The Genetics of Renal Disease

The Genetics of Renal Disease Frances Flinter Eamonn Maher Anand Saggar-Malik Editors OXFORD UNIVERSITY PRESS OXFORD...

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The Genetics of Renal Disease

Frances Flinter Eamonn Maher Anand Saggar-Malik Editors

OXFORD UNIVERSITY PRESS

OXFORD MEDICAL PUBLICATIONS

The Genetics of Renal Disease

OXFORD MONOGRAPHS ON MEDICAL GENETICS General Editors ARNO G. MOTULSKY MARTIN BOBROW PETER S. HARPER CHARLES SCRIVER CHARLES J. EPSTEIN JUDITH G. HALL

16. 18. 21. 22. 24. 25. 26. 27. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49.

C. R. Scriver and B. Child: Garrod’s inborn factors in disease M. Baraitser: The genetics of neurological disorders D. Warburton, J. Byrne, and N. Canki: Chromosome anomalies and prenatal development: an atlas J. J. Nora, K. Berg, and A. H. Nora: Cardiovascular diseases: genetics, epidemiology, and prevention A. E. H. Emery: Duchenne muscular dystrophy, second edition E. G. D. Tuddenham and D. N. Cooper: The molecular genetics of haemostasis and its inherited disorders A. Boué: Foetalmedicine R. E. Stevenson, J. G. Hall, and R. M. Goodman: Human malformations A. S. Teebi and T. I. Farag: Genetic disorders among Arab populations M. M. Cohen, Jr.: The child with multiple birth defects W. W. Weber: Pharmacogenetics V. P. Sybert: Genetic skin disorders M. Baraitser: Genetics of neurological disorders, third edition H. Ostrer: Non-mendelian genetics in humans E. Traboulsi: Genetic diseases of the eye G. L. Semenza: Transcription factors and human disease L. Pinsky, R. P. Erickson, and R. N. Schimke: Genetic disorders of human sexual development R. E. Stevenson, C. E. Schwartz, and R. J. Schroer: X-linked mental retardation M. J. Khoury, W. Burke, and E. Thomson: Genetics and public health in the 21st century J. Weil: Psychosocial genetic counseling R. J. Gorlin, M. M. Cohen, Jr., and R. C. M. Hennekam: Syndromes of the head and neck, fourth edition M. M. Cohen, Jr., G. Neri, and R. Weksberg: Overgrowth syndromes R. A. King, J. I. Rotter, and A. G. Motulsky: The genetic basis of common diseases, second edition G. P. Bates, P. S. Harper, and L. Jones: Huntington’s Disease, third edition R. J. M. Gardner and G. R. Sutherland: Chromosome abnormalities and genetic counselling, third edition I. J. Holt: Genetics of mitochondrial disease F. Flinter, E. Maher, and A. K. Saggar-Malik: The genetics of renal disease C. J. Epstein, R. P. Erickson, and A. Wynshaw-Boris: Inborn errors of development: the molecular basis of clinical disorders of morphogenesis 50. H. V. Toriello, W. Reardon, and R. J. Gorlin: Hereditary hearing loss and its syndromes, second edition

Oxford University Press makes no representation, express or implied, that the drug dosages in this book are correct. Readers must therefore always check the product information and clinical procedures with the most up to date published product information and data sheets provided by the manufacturers and the most recent codes of conduct and safety regulations. The authors and the publishers do not accept responsibility or legal liability for any errors in the text or for the misuse or misapplication of material in this work.

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The Genetics of Renal Disease Edited by

FRANCES FLINTER Clinical Genetics Department, Guy’s Hospital St Thomas Street, London SE1 9RT, UK EAMONN MAHER Clinical Genetics Unit, Birmingham Maternity Hospital, Queen Elizabeth Medical Centre, Edgbaston, Birmingham B15 2ST, UK ANAND SAGGAR-MALIK Clinical Genetics Department, St George’s Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK

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Great Clarendon Street, Oxford OX2 6DP Oxford University Press is a department of the University of Oxford. It furthers the University’s objective of excellence in research, scholarship, and education by publishing worldwide in Oxford New York Auckland Bangkok Buenos Aires Cape Town Chennai Dar es Salaam Delhi Hong Kong Istanbul Karachi Kolkata Kuala Lumpur Madrid Melbourne Mexico City Mumbai Nairobi São Paulo Shanghai Taipei Tokyo Toronto Oxford is a registered trade mark of Oxford University Press in the UK and in certain other countries Published in the United States by Oxford University Press Inc., New York Previously published as The Genetics of Renal Tract Disorders by M. D’A Crawfurd in 1988 © Oxford University Press, 2003 The moral rights of the authors have been asserted Database right Oxford University Press (maker) First published 2003 All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, without the prior permission in writing of Oxford University Press, or as expressly permitted by law, or under terms agreed with the appropriate reprographics rights organization. Enquiries concerning reproduction outside the scope of the above should be sent to the Rights Department, Oxford University Press, at the address above You must not circulate this book in any other binding or cover and you must impose this same condition on any acquirer A catalogue record for this title is available from the British Library ISBN 0 19 263146 2 (Hbk) 10 9 8 7 6 5 4 3 2 1 Typeset by Newgen Imaging Systems (P) Ltd., Chennai, India Printed in Great Britain on acid-free paper by Biddles Ltd, Guildford and King’s Lynn

Preface The first part of the twenty-first century is widely predicted to be the ‘age of genomic medicine’. Indeed, the completion of the first draft of the human genome sequence is revolutionizing human genetics research and we can now look forward to a time when the genetic basis of most monogenic disorders is known. There have certainly been huge changes in our knowledge of medical genetics since 1988 when The Genetics of Renal Tract Disorders by M.D’A Crawfurd (Oxford Monographs on Medical Genetics 14) was first published. Genetic factors are important in the pathogenesis of a wide range of renal diseases and we felt that a more contemporary monograph on this subject would be relevant to a wide range of clinicians and other interested groups. Although Dr Crawfurd single-handedly produced an excellent text, given the extent of current knowledge, we decided that a multi-author approach would be the best option. We have been fortunate to recruit a most distinguished group of authors and wish to thank them for their excellent contributions. As with any group of authors, there are bound to be some instances of repetition. We have not attempted to eliminate all of these as different views of the same subject can be most illuminating! Inevitably there will be some topics that have not been covered in as much detail as some readers would wish, and some may have been overlooked. However we believe that our international collection of co-authors have produced a balanced and authoritative book that will address the needs of nephrologists, clinical geneticists, paediatricians, internal physicians, pathologists and urologists and their co-workers. Frances Flinter Eamonn R Maher Anand Saggar Mallick

London, UK Birmingham, UK London, UK


Contents List of contributors 1 Introduction to clinical genetics Shehla Mohammed 2 Renal function and management of renal disease A. David Makanjuola and John E. Scoble 3 Renal development Paul J. D. Winyard 4 Kidney and lower urinary tract malformations Adrian S. Woolf 5 Urinary tract defects and chromosomal disorders David J. Amor, Lachlan de Crespigny, and R. J. McKinlay Gardner 6 Dysmorphic syndromes with renal involvement Ian D. Young 7 Primary hereditary nephropathies Karl Tryggvason 8 Alport syndrome Frances Flinter 9 Autosomal dominant polycystic kidney disease Anand K. Saggar-Malik and Stefan Somlo 10 Autosomal recessive polycystic kidney disease Lisa M. Guay-Woodford 11 Cystic renal diseases Anand K. Saggar-Malik 12 Primary inherited metabolic diseases of the kidney Margaret Town and William van’t Hoff 13 Genetics of stone forming diseases Pasquale Strazzullo and Pietro Vuotto 14 Disorders of tubular transport David S. Geller, Mark A. J. Devonald, and Fiona E. Karet 15 Tuberous sclerosis complex Astrid P. Weber and Robert F. Mueller 16 Neurofibromatosis Susan M. Huson and Natalie Canham

xi 1 27 57 89 117 147 167 183 203 239 253 261 281 307 337 349

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Contents

17 The Bardet–Biedl and Alström syndromes Philip L. Beales, Patrick S. Parfrey, and Nicholas Katsanis 18 Genetic syndromes with a renal component Richard Sandford and Robin G. Woolfson 19 The genetics of glomerulonephritis and systemic disorders affecting the kidney Stephen H. Powis 20 Wilms tumour and the Wilms tumour predisposition syndromes Richard Grundy 21 Von Hippel–Lindau disease Eamonn R. Maher 22 Inherited predispositions to kidney cancer Berton Zbar, Gladys Glenn, Maria Turner, Gregor Weirich, Peter Choyke, Stephen Hewitt, Michael Nickerson, Naboru Nakaigawa, W. Marston Linehan, and Laura Schmidt 23 Gene therapy in renal disease Anand K. Saggar-Malik 24 Gene therapy for renal cancer Michael J. Gough and Richard G. Vile Index

361 399

417 455 487 497

511 515

557

List of Contributors David J. Amor, Genetic Health Services Victoria, Murdoch Childrens Research Institute, Royal Children’s Hospital, Parkville, Melbourne, Australia Philip L. Beales, Molecular Medicine Unit, Institute of Child Health, University College London, UK Natalie Canham, West Midlands Regional Genetic Service, Birmingham Women’s Hospital, Edgbaston, Birmingham, UK Lachlan de Crespigny, Murdoch Childrens Research Institute, Royal Children’s Hospital, Parkville, Melbourne, Australia Mark A. J. Devonald, Cambridge Institute for Medical Research, Addenbrooke’s Hospital, Cambridge, UK Frances Flinter, Clinical Genetics Department, Guy’s & St Thomas’ NHS Trust, London, UK R. J. McKinlay Gardner, Genetic Health Services Victoria, Murdoch Childrens Research Institute, Royal Children’s Hospital, Parkville, Melbourne, Australia David S. Geller, Yale University School of Medicine, New Haven, Connecticut, USA Michael J. Gough, Molecular Medicine Program, Mayo Clinic, Rochester, Minnesota, USA Richard Grundy, Clinical Genetics Unit, Birmingham Maternity Hospital, Queen Elizabeth Medical Centre, Edgbaston, Birmingham, UK Lisa M. Guay-Woodford, Division of Genetic and Translational Medicine, University of Alabama at Birmingham, USA Susan M. Huson, Clinical Genetics Department, Oxford Radcliffe Hospitals, Oxford, UK Fiona Karet, Cambridge Institute for Medical Research, Addenbrooke’s Hospital, Cambridge, UK Nicholas Katsanis, McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University, Baltimore, Maryland, USA Eamon R. Maher, Clinical Genetics Unit, Birmingham Maternity Hospital, Queen Elizabeth Medical Centre, Edgbaston, Birmingham, UK A. David Makanjuola, Renal Department, St Helier Hospital, Carshalton, Surrey, UK Shehla Mohammed, Clinical Genetics Department, Guy’s & St Thomas’ NHS Trust, London, UK Robert F. Mueller, Clinical Genetics Department, St James’s Hospital, Leeds, UK Patrick S. Parfrey, Patient Research Centre, Health Sciences Centre, Memorial University, St Johns, Newfoundland, Canada Stephen H. Powis, Centre for Nephrology, Royal Free & University College Medical School, London, UK

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List of Contributors

Anand K. Saggar-Malik, Clinical Genetics Department, St George’s Hospital Medical School, London, UK Richard Sandford, Cambridge Institute for Medical Research, Addenbrooke’s Hospital, Cambridge, UK John E. Scoble, Renal Department, Guy’s & St Thomas’ NHS Trust, London, UK Stefan Somlo, Yale University School of Medicine, New Haven, Connecticut, USA Pasquale Strazzullo, Department of Internal Medicine, Federico II University of Naples Medical School, Italy Margaret Town, Nephro-Urology Unit, Institute of Child Health, University College London, UK Karl Tryggvason, Division of Matrix Biology, Karolinska Institutet, Stockholm, Sweden William van’t Hoff, Nephro-Urology Unit, Institute of Child Health, University College London, UK Richard G. Vile, Molecular Medicine Program, Mayo Clinic, Rochester, Minnesota, USA Pietro Vuotto, Federico II University of Naples Medical School, Naples, Italy Astrid P. Weber, St James’s Hospital, Leeds, UK Paul J. D. Winyard, Institute of Child Health and Urology, Great Ormond Street Hospital NHS Trust, London, UK Adrian S. Woolf, Nephro-Urology Unit, Institute of Child Health, University College London, UK Robin G. Woolfson, University College London Hospitals, London, UK Ian D. Young, Clinical Genetics Department, Leicester Royal Infirmary, Leicester, UK Berton Zbar, Laboratory of Immunology, National Cancer Institute, Frederick Cancer Research & Development Center, Frederick, Maryland, USA

1 Introduction to clinical genetics Shehla Mohammed

Human chromosome constitution Introduction This introductory chapter aims to provide some basic information about human genetics, the nature of genetic counselling and to highlight some of the issues and dilemmas that may arise following the diagnosis of a genetic disorder in a family. The double stranded DNA (deoxyribonucleic acid) helix is partitioned into small units called chromosomes, located in the nucleus of each cell: the word chromosome being derived from the Greek, chroma (colour) and soma (body). The chromosomes exist in 23 pairs. Of these 46 chromosomes, the autosome pairs are numbered sequentially from 1 to 22 in descending order of size. The remaining pair, the sex chromosomes, comprises two X chromosomes in females and an X and a Y chromosome in males. The presence of paired chromosomes implies that all somatic cells contain two copies of every gene, also referred to as the diploid genome. In the gametes, each mature egg and sperm contains a single haploid set of genes, thereby ensuring that each individual inherits one chromosome of each pair from each parent. Chromosome structure Each chromosome is comprised of a densely compacted coil of DNA, bonded with protein molecules called histones (Fig. 1.1). This nucleic acid–histone complex is folded in an orderly manner, allowing large stretches of DNA to be accommodated in the finite space of the cell nucleus. Furthermore, the folding of a particular section of the DNA molecule can influence the activity of genes encoded in that region. The length of the DNA loop can vary in size from about 20,000 to 80,000 nucleotide pairs, resulting in an average human chromosome being about 100 m long. Human chromosomes are readily visualized by halting them at a specific stage of the cell cycle and using special stains, which are selectively taken up by the DNA in each chromosome. This is normally done during metaphase, a stage of cell division when the chromosomes are maximally contracted and can therefore be visualized easily by light microscopy (Fig. 1.2(A)). A specific banding pattern gives each chromosome a characteristic appearance (Fig. 1.2(B)). Euchromatin contains actively expressed genes and stains as light bands, while the darker bands comprise heterochromatin, which is believed to contain largely inactive or silent DNA. Each chromosome is comprised of two halves, called chromatids, which are separated from each other along the length of the chromosome, except at a narrowing called the centromere. The size, banding pattern, and position of the centromere identify a particular chromosome.

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Shehla Mohammed Histone “bead” DNA duplex

DNA helix

100 Å Chromatin fibre

Nucleosomes

Loop

Chromosome

Fig. 1.1 Coiling of DNA and association with histone. (Reproduced with kind permission of The Open University from Science: a Second level course in genetics—Units 1 and 2, 1990.)

A

B

1

6

13

2

3

7

8

14

15

9

10

16

4

5

11

12

17

18

19 20 21 22 X Three chromosomes 21 instead of two

Y

Fig. 1.2 A: Chromosomes under light microscopy. B: Ideogram and notation. (See Plate 1 of the Colour Plate Section at the centre of this book.)

The centromere divides each chromosome into short and long arms, the standard notation being ‘p’ for petit, and ‘q’ (the next letter in the alphabet), respectively (Fig. 1.3). The ends of chromosomes are referred to as telomeres, which have an important role in maintaining the integrity of chromosomes by effectively ‘sealing’ their ends. Chromosome notation There is an agreed nomenclature for describing the chromosomal constitution (Paris Conference 1971). The total number of chromosomes per cell is described using an Arabic number, followed by the sex chromosome constitution. If there is an extra chromosome, its number is preceded by ‘’ plus symbol. A missing chromosome is denoted by a minus ‘–’ symbol. Therefore, Down syndrome (trisomy 21 in a female) is notated as 47,XX21. A reciprocal translocation is denoted by ‘t’ followed by parentheses containing the number and arm of the chromosome involved. For example, t (13p; 18q) indicates the exchange of chromosomal material between the short arm of chromosome 13 and the long arm of chromosome 18. More complex structural rearrangements use the centromere as the point of reference and designate the terminal ends as ‘ter’; ‘pter’ being the end of the short and ‘qter’ the end of the long arm.

Introduction to clinical genetics

3

22 21 15.2 15.3 15.1 p 14 13 11.2 12 11.1 11.1 11.22 11.21 11.23

q

21.1 21.2 21.3 22 31.1 31.2 31.3 32 33 34 35 36 7

Fig. 1.3 Chromosome 7 ideogram with division into dark and light bands. (See Plate 2 of the Colour Plate Section at the centre of this book.)

Chromosome abnormalities The frequency of chromosome abnormalities at birth is approximately 9.2 per 1000 (Jacobs 1992). Approximately 50–60 per cent of all spontaneous miscarriages and 8 per cent of all clinically recognized pregnancies have an underlying chromosomal anomaly. These may be numerical or structural abnormalities (Table 1.1). The gain or loss of chromosomal material is referred to as aneuploidy. Aneuploidy of the autosomes tends to be associated with significant learning and/or physical disabilities, whereas an extra sex chromosome (e.g. 47,XXX) tends to have less effect on the phenotype. Structural abnormalities may be ‘balanced’ or ‘unbalanced’. Balanced translocation carriers are, themselves, healthy, but are at risk of having a fetus with an unbalanced chromosome constitution, which may miscarry spontaneously, or be viable, with a significant risk of physical and learning disability. Table 1.1 Numerical and structural abnormalities Numerical abnormalities Structural abnormalities Trisomy Monosomy Polyploidy

Deletions Duplications Inversions Ring chromosomes Translocations Fragile sites

Mendelian inheritance Disorders caused by a defect in a single gene, the so-called ‘Mendelian’ disorders, follow a pattern of inheritance as described by Gregor Mendel. Traits or disorders determined by

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a gene on an autosome show autosomal inheritance. Those determined by a gene on one of the sex chromosomes show sex-linked inheritance. These single, or monogenic, disorders can be subdivided into the following. Autosomal dominant (AD) inheritance In autosomal dominant (AD) inheritance, the disorder manifests in the heterozygote state; that is, in a person who has both an abnormal copy of a gene and a ‘matched’ normal homologous copy. If an individual has an AD condition, each of their offspring has a 50 per cent chance of inheriting the abnormal gene, irrespective of the sex of the parent or the child (Fig. 1.4). Examples of AD disorders include autosomal dominant polycystic kidney disease, tuberous sclerosis, and myotonic dystrophy (Table 1.2). Variable expression In AD disorders, the clinical features can show striking variation from person to person, even within the same family. In conditions that are variably expressive (e.g. neurofibromatosis),

Affected father

Mother

D d

d d

D d

d d

D d

d d

Affected son (25%)

Normal daughter (25%)

Affected daughter (25%)

Normal son (25%)

Fig. 1.4 Segregation of autosomal dominant trait. Reprinted with kind permission of the publisher from Greenwood Genetics Centre (1995). Counseling aids for geneticists, Lippincott, Williams & Wilkins, Greenwood, USA. Table 1.2 Features of autosomal dominant inheritance Multiple generations affected Both males and females are affected with equal frequency and severity Transmission of the disorder from one generation to the next Male-to-male transmission observed Each offspring of an affected parent has a 50% chance of being affected and a 50% chance of being unaffected


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it can be difficult to establish the gene carrier status in apparently unaffected individuals if no direct gene test is available. Variable penetrance Most AD disorders are completely penetrant, with all gene carriers showing some clinical features; however, some conditions show incomplete or reduced penetrance, i.e. a proportion of heterozygotes for a dominant gene may not express the trait. This explains the presence of an apparently unaffected individual who has both a parent and a child with the same AD disorder leading to the impression that a disorder can apparently ‘skip’ a generation (Fig. 1.5). Variable penetrance may also result from the now well-recognized phenomenon of germinal (or gonadal) mosaicism, where an individual’s gonadal cells alone carry a mutation, whilst the somatic cells are normal. This explains the recurrence of a genetically dominant disorder in other children of unaffected parents (e.g. tuberous sclerosis). Penetrance is usually quoted as a percentage. For example, a figure of 90 per cent means that 9 out of 10 of all heterozygotes will exhibit some features of the condition.

