activation of ERK, AKT and STAT5 [32]. In contrast to earlier transient transfection studies [12, 23], there was no requirement for co-expression of Gab1 (or another Gab protein) in these primary cell systems. These results suggest that differential expression of important SHP2-binding proteins may be one reason for the tissue-specific effects of disease-associated PTPN11 mutants. Taken together, these retroviral gene transduction studies have provided firm evidence for the causal role of PTPN11 mutants in leukemogenesis, and identified key structure/function relationships (most notably, the PTP domain requirement) and abnormal biochemical consequences that might be pertinent for development of novel therapeutics. KRAS mutants, which are found in a small percentage of NS and NS/JMML patients [6], also cause cytokine hypersensitivity and increased activation of RAS, MEK and AKT. For further details about the hematological effects of PTPN11 and KRAS mutants, the reader should consult more comprehensive reviews [35, 36]. The effects of CS-associated HRAS mutations have been assessed using fibroblasts from affected individuals. Compared to normal fibroblasts, these have increased proliferation (as assayed by

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BrdU incorporation) in response to epidermal growth factor (EGF) or fetal calf serum [13]. Although the common features of the NCFC syndromes include short stature, facial abnormalities and cardiac defects, there have been few studies of the effects of NCFC mutations on the cell types relevant to these defects (in part, because in most cases, these have not been defined; see below). One exception has been an attempt to assess the effects of an NS mutant on valve development, using so-called ‘AV cushion explant assays’ [37]. Valvulogenesis is a complex process [38], involving at least three cell types, which takes place in specialized structures termed ‘cardiac (or endocardial) cushions’. There, specialized endothelial cells (termed cushion endothelium or cushion endocardium), which rest upon a specialized extracellular matrix (the ‘cardiac jelly’), respond to signals from the subjacent myocardium and undergo an endothelial to mesenchymal transition (EMT). Once transformed, cushion mesenchymal cells invade the cardiac jelly and proliferate. A complex morphogenetic process ensues, which entails cessation of cushion mesenchymal cell proliferation, cell shape changes and substantial apoptosis. The third cell type, the cardiac neural crest (NC), migrates into the developing cushion and is important for proper valve and septum generation [39]. In the mouse, cardiac NC migration occurs at around E10.5 and only involves the outflow tract (OT) valves [40]. The cushion explant assay models the early events of EMT, mesenchymal cell invasion and, to some extent, mesenchymal proliferation. Initially developed for studies of chicken valve development [41], explant assays subsequently were optimized for murine AV and outflow (OT) cushions [42, 43] and can be used to test the effects of various agonists/antagonists on the above processes. Robbins and coworkers found that the PTPN11 mutant Q79R, introduced by adenoviral gene transduction, had no effect on EMT per se, but increased Erk activation and enhanced the

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proliferation of transformed mesenchymal cells in chicken AV cushion explants [37]. These results are consistent with the increased cushion mesenchymal cell proliferation (as assayed by BrdU incorporation) and Erk activation (by anti-pErk immunohistochemistry) seen in a mouse model of NS (see below), and could help explain the hypertrophic valves seen in many NS patients. However, these results are somewhat inconsistent with previous studies of Nf1–/– mice. Nf1 homozygosity is not compatible with life, and thus Nf1–/– mice do not model a specific human syndrome. However NF1 patients have pulmonic stenosis more often than in the general population [44], suggesting that, contingent upon the genetic background, NF1 may be haploinsufficient for vavulogenesis. Consequently, the cardiac phenotype of Nf1–/– embryos may represent a more severe version of the consequences of NF1 heterozygosity in humans. Such embryos exhibit pan-valvular stenosis, atrial and ventricular septal defects, and double outlet right ventricle. Studies using a conditional Nf1 allele indicate that these result selectively from the absence of Nf1 in endothelial cells [43, 45]. Similar to the phenotype of NS mice (see below), Nf1–/– embryos show increased mesenchymal cell proliferation, but in contrast to the above chicken explant studies, Nf1–/– cushion explants reportedly show increased EMT, as do explants from NS mice ([43]; also, see below). This could reflect an intrinsic difference in chicken and murine explant assays (and possibly, differences between the effects of NS mutants in avian and murine systems). Alternatively, and perhaps more likely, the Q79R allele is not expressed early enough to alter EMT in the chicken explant experiments.

Drosophila Models

The strong conservation of the Ras/Erk pathway across evolution allows the study of human disease-associated NCFC mutants in model genetic