I:1

I:2

II:1

II:2

II:3

III:1

III:2

Fig. 1.5 Autosomal dominant pedigree, showing a non-penetrant carrier.

Autosomal recessive (AR) inheritance Autosomal recessive (AR) disorders manifest in the homozygote state, that is, in an individual who has two copies of the abnormal gene (and no normal copy). If an individual has an AR condition, both of his or her parents will be heterozygotes, that is, usually healthy carriers of a single copy of the abnormal gene. If a couple are both carriers of an autosomal recessive gene, each of their offspring has a 25 per cent chance of inheriting a ‘double dose’ of the gene and, hence, being affected with the disorder. Each of their offspring also has a 50 per cent chance of being a healthy carrier and a 25 per cent chance of not inheriting the abnormal gene at all (Fig. 1.6). AR disorders are generally severe and are found in many metabolic conditions and complex malformation syndromes. Examples of autosomal recessive disorders include autosomal recessive polycystic kidney disease, Bardet–Biedl syndrome and cystic fibrosis (Table 1.3).

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Carrier father

Carrier mother

R r

R R

R r

R r

R r

r r

Normal

Carrier

Affected

(25%)

(50%)

(25%)

Fig. 1.6 Segregation of autosomal recessive trait. Reprinted with kind permission of the publisher from Greenwood Genetics Center (1995). Counseling aids for geneticists, Lippincott, Williams & Wilkins, Greenwood, USA. Table 1.3 Features of autosomal recessive inheritance Usually affects only one generation in a single sibship with unaffected parents Males and females affected with equal frequency and severity The recurrence risk for each offspring of carrier parents is 25% Higher incidence of consanguinity Certain recessive disorders are more common in specific ethnic groups, e.g. sickle cell disease and congenital Finnish nephrotic syndrome

X-linked recessive inheritance An X-linked recessive trait is one determined by a gene on the X chromosome and usually manifests itself in males only (Table 1.4). A disorder is transmitted by healthy female heterozygotes to affected males (Fig. 1.7). In each pregnancy, a woman carrying an X-linked recessive disorder has a ● ● ● ●

25 per cent chance of having an affected son 25 per cent chance of having an unaffected son 25 per cent chance of having a carrier daughter 25 per cent chance of having a non-carrier daughter

Rarely, a woman can exhibit signs of an X-linked recessive disorder, because of ‘skewed X-inactivation’. This X-inactivation, or Lyonization, occurs during early embryonic life and


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is random, but only one X chromosome is inactivated. If this inactivation is ‘non-random’ or ‘skewed’, and the X chromosome that remains active in the cells is the one that contains the abnormal gene, carriers of X-linked recessive traits may occasionally develop signs and symptoms of the disorder. Another cause of a manifesting female carrier is the presence of a structural abnormality involving the X chromosome, for example, an X-autosome translocation (the X chromosome which remains active in most of the cells is the one which contains the abnormal gene).

Father

Carrier mother

X Xr

X Y

X X

X Y

X Xr

Normal daughter

Normal son

Carrier daughter

(25%)

(25%)

(25%)

Xr Y

Affected son (25%)

Fig. 1.7 Segregation of X-linked recessive trait. Reprinted with the kind permission of the publisher from Greenwood Genetics Center (1995). Counseling aids for geneticists, Lippincott, Williams & Wilkins, Greenwood, USA.

Table 1.4 Features of X-linked recessive disorders Usually only males affected Maternally related affected males in more than one generation* No male-to-male transmission All daughters of an affected male are obligate gene carriers (and all his sons are unaffected) * A family history may not always be positive, as X-linked disorders have a high incidence of new mutations. This pattern of inheritance, therefore, needs to be considered in the event of an isolated affected male, as female relatives are at risk of being carriers and having affected males irrespective of whom they marry (Kingston 1998).

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Shehla Mohammed

Many X-linked recessive disorders are severe or usually lethal, so that affected males may not survive to reproductive age. The recognition of gonadal mosaicism in X-linked recessive disorders (e.g. Duchenne muscular dystrophy, DMD), means there is an appreciable risk of recurrence (10 per cent) in a situation of an apparently isolated case of DMD. X-linked dominant inheritance These rare disorders are determined by genes on the X chromosome and are manifest in the heterozygote female as well as in the hemizygous male. In some disorders, the effects on males are often so profound that the disorder is usually lethal. Examples include vitamin-Dresistant rickets and Incontinentia Pigmenti (Table 1.5; Fig. 1.8).

Table 1.5 Features of X-linked dominant inheritance Transmission through many generations Both males and females affected but with an excess of affected females Females usually less severely affected than males No male-to-male transmission An affected male transmits the disorder to all his daughters An affected female transmits the disorder to half her daughters and sons

I:1

II:1

male III:1

male III:2

II:2

male III:3

I:2

II:3

III:4

II:4

II:5

III:5

Fig. 1.8 Segregation of X-linked dominant trait. Reprinted with the kind permission of the publisher from Greenwood Genetics Center (1995). Counseling aids for geneticists, Lippincott, Williams & Wilkins, Greenwood, USA.

Y-linked inheritance Traits caused by genes on the Y chromosome (also referred to as holandric inheritance) are only transmitted from father to son. Hairy ears show this pattern of inheritance. In addition, the H–Y histocompatibility antigen and genes involved in spermatogenesis are carried on the Y chromosome. No pathological traits have been described with this mode of inheritance.


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Mapping disease genes Introduction Rapid advances in molecular genetic techniques for the detection of specific DNA sequences have led to an exponential increase in the discovery of human disease genes. This is reflected in the increasing number of genes being identified: 200 in 1986, with more than 9800 currently, with the numbers increasing rapidly and a total of 30,000 genes predicted to be identified on completion of the Human Genome Mapping Project (Cooper et al. 1998, HGMD April 2000 htpp://uwcm.ac.uk/uwcm/mg/hgmdo.html). This section deals briefly with some of the methods used for identifying genes. In lowresolution mapping, a gene is assigned to a particular chromosome or a specific region on a chromosome. More detailed analysis using high-resolution techniques can then be used to isolate the gene of interest. Restriction enzymes: site-specific DNA cleavage Restriction enzymes are endonucleases produced by bacteria. These enzymes, by recognizing specific short ‘palindromic’ DNA sequences, cleave double stranded DNA in a highly specific manner. They act like ‘molecular scissors’, with each particular restriction enzyme yielding the same reproducible double stranded DNA fragments. As change of a single base pair within a recognition site will abolish cleavage, these enzymes can also be used to detect variation in DNA sequence at a specific site. Some endonuclease rare cutter sites are often associated with regions of actively transcribed genes. Recognition of such sites can, therefore, be used as pointers to the sites of these genes. Linkage analysis The first step in identifying a disease gene is usually a linkage study, whereby the chromosomal location of the gene is established. Utilizing this approach, a gene is mapped by localizing its proximity to another nearby locus on the same chromosome. Such loci are said to be ‘linked’. Using polymorphic DNA markers, multiple affected family members are typed and the inheritance of the marker traced through the family until a marker is found which co-segregates with the disease (Fig. 1.9). For conditions with an autosomal pattern of inheritance,

2 3 3 1

1 3 2 2

3 5 1 3

3 5 2 3

4 5 3 3

3 5 1 3

Fig. 1.9 Pedigree showing linkage.

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this will include markers from chromosomes 1–22 and for X-linked diseases, from the X chromosome. A DNA marker of known chromosomal localization, segregating with the disease in multiple families, will identify the location of the gene; however, sometimes it can be difficult to identify a sufficient number of affected families to provide statistical power for the analysis. Linkage analysis relies on estimating the frequency of crossing over (or recombination) that occurs between homologous chromosomes at meiosis. A DNA marker that is not close to a gene on a particular chromosome (or is on a different chromosome) will be inherited independently of the gene. The closer a marker is to the gene, the less likely it is that the two will be separated during meiosis. In practical terms, markers that show less than 5 per cent recombination with a disease gene are useful in carrier detection and prenatal diagnosis. The statistical likelihood that two markers are linked is measured as the logarithm of the odds (LOD). A LOD score of 3 is usually taken as the threshold of significance, while a LOD score below 2 suggests that the two loci are not linked. LODs between 2 and 3 are not significant. Cytogenetic abnormality Some disease genes may be localized if an affected individual has a chromosomal aberration that points to a specific gene location. The astute clinical observation of such rare situations, where an individual has been recognized to have a monogenic disorder and also found to have a constitutional chromosomal abnormality has allowed the isolation of genes for a number of single gene disorders. Although individually rare, this is true for the positional cloning of several important genes in humans, including Duchenne muscular dystrophy (DMD). The DMD gene was localized to the short arm of the X-chromosome, following identification of several females with DMD, who also had an X-autosome translocation (a chromosomal rearrangement between the short arm of the X-chromosome and an autosome). Isolation of DNA clones spanning the region of the X-chromosome involved in the rearrangement provided detailed gene mapping information and the eventual cloning of the DMD gene (the genes for neurofibromatosis and familial polyposis coli were also identified in this manner (Ledbetter et al. 1989, Herrera et al. 1986)). Similarly, the occurrence of individuals with multiple abnormalities may be reflective of several closely occurring genes being involved. It was such a contiguous gene syndrome in an individual with autosomal dominant polycystic kidney disease and tuberous sclerosis that led to the identification of one of the polycystic kidney disease genes (PKD1) on chromosome 16 (Sampson et al. 1997, the European PKD Consortium 1994). Although individuals with a chromosomal abnormality and a single gene disorder are rare, their recognition is important, as this can often provide the first clue for gene mapping studies. Low-resolution mapping Somatic cell hybridization The technique of fusing cells from two different species has long been used as a gene mapping technique. A somatic cell is any cell of an organism that is not a gamete. Fusion of human with mouse or hamster cells is facilitated in the presence of a virus or chemical which causes membrane fusion. The resulting somatic cell hybrids, when grown in continued culture, show preferential loss of human chromosomes from the cells. This loss of chromosomes is random, with no selectivity for individual chromosomes. A panel of hybrid cells can, therefore,


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be produced with different combinations of human chromosomes. By karyotypically characterizing the hybrids, it is possible to identify which human chromosomes have been retained. Genomic DNA is prepared from the hybrid panel and hybridized with the human probe to be mapped. By determining which panel shows a hybridization signal, and comparing it with the karyotype of the cell, it is possible to infer which chromosome carries the gene of interest. In situ hybridization In situ hybridization is another means of mapping a gene. In this technique, a DNA probe is labelled with a fluorochrome and hybridized directly with a metaphase chromosome spread on a microscope slide. Using chromosome-specific probes, fluorescent in situ hybridization (FISH) can distinguish each human chromosome. FISH is particularly useful for determining the respective location of two markers on the same chromosome. By using two probes labelled with different fluorochromes, the technique can be used to order probes along a chromosome when they are separated by an order of one to several megabases (Fig. 1.10). Higher resolution, down to a range of 20–30 kb, is achieved by studying interphase nuclei, as in this phase, the DNA is less tightly packed.

Fig. 1.10 Fluorescent in situ hybridization (FISH). William syndrome is associated with a submicroscopic deletion in the long arm of one homologue of chromosome 7 in band 7q11.23. The deletion is not visible on G-banded chromosome preparations, but can be detected using FISH. (See Plate 3 of the Colour Plate Section at the centre of this book.) (The pictures were provided by C. Mackie Ogilvie and P. N. Scriven, Department of Cytogenetics, Guy’s Hospital, London UK.)

High-resolution mapping Once a gene has been mapped to a particular region of a chromosome, even with closely linked markers, the gene of interest may still reside in a region covering several million base pairs. There are a number of techniques that can be used to narrow down the region of interest and include the following.

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Shehla Mohammed

Pulse field gel electrophoresis (PFGE) Pulse field gel electrophoresis (PFGE) is a technique that permits separation of large fragments of DNA. The extraction of DNA for PFGE is done in situ in the gel, as conventional DNA extraction techniques can result in random shearing and fragmentation of the DNA. Using infrequently cutting restriction enzymes, which have 6–8 bp nucleotide recognition sequences, results in relatively larger DNA fragments. Following the application of pulsed, perpendicular electrical fields to the gel and electrophoresis, long stretches of DNA (several million base pairs in length) become separated as large molecules change direction less rapidly as they move through the agarose gel. The location of a gene within this long-range physical map can be determined by constructing physical maps of large stretches of DNA. Yeast artificial chromosomes (YACs) Yeast artificial chromosomes (YACs) are a cloning vector, which also allow the cloning of large segments of DNA. By inserting a specific segment of DNA into a yeast vector, milligram amounts of DNA can be prepared that will allow detailed analysis of gene structure. YACs can carry between 50 and 1000 kb of foreign DNA. By producing a series of overlapping YAC clone sequences, distal to the starting point, a probe can be defined. High-resolution FISH A technique known as chromatin fibre-FISH, which artificially stretches the chromatin fibres, can resolve distances down to several kilobases. Positional cloning Once a gene has been localized to a particular chromosomal region, it becomes possible to identify the gene by positional cloning. Even after narrowing the candidate region to a relatively small interval of 1 cM or less, there may be several genes that reside in the area. There are several mechanisms that can be used to identify the causative gene, even in the absence of any information about the gene product or its function. This approach has been possible since the 1980s and has accelerated rapidly with the mapping tools provided by the Human Genome Project. Candidate genes The availability of sequence databases means that once a disease is mapped to a particular chromosomal region, genes located in or near the region can be scrutinized closely as potential candidate genes. Confirmation or exclusion of specific mutations in the gene can then rapidly establish its status as the causative gene. This ‘positional candidate gene’ approach is rapidly replacing the longer gene mapping strategies. The time interval between mapping a locus to a chromosomal site and identification of the disease-causing gene and pathogenic mutations is considerably shortened as a result. Similarly, searching databases for genes already cloned and that may have functions likely to be involved in the pathogenesis of the disorder under consideration, may be another way of identifying a likely candidate gene. Observing a similar disease phenotype in another species, such as the mouse, may suggest other candidate genes and allows human–mouse homologies to be investigated. Computer databases There are a number of robust computer resources for accessing information about human genes. These include the following.


13

1. Online Mendelian Inheritance in Man (OMIM) (http://www.ncbi.nlm.nih.gov/Omim): Lists and catalogues all known inherited human disorders and currently contains entries for approximately 7720 distinct genetic loci. 2. The London Dysmorphology Database (LDDB) and London Neurogenetics Database (LNDB) (http://www.lmdatabases.com): Contains information on over 3000 nonchromosomal multiple congenital anomaly syndromes with references and a photo-library on CD Rom. The companion LNDB covers clinical neurological features, including neuroradiology and neuropathology with comprehensive referencing. 3. Genome Database (GDB) (http://gdbwww.gdb.org): Contains information about genetic markers and location of known genes. 4. The Dysmorphology Human–Mouse Homology Database (DHMHD) (http://www. hgmp.mrc.ac.uk/DHMHD/dysmorph.html): Lists phenotypic features of human dysmorphic syndromes, mouse syndromes, and human cytogenetic abnormalities as well as having search facilities for human–mouse homologies. Functional cloning This approach correlates the basic biochemical defect with the causative gene. If the protein product of a gene has been characterized, the responsible gene can be identified using the cloning methods described (Fig. 1.11); however, this process has limited applicability, as there are only a minority of single-gene disorders where a biochemical defect has already been identified. Cytogenetic abnormality

Families Genetic markers

Finer genetic mapping

Physical mapping and cloning

Mutation search Transcript identification

Normal mutation

YACs and BACs Positional candidate approach

Fig. 1.11 Functional versus positional cloning. (Reprinted with the kind permission of the publisher from Mueller, R. F. and Young, I. D. (1998). Emery’s elements of medical genetics, Churchill Livingstone, London.)

Mutational analysis Once a disease gene has been identified, it must fulfil a number of criteria before it is confirmed to be responsible for that particular disorder. The first is to identify mutations in the candidate gene. A variety of mutation scanning methods have been developed (Grompe

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Shehla Mohammed

1994) and the mutation then requires confirmation by sequencing; however, any detectable mutation requires careful interpretation. For example, large deletions that remove part of the coding sequence of the gene, or sequence changes that are likely to have a functional effect by causing premature termination of translation by generating a stop codon, are likely to be particularly convincing. However, a missense mutation (which alters a single amino acid residue) may be a genuine mutation or a normal variant, the latter having no effects on protein function. The pathogenic credentials of an identified mutation are strengthened further if it is shown to co-segregate with the disease in the family, be absent in 100 ethnically matched controls and predicted to have a significant effect on the protein structure. Further support is provided if the candidate gene can be shown to be expressed in the appropriate tissues and at the relevant stages of development. In vitro restoration of the normal phenotype, by transfecting the normal gene in a cell line or the creation of a transgenic model by introducing the mutation in a homologous gene, adds further evidence for the relevance of a mutation. Genetic counselling Introduction Genetic counselling is a communication process that deals with the human problems associated with the occurrence, or risk of occurrence, of a genetic disorder in a family. It may also be defined as what happens when an individual, a couple or a family ask questions of a genetic counsellor about a medical condition or disease that is, or may be, genetic in origin (Clarke 1994). It involves an attempt to help the individual or family: ● ●

● ● ● ●

Comprehend the medical facts about a disorder. Appreciate the manner in which heredity contributes to the disorder and to the risk of recurrence. Understand the choices and options available for dealing with the risk of recurrence. Choose a course of action that seems most appropriate to them. Make the best possible adjustment to the disorder in an affected member. It has a strong communicative and supportive element.