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organisms. When such mutants evoke relevant phenotypes, such organisms as Drosophila melanogaster provide the potentially big advantage of rapidly deciphering key pathways using genetic analysis and the large number of hyper- or hypomorphic lines already established. Flies also have some clear limitations: obviously, key syndromic features such as cardiac, facial and stature abnormalities cannot be modeled accurately. Drosophila does, however, have a rudimentary hematopoietic system, and earlier work established that expression of activated RAS mutants causes excessive hemocyte production [46]. Similarly, expression of the leukemia-associated mutant PTPN11 E76K under the control of a hemocyte-selective promoter causes an ~6-fold increase in the number of plasmatocytes, which are myeloid-like cells that comprise the major circulating blood cells in flies [32]. The mutant PTPN11 allele also alters cellular morphology, suggesting an additional effect on myeloid differentiation. These effects are qualitatively similar, although considerably weaker, than those evoked by activated RAS. It is unclear if this reflects a lower leukemogenic potential of mutant SHP2 compared to RAS, or that human SHP2 is less leukemogenic than the cognate mutation in the fly SHP2 ortholog, corkscrew (csw) might be. Notably, though, mutant Ras also is more potently leukemogenic than mutant Ptpn11 in mice. Oishi et al. generated transgenic flies with GAL4-inducible expression of wild type csw or a series of csw mutants, corresponding to PTPN11 E76K, A72S and N308D, which show different degrees of catalytic activation (E76K>A72S>N308D) [47]. Interestingly, ubiquitous expression of the A72S or E76K mutants causes lethality, but the N308D mutant was compatible with viability. Doubling N308D gene dosage also resulted in lethality, suggesting that the extent of catalytic activation – or at least the degree to which a mutation causes SHP2 to reside in the open state – helps determine the disease phenotype. Similar observations have been made using mouse model

Animal Models for Noonan Syndrome and Related Disorders

of NS ([48], and T. A., G. C., and B.G. N., manuscript in preparation; see below). Much like the selective effects of NS mutants on human (and mouse; see below) development, the phenotypes evoked by the cognate csw mutants do not reflect universal abnormality of Drosophila tyrosine kinase signaling. For example, the N308D mutant causes a wing vein phenotype similar to that evoked by gain-of-function mutants in the Drosophila EGFR, and this phenotype is rescued by loss-of-function mutants in the EGFR signaling pathway. Epistasis studies identify additional genetic interactions between N308D and the Notch, BMP and Jak/Stat pathways, respectively, which may have important implications for the pathogenesis of key NS phenotypes. For example, EGFR, Notch and BMP signaling are important for valvulogenesis [38], whereas the Jak/Stat pathway mediates the effects of cytokines such as GM-CSF and IL-3, and may therefore be relevant to the pathogenesis of MPD evoked by PTPN11 mutants.

Zebrafish Model

Very recently, Jopling et al. compared the effects of NS and LS mutants of PTPN11 in zebrafish [49]. Injection of either type of mutant results in significantly shorter embryos at 4 dpf without affecting cell specification. These results suggest impaired gastrulation, and indeed, cell tracing experiments indicate that both extension and convergence movements are reduced significantly upon injection of the NS mutant. NS and LS mutant embryos also showed wide-set eyes as well as edematous hearts, although these defects were not characterized further in this report. In general, the phenotypes caused by NS or LS mutants were indistinguishable, which is similar to the human situation. This is, of course, surprising, given that NS and LS alleles have opposite effects on SHP2 catalytic activity. However, the effects of the two mutants were neither additive

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nor synergistic, which the authors interpreted as indicating opposing actions on the same pathway. On the other hand, the two mutants did not rescue each other, so their precise mode of action remains unclear. Lowering endogenous Shp2 levels by means of morpholinos resulted in a gastrulation defect, similar to the effects of dominant negative Shp2 expression in Xenopus embryos [50] or homozygosity of a Ptpn11 allele that results in an N-terminal Shp2 truncation in the mouse [51]. The effects of zebrafish Shp2 deficiency were attributed to defective Src activity and Rho activation. The effects of the NS and LS mutants on these pathways were not reported, however. In unpublished studies, we (R. Stewart, M. Kontaridis, K. Swanson, B.G. N. and A. T. Look) have also characterized the effects of NS and LS mutants, compared to those of zebrafish Ptpn11 morphants. Similar to the above results, embryos injected with NS and LS mutants, as well as morphants, have defective gastrulation. However, we also find distinct effects of these three treatments on NC development, which suggest that SHP2 has PTP-dependent and independent effects on key NC developmental pathways. The differential ability of NS and LS mutants to mediate these pathways may help explain the phenotypic similarities and differences between LS and NS.

Mouse Models

Over the past decade, several groups have developed mouse models of NF1, by means of conventional and conditional Nf1 gene inactivation. As noted above, Nf1–/– mice are not viable. In contrast, Nf1+/– mice, which are genetically similar to NF1 patients, are healthy at birth but succumb to leukemia or pheochromocytoma (both of which are NF1 characteristics) by 15–18 months of age [52]. Nf1+/– mice also have learning defects, which can be rescued by genetic and pharmacological manipulations that decrease Ras function [53,

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54]. Notably, however, these mice develop neither neurofibromas nor astrocytomas, the hallmark features of Type 1 NF. Homozygotic Nf1 inactivation in Schwann cells (peripheral nervous system) or astrocytes (central nervous system) also fails to cause tumors. By contrast, Nf1 deletion in hematopoietic cells results in a progressive myeloproliferative disorder that resembles JMML [55]. Parada and colleagues resolved this paradox by realizing that in NF1 patients, neurofibromas, which are of Schwann cell origin, are generated in the context of NF1 heterozygous tissues. Remarkably, they found that homozygous Nf1 deletion in Schwann cells, in the background of Nf1 heterozygosity results in fusiform paraspinal masses with histological features of plexiform neurofibroma [56]. Nf1+/– mice lacking neurofibromin in astroglial precursors developed fusiform masses of the optic nerve and chiasm, resembling optic nerve gliomas in children with NF1 [57]. These results indicate that NF1+/– cells in these environments cooperate with NF1–/– Schwann or astroglia cells to cause neurofibromas or astrocytomas, respectively. Parada and colleagues subsequently found that Nf1–/– Schwann cells secrete mast cell chemotactic factors, including Kit Ligand. In response to such factors, Nf1+/– mast cells are recruited to the vicinity of Nf1–/– Schwann cells to facilitate neoplastic transformation [58]. Our group has generated and characterized knock-in mice expressing the NS mutant Ptpn11D61G (hereafter, DG) [48]. Homozygous DG mice die at mid-gestation, with a phenotype similar to global Nf1 deletion. At E13.5, DG/DG embryos exhibit severe cardiac defects, including atrial, ventricular, or atrio-ventricular septal defects, double-outlet right ventricle, enlargement of AV and OT endocardial cushions and markedly thinned myocardium. These abnormalities, with the possible exception of myocardial thinning (see below), represent severe versions of cardiac phenotypes seen in NS patients (and in DG/+ mice).