Genetic counselling is practised within clinical genetics, a unique branch of medicine that draws upon the resources of high technology and applies them to the problems of those who are at risk of genetic disease, either in themselves or their offspring (Berry 1994). The goal of genetic counselling is to improve the quality of life of the families that seek such help (Twiss 1979). The ethos is for the professional to respond to the concerns of the clients and not to impose his/her own understanding on solutions to the problem. The manner in which the information provided might be received by a consultand and utilized in arriving at a decision, will depend on a number of factors. These include previous experiences, prior knowledge and understanding, beliefs, values and social obligations and a sense of personal identity (Clarke, A. 1997). Organization of clinical genetics services In some countries, regional genetic centres tend to be based in main teaching centres, with most holding central clinics on site, as well as peripheral or satellite clinics in other hospitals in their regions. Specialist laboratories providing molecular, cytogenetic, and biochemical services are an integral part of the provision of a clinical genetics service (Table 1.6).


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Table 1.6 The main sources of referrals to clinical genetic services Paediatricians Obstetricians General practitioners Community child health services Other hospital specialities Adoption agencies Self-referrals

Clinic staff The clinical team normally includes a number of health professionals, often with both NHS and academic affiliations. Apart from clinical geneticists and specialist registrars, genetic counsellors or associates are an increasingly important part of the clinical team. Many have a specialist nursing background, with others having a postgraduate qualification in genetic counselling in addition, or as an alternative, to a nursing qualification. Clinic environment The personal appointment, which is likely to last 40 min or longer, needs a quiet surrounding, conducive to the nature of the consultation, ensuring privacy and allowing ample time for discussions and questions. It is generally inappropriate for more than one observer to be present and then only with the permission of the family. The consultation An accurate diagnosis is the essential basis of genetic counselling. As in any field of medicine, reaching a diagnosis involves taking a history, carrying out an examination and, if appropriate, undertaking necessary investigations. Referral letters may contain varying amounts of information, which will require clarification. The standard way of recording genetic information is by constructing a family tree (pedigree). The main symbols/notations used and a sample pedigree are shown in Fig. 1.12. Specific enquiries pertaining to miscarriages, stillbirths, infant deaths, and consanguinity need to be made with sensitivity, as this information may not always be volunteered. A clinical examination may yield diagnostic information about the disorder in the individual seeking advice (e.g. neurocutaneous signs of tuberous sclerosis) and may require further investigation. An estimation of the genetic risk may be possible and is dependent on the pattern of inheritance of the disorder in the family. The availability of an accurate test and interpretation of the findings is obviously desirable when advising the family. It may be possible to quantify both the risk of developing and transmitting the disorder on the basis of the family tree, whereas specific tests may be required to identify gene carriers of other conditions (e.g. a renal ultrasound scan in an individual at risk of polycystic kidney disease). As most genetic diseases are individually rare, detailed literature searches and the use of computerized databases are often required. With the rapid pace of gene identification, conditions for which molecular tests were not possible until a few months ago may now be available. In many instances, it may be necessary to examine the affected person in the family or obtain the medical records.

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Shehla Mohammed Male

Female

Sex unknown

Pregnancy

Individual Key: example Affected individual

Colour blindness

Two or more conditions

cystic fibrosis

Multiple individuals

5

2

6

d.35 yr

d.4 mo.

d.1936

SB 30 wk

SB 34 wk

Number inside symbol

Deceased individual

Stillbirth SB 28 wk Consultand (individual seeking genetic counselling) Proband (First affected member coming to medical attention) P Obligate carrier (Will not develop condition) ∆F508

P

Record any information for example, known mutation under symbol

45,XX,t(14 : 21)

Spontaneous abortion 11wk Affected spontaneous abortion

8wk

Termination of pregnancy Affected termination of pregnancy Partnership Previous Twins

Consanguineous ?

Monozygotic Dizygotic Unknown (id i l) ( id i l) Fig. 1.12 Pedigree symbols and sample pedigree. (Reprinted with the kind permission of the publisher. From Bonthron D., FitzPatrick D., Porteus M., and Trainer A. (1998). Clinical genetics: a case-based approach, W. B. Saunders, London.)

It is prudent to establish whether there may be a current pregnancy in the individual seeking advice, or in a family member, who may be at genetic risk and to discuss the need and organization of family studies if appropriate. Who attends? The need for genetic counselling is well recognized for a number of common genetic disorders; however, the need for genetic advice in many other situations may be equally important.


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Table 1.7 Indications for referral to a genetics clinic Those who have had a child affected with a genetic condition Those who have a family history of a genetic condition Those who are, themselves, at risk of developing a genetic condition Investigation and diagnosis of possible genetic disease Diagnosis or family history of learning difficulties/physical handicap History of neonatal abnormalities or stillbirths Related couples, e.g. first cousins Investigation of recurrent pregnancy loss Advice regarding management of pregnancies at an increased genetic risk Interpretation of an abnormal prenatal result Those whose ethnic background indicates an increased chance of carrying a specific gene Pregnant couples/individuals who fall into any of the above categories

For example, a birth defect or learning difficulties can occur as part of a single gene disorder, chromosomal abnormality or from an interaction of environmental and genetic factors. Some of the indications for referral to a genetics clinic are outlined in Table 1.7. Information giving The nature and needs of the person requesting information need to be explored, so that the technical aspects of the condition can be communicated in an appropriate and sensitive manner, without resorting to jargon. Many genetic conditions, particularly those inherited in an autosomal dominant manner, show a variable phenotype. Portrayal of the variable nature of the condition is an important part of the counselling process. For example, an individual who may exhibit only mild signs of neurofibromatosis needs to be aware of the full range of potential manifestations, so that they can decide whether or not to take the 50 per cent risk of transmitting the disorder to future offspring, in whom it will be impossible to predict the severity of the disease. The diagnosis of a genetic disorder is often accompanied by feelings of grief, denial and/or anger in the individual or family and such feelings need to be acknowledged (see below). Follow-up arrangements Most genetic consultations are followed up with a summary letter, sent to the individual/ couple, as well as correspondence with the referring clinician. Any outstanding issues are covered in a follow-up appointment and, where appropriate, by a home visit. Concerns/issues for families The diagnosis of a genetic disorder can often be accompanied by considerable emotional stress and trauma for the family. The impact and long-term burden of a disorder need to be discussed, particularly the natural history and prognosis of the condition and its medical management, as well as the psychosocial implications. Inevitably, the lack of specific treatment and cure for most genetic conditions can be particularly difficult. For many families, an important question concerns the recurrence risk. If a clear diagnosis has been established, then talking to families about their risk is usually straightforward, at least mathematically (Table 1.8). For other families, however, the recurrence risk is less clear, either owing to the lack of a clear diagnosis, an unknown inheritance pattern or the unavailability of samples from other family members. In such situations, the best that may be available is an empirical risk. Many families are faced with making decisions about future reproduction, often not only in the light of worrying recurrence risks, but in the knowledge that a prenatal test may lead

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Shehla Mohammed Table 1.8 Offspring risk for carriers Inheritance

Risks to offspring

Autosomal recessive Autosomal dominant X-linked recessive

Very low (unless partner also a carrier) 50% (risk of overt disease may vary) 50% (if male offspring)

Table 1.9 Problems arising from genetic counselling Guilt and loss of self-esteem Anxiety regarding personal health Marital stress Family disruption and ill-feeling Risks to future children and grandchildren Ethical decisions

to difficult decisions about the future of the pregnancy. Prenatal diagnosis is available for an increasing number of genetic conditions; however, for many individuals, testing is not an option due to religious or personal reservations about termination of pregnancy. It is important for the clinician to remember this when dealing with families who may be considering extending their family in order to prevent assumptions being made and to ensure that all avenues are explored with the couple. For some, the answer lies in the use of other techniques such as donor insemination, ovum donation, or preimplantation genetic diagnosis (PGD). The geneticist is exposed to complex feelings of guilt and loss, which may take a variety of forms in numerous circumstances. As well as some of the predictable emotions experienced by those who pass on ‘disease causing’ genes to their offspring or experience the loss of an affected child or pregnancy, the guilt of the ‘survivor’ is often felt by family members who find that they do not carry the gene for the disorder in the family, whilst others experience a significant loss of ‘normality’ after learning they carry a ‘faulty’ gene (Table 1.9). Many families may find a specific genetic diagnosis helpful, whilst for others, a ‘label’ is associated with a negative experience of other peoples’ assumptions about what the condition will entail. This is particularly difficult for affected individuals where the clinical phenotype is widely variable or where there is a significant degree of public ignorance about the genetic condition in question. Professional concerns Although clinical genetics is often viewed as being at the forefront of a rapidly developing discipline, testing for genetic disorders brings with it many ethical dilemmas. For example, it is now possible to test a healthy individual for a late-onset incurable condition like Huntington disease. Whilst such testing can dispel uncertainty and enable individuals to make appropriate plans for the future, it can result in significant emotional turmoil and has implications not only for the individual seeking testing, but also for the wider family. These issues and those surrounding pre-symptomatic testing of children are covered in more detail in the Section on ‘Ethical issues and presymptomatic testing’. As already mentioned, the aim of genetic counselling is to provide information and to help support individuals and families to make choices. Most geneticists would view their roles as


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providers of information, rather than dispensers of definitive advice about emotive and highly personal issues (Berry 1994). This ‘non-directive’ approach can, however, prove difficult for some families who may request specific direction and advice from the counsellor. Providing an opportunity for careful reflection on different options available may be helpful in such situations. This is particularly important when there are reproductive issues to consider, with potentially irreversible decisions, such as sterilization. The inherent nature of genetic disorders means that there are often ramifications for the wider family. Varying family relationships and long-term estrangements in some families can sometimes lead to conflicting rights of individuals when access to genetic information is needed. Whilst many such situations can be resolved, it is worth remembering that while individuals have wider moral concerns for others with whom they have close ties, these are for the individual to consider, rather than for professionals to enforce (BMA 1995). In the meantime, geneticists must continue to remain sensitive to the interests of those who consult them as well as to their extended family (Berry 1994). Prenatal diagnosis Introduction Over the past three decades there has been a considerable increase in the use of prenatal diagnosis, the ability to detect abnormalities in an unborn child. Many couples facing a high genetic risk will consider embarking on a pregnancy only if a prenatal test is available. Until very recently, such couples would have had to choose between accepting the risk, having no further children or considering gamete donation or adoption. The availability of a prenatal test means that couples facing the risk of having a child with a genetic disorder have the option of preparing for the birth of an affected child and, if appropriate, opt for in utero treatment. It also enables them, if they wish, to choose termination of pregnancy. Equally, prenatal testing may be appropriate for low-risk pregnancies in the presence of parental anxiety. Apart from pregnancies that are at known genetic risk, some may be identified following the screening, which is often offered as a routine part of antenatal care. In many situations, as this is accepted with a view to ‘reassurance’ and with the expectation of a normal result, the detection of fetal abnormality can be particularly difficult to cope with. Many of the common developmental renal abnormalities may be detected in this manner. Specific fetal renal abnormalities are mentioned elsewhere, so that the general principles of prenatal diagnosis are outlined below. Indications for prenatal diagnosis There are approximately 100 genetic disorders with renal involvement (Winter and Baraitser 2001), which are not detailed here, but many are mentioned in relevant chapters. Broadly speaking, these comprise chromosomal disorders (e.g. trisomy 13, 18, and 21, Turner syndrome), Mendelian conditions (Alport disease, polycystic kidney disease, and tuberous sclerosis), and non-Mendelian or developmental disorders (Lowe syndrome, renal agenesis, Prune-Belly syndrome, and VATER association). Indications for prenatal diagnosis are summarized in Table 1.10 Methods for prenatal diagnosis A variety of techniques exist by which prenatal diagnosis can be achieved (Table 1.11).

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Shehla Mohammed Table 1.10 Indications for prenatal diagnosis Advanced maternal age Previous history of chromosomal abnormality Single gene disorder Neural tube defect Congenital structural abnormalities Other high risk factors: consanguinity, maternal diabetes

Table 1.11 Non-invasive techniques Technique type

Timing

Miscarriage risk (%)

Detection of abnormality

Non-invasive techniques Ultrasonography Nuchal scan

2nd trimester 11 weeks

No risk

Structural abnormalities Marker for chromosomal/ structural abnormality

Invasive techniques Amniocentesis

14–19 weeks

1

Chorion biopsy

11–12 weeks

2

Cordocentesis

2nd trimester

3

Chromosomal, biochemical disorders DNA, enzyme analysis, chromosomal disorders DNA, chromosomal, haematological disorders

Placental biopsy Fetoscopy

2nd trimester 2nd trimester

2–3 4–5 2–3

Fetal tissue sampling Viewing

Ultrasonography Fetal ultrasonography is an increasingly sensitive method for detecting developmental abnormalities. The sensitivity of the method is operator-dependent and increased in known highrisk cases than on routine screening (Chitty et al. 1991). Accordingly, the interpretation of an abnormality may not be straightforward and may require further detailed assessments in a specialist unit. Some of the urinary tract abnormalities detectable on scanning include: ● ● ● ● ● ● ●

Renal agenesis Hydronephrosis, secondary to pelviureteric junction obstruction or urethral valves Polycystic kidneys Dysplastic kidneys Bladder exstrophy Duplication abnormalities Horseshoe kidney

In addition to structural abnormalities, oligohydramnios or polyhydramnios may also indicate an underlying renal abnormality.


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Amniocentesis Amniocentesis is a widely available and long-established procedure, which is normally performed between 14 and 19 weeks gestation. Although a relatively safe procedure, it does carry a miscarriage risk, generally thought to be around 0.5–1 per cent. Around 20 ml of amniotic fluid is aspirated under ultrasonic control, following placental localization (Fig. 1.13). The fluid is normally clear and straw-coloured, with suspended amniotic (fetal) cells, which require culturing. A bloodstained sample indicates damage to the placenta, while discoloration may indicate fetal death. The sample is spun and duplicate cell cultures established which normally require between 2 and 3 weeks of growth to obtain sufficient cells. The main indication for amniocentesis is for chromosomal or biochemical analysis. Although generally thought to have a lower associated miscarriage risk, the timing of the test and results necessitates a second trimester termination in the event of a detectable abnormality. Probe Placenta

Uterus

Amniotic fluid Symphysis pubis

Sacrum

Cervix Vagina

Fig. 1.13 Amniocentesis.

Chorion biopsy/chorionic villus sampling Chorionic villus sampling (CVS) allows first trimester prenatal diagnosis, using fetal tissue obtained from chorionic villi. The sample is normally obtained around 11–12 weeks of pregnancy by a transcervical or transabdominal approach under ultrasonic guidance (Fig. 1.14). The maternal decidual tissue is dissected away microscopically, leaving villi, consisting of tissue that is representative of the fetus for analysis. Uncultured chorionic villi are the most suitable source of fetal DNA and can be used for molecular testing for any disorder for which such molecular testing is feasible. Chorion biopsy is also used for the detection of chromosomal disorders, either following cultured preparations, but increasingly, for rapid analysis using FISH. Prior to pregnancy Pre-implantation diagnosis Pre-implantation genetic diagnosis (PGD) is now becoming feasible. This technique relies on in vitro fertilization and embryo culture, followed by biopsy at the 6–8 cell stage of

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Shehla Mohammed Probe Speculum

Chorion

Cannula

Fig. 1.14 Chorion villus sampling.

embryonic development. Analysis of a single cell, by molecular testing or chromosomal in situ hybridization, is followed by the reimplantation of normal embryos. This method may be more acceptable to some, as it obviates the dilemmas of termination of pregnancy. However, the procedure is associated with a low rate of successful pregnancies because of the technical complexities and the need for IVF. Refinement of the techniques involved should enable PGD to be offered for many genetic disorders in the future. Prenatal treatment Experience of prenatal treatment is limited, but some cases of urinary tract obstruction are amenable to intrauterine treatment by the insertion of a vesicoamniotic shunt (Freedman et al. 1999). Termination of pregnancy The increasing availability of genetic and ultrasound technology can confer many benefits to those who face pregnancies with potential genetic problems with the relief that comes from a reassuringly normal result; however, the availability of such testing also presents couples and clinicians with often complex decisions. The detection of a serious fetal abnormality may be unexpected and the decision to terminate or continue the pregnancy is usually exceedingly difficult. Couples may experience disbelief, shock, and anger and it is, therefore, not surprising that decisions made in this frame of mind can be followed by doubts about the wisdom of such an emotive decision. After termination of pregnancy, grief and guilt may be compounded by low self-esteem and awareness of a personal contribution to the loss, particularly when there are painful reminders of other, continuing pregnancies in friends and relatives. Individuals or couples will need continuing help and support in coping with a loss that is ‘chosen’, but unavoidable. Regaining equilibrium takes time, with continuing sadness about the loss of a wanted pregnancy and anxiety about recurrence in the future (White van Mourik 1994). Professionals have a responsibility to understand the grief such pregnancy loss brings and to recognize the need for professional counselling if appropriate. Discussions about prenatal diagnosis should include preparation for an abnormal result. Although painful, it is appropriate


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to allude to the nature of termination that may be available if requested. This might entail a surgical first-trimester termination under an anaesthetic, but with a later termination, a woman would experience labour and delivery. It is worth remembering that whilst the majority of couples may opt for termination in the face of a serious abnormality, for some, continuation is a better option than termination, with nature being allowed to take its course. There is some evidence to suggest that those who choose this option have less guilt and distress than those who choose termination (Chitty et al. 1996). Ethical issues and presymptomatic testing Recent advances in medical genetics bring with them potentially huge benefits for those at risk of genetic disease, giving them greater control over their lives. All disciplines of medicine encounter ethical issues, but none more so than clinical genetics, which arise because a genetic diagnosis impinges not only on an individual, but also on the wider family. Inevitably, the benefits to one individual may generate conflict for other relatives who may be the unwilling recipients of information they had not sought. For example, an identical twin at 50 per cent risk of autosomal dominant polycystic kidney disease, by clarifying his/her own genetic status and receiving a positive result, will inevitably have established that his/her twin is also at risk, when this individual may not choose to have this information. Whilst such advances may offer more informed choices, it might bring burdens for others. The diagnosis of a lethal X-linked recessive disorder in a boy, impacts on his female relatives, who may not only have concerns for the health of their own sons, but may be faced with difficult reproductive choices. Some of the common situations where ethical issues often arise are those around informed consent, confidentiality, paternity, and pre-symptomatic testing. These are discussed briefly here, but are also the subject of many excellent reviews (Harper and Clarke 1997, Clark 1998, Marteau and Richards 1996). Informed consent There is general agreement that any form of testing or treatment in medicine is based upon the individual concerned giving free and informed consent. This necessitates the provision of accurate information, so that consent can be valid. In a research setting, the nature of consent is usually stipulated by ethical committees approving the research project. More recently, the issue of written consent has received a higher profile and it is now recommended that a consent form should be adopted as a means of formalizing and recording consent to share family information (Genetic Interest Group 1998). Initially, these will focus on molecular and other laboratory data, but eventually they may cover other aspects of the counselling session. Clinicians need to be aware that individuals may be consenting to a genetic test under pressure from partners, relatives or even other health professionals. Where there are grounds to suspect that undue pressure is being applied, there is a need to address and resolve these issues before proceeding with such testing. If it emerges that valid consent has not been given, testing should be postponed, at least until the conditions under which consent has been ‘given’ can be properly explored.

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Shehla Mohammed

Confidentiality Confidentiality has been a long-standing cornerstone of good medical practice. In clinical genetics, as testing one individual can have direct relevance to the extended family, difficulties and conflicts with regard to confidentiality often arise. As the ability to give accurate information to those seeking advice relies on an accurate diagnosis, access to medical records is often essential. Although, ideally, the release of medical information should require consent from the affected individual, in practice, this can be difficult. The affected person may have died or be incapable of giving consent. This may be compounded by the client’s wish not to disclose to other family members that they are seeking genetic advice, particularly if there is an ongoing pregnancy. At present, there is a degree of medical acceptance that information can be passed from one medical practitioner to another, but this may change in the future with the demise of medical paternalism (Berry 1994). In some situations, information may be deliberately withheld. Whilst most couples or individuals will recognize their responsibilities to other family members, others may have an immediate and understandable desire to keep the information a ‘secret’. It is unusual, however, for individuals to refuse to transmit information to relevant family members once they have had a chance to think through carefully the implications of not disclosing this information. Withholding information, or refusing to supply key biological samples, may reflect longstanding family disputes or a reluctance to exacerbate already strained relationships. In such situations, clinicians may have an obligation to override confidentiality and pass on such information. Reports from the Royal College of Physicians (1991), the Nuffield Council of Bioethics (1993), and the Genetic Interest Group (1998) suggest that there may be situations where the breaching of confidentiality is justified. Others argue against the principle of involuntary disclosure on the premise that it diminishes medical respect for genetic privacy and that the imposition of duty on families and professionals to pass on genetic information, rather than recommending it as good practice, would lend support to the concept of genetic harm (Clarke, A.J. 1997). Issues surrounding inadvertent disclosure can sometimes arise during the course of genetic testing. This is particularly likely if the laboratory needs to undertake linkage studies to answer a diagnostic or pre-symptomatic question, which will often require samples from both affected and unaffected family members. To avoid such studies revealing the genetic status of relatives who may have given samples only to assist with molecular studies, it is important to ensure that the laboratory only generates information that has been requested. With the advent of direct mutational analysis, such situations arise less often, but need to be borne in mind. Paternity issues Non-paternity is sometimes discovered during the course of a routine genetic test or is potentially recognized if there is a puzzling pedigree for a condition with a recognized inheritance pattern. This is particularly likely if linkage analysis is used which then shows lack of transmission of a genetic marker by the father. Results can sometimes still be given independent of paternity, but the reason for difficulty in interpreting results may well have to be discussed in confidence with the appropriate individual. Predictive testing Individuals at risk of a late-onset disorder, for which genetic testing is possible, now have the choice to clarify their own risk so that they can make appropriate life plans for the future. The


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extensive experience of pre-symptomatic testing for Huntington disease is now being adapted for use in other dominantly inherited late-onset disorders and testing for conditions predisposing to cancer susceptibility. As most disorders for which such testing is now possible are progressive in nature (and often untreatable), careful consideration is required of a number of issues prior to testing. Accordingly, protocols for such testing have been designed to include a minimum of two counselling sessions some months apart. Apart from covering the inheritance and medical facts about the disorder, these sessions also enable discussions about the reasons for requesting testing, ways of coping with the result, reviewing professional and social support, as well as practical implications for life insurance and careers. One important dilemma that is raised follows the request for testing by individuals who are at 25 per cent risk of being gene carriers, that is, those who have an affected grandparent, but the intervening parent remains healthy. By inference, the identification of a mutation in the individual at 25 per cent risk means that an inadvertent predictive result will have been made on the intervening parent, when they may not have wished to have this information. These situations can be resolved by appropriate discussions including all parties. If not, then a decision has to be made as to whether the right of the individual at 25 per cent risk to know their genetic status outweighs the right of the intervening parent ‘not to know’. Similar issues surround the pre-symptomatic testing of children. The genetic testing of children can be uncontroversial, for example, the identification of a particular faulty gene can lead to life-saving surgery in a child with a potentially lethal inherited condition. Equally, if a genetic test proves that a young person does not carry the disease-causing mutation for an inherited type of bowel cancer, the child is spared many distressing medical investigations. The central problems with the use of genetic testing in childhood lie in two main areas. First, in situations where the medical benefits of testing are unproven. Second, and perhaps most controversially, where the medical benefits of testing are clearly non-existent. In the latter situation, testing can only be justified if it will result in psychological or social benefit and, at the very least, do the child no harm. However, there is little evidence as to whether carrier testing is harmful or beneficial to the child or the family and research studies in this area are scarce. One central anxiety arises from potential damage caused by possible changes in the attitude of a parent towards a child who is identified as genetically ‘different’. Childhood testing raises many issues to do with parental rights and responsibilities. It also brings into question such issues as the autonomy of a child as a future adult and medical power in allowing or withholding tests requested by parents on behalf of their children (Clarke, A. 1997). References Berry, A. C. (1994). Genetic Counselling: a medical perspective. In Genetic counselling: practice and principles (ed. A. Clarke), pp. 1–28. Routledge, London and New York, NY. British Medical Association (1995). Human Genetics; choice and responsibility. Oxford University Press, Oxford. Chitty, L. S., Barnes, C. A., and Berry, A. C. (1996). Continuing with pregnancy after a diagnosis of lethal abnormality: experience of five couples and recommendations for management. British Medical Journal, 313, 478–80. Chitty, L. S., Hunt, G. H., Moore, I., and Lobb, M. O. (1991). Effectiveness of routine ultrasonography in detecting foetal structural abnormalities in a low-risk population. British Medical Journal, 303, 1165. Clarke, A. (1997). Introduction. In Culture, kinship and genes. Towards cross-cultural genetics (ed. A. Clark and E. Parsons). Macmillan Press, London and St Martins Press, New York, NY.

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Shehla Mohammed

Clarke, A. (1994). Introduction. In Genetic counselling: practice and principles (ed. A. Clarke), pp. 1–28. Routledge, London and New York, NY. Clarke, A. J. (1997). Challenges to genetic privacy. In Genetics, society and clinical practice (ed. P.S. Harper and A.E. Clarke). BIOS Scientific Publishers Ltd, Oxford. Clarke, A. J. (1998). The genetic testing of children. BIOS Scientific Publishers, Oxford. Collins, F. S. (1992). Positional cloning: let’s not call it reverse any more. Nature Genetics, 1, 3–6. Cooper, D. N., Ball, E. V., and Krawczack, M. (1998). The human gene mutation database. Nucleic Acids Research, 26, 285–7. Freedman, A. L., Johnson, M. P., Smith, C. A., Gonzales, R., and Evans, M. I. (1999). Long-term outcome in children after antenatal intervention for obstructive uropathies. Lancet, 354(9176), 374–7. Genetic Interest Group (1998). Confidentiality guidelines—Confidentiality and medical genetics. Grompe, M. (1994). The rapid detection of unknown mutations in nucleic acids. Nature Genetics, 5, 111–7. Harper, P. S. and Clarke, A. J. (1997). Genetics, Society and Clinical Practice. BIOS Scientific Publishers, Oxford. Herrera, L., Kakati, S., Gibas, L., Pietzrak, E., and Sandberg, A. A. (1986). Gardner syndrome in a man with an interstitial deletion of 5q. American Journal of Human Genetics, 25(3), 473–6. Jacobs, P. A., Browne, C., Gregson, N., Joyce, C., and White, H. (1992). Estimates of the frequency of chromosome abnormalities detectable using moderate levels of banding. Journal of Medical Genetics, 29, 103–8. Kessler, S. (1990). Current psychological issues in genetic counselling. Journal of Psychosomatic Obstetrics and Gynaecology, 11, 5–13. Ledbetter, D. H., Rich, D. C., O’Connell, P., Leppert, M., and Carey, J. C. (1989). Precise localization of NF1 to 17q11.2 by balanced translocation. American Journal of Human Genetics, 44(1), 20–4. Marteau, T. M. and Richards, M. (1996). The troubled helix-social and psychological impact of the new human genetics. Cambridge University Press, Cambridge. Paris Conference (1971). Birth defects original article series (1972) 8, 7: Paris Conference 1971: Standardization in Human Cytogenetics. National Foundation – March of Dimes, New York, NY. Sampson, J. R., Maheshwar, M. M., Aspinall, R., Cheadle, J. P., Ravine, D., Roy, S., et al. (1997). Renal cystic disease in tuberous sclerosis: the role of the polycystic kidney disease 1 gene. American Journal of Human Genetics, 61, 843–51. The European Polycystic Kidney Disease Consortium (1994). The PKD1 gene encodes a 14 kb transcript and lies within a duplicated region of chromosome 16. Cell, 77, 881–94. Twiss, S. B. (1979). Problems of social justice in applied human genetics. In Genetic counselling: facts, values and norms (ed. A. M. Capron, R. F. Laper, T. M. Murray, T. M. Powledge, and D. Bergsma). Alan R. Liss, Inc., New York, NY. White van Mourik, M. (1994). Termination of a second trimester pregnancy for fetal abnormality— psycho-social aspects. In Genetic counselling: practice and principles (ed. A. Clarke), pp. 1–28. Routledge, London and New York. Winter, R. and Baraitser, M. (2001). Oxford Medical Database. Version 3.0. www.oup.com/uk/omd

Suggested reading Abramsky, L. and Chaple, J. (1994). Prenatal diagnosis—the human side. Chapman and Hall, London. Strachan, T. and Read, A. P. (1999). Human molecular genetics. BIOS Scientific Publishers Limited, Oxford. Gardner, R. J. M. and Sutherland, G. R. (1966). Chromosome abnormalities and Genetic Counselling. Oxford University Press, Oxford. Kingston, H. M. (1996). ABC of Clinical Genetics, Chapter 1. BMJ Publishing Group, London.

2 Renal function and management of renal disease A. David Makanjuola and John E. Scoble

Introduction The kidney inspires awe in all those to attempt to understand its functions. The concepts of its actions are simple. Blood is passed through a filter which has some specific size and charge characteristics. This produces an ultrafiltrate which is then reabsorbed. Figure 2.1, shows a physician and his glomerular filtrate to his left. It can immediately be seen that with normal function we each filter more than twice our body weight each day. In fact, the physician holds the urine volume which is less than 2 per cent of the amount he has filtered in a 24-h period. Conceptually, it has been easy to imagine a pump such as the heart involved in work of expelling a certain volume every minute. However, because the kidney is silent in its functions it is more difficult to imagine the ‘work’ involved in the reabsorption of 178 kg of fluid

Fig. 2.1 A physician and his glomerular filtrate. This shows a renal physician on the right with peritoneal dialysis fluid of 180 litres which is equal to his glomerular filtrate, and he holds in his hands 2 litres which represents his urinary output. This shows both the volume filtered and the work performed by the kidney each day and the small proportion which results in urine production.

A. David Makanjuola and John E. Scoble

Kidney function (%)

28

100 90 80 70 60 50 40 30 20 10 0 0

20

40 60 Time (min)

80

100

Fig. 2.2 Renal function and symptoms. Patients rarely exhibit symptoms until they enter the grey zone below a GFR of 20 ml/min but as soon as the GFR passes below 10 ml/min it is potentially lethal.

under normal conditions. However, the vast volumes that are filtered and reabsorbed suggest the important impact that small changes in the reabsorptive processes can have in producing pathophysiological changes. At the same time, it can be appreciated that significant changes in excretory function can occur without a change in urine volume or significant symptoms. Symptoms rarely occur until the overall renal function has decreased to less than 20 per cent whereas this fall in function for the heart would result in severe and disabling symptoms, Fig. 2.2. This results in many patients with progressive renal disease of whatever cause presenting as a medical emergency with less than 10 per cent of normal renal function. In practice, the planning of renal replacement therapy in many patients with genetic renal disease occurs when their glomerular filtration rate does not give them any symptoms. Renal physiology The kidney provides a highly efficient mechanism for waste product excretion which cannot be reproduced by even the most modern dialysis mechanisms. This is because it filters an enormous volume of plasma per day and has exquisitely effective mechanisms for the reabsorption of all but the smallest amount. Figures 2.3–2.8 show that the functions of the kidney can be divided into simple parameters. The proximal tubule is an isotonic reabsorptive segment with the consequent sodium transport process and water permeability. However, the thick ascending limb is a sodium reabsorbing segment without water permeability. As will become clear with the subsequent chapters, these specific mechanisms are the consequence of specific transport proteins which have biological variation. The glomerular filtrate is the consequence of the hydrostatic forces within the glomerulus which is a hemi-arteriole and the physical characteristics of the glomerular basement membrane. In certain pathophysiological situations these forces can be altered by extrinsic factors. For instance, in the renovascular disease associated with neurofibromatosis, the decreased afferent arteriolar pressure can be offset by increased angiotensin II production and increased efferent arteriolar tone. However, once the filtrate is formed it is under the control of the tubular reabsorptive mechanisms.

Renal function and management of renal disease

H2O permeable H2O permeable + Na transporting H2O permeable + Na transporting +/– H2O permeable + Na transporting

29

Fig. 2.3 The renal tubule and the function of the individual segments with the hypertonicity of the inner medulla generated by the thick ascending limb.

H+ Na+ Gl Na+

ATP K+ Na+

Phos Na+ AA Na+

Cl– HCO3–

H2O

Fig. 2.4 The proximal tubular cell and its transport mechanisms.

Loop diuretics

ATP K+ Na+

Na+ K+ 2Cl– Cl– K+

Fig. 2.5 The thick ascending limb and its transport mechanisms.

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Thiazide diuretics

ATP K+ Na+

Na+ Cl– Cl–

Fig. 2.6 The early distal tubule and its transport mechanisms.

ATP K+

Na+

Aldost.

Na+ K+

Principal cell

Cl– Band 3 protein

ATP

ATP K+ Na+

H+ Intercalated cell

Fig. 2.7 The distal tubule and its transport mechanisms.

Although the array of reabsorptive mechanisms is enormous when individually analysed the number of processes in each individual segment of the renal tubule is small as shown in Fig. 2.2. Figures 2.3–2.8 show the mechanisms which are important in the individual segments of the renal tubule.


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ADH cAMP

H2O

Fig. 2.8 The collecting tubule and its transport mechanisms.

The proximal tubule is responsible for bulk reclamation of the ultrafiltrate. This is achieved by sodium transport with anion transport of bicarbonate (Fig. 2.4). Also obvious from Fig. 2.4 is the fact that all the substances which require absolute reabsorption under normal circumstances have sodium co-transporters. Thus glucose and amino acid reabsorption is linked to sodium reabsorption. Although previously thought to depend on paracellular water transport, it is now clear that water movement is through the aquaporin 1 (CHIP) water channels through the proximal tubular cell (Yamamoto and Sasaki 1998). It is important to note that at the end of the proximal tubule the tubular osmolality is unaltered even though twothirds of the filtered fluid volume has been reabsorbed. In spite of bicarbonate reabsorption there is little alteration in tubular pH. Thus the proximal tubule reabsorbs the precious commodities and two-thirds of the salt and water without altering tubular fluid osmolality or pH. At the end of the proximal tubule, the tubular fluid osmolality is unchanged at approximately 300 mosm/kg. The tubular fluid pH is also unchanged. Thus bulk fluid reclamation has occurred with specific reabsorption of essential moieties. Tubular fluid then descends to the thin ascending limb to the centre of the osmotic gradient and the tip of the renal medulla. In humans, the interstitial osmolality reaches 1200 mosm/kg we believe. As can be seen from Fig. 2.3 the interstitial osmolality increases with the nearing of the tip of the medulla. In the thin ascending limb, there are no active transport mechanisms but at the same time this segment is entirely permeable to both salt and water. However, as can be seen from Fig. 2.5 in the thick ascending limb there are active reabsorptive mechanisms. At this point there is the NaK/2Cl co-transporter. This segment is also known as the diluting segment. Because, as is shown in Fig. 2.4, in this segment the tubule is impermeable to water, any electrolyte reabsorption results in a decrease in the tubular fluid osmolality. The maximum that a human can achieve is about 70 osmo/kg. As can be seen there is a leak back of K into the tubular fluid via the renal outer medullary K (RomK) channel (Kurtz 1998). The early distal tubule, Fig. 2.6, has a sodium reabsorptive transporter in the Na/Cl co-transporter. It is unclear at the present time how this transporter is related to calcium and magnesium reabsorption (Kurtz 1998). However it is clinically apparent that ‘Loop diuretics’ which inhibit the NaK/2Cl co-transporter also result in a loss of urinary calcium and magnesium. It is also clear that thiazide diuretics which inhibit the Na/Cl co-transporter are also associated with both an increase in urinary magnesium excretion but also an increase in calcium reabsorption. This has been used as a diagnostic test for hyperparathyroidism.