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Mid-gestation DG/DG embryos also are markedly edematous (probably due to their severe cardiac defects), and show evidence of hemorrhage and liver necrosis. Bleeding and clotting abnormalities are seen with variable penetrance in NS patients [59, 60]; hence, the hemorrhage seen in DG/ DG embryos might reflect NS-associated platelet or clotting factor defects. Liver abnormalities are not a known feature of NS, however, and hemorrhage could be an indirect consequence of liver and/or cardiac defects in these embryos. Although we have not yet pursued the molecular basis of the liver defects in DG/DG embryos (mainly because it is not a feature of the human syndrome), midgestation hepatic necrosis is a classical manifestation of defects in the NF-κB pathway (which is required to prevent apoptosis in response to TNFα produced at that time). There has been a report that SHP2 regulates NFκB activation [61]. As TNF family receptors regulate a wide array of physiological functions that could contribute to bona fide NS phenotypes, further exploration of the molecular pathophysiology of hepatic necrosis in DG/DG mice might prove informative. The relevance of the myocardial thinning seen in DG/DG mice to NS pathogenesis also is unclear. Hypertrophic cardiomyopathy (HCM) is one of the features of human NS [62], but in one of the few clear genotype/phenotype correlations reported, HCM was found to be less common in NS caused by PTPN11 mutations [63]; indeed, recent studies show markedly increased incidence of HCM in NS caused by specific RAF1 alleles [9, 10]. In any event, it appears that high levels of SHP2 activation cause the opposite phenotype (myocardial thinning), at least in mice. Notably, however, ventricular noncompaction or hypoplasia has been reported in some NS patients [64–66], although the genotypes of these individuals have not been reported. Transgenic expression of an NS mutant causes a similar phenotype [67], although as discussed below, the relevance of that system is unclear. In contrast to the uniform lethality of DG/ DG embryos, ~50% of D61G/+ mice (on 129Sv ×

Animal Models for Noonan Syndrome and Related Disorders

C57BL6/J background) die in late gestation or perinatally with multiple cardiac defects. At E13.5, D61G/+ embryos are obtained at the expected Mendelian ratio, but fall into 2 groups: severely affected or mildly affected. Severely affected embryos have ventricular septal defects, double-outlet right ventricle and increased size of all valve primordia. These phenotypes are similar to, although less severe than, those found in DG/DG embryos; also, unlike the latter, DG/+ embryos have normal myocardial thickness (and no edema or hepatic necrosis). BrdU labeling experiments show increased mesenchymal cell proliferation and decreased apoptosis in DG/+, compared to WT embryos. All of these cardiac defects resemble those seen in Nf1–/– mice, suggesting that they result from increased activity of the Ras/Erk pathway. Indeed, increased numbers of pErk-positive cells are found in DG/+ [48] and Nf1–/– [45] endocardial cushions. Overall, DG/+ mice provide a reasonable model for the cardiac defects in NS. There are, however, some important caveats. First, the most common cardiac defect in human NS patients is pulmonic stenosis, whereas AV valve hyperplasia is more common in the murine model. This could reflect differences in NC function in human and mouse valvulogenesis, and raises the possibility that NS alleles may have effects in human cardiac NC that are not adequately modeled in the mouse (see below). In addition, human patients typically have some, but rarely all, of the cardiac defects seen in DG/+ mice. Furthermore, the cardiac defects in DG/+ mice (as well as more recent models that we have generated; see below), appear in an ‘all-or-none’ fashion. This could indicate some fundamental property of murine valvuloseptal development that will limit the conclusions that can be drawn from this and other NCFC models. Nevertheless, it seems likely that the fundamental cellular and biochemical abnormalities revealed by these models will be relevant to pathogenesis of the human syndromes. Although enhanced Erk activation (as assessed by whole mount immunohistochemistry) is seen in

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a few other sites in DG/+ embryos – most notably those, such as the developing face and limb bud, in which other NS-like defects are observed subsequently (see below) – Erk hyper-activation is not uniform. Furthermore, primary mouse embryo fibroblasts fail to show increased Erk activation in response to low or high doses of EGF, FGF, IGF or PDGF. Thus, as in the Drosophila model, NS mutants have a cell/tissue-selective ability to increase Erk activation. The reason for this selectivity remains unknown, although possible explanations include differential levels of SHP2 binding proteins (discussed above) or substrates or differential activity of homeostatic feedback pathways able to diminish the effects of increased SHP2 activity. Severely affected DG/+ embryos survive to at least E18.5 (the last developmental time point analyzed), and probably die perinatally. In contrast, mildly affected DG/+ mice survive to adulthood and manifest other NS features, including facial abnormalities and proportionate short stature. These mice also exhibit an initially mild MPD, characterized by increased splenic size, mild myeloid hyperplasia, and factor-independent colony production by BM and spleen cells. Although this MPD is well-tolerated at first, DG/+ mice eventually die much earlier (at 12–15 months) than their WT counterparts (T.A. and B.G.N., unpublished). Notably in this regard, the D61G allele, which initially was reported only in NS patients, was subsequently observed in JMML as well [68]. The effects of D61G on neurogenesis have been analyzed by means of combined ex vivo and in vivo approaches [69]. Over-expression of D61G in cortical precursors promotes neurogenesis and inhibits astrogenesis. There also is a small, but statistically significant, increase in neurogenesis and decrease in astrogenesis in the dorsal cortex and hippocampus of DG/+ mice. These alterations may perturb neural circuit formation to cause the cognitive deficits seen in NS patients. In summary, DG/+ mice recapitulate many of the main features of human NS. In addition to providing avenues for exploring the pathogenesis of

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NS phenotypes, these mice also raise several new questions. First, the effects of genetic background on phenotypes are not clear: e.g., it is unclear whether the incomplete penetrance of the DG/+ cardiac phenotype is stochastic or reflects modifier loci segregating in the mixed background used for the initial studies. Second, biochemical analyses show that D61G is one of, if not the, most highly activated PTPN11 alleles associated with NS [15]. Given that increasing gene dosage enhances phenotypic severity caused by activated Shp2 in flies and mice (see above), mutants with varying degrees of catalytic activation might also be expected to have phenotypic differences. Finally, the key cell types in which PTPN11 mutants act to cause NS phenotypes have not been defined. We have begun to address several of these issues using both our initial DG mice and several new mouse models, including knock-in mice expressing Ptpn11N308D and inducible knockin mice expressing Ptpn11D61Y (T.A., G.C., and B.G.N., manuscript in preparation). By crossing the DG allele onto 129S6/SvEv, C57BL6/J or Balb/c, we have found that the genetic background strongly influences the NS phenotype. It is not yet clear, however, if cloneable modifiers exist or if these strain differences are attributable to heterosis. Our data also indicate, however, that, as in the Drosophila studies, the specific Ptpn11 allele (on the same genetic background) can strongly influence the NS phenotype. This, in turn, may depend on the degree of Shp2 hyper-activation; indeed, there appears to be a hierarchy of phenotypes contingent on increasing Shp2 activity, with the lowest levels of Shp2 hyper-activation capable of affecting growth, while increasingly higher levels are required to evoke facial abnormalities, cardiac defects and fatal MPD, respectively. Studies using tissue-specific Cre recombinase lines to activate the Ptpn11D61Y allele selectively show that the facial abnormalities result from mutant expression in NC-derived cells, whereas all cardiac defects are caused by mutant expression in

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endocardium/endothelium, not the NC or myocardium. Notably, tissue-specific deletion of Nf1 using the same Cre lines has similar phenotypic consequences [45]. Studies to elucidate the cell(s) responsible for NS growth defects are ongoing. Finally, using explant assays from DG/+ embryos, we have found that NS mutants extend the normal interval during which EMT occurs, and as a consequence of the increased Erk activation that they evoke. In contrast to the chicken explant studies [37], we do not observe increased mesenchymal proliferation in mouse AV cushion explants. This could reflect differences between the chicken and mouse systems. Given that cushion mesenchymal cell proliferation is enhanced in DG/+ embryos, though, it is perhaps more likely that the mouse explants are inadequate to model increased proliferation for technical reasons. Notably, mesenchymal outgrowths are observed in these explants when growth factors (e.g., PDGF, FGF, Neuregulin) are added exogenously, suggesting that endogenous growth factors may be limiting in mouse explants (but not in chicken). The emerging picture, combining the mouse and chicken studies, is that NS mutants may both extend the normal interval for EMT and cause excess proliferation of cushion mesenchymal cells. Although both of these defects appear to result from enhanced Erk activation, it is unclear if the same upstream (i.e., growth factor, cytokine, integrin) signals mediate both effects. Also unclear is how increased Erk activation translates into enhanced EMT and proliferation. One attractive possibility is that NS alleles hyper-activate the transcription factor Sox9. Sox9 is known to be an immediate-early gene dependent on Erk activation [70], and Sox9-deficient mice have hypoplastic cardiac valves due to defective EMT [71], the converse phenotype to NS mice. Altered Sox9 activity also could explain other NS phenotypes (e.g., facial and stature abnormalities), given that Sox9 is also required for chondrogenesis [72], and that Erk activity reportedly affects Sox9 activity differentially in micromass cultures from chicken facial NC [73].