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If a patient’s plasma calcium increases on a thiazide diuretic, then this is seen as a diagnostic criterion for primary hyperparathyroidism. The later distal tubule (Fig. 2.7), is the segment responsible for pH alteration and also for potassium regulation. This is a segment which was recently thought to possibly be responsible for essential hypertension (Stokes 1999). The transport in this segment revolves around the Na channel. Abnormalities of this may result in Liddle syndrome (Stokes 1999) but are also physiologically controlled by aldosterone and thus abnormal in Addison disease and Conn syndrome. It is also the segment responsible for acidification of the urine which is achieved by the H-ATPase. Abnormalities of this or the basolateral HCO3/Cl can result in a deficiency of urinary acidification, distal renal tubular acidosis. It is also clear that extra sodium reabsorption, as in Conn syndrome, will result in hypertension and because of the resultant intraluminal positive electrical gradient, an alkalosis and hypokalaemia. The intriguing feature is whether minor defects short of those in Liddle syndrome might be responsible for essential hypertension. The collecting tubule’s essential function is water homeostasis. The inner medullary hypertonicity is connected to the contents of the collecting tubule if there is water channel insertion in this segment. In the absence of water channel insertion, there is the syndrome of diabetes insipidus. Classically, this is due to the lack of pituitary derived vasopressin. However, this syndrome can also be produced by defects in the effector mechanisms in the collecting duct in nephrogenic diabetes insipidus. Various genetic abnormalities of the insertion of the water channels have been described as well as damage due to drugs such as lithium (Bichet 1998). Figure 2.3 shows the properties of the various segments of the renal tubule with regard to a small number of parameters. The proximal tubule is sodium transporting and water permeable. The thick ascending limb is sodium transporting but water impermeable. The collecting duct is variably water permeable. The action of the renal tubule is dependent on the insertion and action of a small number of transport proteins. It is not surprising that genetic defects in these result in a large number of clinical syndromes.

Non-excretory renal function Although the kidney is an organ of excretory function as well as controlling the fluid status of the individual there are a number of other functions. The kidney is the site of erythropoeitin production (Macdougall and Eckardt 1998). The association of anaemia and renal failure has long been recognized. There have been many suggestions as to the basis metabolic problem ranging from a uraemic toxic effect on the bone marrow to gut iron loss from the prolonged bleeding time in renal failure. It was also recognized that in certain forms of renal disease anaemia was less common. In autosomal dominant polycystic kidney disease (APKD) even patients on dialysis could have well-maintained haemoglobins. The introduction of the recombinant erythropoeitin and its dramatic effect in correcting the anaemia seen in patients with renal failure showed that this was the most important defect. It appears that in adults erythropoeitin production occurs in cells within the interstitium. The importance of this site for production is unclear but the relatively large blood supply to the kidney may be a reason. However, it is clear that as renal function deteriorates in most forms of renal disease then anaemia is an absolute consequence. It is now routine practice to start patients on erythropoeitin prior to dialysis if they are iron-replete and anaemic.


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The kidney is the site of the hydroxylation of 25-hydroxycholecalciferol to the active metabolite 1,25-dihydroxycholecalciferol. This is vitally important to calcium phosphate homeostasis and its lack leads to the complex process of renal osteodystrophy. Activation of the hepatically produced 25-hydroxycholecalciferol occurs in the proximal tubular cells. With the decline in renal function the lack of 1,25-dihydroxycholecalciferol produces significant changes in calcium homeostasis. Bone matrix calcification fails with the increase in uncalcified osteoid resulting in osteomalacia. Gut absorption of calcium is reduced leading to low plasma calcium levels. At the same time 1,25-dihydroxycholecalciferol has a direct effect in inhibiting the release of parathyroid hormone from the parathyroid cells. The low plasma calcium levels and the lack of negative feedback result in the increase in parathyroid hormone excretion. In health, this hormone has many effects including the specific stimulation of the 1-hydroxylase in the proximal tubular cell to increase production of 1,25-dihydroxycholecalciferol. As with erythropoeitin production synthetic 1,25-dihydroxycholecalciferol can be administered to patients with renal failure and abrogate the hyperparathyroidism seen in endstage renal failure. The kidney is also the organ of metabolism of all the small peptide hormones. These are filtered and degraded in the brush border of the proximal tubular cell. The most clinically important effect of this is in diabetes mellitus where a consequence of progressive renal impairment is a decreased insulin requirement. This may mean that the first sign of renal impairment in a stable diabetic is the new occurrence of hypoglycaemic attacks. Most small polypeptides are found in high concentrations in patients on haemodialysis. The kidney is intimately involved in acid–base homeostasis. The predominant organ which controls acid–base homeostasis however is the liver (Atkinson and Bourke 1987). This alters the degradation of amino acids to produce either urea which does not lead to bicarbonate generation or glutamine which can be metabolized in the proximal tubule to generate bicarbonate. This results in bicarbonate which can be reclaimed to the circulation and excretion of ammonium into the urine. A consequence of this metabolic function is the production of Acetyl CoA. This is then synthesized into glucose. The kidney is the only organ other than the liver to have the property of gluconeogenesis. The common fallacy that metabolism results in the net generation of an acid load although much discussed in older text books can no longer be justified (Atkinson and Bourke 1987).

Investigations of renal disease The investigations of renal disease can be divided into two avenues. The first is the gross anatomy and function of the kidney. The second is the renal parenchymal architecture and includes examination of the renal histology. Imaging procedures can provide data on the size and configuration of each kidney. These are shown in Table 2.1. The advantages and disadvantages of the various techniques are shown. Each technique provides complementary information about the renal vasculature as well as renal parenchyma. At present, there are no techniques which provide all the information required. An example of the change in practice is APKD. Previously, radiological investigations involving the use of radiocontrast agents and radiation were required to diagnose this condition. However, with the advent of ultrasound it is now simple and non-invasive to diagnose this condition after the age of 20 years. Figure 2.9 shows the detail a CT scan can

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Table 2.1 Imaging procedures for determining kidney size and configuration Technique

Advantage

Disadvantage

Plain abdominal film

Simple Low X-ray dose Good delineation of collecting system and ureter Non-invasive Good delineation of intrarenal lesions and dilatation Excellent delineation of renal artery

Only relevant in radio-opaque renal stone disease Contrast load Poor delineation of intrarenal lesions Poor views of ureter Operative dependent

Intravenous urogram Renal ultrasound Intra-arterial angiography Spiral CT scan MRI angiography

No intra-arterial instrumentation Three dimensional images of renal vasculature No contrast load

DTPA scanning DMSA* scanning

Dynamic view of renal function Static view of functional renal tissue

*DMSA Dimercapto

Contrast load dependent on angle of view Intra-arterial instrumentation Contrast load Over-interpretation of lesions Patient compliance Dependent on initial bolus Not related to renal blood flow

succinic acid.

Fig. 2.9 A CT scan of a patient with both polycystic kidney disease and a horseshoe kidney.


35

A

B

Fig. 2.10 Intra-arterial angiography, A: in the same patient compared with MRI angiography, B: in between the first and second scan the patient had occluded his aorta below the level of the renal arteries.

provide in APKD with the congenital variant of a horseshoe kidney. Figure 2.10 shows the use of magnetic resonance imaging (MRI) in atherosclerotic renal artery stenosis compared with conventional angiography in the same patient. It is apparent that at present a number of investigations will provide anatomical clarity of the renal tract. With time those investigations which provide the greatest clarity with the least radiation exposure will become the most widely used.

36


The advantages and disadvantages of the various tests to determine renal function are shown in Table 2.2 (Cameron and Greger 1998). The nuclear medicine investigations do not provide excellent anatomical clarity but do reflect renal function. The diethylene triamine penta acetate (DTPA) nuclear medicine scan can show obstruction to urinary flow. It can also confirm renal artery stenosis when combined with administration of an angiotensin converting enzyme inhibitor (ACEI) such as captopril as shown in Fig. 2.11. Other parameters which are important include 24-h urinary protein excretion. Specific investigations relating to the Table 2.2 Techniques for determining renal function Technique

Advantage

Disadvantage

Plasma creatinine

Simple Single test Estimation of glomerular filtration rate Cheap No radiation Single blood test Precise Not dependent on complete urine collection Precise Not dependent on complete urine collection No chemical assay

Dependent on muscle mass

Creatinine clearance

Inulin clearance Cr–EDTA clearance

Incomplete collection common

Needs multiple blood tests Needs specific laboratory analysis Radioactive Needs multiple blood tests

Fig. 2.11 The DTPA scan shows the effect of a single administration of 25 mg of the ACEI captopril on excretion in a patient with bilateral renal artery stenosis. This shows that the renal artery stenosis was critical to function. The patient had ‘flash pulmonary oedema’ and was cured by bilateral renal angioplasty.


37

disease causing the renal disease will be important for patient management but do not relate to renal function. In many conditions after the initial diagnosis is made the most important parameter is the plasma creatinine as this is an imperfect but reproducible measure of renal function. However, this does presume that creatinine is filtered and neither excreted nor reabsorbed by the renal tubule. This may not be true in situations of low renal function. In normal clinical practice, the plasma creatinine is used as a simple measure of renal function. However, as the relationship between plasma creatinine and creatinine clearance is a reciprocal one it can be difficult to grasp the changes in renal function. For instance, every time the plasma creatinine doubles the creatinine clearance halves. For an individual with a creatinine clearance of 100 ml/min, a plasma creatinine of 100 mol/l then a rise of plasma creatinine to 200 mol/l represents a halving of renal function. That is a loss of 50 ml/min. If, however, the creatinine then doubles to 400 mol/l this only represents the loss of a further 25 ml/min of glomerular filtration rate. The use of the creatinine clearance will provide a measure of glomerular filtration but incomplete 24-h urinary collection will lead to underestimation of renal function. The most specific measures of renal function use either inulin clearance or Cr–EDTA clearance (Maisey and Britton 1998) as shown in Table 2.2. These techniques require repeated blood samples and in the case of Cr–EDTA use of a radionuclide. This is because most renal disease is bilateral and affects both kidneys equally. The new technique of single kidney glomerular filtration rate has proved useful in asymmetrical renal disease (Scoble and Cook 1998). The measurement of overall renal function can be achieved by a number of techniques. These tests are applicable as most causes of renal dysfunction affect both kidneys equally. Table 2.2 shows the advantages and disadvantages of each. However, the difficulty with this parameter is that it does not correlate with symptoms until very late in the disease course, Fig. 2.2. Newer investigations such as MRI may provide non-invasive imaging in conditions affecting the renal vasculature as well as functional assessment in a single test. The single test which will provide evidence of specific changes in the glomerular or tubular compartment is the renal biopsy (Ponticelli and Mihatsch 1998). This technique is universally used and provides very precise recognition of patterns of renal disease. There is however a risk of bleeding produced by the biopsy and rarely nephrectomy can be required. The risks of bleeding increase with the worsening of renal function as there is prolongation of bleeding time with significant renal dysfunction. A single histological pattern may result from a number of different causes. For instance focal glomerulosclerosis can be related to a familial condition, renal artery stenosis, gross obesity, or an idiopathic cause! The importance of the various patterns of renal histology will be discussed below but this investigation remains the cornerstone of the investigation of most forms of renal disease.

Management of renal disease The major issue with renal physicians is that symptoms with renal disease are rare below a glomerular filtration rate (GFR) of 30 ml/min, Fig. 2.2. It is unclear why cardiac disease will produce symptoms at marginal changes in function whereas in renal disease the vast majority of renal function has to be lost before the patient feels any abnormality. This explains why

38


in a number of conditions abnormalities of urine testing, such as haematuria in Alport syndrome, are common presentations of renal disease. Although changes in glomerular filtration present late, changes in tubular electrolyte handling will present much earlier as is seen in Bartter syndrome. The management of renal disease can be divided into the general aspects of the management of a patient with a reduced glomerular filtration rate and the specific management of the pathological processes in a specific condition. There are a number of conditions such as focal and segmental glomerulonephritis where there are a number of aetiological factors including a genetic element where the general management is the same irrespective of the cause. There are also a number of conditions where a genetic element in the aetiology has only recently been recognized such as reflux nephropathy. Presumably, in the future there will be a number of idiopathic conditions described here which will be found to have a genetic element. The general management of patients with renal disease has evolved from simple dietary restriction or alteration to focus on factors which may lead to progression (Locatelli and Del Vecchio 2000). The most important of these is the management of hypertension (Dworkin and Weir 2000). Renal disease can obviously cause hypertension and hypertension can cause renal disease. In its most severe form hypertension can cause acute dialysis dependence which may not be reversible. However, it is now clear that in all forms of renal disease, control of hypertension is the single most effective treatment to halt the progression. The work that has established this has come from the investigation of patients with juvenile onset diabetes mellitus. The work of Parving et al. (1983) has shown that tight control of hypertension can cause a decrease in the rate of decline of renal function. This group of patients has been the most easy to study given its homogeneity. The results appear applicable to all forms of renal damage including post-transplantation. In the clinical setting, for conditions such as APKD where there are no specific ameliorative gene therapies at present, we can offer the patient the possibility of altering the rate of decline of renal function by tight blood pressure control. This may however mean the use of multiple agents to achieve the blood pressure guidelines equal to diabetic patients of 130/85 which are now advised. The work of Lewis et al. (1993) clearly showed that for patients with juvenile onset diabetes mellitus (JODM) there was an additive effect of the use of ACEI to decrease the reaching of a combined end-point of death or renal failure. The important feature to note is that in this trial this was achieved against a placebo group with good blood pressure control. A subsequent study in a large group of patients with maturity onset diabetes mellitus (MODM) has suggested that control of blood pressure with or without an ACEI is the most important therapeutic manoeuvre (UK Prospective Diabetes Study Group 1998). However, other studies have suggested that in renal disease the use of an ACEI in the management of all forms of progressive renal disease does confer a beneficial outcome (Maschio et al. 1996). In proteinuric states ACEI will also beneficially reduce the proteinuria in an effect separate from control of hypertension which has been linked to the reduction in intraglomerular pressure. In all forms of renal disease, the management of hypertension is a primary aim of therapy and it is probable that in most forms the use of an ACEI will also be beneficial. The general management of renal disease will also include dietary manipulation. There has been considerable experimental evidence from animal models of a beneficial effect of protein restriction on the progression of renal disease. However, in clinical practice it is difficult to severely protein restrict many of our patients. The results of a large trial have been disappointing in not showing an overall effect of protein restriction in the progression of renal


39

disease (Klahr et al. 1994). However in individual patients control of protein, potassium, and phosphate intake will prolong the time to dialysis. Irrespective of the progression of renal disease, these individual parameters may require urgent dialysis in individual patients. General management of patients with progressive renal disease now include measures to improve symptomatology. The use of erythropoeitin prior to dialysis is now widespread in patients approaching end-stage renal failure. At the same time, the use of 1,25-dihydroxycholecalciferol or an analogue will also be routine practice in pre-dialysis patients to prevent renal osteodystrophy. However, at present there are no routine measures to try to prevent the extremely high initial cardiovascular mortality of patients entering the end-stage programme compared with age matched controls (Parfrey and Foley 1999). This is a frequent cause of both morbidity and mortality in this group of patients. It is important to state that the 5-year survival of patients over the age of 64 years entering a dialysis programme is worse than similar patients with colonic cancer (Parfrey and Foley 1999)! The management of renal disease in specific conditions will depend on the condition. The causes of renal disease can be simply analysed as pre-renal, parenchymal renal, and post-renal disease. Pre-renal disease Pathological processes affecting the renal artery or more rarely the renal venous supply will effect renal function. The causes of renal artery disease are shown in Table 2.3. The various conditions are very disparate. The original work of Goldblatt et al. (1934) suggested that the relief of renal artery narrowing will cure the hypertension the renal ischaemia induces. A major focus on the management of these conditions is to re-establish the normal blood flow to the kidney. Initially the early reports were of nephrectomy or renal artery surgery. The introduction of renal angioplasty and more recently renal artery stenting has meant that renal revascularization can be achieved without a significant surgical operation. However, the use of this procedure has shown how complex the processes involved in renal artery narrowing are. It is clear from the artery reports that acute renal artery occlusion can be tolerated with the recovery of renal function even after anuria. The situation in chronic renal artery narrowing is less clear. In conditions where there is a systemic component such as Takayasu syndrome or atherosclerotic management of the non-renal manifestations may be most important in patient outcome. The group also represents a heterogeneous pathology of renal disease. The cure of hypertension was an early goal and the use of angioplasty has produced good results in fibromuscular dysplasia even in the early reviews of data. However, later studies have shown a less good result for atherosclerotic disease. The results from intervention in atherosclerotic disease which is the most common in the UK and the USA suggest a variable response with only about a third of patients benefiting from intervention. The medical management of these patients is made more difficult by the inability to use ACEI as this causes a decline in renal function. The major causes of renal vascular disease are discussed. Fibromuscular dysplasia This condition appears to affect the renal arteries and less commonly other territories including the carotid and iliac arteries (Stanley 1995). It has a predominantly female bias in

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A. David Makanjuola and John E. Scoble Table 2.3 Causes of renal artery diseases Renal artery occlusion Acute Embolus from central source; thrombus, bullet Trauma Acute on chronic with pre-existing renal artery stenosis, e.g. with ACEI inhibitors and diuretics Renal artery aneurysm Aortic dissection Aortic occlusion and retrograde thrombosis leading to renal artery occlusion Secondary to intervention e.g. angioplasty Clotting disorder Antiphospholipid antibody syndrome Acute vascular transplant rejection Spontaneous Chronic Atherosclerotic Fibromuscular dysplasia Takayasu disease Middle Aortic syndrome Renal transplant renal artery stenosis Renal vein occlusion Acute Nephrotic syndrome Clotting disorders Antiphospholipid antibody syndrome Secondary to intervention e.g. renal venography or surgery Trauma Inferior vena cava occlusion Acute vascular transplant rejection Spontaneous Chronic Nephrotic syndrome especially membranous glomerulonephritis Tumour Retroperitoneal fibrosis Veno occlusive diseases

incidence and is also very rare in certain ethnic groups such as the Afro-Caribbean population. Its presentation is of hypertension at an early age and must be suspected in malignant hypertension at an early age. It appears that the lesions progress rarely in later life although the long-term data are unclear on this aspect. A decline in renal function appears also to be rare although patients may present with another manifestation of renal artery disease, acute unprovoked pulmonary oedema. Management of this condition will include control of hypertension as well as angioplasty. In a number of patients the renal artery lesions may be very distal, and require bench surgery and auto-transplantation. There are no systemic manifestations of this disease as is the case of the rarer condition of the Middle Aortic syndrome.


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Takayasu syndrome This condition was first completely described during the Second World War in Japan. It is an arteritis affecting the aorta and major arteries resulting in its description as the pulseless disease. The renal artery lesion is progressive and can lead to dialysis requirement. There are manifestations of systemic disease with a raised erythrocyte sedimentation rate (ESR). The management of this condition involves the suppression of the arteritis with steroids and then the revascularization of the arterial tree affected. Renal angioplasty has been effective and more recently it has been suggested that mycophenolate mofetil is useful in the long-term management (Daina et al. 1999). Atherosclerotic renal artery stenosis This is the most common cause of renal artery stenosis in the UK and USA (Scoble 2000). It is predominantly a disease of the older population and is intimately related to peripheral vascular disease. It can present with hypertension, renal impairment, or flash pulmonary oedema. It is also a marker of enormously high cardiovascular mortality. Although it is possible to revascularize the kidneys the results for hypertension are disappointing and the results for renal function suggest only a minority of patients, who cannot be identified prior to intervention can be identified. However, in anecdotal reports and in the minority of patients in series of intervention, dialysis-dependent patients may be rendered dialysis-independent. Glomerular disease The spectrum of glomerular disease extends from patients with mild urinary abnormalities to those with the full-blown nephritic or nephrotic syndrome, chronic glomerulonephritis, and end-stage renal failure. The major clinical syndromes of glomerular disease are acute glomerulonephritis, rapidly progressive glomerulonephritis, chronic glomerulonephritis, nephrotic syndrome, and asymptomatic haematuria with or without proteinuria. Acute glomerulonephritis These patients have an abrupt onset of haematuria, proteinuria, oedema, oliguria, hypertension, with a reduction in GFR, and uraemia. Haematuria refers to the excretion of abnormal amounts of erythrocytes in the urine. This is often first brought to light on dipstick examination of the urine (microscopic haematuria), or it might be obvious to the naked eye either as dark brown or bright red urine (macroscopic haematuria). The haematuria may either be persistent or intermittent, and it might be symptomatic or asymptomatic (painless). Patients with acute glomerulonephritis often have red cell casts in the urinary sediment, and the proteinuria is usually less than 3 g per day. Some of the causes of acute glomerulonephritis are shown in Table 2.4. There is often an immunological basis to the glomerular injury, which then triggers secondary events such as complement activation, fibrin deposition, platelet aggregation, and

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A. David Makanjuola and John E. Scoble Table 2.4 Some causes of acute glomerulonephritis Familial Alport syndrome Nail-patella syndrome Fabry disease Primary Fibrillary glomerulonephritis Post-streptococcal glomerulonephritis IgA nephropathy Idiopathic rapidly proliferative glomerulonephritis Membranous nephropathy Focal and segmental glomerulosclerosis Secondary Systemic lupus erythematosus Goodpasture syndrome Henoch–Schonlein purpura Mixed essential cryoglobulinaemia

activation of kinin systems. These result in increased capillary permeability and glomerular damage. The kidneys, in acute glomerulonephritis, are usually of normal size, but may be enlarged and oedematous, and the surface of the kidney may show punctate haemorrhages. In the clinical setting, it is often important to distinguish between the patient who has an acute exacerbation of an underlying chronic renal disorder from the patient with previously normal renal function. It would be reasonable to expect the latter patient to regain normal excretory renal function perhaps with residual asymptomatic haematuria or proteinuria; although it is possible that they may develop chronic renal impairment, or pursue a rapidly progressive course, leading to end-stage renal failure. Rapidly progressive glomerulonephritis (RPGN) These patients are characterized by a rapid, often inexorable progression towards end-stage renal failure without (and often in spite of) treatment. Chronic glomerulonephritis This is the persistence and/or progression of an initial glomerular insult, which will ultimately lead to end-stage renal failure. It may take years before patients end up requiring renal replacement therapy, and the need for this is often precipitated by other factors, such as poorly controlled hypertension. In chronic glomerulonephritis, the kidneys are often small, with fine granular scarring. Nephrotic syndrome Protein is normally present in only small amounts in the urine (Cameron 1998). This is largely due to the fact that in spite of the huge amounts delivered to the nephrons the glomerulus significantly restricts the passage of proteins across its membrane, and the tubules reabsorb most of the protein that manages to traverse the glomerular basement membrane. About 0.5–1 g of


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albumin is filtered by the glomeruli each day, but after tubular reabsorption, only 20–40 mg is passed in the urine. In the healthy individual, 24-h protein excretion is usually less than 150 g, but glomerular and tubular diseases often lead to a significant increase in the amount of protein excreted. In the absence of urinary tract infections, it is fair to say that proteinuria is almost always of glomerular origin, whereas haematuria may be due to lesions anywhere along the renal tract. The nephrotic syndrome is the consequence of persistent heavy proteinuria. Its cardinal features are proteinuria, which by definition is in excess of 3.5 g/1.73 m2 body surface area or 50 mg/kg body weight, hypoalbuminaemia, oedema, hyperlipidaemia, and hypercoagulability (Cameron 1998). These patients also have some degree of immunodeficiency. Proteinuria secondary to glomerular disease is believed to arise as a result of compromise of the filtration barrier, which alters its charge selectivity (minimal change nephropathy), or alters the pore sizes (proliferative glomerulonephritis). This allows plasma proteins, especially albumin to leak into the filtrate in quantities that exceed the tubules’ reabsorptive capacity, and thus end up in the urine. Hypoalbuminaemia is due to a variety of factors, which include excessive loss of albumin in the urine; increased renal tubular catabolism of albumin, and an inappropriately low rate of albumin synthesis by the liver. Oedema is partly due to the reduced plasma oncotic pressure as a consequence of the hypoalbuminaemia, but this is by no means the sole explanation. These patients exhibit a significant avidity for sodium, with consequent sodium retention by the kidneys, the cause/s of which have not been fully elucidated. Hyperlipidaemia is due to increased liver synthesis of lipoproteins. The hypoalbuminaemia and increased albumin synthesis by the liver seem to be accompanied by increased delivery of the cholesterol precursor mevalonic acid to the liver, and stimulation of lipoprotein synthesis, especially very low density lipoprotein (VLDL). Reduced lipoprotein catabolism also contributes to the hyperlipidaemia. Lipiduria is another common feature. It is possible that these patients are at increased risk of ischaemic heart disease, and in patients with other risk factors for ischaemic heart disease, lipid-lowering therapy ought to be instituted. Hypercoagulability usually manifests as thrombo-embolism. This is predominantly venous, but can also affect the arterial tree. The most common manifestation is renal vein thrombosis, but close to one-fifth of all patients with the nephrotic syndrome develop deep venous thromboses and pulmonary emboli. Low levels of antithrombin III seem to correlate closely with the increased risk of thrombo-embolism. Some nephrologists advocate prophylactic anticoagulation once the serum albumin levels fall below 20 mg/dl. Immunodeficiency is believed to be due to losses of immunoglobulins in the urine, as well as increased catabolism and depressed synthesis. Serum immunoglobulin (Ig) levels are reduced, and there are defects in cell-mediated immunity. These people are prone to serious infections, and this is often compounded by the fact that treatment regimes often involve the use of steroids and other immunosuppressive agents. Virtually any glomerular lesion, if it generates enough proteinuria, will produce the features of this syndrome but only a small number of disorders constitute the bulk of the cases encountered in clinical practice. Secondary causes of the nephrotic syndrome include diabetic nephropathy, amyloidosis, systemic lupus erythematosus, and neoplasia. Primary (idiopathic) causes include minimal change nephropathy, focal and segmental glomerulosclerosis, and membranous nephropathy.

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Patients with the nephrotic syndrome may also have features of acute glomerulonephritis, and may progress to chronic glomerulonephritis. Tubular proteinuria is usually of lesser magnitude, and in a patient known to have tubulointerstitial disease, development of nephrotic range proteinuria strongly suggests co-existent glomerular pathology. Histopathological types of glomerular diseases The histological response to immunological insults varies, and is nonspecific. Identical histological appearances may be the result of very different insults, but on the other hand, a condition such as systemic lupus erythematosus is associated with many different histological changes. Clinical manifestations do not correlate closely with the histological changes, but nonetheless, it is often possible to predict with some degree of accuracy what the clinical outcome is likely to be after reviewing the renal biopsy, by light microscopy, electron microscopy, and immunofluorescence. Minimal change nephropathy (minimal change disease) As the name suggests, the glomerular appearances are almost normal (Broyer et al. 1998). On light microscopy, there is usually no abnormality of note, and electron microscopy shows fusion of the epithelial cell foot processes. Immunofluorescence is negative. Its aetiology is unknown, but an immunological basis is suggested by the fact that it is usually steroid responsive, there is an increased incidence of atopy in patients as well as family members, and depressed levels of IgG subclasses and elevated IgM levels have been noted. The fact that it occurs in some patients with T-lymphocyte dysfunction such as Hodgkin disease and that it remits on treatment of the malignancy. Minimal change disease is the most frequent chronic renal disease in childhood, and its relative incidence decreases with advancing age in patients which manifest clinically with the nephrotic syndrome. The proteinuria is believed to be due to increased glomerular permeability, and some authors have suggested that one or more lymphocyte-derived factors could cause this by inducing electrostatic changes on the glomerular capillaries. There is a possibility that a more generalized increase in capillary permeability is present in children with minimal change disease, as they often have more oedema than other patients with comparable degrees of hypoproteinaemia due to other glomerular lesions. The clinical course is characterized by relapses of varying frequency. Eighty per cent of children have a rapid response to an initial course of steroid therapy, but relapses occurred in the majority of them. The frequency of relapses is unpredictable for the individual patient, but in general, relapses are more common if the onset of disease was in early childhood. Progressive disease leading to end-stage renal failure is uncommon in children. Mild to moderate degrees of renal impairment is seen in some patients and is often due to intravascular volume depletion, or sepsis. Idiopathic focal and segmental glomerulosclerosis (FSGS) This condition, described initially is seen in 10–15 per cent of people with idiopathic nephrotic syndrome, and is the most common cause of steroid-resistant nephrotic syndrome


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in children. It has been suggested that there is a circulating factor able to elute from the serum of these patients which might be responsible for the increased glomerular permeability, and this might explain the tendency for this condition to recur in kidneys transplanted into affected individuals sometimes within hours of transplantation (Korbet 1998). Light microscopy in FSGS shows focal and segmental glomerular sclerosis, varying degrees of mesangial proliferation and often, subendothelial areas of hyalinosis and lipid droplets. Other portions of the glomeruli appear normal. The process seems to begin in the juxta-medullary glomeruli and then progresses to involve the more peripheral glomeruli. The tubules may be atrophic and there might be an interstitial mononuclear cell infiltrate. Immunofluorescence shows mesangial deposits of IgM and C3, usually within sclerotic segments. These histological findings are indistinguishable from those seen in patients with secondary FSGS, suggesting that the histologic picture is a common final pathway for several kinds of glomerular damage (Korbet 1998). These patients, as mentioned earlier, tend to be steroid unresponsive. There is also a greater incidence of hypertension, azotaemia, and progression to chronic renal impairment than is seen in patients with minimal change disease. Diffuse proliferative glomerulonephritis This histological lesion is often seen in patients with acute post-streptococcal glomerulonephritis, and can also be seen in systemic lupus erythromatosus (SLE). It usually manifests clinically as an acute nephritis, with haematuria, proteinuria, hypertension, oedema, and azotaemia. On light microscopy, the glomerulus appears swollen, with proliferation of endothelial and mesangial cells and several polymorphonuclear leucocytes. Electron microscopy shows subepithelial humps on the glomerular basement membrane (GBM), and immunofluorescence shows granular deposits of immunoglobulins and C3. Focal proliferative glomerulonephritis This condition is seen SLE, IgA nephropathy, Henoch–Schonlein purpura, anti-neutrophil cytoplasmic antibody (ANCA)-positive vasculitis, and infective endocarditis. Only some of the glomeruli are involved, and the affected glomeruli show segmental proliferative changes. Clinical manifestations are varied, and range from minor urinary abnormalities to acute nephritis, or the nephrotic syndrome. Crescentic (rapidly progressive) glomerulonephritis Extensive glomerular crescent formation (usually more than 50 per cent) is the main histological finding (Cameron and Rees 1998). It is a particularly severe form of many glomerular diseases, and is quite often seen in anti-GBM disease, microscopic polyarteritis and Wegener granulomatosis. The crescents may be partially or totally circumferential, and may be cellular, fibrocellular, or fibrotic. Prominent endocapillary proliferation suggests a post-infectious or multisystem disease. The presence of a vasculitis and necrotizing glomerular lesions suggests systemic disease. Immunofluorescence will show linear staining of the GBM with IgG, in anti-GBM disease. Idiopathic RPGN is associated with granular deposits of IgG and C3 in mesangial areas and along the subendothelial aspect of the capillary wall.

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As the name suggests, renal function declines rapidly, with end-stage renal failure ensuing within anything from 1 week to 3 months from diagnosis or the onset of symptoms in the absence of treatment. The outlook has improved significantly since the advent of treatment with high dose steroids, plasma exchange, and cytotoxic agents such as cyclophosphamide. Membranous glomerulonephritis This condition occurs mainly in adults and predominantly in males (Zucchelli and Pasquali 1998). These patients typically present with proteinuria, and in the great majority, the nephrotic syndrome. Light microscopy shows diffuse and uniform thickening of the GBM. Mesangial proliferation and cellular infiltration may occur in patients with underlying disease, but are often absent in the idiopathic form. Immunofluorescence in well-developed cases shows diffuse granular staining with IgG and usually C3 in capillary walls. Silver staining outlines the GBM but not immune deposits, and displays the characteristic ‘spikes’. Electron microscopy reveals subepithelial deposits. One half of the patients will achieve partial or complete remission, and the other half will have chronic or end-stage renal failure. Young age, female sex, normal renal function, and the absence of the full-blown nephrotic syndrome at presentation are associated with a good prognosis. The presence of tubular atrophy and interstitial fibrosis on renal biopsy are adverse prognostic features. Mesangio-capillary (membrano-proliferative) glomerulonephritis (MCGN) This glomerular lesion is associated with a chronic, progressive glomerulonephritis, which tends to affect adolescents and young adults (Williams 1998). Secondary forms occur, usually in association with systemic, infectious, and neoplastic disorders. Idiopathic MCGN is divided into three types based on histo- and immuno-pathologic features. In Type 1, there are subendothelial and mesangial deposits of immunoglobulin and C3. This is the most common form. It is also the histological pattern most commonly seen in association with secondary forms. The most common clinical manifestation is the nephrotic syndrome, though some patients may present with an acute nephritis. C3 levels are low, and both the classical and alternative pathways of complement activation are involved. Type 2 is characterized by electron-dense deposits and linear C3 deposits situated in intramembranous and mesangial areas. It is also known as dense deposit disease. These patients tend to have a younger age of onset, and the typical presentation is with acute nephritis. Partial lipodystrophy may be seen. C3 levels are low, and a unique IgG auto-antibody— the C3 nephritic factor—causes complement activation via the alternative pathway. In Type 3 there is a rare group of patients who exhibit features of both Type 1 and Type 2. This category is however, not universally accepted. Secondary causes of MCGN include hepatitis C, SLE, and mixed essential cryoglobulinaemia. Type 1 MCGN is slowly progressive, but Type 2 is much more aggressive, with patients progressing to end stage renal failure from 5 to 10 years following diagnosis. Immunoglobulin A (IgA) nephropathy (Berger disease) Originally described in 1968, this is an idiopathic glomerular disease characterized by diffuse mesangial deposition of IgA (Topham et al. 1994). It appears to be the most frequently


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encountered glomerular disease worldwide. It may have a genetic component as the incidence is much lower in blacks in the USA, but seems to be much more common in the American Indians of New Mexico and familial clustering has been reported. The HLA B35 is reported to be more frequent in a large population of French patients, in whom it seems to correlate with progression to end-stage renal failure. The diagnostic hallmark of the disease is mesangial deposition of IgA. Immunofluorescence on renal biopsies shows IgA to be the predominant or sole immunoglobulin, although in most cases, C3 and IgG are also present, though with a lesser degree of immunofluorescence. Light microscopy shows mesangial enlargement, usually in a focal and segmental distribution, but in some cases, the appearance may be that of a diffuse proliferative process. The pathogenesis is not clear, but polymeric IgA1 of mucosal origin is believed to be the immunoglobulin acting as antibody. Clinically, patients manifest with asymptomatic microscopic haematuria with or without proteinuria. These patients are usually detected by chance at routine medical examinations. Macroscopic haematuria, when it occurs, is usually temporally related to a respiratory, gastro-intestinal, or less commonly a urinary tract infection. Proteinuria is often mild, but may occasionally be severe enough to cause the nephrotic syndrome. As many of these patients are asymptomatic, the true onset of the disease is not often known. Renal survival rates are estimated at 78–91 per cent at 10 years and 75–80 per cent at 20 years from apparent onset in some large series. The rate of progression from diagnosis to end-stage renal failure varies from a few months to over 30 years. Poor prognostic factors include male sex, elevated serum creatinine at time of diagnosis, hypertension, protracted severe proteinuria, and absence of a history of recurrent macroscopic haematuria.

Tubulo-interstitial disease Tubulo-interstitial disease is any disorder resulting in structural and/or functional derangements of the tubulo-interstitial constituents of the kidney (Michel and Kelly 1998). The causes of this histological pattern are many. Tables 2.5–2.7 show the drug, infectious, and metabolic causes of this pattern of renal disease. Tubulo-interstitial disease can be divided into acute and chronic types. About 15 per cent of acute renal failure and 25 per cent of end-stage renal failure is caused by primary tubulointerstitial disease. The pathogenesis of tubulo-interstitial disease can involve a variety of mechanisms, which include direct invasion by microbial organisms, direct toxicity as with heavy metal poisoning, immunological insults, metabolic diseases, and hereditary disorders such as cystic diseases and Alport syndrome. Acute tubulo-interstitial nephritis Councilman used this term in 1898 as a histopathological diagnosis for the acute renal inflammatory changes seen in autopsy specimens of patients with diphtheria and scarlet fever (Michel and Kelly 1998). Infections are less commonly responsible for acute interstitial nephritis nowadays and most cases are drug induced. In a few cases, no definite cause is found and in this ‘idiopathic’ group, no clear sex or age predisposition has been found.

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A. David Makanjuola and John E. Scoble Table 2.5 Drugs associated with acute interstitial nephritis Antibiotics Beta-lactam Ampicillin Methicillin Flucloxacillin Carbenicillin Amoxycillin Cefotaxime Cephalexin Cephloridine Cefoxitin

Anti-convulsants Phenytoin Sodium valproate Carbamazepine Phenobarbitone

NSAIDs* Ibuprofen Indomethacin Fenoprofen Naproxen Diclofenac Piroxicam

Others Rifampicin Sulphonamides Septrin Ciprofloxacin Erythromycin Vancomycin Ethambutol

Diuretics Thiazides Frusemide Triamterene

Others Cimetidine Allopurinol Haloperidol Captopril Diazepam Propranolol Amphetamines Aspirin Clofibrate

*NSAIDs Non-steroidal anti-inflammatory drugs.