Animal Models for Noonan Syndrome and Related Disorders

In contrast to the above observations using Ptpn11 knock-in mice, Nakamura et al. analyzed transgenic mice expressing Ptpn11Q79R in the myocardium under the control of the αMHC and βMHC promoters, respectively [67]. Because the βMHC promoter becomes active in early gestation, whereas αMHC turns on postnatally, mutant Shp2 expression occurs during different time windows in each line. Embryonic expression of Q79R resulted in altered cardiomyocyte cell proliferation, ventricular non-compaction, and ventricular septal defects. In contrast, postnatal expression of Q79R mutant had no apparent effect. Erk activation was increased in mutant hearts, and decreasing expression of Erk1 or 2 (by crossing to Erk1+/– or Erk2+/– mice) ablated the cardiac defects caused by embryonic Q79R expression. It is difficult to reconcile these findings with our observations that myocardial expression of the even more potently activated Ptpn11 mutant DY (see above), or myocardial-specific Nf1 deletion [45], has no phenotypic consequences, whereas endothelialdriven expression phenocopies all aspects of the mouse NS phenotype (including ventricular thinning). Conceivably, the total level of myocardial SHP2 activity in the transgenic model (caused by over-expression of the protein plus its increased activity) is greater than that in mice with myocardial-specific DY expression. Indeed, there was a Q79R dosage-dependent difference in penetrance of the myocardial phenotype in different transgenic lines generated by Nakamura et al. Alternatively, owing to position effects, the Q79R allele could have been expressed gratuitously in the developing endocardial cells in the transgenic mice.

Related Animal Models

Jacks’ group has generated inducible knock-in mice expressing the strongly activated KrasG12D mutant [74]. Although more potently activated than the KRAS alleles found in NS patients, the effects of G12D are likely to be qualitatively

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similar (though more severe). Global G12D expression causes early embryonic lethality due to trophoblast defects. The early lethality can be bypassed by evoking expression in the epiblast only, using Mox2-Cre mice, but mutant embryos then succumb to cardiovascular defects quite similar to those seen in DG/DG (and Nf1–/–) mice [75]. Mutant embryos also demonstrate hematopoietic abnormalities and a profound defect in lung branching morphogenesis, associated with upregulation of Sprouty-2, a member of the Spry family (Sprouty 1–4) of poorly understood feedback inhibitors of Ras/Erk pathway. Although defective lung branching morphogenesis is not characteristic of NCFC syndromes, these findings nevertheless suggest that Spry proteins and their relatives, the Spreds (Spred1–3) may be important modifiers of hyper-activated RAS/ERK pathway components, and thus may help explain phenotypic variation in the NCFC. Consistent with this notion, Spred1 and Spred2 knockout mice exhibit features similar to NS and other NCFC [76]. Indeed, SPRED1 should probably be added to the list of NCFC genes, given the recent finding of SPRED1 mutations in a group of patients with a neurofibromatosis-like syndrome [29].

Conclusions and Perspectives

Existing mouse models should provide fertile ground for future examination of the pathophysiological basis of NS and NF, as they appear to reproduce many important syndromic features. The challenge now is to explore the cellular and molecular basis of these defects in detail by determining the precise upstream signaling pathways affected and how aberrant signaling by these pathways is translated into abnormal morphogenesis. Further analysis of more genetically tractable organisms such as the fly and fish may provide valuable insights into such pathways, and act as hypothesis generators for studies in more complex systems. It will also be important to define the precise temporal windows during which disease-associated mutants act, as these may suggest (or certainly impose limits on) timing for potential therapeutic interventions. Finally, mouse models for other NS genes, as well as for other NCFC syndromes, will be critical if we are to elucidate the molecular basis for the similarities and differences between these fascinating syndromes.

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68 Kratz CP, Niemeyer CM, Castleberry RP, Cetin M, Bergstrasser E, et al: The mutational spectrum of PTPN11 in juvenile myelomonocytic leukemia and Noonan syndrome/myeloproliferative disease. Blood 2005;106: 2183–2185. 69 Gauthier AS, Furstoss O, Araki T, Chan R, Neel BG, Kaplan DR, Miller FD: Control of CNS cell-fate decisions by SHP-2 and its dysregulation in Noonan syndrome. Neuron 2007;54: 245–262. 70 Murakami S, Kan M, McKeehan WL, de Crombrugghe B: Up-regulation of the chondrogenic Sox9 gene by fibroblast growth factors is mediated by the mitogen-activated protein kinase pathway. Proc Natl Acad Sci USA 2000;97:1113–1118. 71 Akiyama H, Chaboissier MC, Behringer RR, Rowitch DH, Schedl A, Epstein JA, de Crombrugghe B: Essential role of Sox9 in the pathway that controls formation of cardiac valves and septa. Proc Natl Acad Sci USA 2004;101: 6502–6507. 72 Bi W, Deng JM, Zhang Z, Behringer RR, de Crombrugghe B: Sox9 is required for cartilage formation. Nat Genet 1999;22:85–89. 73 Kulyk WM, Franklin JL, Hoffman LM: Sox9 expression during chondrogenesis in micromass cultures of embryonic limb mesenchyme. Exp Cell Res 2000;255:327–332. 74 Tuveson DA, Shaw AT, Willis NA, Silver DP, Jackson EL, et al: Endogenous oncogenic K-ras(G12D) stimulates proliferation and widespread neoplastic and developmental defects. Cancer Cell 2004;5:375–387. 75 Shaw AT, Meissner A, Dowdle JA, Crowley D, Magendantz M, et al: Sprouty-2 regulates oncogenic K-ras in lung development and tumorigenesis. Genes Dev 2007;21:694–707. 76 Bundschu K, Walter U, Schuh K: Getting a first clue about SPRED functions. Bioessays 2007;29:897–907.

Benjamin G. Neel Ontario Cancer Institute, University Health Network 101 College Street, TMDT8–355 Toronto, ON, M5G1L7 (Canada) Tel. +1 416 581 7757, Fax +1 416 581 7698, E-Mail [email protected]

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Towards a Treatment for RAS-MAPK Pathway Disorders V.A. Joshia,b  A.E. Robertsa,c  R. Kucherlapatia aHarvard

Medical School – Partners HealthCare Center for Genetics and Genomics, Boston, Mass., of Pathology, Massachusetts General Hospital, Boston, Mass., cDepartment of Cardiology, Children’s Hospital Boston, Boston, Mass., USA bDepartment

Abstract The molecular pathogenesis of Noonan, Costello, and Cardio-facio-cutaneous syndromes has recently been described. All of these disorders result from an abnormal activation of the RAS-MAPK pathway. RAS-MAPK pathway activation is a common occurrence in tumor cells, and much effort has been made to develop inhibitors of this pathway to treat cancer. This chapter will describe several different strategies of RAS-MAPK pathway inhibition that are being evaluated in clinical trials. The potential application of these inhibitors to individuals with RAS-MAPK developmental disorders will also be discussed. Copyright © 2009 S. Karger AG, Basel

Human genetic disorders can be classified into monogenic and complex disorders. Monogenic disorders, in turn, are classified based upon their inheritance patterns: autosomal dominant, autosomal recessive and sex-linked being the most common. A large number of monogenic disorders have been described (OMIM). In most cases of autosomal recessive disorders, both parents are carriers of a recessive allele. In autosomal dominant disorders, one of the parents may be affected and pass on the dominant allele to their offspring. Alternatively, these disorders may also result from spontaneous mutations in the germ

cells. Noonan syndrome (NS; MIM 163950) and LEOPARD syndrome (LS; MIM 151100) are inherited in an autosomal dominant fashion and individuals with a mutation in one of several genes (see below) are affected. Cardio-faciocutaneous syndrome (CFC; MIM 115150) and Costello syndrome (CS; MIM 218040), by contrast, typically occur sporadically in families. Newborns with one of these syndromes can often be diagnosed based on the manifestations of the syndrome. Accurate diagnosis helps with prediction of the course and the severity of the disorders and thus can assist with management of the patients. Although many of the later onset symptoms can be predicted based on the diagnosis and examination of the nature of the mutations in the causal gene, there are no curative therapies for these disorders. The identification of several genes involved in these disorders and an understanding of the pathways in which these genes function now provides a possible approach for developing therapeutics. Because of the relative rarity of these monogenic disorders, pharmaceutical companies, who have the expertise to develop new drug entities and test them in patients, are unlikely to be interested in developing

drug based therapies. However, these syndromes have unique properties that may allow them to take advantage of drugs that are already in development by several major pharmaceutical companies. The majority of NS, LS, CFC and CS result from dominant mutations in components of the RAS-MAPK pathway. RAS is an important upstream member of this pathway and specific activating mutations in the RAS genes are detected in more than 30% of all solid tumors [1]. Activating mutations in the RAS genes lead to a cascade of events, among which is the activation of ERK and MEK. Because of the involvement of RAS mutations in a large proportion of human cancers, many pharmaceutical companies are developing inhibitors that aim to block the activation of the RAS-MAPK signaling pathway with the expectation that such inhibition would halt tumor progression and reverse the growth of the tumor. We will consider the possibility of developing targeted therapies for NS, LS, CFC, and CS and what type of drug development pathway may be undertaken.

Clinical Features and Management of RAS-MAPK Developmental Disorders

Individuals with NS, LS, CFC, and CS share clinical features that result from defects in the RASMAPK signaling pathway. NS is characterized by variable developmental delay, short stature, webbed neck, pectus abnormalities, coagulation defects, and cryptorchidism, with characteristic facial features. Congenital heart defects, primarily pulmonary valve stenosis and hypertrophic cardiomyopathy, which affect 20–50% and 20–30% of individuals, respectively, are the primary cause of morbidity and mortality. Juvenile myelomonocytic leukemia (JMML) and acute lymphoblastic leukemia (ALL) have been associated with NS [2]. In one longitudinal study of the natural history of NS, only one case of breast cancer was reported,

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with no additional cases of any cancer [3]. Based on the current understanding of the molecular pathogenesis of NS, this is somewhat surprising. However, the mean age of the cohort examined was 25.3 years, with a mean follow-up of 12.02 years, and PTPN11 mutations were only identified in 35% of individuals. It is possible, therefore, that this cohort is not completely representative of the adult NS population. Additional longitudinal studies should help clarify the risk of neoplasia associated with NS. NS can be inherited in an autosomal dominant fashion, but sporadic cases are common. It is estimated that as many as 1/1,000 individuals are affected with NS [4, 5]. LS, CFC, and CS share with NS similar facial features, congenital heart defects, growth retardation, and developmental delay or mental retardation. LEOPARD (Lentigines, Electrocardiographic conduction defects, Ocular hypertelorism, Pulmonary stenosis, Abnormalities of the genitals, Retarded growth resulting in short stature, and Deafness) is an allelic disorder of NS. The multiple lentigines and deafness observed in these individuals are characteristic and unique. CFC is distinctive in that individuals typically have hair and skin abnormalities such as sparse curly hair, absent eyelashes, patchy alopecia, ichthyosis, hyperkeratosis, and ulerythema ophryogenes (absent eyebrows with hyperkeratosis). Individuals with CFC and CS typically have more significant cognitive delays than those with LS or NS. One of the distinctive features of CS is the risk of neoplasia. Papillomas, benign tumors, frequently develop around the mouth and anus. The most common malignant tumor is rhabdomyosarcoma, although neuroblastoma and bladder cancer have also been observed in multiple individuals [6]. Because the cancer risk is as high as 17%, a screening protocol has been recommended for individuals with CS consisting of ultrasound examination of the abdomen and pelvis, urine catecholamine metabolite analysis, and urinalysis [7].