Table 2.6 Infectious causes of acute interstitial nephritis Bacteria Escherichia coli Proteus species Enterococci Klebsiella aerobacter Staphylococci Leptospira species Brucella species Legionella species Viruses Epstein Barr virus Coxsackie B virus Fungi Candida species Coccidioidomycosis Protozoa Falciparum malaria Toxoplasmosis


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Table 2.7 Causes of chronic interstitial nephritis Metabolic abnormalities Hyperuricaemia Oxalosis Hypercalcaemia Hypokalaemia Cystinosis Hereditary disorders Sickle cell disease Alport syndrome Polycystic kidney disease Others Balkan nephropathy Radiation Allograft rejection Tuberculosis Leprosy Sjogren syndrome Obstructive uropathy Reflux nephropathy Chronic pyelonephritis

Neoplasia Multiple myeloma Lymphoma Leukaemia

Systemic disorders Systemic lupus erythematosus Polyarteritis nodosa Wegener granulomatosis Cryoglobulinaemia Exogenous toxins Lead Cadmium Gold Cyclosporin cis-Platinum Lithium

Clinically, patients with acute tubulo-interstitial nephritis manifest with an abrupt deterioration in renal function characterized histologically by inflammation and oedema of the renal interstitium. Drug-induced acute interstitial nephritis There are a significant number of drugs, which have been reported as causative agents. The beta lactam antibiotics and non-steroidal anti-inflammatory drugs are well recognized causes. The clinical manifestations depend on the drug producing the adverse reaction. With beta lactam antibiotics, the hypersensitivity triad of rash, fever, and eosinophilia dominates the picture. Patients may also have arthralgia, eosinophilluria, lymphadenopathy, and hepatitis. Sterile pyuria and haematuria may occur. These manifestations usually occur in patients who have received high doses of the beta lactam for a prolonged period of time and can occur either in the presence of active infection or when the drug is being used prophylactically. Non-steroidal anti-inflammatory drugs are frequently associated with very heavy proteinuria and minimal change nephrotic syndrome. The onset of the nephrotic syndrome typically coincides with the onset of the acute interstitial nephritis and acute renal failure, all of which tend to settle on cessation of the drug. Infectious causes The causative agents, Table 2.6 may be bacterial, viral, fungal, or parasitic in nature. Acute renal failure is prominent, but the hypersensitivity triad is not common. The manifestation of

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the basic infective process often predominates, and the renal failure usually remits as the infection settles. The diagnosis of acute tubulo-interstitial nephritis is conclusively made on renal biopsy. However, a classical clinical picture in association with non-oliguric renal failure in a patient on a medication known to cause this is highly suggestive, especially if the renal failure improves following cessation of the drug. Chronic tubulo-interstitial nephritis This is characterized by progressive renal insufficiency, associated with non-nephrotic range proteinuria and functional tubular abnormalities. The primary site of injury is the tubule and surrounding interstitium. In far advanced disease, peri-glomerular fibrosis, and glomerulosclerosis may be seen. The tubules are often atrophic and dilated. Clinical manifestations vary, and depend to some degree on the causative factor. Conditions such as heavy metal poisoning and multiple myeloma affect the proximal tubule primarily, and may present with renal tubular acidosis, glycosuria aminoaciduria, and uricosuria. Chronic obstructive uropathy or amyloidosis may cause isolated distal tubular damage; while sickle cell disease, analgesic nephropathy, or polycystic disease of the kidney may affect the medulla and lead to a defect in concentrating ability. Hypertension is usually a late finding and oedema is often absent. Avoidance of the nephritogenic agent or correction of the predisposing factor may prevent progression or even reverse the renal insufficiency. Hypertension where present should be treated aggressively. Post-renal disease This is an important area where the causes of renal dysfunction are geographically very different. Table 2.8 shows that the causes of this are many and can be divided into malignant and non-malignant. Just as in renal arterial obstruction relief of the obstruction is important, then even more so in renal outflow obstruction, relief is vitally important and may enable patients to come off dialysis. Pelvi-ureteric obstruction is usually not complete and often is precipitated by fluid challenge. Its relief is by surgical plication of the pelvi-ureteric junction. Renal stones may cause obstruction to the urinary tract from the calyx to the urethra but most commonly lodge at the pelvi-calyceal junction or at the uretero-vesical junction. The causes of renal stones will be discussed elsewhere. However, once formed their management is by their removal. This can be achieved via lithotripsy or surgical removal directly or endoureterically. The management of malignant obstructive disease depends on the cause. In prostatic malignancy, obstruction can occur at both the urethral level as well as ureteric level. Hormone manipulation can provide a dramatic response. For other malignancies such as cervical cancer the outcome is more gloomy and hormonal manipulation is not possible. In the infective causes of obstruction the principle of therapy is treatment of the disease and then surgical treatment of the urinary tract to provide adequate drainage. The management of congenital obstructive disease is to reverse the obstruction at the earliest possible opportunity although the outcome of this is variable. It is clear that complex


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Table 2.8 Causes of obstructive uropathy Non-malignant Pelvi-ureteric obstruction Urethral valves Benign prostatic hypertrophy Stone disease Retroperitoneal fibrosis Ureteric fibrosis—tuberculosis Bladder fibrosis—Bilharzia Malignant Retroperitoneal fibrosis—lymphoma Prostatic malignancy Cervical malignancy Urothelial malignancy

pathological processes are unleashed by urinary obstruction and it is not a simple matter of mechanical obstruction (Klahr 1983). Treatment of end-stage renal failure This book seeks to enlighten us about the genetic causes of renal failure. It is also about the prevention of end-stage renal failure by treatment of the genetic diseases if possible. Although it is unnecessary to document the technicalities of the treatment of end-stage renal failure, there are some specific points which need to be made. The first patient to receive renal dialysis treatment was Sophia Schafstadt in the Second World War in Nazi occupied Holland. Kolff performed this first dialysis and Fig. 2.12 shows a later version of the original machine. Although there has been considerable development of dialysis machines the basic principles remain as described by Kolff. Initial use of dialysis was for conditions which would recover. Regular haemodialysis only became possible with the development of the Scribner shunt and this was then superseded by the Cimino fistula which remains to this day the optimum vascular access. Peritoneal dialysis which originated as a form of acute dialysis with a ‘hard catheter’ developed into chronic ambulatory peritoneal dialysis (CAPD) with the use of the soft Tenkhoff catheter. It is important to underline that both haemodialysis and CAPD provide only the equivalent to 10 per cent of normal glomerular filtration rate. This fact relates to Fig. 2.1. This shows a healthy nephrologist and his glomerular filtrate. If he had end-stage renal failure he may be assigned to CAPD and would do four exchanges a day totalling 8 l. This would prevent him from dying due to hypervolaemia or hyperkalaemia but would not give him by any estimation normal plasma biochemistry. The only form of treatment of end-stage renal failure which provides for treatment which will reproduce normal renal function is renal transplantation. It is probably best to describe dialysis as preventing death rather than replacing normal renal function which can today only be achieved by transplantation. Renal transplantation has evolved from the procedure only applicable to identical twins to a process which may provide excellent life expectancy to many potential recipients. In the majority of instances transplantation is from cadaveric donors who have died due

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Fig. 2.12 A Kolff dialysis machine similar to the original machines used for haemodialysis.

100 95 90

LRDT-identical Parent LURT CAD-identical CAD-all

85 80 75 70 65 60 Fig. 2.13 The outcome of 3-year renal graft survival where LRDT = live related donor transplant; LURT = live unrelated donor transplant; and CAD = cadaveric donor. UCLA data of 28,945 patients in the USA.

to a number of reasons. Figure 2.13 shows the outcome for renal transplantation in the USA published in 1995 (Terasaki et al. 1995). The use of initially Azathioprine and Prednisolone achieved useful results. The introduction of more powerful agents such as cyclosporin A and more recently Tacrolimus and Mycophenalate moefitil have enabled the results shown in Fig. 2.13 to be achieved. However, what is clear from these data is that the donor origin is an important determinant of graft outcome. This is especially relevant in inherited renal disease as related donors must be excluded from having renal disease. There is a lack of cadaveric


53

donors and this means that the waiting list for transplantation in the UK increases year by year. The only country in Europe which has not had an increase in its transplant waiting list is Norway. This has been achieved by 40 per cent of transplants occurring from living donors. This compares with the rate for 1998 in the UK of 15 per cent. However with regard to endstage renal failure due to genetic disease this is a very difficult area as the most common donors are first degree relatives. The only group unaffected by potential genetic disease is spousal donors. The figures in Fig. 2.11 show that organ donation from living donors has a better outcome even in genetically unrelated spousal donors with the best figures being in HLA identical siblings. This is obviously a very difficult area for patients with end-sage renal disease due genetic causes. Renal transplantation in spite of the risks of malignancy and opportunistic infection still provides the best replacement of renal function. At the same time, if successful it also provides the longest life expectancy for the individual. Conclusion There are many forms of genetic disease which affect the renal tact and may lead to total failure of excretory function. A few have specific treatments which may help but for the majority of patients the investigations and treatments will be the same as for any renal disease. It is clear that with time the proportion of diseases which have a genetic basis or altered susceptibility on the basis of genetic factors will increase. However the management of most conditions revolves around tight blood pressure control prior to end-stage renal failure and then renal replacement therapy by transplantation.

References Atkinson, D. E. and Bourke, E. (1987). Metabolic apsects of the regulation of systemic pH. American Journal of Physiology, 252, F947–56. Bichet, D. G. (1998). Nephrogenic diabetes insipidus. In Oxford textbook of clinical nephrology (ed. A. M. Davison, J. S. Cameron, J. P. Grunfeld, D. N. S. Kerr, E. Ritz, and C. G. Winerals), Vol. 2, pp. 1095–109. Oxford University Press, Oxford. Broyer, M., Meyrier, A., Niaudat, P., and Habib, R. (1998). Minimal changes and focal segmental gloemruloslerosis. In Oxford textbook of clinical nephrology (ed. A. M. Davison, J. S. Cameron, J. P. Grunfeld, D. N. S. Kerr, E. Ritz, and C. G. Winerals), Vol. 1, pp. 493–536. Oxford University Press, Oxford. Cameron, J. S. (1998). The nephrotic syndrome: management, complications and pathophysiology. In Oxford textbook of clinical nephrology (ed. A. M. Davison, J. S. Cameron, J. P. Grunfeld, D. N. S. Kerr, E. Ritz, and C. G. Winerals), Vol. 1, pp. 461–93. Oxford University Press, Oxford. Cameron, J. S. and Greger, R. (1998). Renal function and testing of function. In Oxford textbook of clinical nephrology (ed. A. M. Davison, J. S. Cameron, J. P. Grunfeld, D. N. S. Kerr, E. Ritz, and C. G. Winerals), Vol. 1, pp. 39–69. Oxford University Press, Oxford. Cameron, J. S. and Rees, A. J. (1998). Crescentic glomerulonephritis. In Oxford textbook of clinical nephrology (ed. A. M. Davison, J. S. Cameron, J. P. Grunfeld, D. N. S. Kerr, E. Ritz, and C. G. Winerals), Vol. 1, pp. 625–46. Oxford University Press, Oxford. Daina, E., Shieppati, A., and Remuzzi, G. (1999). Mycophenolate mofetil for the treament of Takayasu arteritis: report of three cases. Annals of Internal Medicine, 130, 422–6.

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Dworkin, L. D. and Weir, M. R. (2000). Hypertension in renal parenchymal disease: role in progression. In Mechanisms and clinical management of chronic renal failure (ed. A. M. El Nahas, K. P. G. Harris, and S. Anderson), pp. 173–210. Oxford University Press, Oxford. Goldblatt, H., Lynch, J., Hanzal, R. F., and Summerville, W. W. (1934). The production of persistent elevation of systolic blood pressure by means of renal ischemia. Journal of Experimental Medicine, 59, 347–78. Klahr, S. (1983). Pathophysiology of obstructive nephropathy. Kidney International, 27, 690–702. Klahr, S., Levey, A. S., Beck, G. J., et al. (1994). The effects of dietary protein restriction and blood pressure control on the progression of chronic renal disease. New England Journal of Medicine, 330, 877–84. Korbet, S. M. (1998). Primary focal segmental glomerulosclerosis. Journal of American Society of Nephrology, 9, 1333–40. Kurtz, I. (1998). Molecular pathogenesis of Barrter’s syndrome and Gitelman’s syndromes. Kidney International, 54, 1396–1410. Lewis, E. J., Hunsicker, L. G., Bain, R. P., and Rohde, R. D. (1993). The effect of angiotensinconverting-enzyme inhibition on diabetic nephropathy. New England Journal of Medicine, 329, 1456–62. Locatelli, F. and Del Vecchio, L. (2000). Natural history and factors affecting the progression of human renal diseases. In Mechanisms and management of chronic renal failure (ed. A. M. El Nahas, K. P. G. Harris, and S. Anderson), pp. 20–79. Oxford University Press, Oxford. Macdougall, I. C. and Eckardt, K. U. (1998). Haematological disorders. In Oxford textbook of clinical nephrology (ed. A. M. Davison, J. S. Cameron, J. P. Grunfeld, D. N. S. Kerr, E. Ritz, and C. G. Winerals), Vol. 3, pp. 1935–53. Oxford University Press, Oxford. Maisey, M. and Britton, K. E. (1998). Nuclear imaging in nephrology. In Oxford textbook of clinical nephrology (ed. A. M. Davison, J. S. Cameron, J. P. Grunfeld, D. N. S. Kerr, E. Ritz, and C. G. Winerals), Vol. 1, pp. 137–49. Oxford University Press, Oxford. Maschio, G., Alberti, D., Janin, G., Locatelli, F., Mann, J. F., Motolese, M. et al. (1996). Effect of the angiotensin-converting-enzyme inhibitor benazapril on the progression of chronic renal insuffficiency. New England Journal of Medicine, 334, 939–45. Michel, D. M. and Kelly, C. J. (1998). Acute interstitial nephritis. American Journal of Kidney Diseases, 9, 506–15. Parfrey, P. S. and Foley, R. N. (1999). The clinical epidemiology of cardiac disease in chronic renal failure. Journal of American Society of Nephrology, 10, 1606–15. Parving, H. H., Andersen, A. R., Smidt, U. M., and Svendsen, P. A. (1983). Early aggressive antihypertensive treatment reduces the rate of decline in kidney function in diabetic nephropathy. Lancet, 1, 1175–9. Ponticelli, C. and Mihatsch, M. J. (1998). Imbasciata. Renal biopsy: performance and interpretation. In Oxford textbook of clinical nephrology (ed. A. M. Davison, J. S. Cameron, J. P. Grunfeld, D. N. S. Kerr, E. Ritz, and C. G. Winerals), Vol. 1, pp. 157–71. Oxford University Press, Oxford. Scoble, J. E. (2000). The natural history and management of renovascular disease. In Mechanisms of chronic renal failure (ed. A. M. El Nahas, K. P. G. Harris, and S. Anderson), pp. 265–301. Oxford University Press, Oxford. Scoble, J. E. and Cook, G. J. R. (1998). Individual kidney function in atherosclertotic nephropathy. Nephrology, Dialysis and Transplantation, 13, 842–4. Stanley, J. C. (1995). Renal artery fibrodysplasia. In Renal vascular disease (ed. A. Novick, J. Scoble, and G. Hamilton), pp. 21–33. W. B. Saunders, London. Stokes, J. B. (1999). Disorders of the epithelial sodium channel: insights into the regulation of extracellular volume and blood pressure. Kidney International, 56, 2318–33. Terasaki, P. I., Cecka, J. M., Gjertson, D. W., and Takemoto, S. (1995). High survival rates of kidney transplants from spousal and living unrelated donors. New England Journal of Medicine, 333, 333–6.


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Topham, P. S., Harper, S. J., Furness, P. N., Harris, K. P. G., Walls, J., and Feehally, J. (1994). Glomerular disease as a cause of isolated microscopic haematuria. Quarterly Journal of Medicine, 87, 329–35. UK Prospective Diabetes Study Group. (1998). Tight blood pressure control and risk of macrovascular and microvascular complications in type 2 diabetes: UKPDS 38. British Medical Journal, 317, 703–13. Williams, D. G. (1998). Mesangiocapillary glomerulonephritis. In Oxford textbook of clinical nephrology (ed. A. M. Davison, J. S. Cameron, J. P. Grunfeld, D. N. S. Kerr, E. Ritz, and C. G. Winerals), Vol. 1, pp. 591–612. Oxford University Press, Oxford. Yamamoto, T. and Sasaki, S. (1998). Aquaporins in the kidney: emerging new aspects. Kidney International, 54, 10141–51. Zucchelli, P. and Pasquali, S. (1998). Membranous nephropathy. In Oxford textbook of clinical nephrology (ed. A. M. Davison, J. S. Cameron, J. P. Grunfeld, D. N. S. Kerr, E. Ritz, and C. G. Winerals), Vol. 1, pp. 571–90. Oxford University Press, Oxford.


3 Renal development Paul J. D. Winyard

Introduction Mammalian kidneys perform a number of functions that are essential for normal postnatal life including excretion of nitrogenous waste products, homeostasis of water, electrolytes and acid–base balance, and the production of hormones. Mature human kidneys contain around a million nephrons, each consisting of specialized segments including glomerulus, proximal tubule, loop of Henle, and distal tubule, connected to the tree-like collecting duct system and intimately associated with the vascular supply. The precursor of the adult organ, the metanephros, arises in the fifth week of human development and consists of only two cell types: epithelia of the ureteric bud and mesenchyme of the metanephric blastema. Mutual interactions between these two tissues are essential for normal kidney development, or nephrogenesis, which involves precisely coordinated cell proliferation and death, morphogenesis and differentiation. A number of key nephrogenic molecules have recently been identified using descriptive studies of normal and abnormal development and, increasingly, from mice with targeted null mutations which disrupt nephrogenesis. These include the transcription factors PAX2 and WT1, growth factor glial cell line-derived neurotrophic factor (GDNF), and survival factor BCL2. The majority of this chapter is taken up with a review of the cell biology of kidney development, with particular emphasis on these important nephrogenic molecules, after first considering the structure of the mature organ and the anatomy of normal human development.