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The medical and developmental issues seen in these disorders are treated symptomatically as there is no curative treatment for the underlying molecular perturbation of the RAS-MAPK pathway. The treatment of cardiac manifestations is largely the same as in the general population. The pulmonary valve stenosis varies in severity from mild, requiring no intervention, to severe, requiring surgery. Dysplastic pulmonary valves respond less often to balloon dilation than pulmonary stenosis without valve dysplasia [8]. Hypertrophic cardiomyopathy may be treated pharmacologically with beta blockers or calcium channel blockers, may require myomectomy or, less commonly, progress to the point of requiring heart transplant. Arrhythmias (usually supraventricular or paroxysmal tachycardia, most distinctively ectopic atrial tachycardia) are most common in CS though reported in all three disorders [9]. Anyone with a disorder of the RAS-MAPK pathway should be followed by a cardiologist throughout both childhood and adulthood. Certain congenital heart defects require antibiotic prophylaxis for subacute bacterial endocarditis. Feeding problems are common and a majority of children require treatment for gastroesophageal reflux. Most children with CFC and CS require nasogastric or gastrostomy feeding and Nissen fundoplication may be required. These interventions are less commonly indicated in NS. Short stature is prevalent in these disorders and growth hormone deficiency is documented, though not in all children with short stature. It appears that children with NS and a PTPN11 mutation have relative growth hormone resistance and there is some thought that augmentation of growth hormone replacement with IGF-1 may yield a better linear growth response [10]. Growth velocity appears to increase during the first three years of treatment with the greatest increase in growth velocity in the first year [11]. Growth hormone deficiency may present as hypoglycemic seizure in CS [12]. Hypertrophic cardiomyopathy

Towards a Treatment for RAS-MAPK Pathway Disorders

is considered a relative contraindication by some to growth hormone therapy though no impact on ventricular wall size has been documented. A variety of skeletal issues have been observed. Scoliosis and kyphoscoliosis most often respond to bracing, though surgical intervention with rod placement may be required. The ulnar deviation of the wrists and fingers in CS is treated with bracing, physical therapy, and occupational therapy. Large joint extension might be limited and requires physical therapy and occasionally surgical tendon lengthening. Only very rarely does the pectus carinatum or excavatum of NS require surgical correction. Seizures can occur in any of the RAS-MAPK disorders and are treated as in the general population though hypoglycemia, low serum cortisone, and hydrocephalus need to be ruled out as potential causes [13]. Symptomatic Arnold Chiari malformation has been reported in NS and often responds to decompression surgery. Hydrocephalus may require shunting. Malignant hyperthermia has been reported in NS, though it is not clear if these cases are coincidental or truly related to the NS diagnosis. It appears that the risk is greatest if there is myopathy or an elevated serum CK level and thus dantrolene prophylaxis is suggested during surgery when CK levels are elevated or if there is a clinical suspicion of malignant hyperthermia or myopathy [14]. Early developmental milestones are often delayed; this may be more profound in CFC and CS. Gross and fine motor delays are often attributable to low muscle tone and respond well to early intervention with occupational and physical therapy. Articulation difficulties respond well to hearing aids when indicated and speech therapy. In general, IQ falls within the normal range for children with NS and in the mild to moderate mental retardation range for children with CFC and CS. As there is no specific cognitive profile recognized in any of these disorders, children are best served by having regular, detailed neurocognitive evaluations with an individualized education plan.

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Maximization of vision and hearing will help children reach their full developmental potential. Visual problems including myopia, hyperopia, strabismus, and astigmatism can be treated as in the general population [15]. Recurrent ear infections can be complicated by middle ear effusion causing conductive hearing loss and may require placement of pressure equalization tubes. The xerosis and pruritis of CFC syndrome can be treated with an increase in ambient humidity and the use of hydrating lotions [16]. The papillomata of CS can cause irritation or inflammation and can be removed surgically or, in the facial region, treated with cryotherapy. Local medications for keratosis pilaris atrophicans faciei is not usually effective [14]. Skin, particularly in areas of lymphedema, can be prone to infection and is treated with antibiotics as indicated. The multiple nevi often seen in NS and CFC are not thought to be at increased risk for malignant transformation though periodic dermatologic evaluation may be indicated until the natural history is more definitively understood. Children with NS are at increased risk for a bleeding disorder. Platelet aggregation abnormalities, factor deficiencies (most commonly factors V, VIII, XI, and XII, and Protein C), von Willebrand disease, and thrombocytopenia have all been reported. Aspirin and aspirin-containing medications should be avoided. The need for surgical pre-treatment should be assessed by a hematologist. Lymphatic abnormalities including peripheral edema, pulmonary lymphangiectasia, and intestinal lymphangiectasia are reported in a minority of cases but when present can cause significant morbidity and mortality. Support stockings and careful foot hygiene are important for lower extremity edema. Chylothorax may require surgical drainage and/or respond to low-fat diet. Treatment with prednisone has also been reported to be effective [14]. Cryptorchidism and genitourinary reflux are treated as in the general population.

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Molecular Pathogenesis of RAS-MAPK Developmental Disorders

Over the past several years, the molecular defect in each of these four disorders has been at least partially revealed. Mutations in PTPN11 have been identified in 50% of cases of NS and the majority of LS, but were not found in individuals with CFC or CS [17–20]. This discovery set off a flurry of studies with aims to identify other NS genes and the genes responsible for CFC and CS. Using a candidate gene approach, it was shown that mutations in KRAS, SOS1, and RAF1 cause 1, 10, and 3–17% of cases of NS respectively [21–26]. A fraction of cases of LS also have mutations in RAF1 [22]. Mutations in BRAF, KRAS, MEK1, and MEK2 cause 37–78, 7, 9, and 4%, respectively, of cases of CFC [27, 28]. Mutations in HRAS cause up to 92% of CS [29]. Knowledge of the underlying molecular pathogenesis of these disorders could help guide the selection and development of therapies that can be used to treat affected individuals. All of these genes encode proteins that are components of the RAS-MAPK signal transduction pathway. This pathway is responsible for the communication of extracellular growth signals to the nucleus through a complex phosphorylation cascade. Activation of this pathway initiates transcription of genes involved in cell proliferation, inhibition of apoptosis, and metastasis. It has been recognized for some time that upregulation of this pathway is a common feature of tumorigenesis. Somatic activating BRAF mutations, for example, are present in 66% of malignant melanomas and a significant fraction of other tumor types. Activating KRAS mutations are present in up to 21% of all human tumors [30, 31]. Mutations in the eight genes implicated in the RAS-MAPK developmental disorders also typically activate the pathway, although often to a lesser extent than observed by somatic changes. NS-associated mutations in PTPN11 typically lie at the interface of the N-SH2 and PTP interacting

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surfaces, an interaction which is critical for the basal inactive state of the protein [32]. Disruption of the autoinhibited state leads to increased phosphatase activity and activation of the pathway. Similarly, NS mutant alleles of SOS1 show increased ERK and RAS activation [23, 24]. The majority of CS causing HRAS mutations thus far reported have also been identified as somatic mutations in tumors and are known to be activating [29]. The MEK mutations identified in individuals with CFC also stimulate ERK phosphorylation [28]. Exceptions to the gain-of-function mechanism of disease pathogenesis have been described. The majority of the mutations in PTPN11 that cause LS do not show an increase in protein tyrosine phosphatase activity in vitro [33]. RAF1 mutations have also been reported to cause LS [22]. Some RAF1 mutations showed an increase in kinase activity with a consummate increase in ERK activation, whereas others had reduced or absent kinase activity and a decrease in ERK activation [21, 22]. LS-associated RAF1 mutations have increased kinase activity as compared to wild type [22]. In addition, unlike LS-associated mutations in PTPN11 which do not show overlap with NS-associated mutations, RAF1 mutations have been associated with both NS and LS. Therefore, strict loss or gain of function as a mechanism of either NS or LS clinical features may not be the case. BRAF mutations in CFC cluster in either the cysteine-rich domain of the conserved region 1 or in the protein kinase domain [28]. This is in contrast to that observed in most tumors, where the majority of mutations affect a small number of codons; the V600E mutation has been identified in up to 19% of tumors examined and is found in up to 12% of tumors of the large intestine [31]. The kinase activity of CFC-associated BRAF mutants was, in some cases, as activating as the V600E mutant, whereas other CFC mutations impaired kinase activity [27, 28]. This is similar to what has been observed with some somatic mutations [34]. Likewise, activated and impaired

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kinase activity were observed with two different CFC-associated KRAS mutations [27]. While it is possible that these results can be derived from technical differences in the assays used, it is quite possible that both loss and gain of function result in similar RAS-MAPK pathway signaling defects. This is very important to keep in mind as consideration is made for targeted treatment for these disorders.

RAS-MAPK Pathway Inhibitors in Development

Because of their prominent role in tumorigenesis, components of the RAS-MAPK cascade have become attractive targets for inhibition in the treatment of cancer. Inhibition strategies fall into several different categories, including antibody or small molecule inhibitors that target receptors, inhibitors that block post-translational modifications, and small molecule inhibitors of protein kinases. Antibody and small molecule inhibitors of receptors have been well described and are currently in use or under evaluation for the treatment of many solid tumors. However, the targets of these inhibitors lie upstream of the proteins affected by RAS-MAPK developmental disorders, and may therefore not be the best candidates for the treatment of these disorders. Many different inhibitors and inhibition strategies are in various stages of development; only the most advanced inhibitors will be discussed here (table 1, fig. 1, for a comprehensive review see [35]). RAF proteins are serine/threonine kinases that directly phosphorylate MEK1 and MEK2 kinases. The RAF family is comprised of A-RAF, BRAF, and RAF1 (C-RAF). A-RAF mutations have not been found in individuals with NS, and only rarely in tumor cells [21, 31]. Whereas both RAF1 and BRAF are commonly mutated in individuals with NS or CFC, respectively, in tumor cells, RAF1 is rarely mutated (