Structure A brief review of normal mature renal structures is given below. Readers requiring a more comprehensive account should refer to standard anatomical or pathological texts (Ventatachalam and Kriz 1998). Mature human kidneys from adult males are around 11 6 2.5 cm3 in size and weigh up to 170 g. Females have slightly smaller organs. The paired kidneys are located in the retroperitoneum and extend between, but lateral to the twelfth thoracic and third lumbar vertebrae. The renal pelvis lies medially and tapers into the ureter, which connects inferiorly to the bladder. The kidney parenchyma consists of nephrons, collecting ducts, blood vessels, lymphatics, nerves, and interstitium. Each nephron consists of a glomerulus, proximal tubule, loop of Henle, and a distal tubule, which is joined to a collecting duct via the connecting tubule (Fig. 3.1A). All of the glomeruli are located in the cortex, a 1-cm thick strip which forms the

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A

B 1

6 7

2

Cortex

Epithelial cells (podocytes)

7 3

5

2

6 1 3

4

Medulla

4

5

Endothelial cell Fenestrated endothelium

Podocyte foot processes

Basement membrane Fig. 3.1 Schematic of nephron and glomerular structure. A: schematic view of nephron and glomerular structures. A: Short superficial glomerulus (left) and a longer juxtamedullary glomerulus (right), with cortex in the upper part of the panel and medulla in the lower part, separated by the dotted line. Segments are: 1—glomerulus, 2—proximal convoluted tubule, 3—proximal straight tubule, 4—loop of Henle, 5—distal straight tubule, 6—distal convoluted tubule, and 7—connecting tubule. Macula densa in distal straight tubule is shown in black and collecting ducts are shaded grey. B: Glomerulus showing intimate relationship between fenestrated endothelium of endothelial cells, basement membrane, and foot processes of visceral epithelial/podocyte cells. Not drawn to scale.

outermost part of the kidney, whereas other nephron components extend into the medulla towards the centre of the organ. In humans, the cortex is continuous, whereas the medulla consists of around 14 discrete pyramids. This is termed ‘multipapillary’ and contrasts with the ‘unipapillary’ kidneys found in rats and rabbits. Nephrons Glomerulus Human glomeruli are around 200 m in diameter. They consist of mesangial cells and matrix supporting a tuft of capillaries directly surrounded by glomerular basement membrane and visceral epithelial cells (podocytes) separated from the parietal epithelium lining the inner surface of Bowman’s capsule by Bowman’s space (Ventatachalam and Kriz 1998) (Fig. 3.1B). The intimate relationship between the fenestrated capillary endothelium, basement membrane, and podocyte foot processes facilitates glomerular filtration but restricts protein losses in healthy glomeruli. Disruption of any of these components has significant effects on urine composition, as described in Chapters 4–7. Proximal tubule The proximal tubule consists of a convoluted portion, the pars convoluta, followed by a straight portion, the pars recta. Bowman’s space and the lumen of the proximal tubule are continuous, but there is an abrupt transition in cellular phenotype from flattened parietal

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epithelium to tall proximal tubule cells. The latter have a characteristic appearance with a well-developed brush border, to increase the surface area for reabsorption, and numerous mitochondria and lysosomes which reflect their high metabolic rate. Basolateral surface area is also increased by interdigitaiting cytoplasmic protrusions and ridges and this is the major location for NA, K–ATPase activity. The main role of the proximal convoluted tubule is to , PO3), water, and organic solutes such reabsorb minerals, ions (Na, HCO 3 , Cl , K , Ca 4 as glucose and amino acids. Approximately two-thirds of the ultrafiltrate is reabsorbed in the proximal tubule. Loop of Henle The descending thin limb of the loop of Henle arises from the proximal tubule at the junction between the inner and outer stripes, of the outer medulla; loop epithelial cells can be easily distinguished by their flattened appearance and lack of brush border. Some ‘long’ loops descend deep into the medulla and turn back as ascending thin limbs before becoming the thick ascending limb, whereas this transition occurs in the vicinity of the bend in ‘short’ loops. At least four different types of thin limb segments have been described in the rat but there are only a few detailed studies in humans (Kriz 1981). The main role of the loops is to generate an osmolar gradient which facilitates reabsorption of water by the collecting ducts. Distal straight tubule (thick ascending limb of Loop of Henle) The distal straight tubule arises, in humans, as a gradual transition from the thin limb at the junction of the inner and outer medulla; straight tubule epithelial cells are taller and basolateral cell processes interdigitate with neighbouring cells. Rabbits also have a gradual transition, whereas there is a sharp change in the rat kidney. The straight tubule extends into the cortex just beyond the macula densa (described in ‘juxtaglomerular apparatus’ below). The main role of this segment is active, Na, K–ATPase-driven ion transport, particularly sodium chloride via the Na, K, 2Cl co-transporter. Defects in this frusemide sensitive transporter have been implicated in some cases of Bartter syndrome (see Chapter 14) (Guay-Woodford 1998). Distal convoluted tubule Distal convoluted tubules arise in the cortex and the junction of distal straight and convoluted tubules marked by an abrupt increase in epithelial height; cells in the initial segments are otherwise similar to the distal straight tubule, but increasing numbers of intercalated cells are observed in the terminal portion. Connecting tubule The distal tubule is attached to the collecting duct by the connecting tubule. This segment is well defined in the rabbit, with cells which are intermediate in phenotype between distal convoluted tubule and collecting duct cells, but is not so easily distinguishable in other species. Collecting duct Human collecting ducts arise in the cortex as a gradual transition from the connecting tubule and consist of cortical, outer medullary, and inner medullary segments. The initial collecting ducts drain an average of 11 nephrons in humans and successive fusion of these structures generates larger calibre ducts as they pass into the medulla. The largest collecting ducts, the terminal papillary collecting ducts of Bellini, open out into the renal calyces at the papillary tips. Collecting duct epithelia consist of principal and intercalated cells. Principal cells have

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characteristic basal infoldings, rare apical microvilli, and few mitochondria. The apical cytoplasm contains numerous vesicular structures containing aquaporin 2 water channels which increase water permeability by fusing with the apical plasma membrane following vasopressin binding to V2 receptors. Most cases of congenital nephrogenic diabetes insipidus are caused by mutations of these receptors or aquaporin 2 (Bichet 1998; see Chapter 14). Intercalated cells have few, if any, basal infoldings, variable numbers of apical microfolds, and contain dark mitochondria-rich cytoplasm. These cells contain H–ATPase in either the apical or basolateral plasma membranes, and are termed alpha- and beta intercalated cells, respectively. Alpha intercalated cells are responsible for proton secretion whereas beta intercalated cells mediate bicarbonate secretion. Juxtaglomerular apparatus This composite structure comprises the terminal segment of the afferent arteriole and the proximal segment of the efferent arteriole, with the extraglomerular mesangium between them plus the macula densa of the same nephron. The distinctive tall, columnar cells of the macula densa detect sodium chloride delivery to the distal tubule and modulate renin secretion via specialized granular myoepithelial cells in the afferent arteriole. Regulation of glomerular haemodynamics by the juxtaglomerular apparatus is influenced by this local mechanism, along with systemic blood pressure, hormones, and autonomic nerves. Development The process of renal development is conventionally called ‘nephrogenesis’, although this term more correctly refers to differentiation of the nephron tubules alone. The anatomy of normal human nephrogenesis has been described in detail by a number of authors (Kampmeier 1926; Potter 1972), but much less is known about its molecular basis. Paradoxically, recent studies of abnormal development in congenital malformations such as renal dysplasia (Woolf and Winyard 2000) and genetic syndromes (reviewed by Woolf in Chapter 4) have increased our understanding of the normal processes and molecules expressed during human nephrogenesis. There is still, however, significantly more information and a greater understanding of nephrogenesis in mice and other animals because large quantities of embryonic/fetal material are available and it is possible to use a variety of experimental strategies to define the roles of specific genes. This section will therefore consider methods used to study nephrogenesis, basic mechanisms of development, the anatomy of normal nephrogenesis, and molecules expressed during nephrogenesis in both human and non-human species. Methods for studying nephrogenesis There are two complementary ways in which nephrogenesis has been investigated: descriptive studies have traditionally been used to define gross anatomy, but can now also be used to determine the temporal and spatial expression of mRNA and protein, whilst functional studies establish the role of specific genes using in vivo and in vitro methodologies. Descriptive studies Most of the information on human renal development has been derived from descriptive studies since it is difficult to obtain sufficient material for repeated functional studies and

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there are ethical limits on human experiments. In their simplest form, descriptive studies document the gross anatomy and developmental stage of the normal kidney and renal malformations. They also describe patterns of gene expression by defining the distribution of mRNA by in situ hybridization and protein by immunohistochemistry. This may be at a specific timepoint, or at a series of different times/stages. These experiments may identify candidate genes which potentially play an essential role in nephrogenesis, by virtue of their temporal and spatial expression patterns, although animal functional studies are often required to confirm this role. Functional studies in vitro Isolated metanephroi can be grown for several days in organ culture. These organs develop in a morphologically normal manner with serial branching of the ureteric bud and new nephron formation, but the rate of development is slower than normal and glomeruli are avascular (Bernstein et al. 1981). Grobstein used this technique nearly 50 years ago to demonstrate that the mesenchyme and ureteric bud fail to develop when cultured separately, although some nephrons do form when mesenchyme is co-cultured with spinal cord (Grobstein 1955)— hence proving that interactions between the bud and mesenchyme are essential for normal development and that other tissues have the potential to induce normal development. It is possible to manipulate cultured metanephroi by adding exogenous substances to the culture medium. Examples are growth factors, blocking antibodies to growth factors or cell adhesion molecules, and antisense oligonucleotides which block expression of specific genes (Rogers et al. 1992, 1993; Rothenpieler and Dressler 1993). Individual cells have also been labelled using retroviral mediated gene transfer to follow their subsequent fate using in vitro lineage analysis (Herzlinger et al. 1993; Qiao et al. 1995). Cell lines can also be generated from the developing (and mature) kidney. These can be studied in primary culture or transduced with potentially immortalizing constructs such as the SV 40 T antigen to facilitate long-term studies. Cells can then be characterized on the basis of their morphology, membrane constituents, intracellular proteins, synthesis, and release of specific products such as growth factors, and extracellular matrix proteins cells (Burrow and Wilson 1993; Woolf et al. 1995). In this way it is possible to isolate homogeneous populations of cells which have a defined lineage, and these are the ideal populations to assess the response of specific cell types to exogenous factors (as above). Biochemical pathways can also be investigated such as ligand binding, receptor kinetics, and intercellular signalling pathways. Functional studies in vivo Nephrogenesis can be manipulated in vivo in animals using a number of techniques including exposure to teratogens (Gage and Sulik 1991) and obstruction of the developing urinary tract (Beck 1971; Attar et al. 1998). Increasingly, however, transgenic technology is being used to either ablate or overexpress specific molecules. These techniques may well become the standard tools for studying nephrogenesis over the next few years and a detailed description of this technology can be obtained from recent text books and review articles (Rossant and Nagy 1995; Gilbert 1997). A list of null mutant (‘knock out’) mice with abnormal renal development is shown in Table 4.2. Interpretation of functional studies Functional studies must be interpreted with caution since conflicting results can be generated using different techniques. A classic example occurs when organ culture studies implicate a molecule in nephrogenesis in vitro, yet null mutant mice have normal kidney development

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in vivo. There are several possible explanations for this discrepancy. Organ culture, for example may be considered a ‘stressful’ situation which is reliant on molecules that are of little significance in vivo. Alternatively, there may be circulating or maternal factors which compensate for the deficient factor and rescue the phenotype in vivo (Letterio et al. 1994). There may also be functional redundancy in vivo (Sariola 1996) and more than one gene may need to be knocked out to produce a phenotype (Mendelsohn et al. 1994; Davis et al. 1995). It should also be noted that many mice experiments are performed on inbred strains. This may be relevant when extrapolating murine results to out bred human populations since genetic background can influence the final kidney phenotype, suggesting that multiple genes may modulate the same developmental pathway (Threadgill et al. 1995). Anatomy of nephrogenesis There are three pairs of ‘kidneys’ in the mammalian embryo: the pronephros, mesonephros, and metanephros. These arise sequentially from intermediate mesoderm on the dorsal body wall (Larsen 1993). The pronephros and mesonephros degenerate during mammalian fetal life whilst the metanephros develops into the adult kidney. In contrast, the pronephros is the functioning kidney in the adult hagfish and some amphibians whereas the mesonephros is the excretory organ in adult lampreys, some fish, and amphibians. The pronephros It is possible to detect the first evidence of the pronephros at the 10 somite stage on day 22 after fertilization in humans, which is morphologically equivalent to embryonic day 9 in mice. At this stage, it comprises a small group of nephrotomes with segmental condensations, grooves, and vesicles between the second and sixth somites. The nephrotomes are nonfunctional and most likely represent vestiges of the pronephric kidney of lower vertebrates. The pronephric duct develops from the intermediate mesoderm lateral to the notochord (Gilbert 1997) from around the level of the ninth somite. The duct elongates caudally and reaches the cloacal wall on day 26. It is renamed as the mesonephric, or Wolffian duct, as mesonephric tubules develop. The nephrotomes and pronephric part of the duct involute and cannot be identified by day 24 or 25 after fertilization (Gilbert 1997). The mesonephros In humans, the long sausage-shaped mesonephros develops from around 24 days of gestation and consists of the mesonephric duct and adjacent mesonephric tubules (Fig. 3.2A and B). The mesonephric duct begins as a solid rod of cells which canalizes in a caudocranial direction after fusion with the cloaca. Mesonephric tubules develop from intermediate mesoderm medial to the duct by ‘mesenchymal to epithelial’ transformation, a process which is subsequently reiterated during nephron formation in metanephric development. In humans, a total of around 40 mesonephric tubules are produced (several per somite), but the cranial tubules regress at the same time as caudal ones are forming so that there are never more than 30 pairs at any time. Each human mesonephric tubule consists of a medial cup-shaped sac encasing a knot of capillaries, respectively analagous to the Bowman’s capsule and glomerulus of the mature kidney, and a lateral portion in continuity with the mesonephric duct. Other segments of the tubule resemble mature proximal and distal tubules histologically but there is no loop of Henle. The human mesonephros is reported to produce small quantities of urine between

Renal development A

go

63

B

go gl

gl

C

go meso

meta n E

D

l c b b

c F

G

b

c b

s

b b c gl

com

Fig. 3.2 Histology of normal human renal development. Haematoxylin and eosin stained sections of developing human kidneys at A: 38; B–E: 42; F and G: 56 days of gestation. A: Transverse section showing neural tube (n), mesonephroi (enclosed by dotted lines) and gonadal ridges (go). B: High power of mesonephros showing large glomeruli (gl). C: Close anatomical relationship between gonad, mesonephros (meso) and metanephros (meta). D and E: Early metanephros showing central ureteric bud (arrow) with peripheral branches (b) surrounded by condensing mesenchyme (c). F: and G: More mature metanephros showing nephron precursors including comma (com) and S-shaped bodies (s), and an immature glomerulus (gl). Bar (m) corresponds to 500 in A, 250 in C, 150 in D and F, and 60 in the remainder.

weeks 6 and 10 which drains via the mesonephric duct, whereas the murine organ is much more rudimentary and does not contain well-differentiated glomeruli. Mesonephric structures involute during the third month of gestation in humans, although caudal mesonephric tubules contribute to the efferent ducts of the epididymis and the mesonephric duct forms the duct of the epididymis, the seminal vesicle and ejaculatory duct is retained as the vas deferens (Moore and Persaud 1998).

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The metanephros The adult human kidney develops from the metanephros which consists of two cell types at its inception: the epithelial cells of the ureteric bud, and the mesenchyme cells of the metanephric mesenchyme. A series of reciprocal interactions between these tissues cause the ureteric bud to branch sequentially to form the ureter, renal pelvis, calyces, and collecting tubules whilst the mesenchyme undergoes an epithelial conversion to form the nephrons from glomerulus to distal tubule. This process is depicted graphically in Fig. 3.3. In addition, a third cell type, the interstitial cells are also thought to be derived from the mesenchyme.

A

B m u

u

mc

d

C

D c

u

s

gl

Fig. 3.3 Early branching of the ureteric bud and nephron formation. Schematic view with mesonephric duct (d) and ureteric bud (u) shown in black, uninduced mesenchyme (m) in white, and mesenchymal condensates (mc) and nephron precursors, including comma shapes (c), S-shaped bodies (s) and immature glomeruli (gl), in grey. A: In the fifth week of gestation the ureteric bud grows out from the mesonephric duct into metanephric mesenchyme. B: In week 6 the bud branches once and mesenchyme condenses around the ampullae. C: Comma and S-shaped bodies are formed by the eighth week. D: The first glomeruli are formed by the ninth week; further branching of the ureteric bud and mesenchymal condensation continues in the nephrogenic cortex.

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Timing of nephrogenic events In humans, metanephric kidney development begins at day 28 after fertilization when the ureteric bud sprouts from the distal part of the mesonephric duct (Larsen 1993). By day 32, the tip (ampulla) of the bud penetrates a portion of sacral intermediate mesenchyme called the metanephric blastema, and this condenses around the growing ampulla. The first glomeruli form by 8–9 weeks and nephrogenesis continues in the cortex of the fetal kidney until 34 weeks (Potter 1972). Nephrons elongate and continue to differentiate postnatally but no new nephrons are formed. In mice, the ureteric bud enters the metanephric mesenchyme by embryonic day 11.5, the first glomeruli form by embryonic day 14 and nephrogenesis continues for 14 days after birth. The timing of nephrogenesis in humans/mice is shown in Table 3.1. Differentiation of the ureteric bud and its derivatives Branching of the ureteric bud. As the ureteric bud grows into the metanephric blastema it becomes invested with condensed mesenchyme and the ampullary tip begins to divide. This process of growth and branching occurs repeatedly during nephrogenesis, particularly in the outer or nephrogenic cortex where new nephric units are formed until around 34 weeks of human gestation (Potter 1972). It leads to an arborialized (tree-like) collecting duct system connected to nephrons which develop concurrently from mesenchymal condensates adjacent to the ampullary tips (see below). There is great inter-species variation in the number of nephrons which reflects the number of branches of the ureteric bud required to both induce these nephrons and form the collecting ducts to drain urine from them. It has been estimated that it requires 9–10 branching generations in mice to obtain 10–20,000 nephrons and a further 10 generations to give rise to one million nephrons in each human kidney (Ekblom et al. 1994). Formation of the renal pelvis and calyces. The mature collecting ducts drain into minor calyces which in turn connect to the major calyces of the renal pelvis before the ureter. These intervening structures are formed by the coalescence of many of the early branches of the ureteric bud. This concept of remodelling has been recognized since Kampmeier (1926) Table 3.1 Timing of nephrogenic events Structure Pronephros Appears Regresses Mesonephros Appears Regresses Metanephros Renal pelvis Collecting tubules/nephrons Glomeruli Nephrogenesis ceases Length of gestation *Time

Human

Mouse

22 days 25 days

9 days 10 days

24 days 16 weeks 32 days 33 days 44 days 9 weeks 34–36 weeks 40 weeks

10 days 14 days 11.5 days 12.5 days 13 days 14 days 14 days after birth 20 days

of first appearance of renal structures during human and murine nephrogenesis, unless otherwise stated.

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described ‘vestigial’ and ‘provisional’ zones in the human medulla in which nephrons initially developed but subsequently degenerated. The exact number of generations of branches which are remodeled is unknown although Potter (1972) estimated that the first 3–5 generations form the pelvis and the next 3–5 give rise to the minor calyces and papillae. Nephrons which were initially attached to these early branches were described by Potter to either transfer to a later branch or degenerate during development. Differentiation of the mesenchyme and its derivatives. Each nephron develops from mesenchyme adjacent to an ampullary tip of the ureteric bud. The mesenchyme is initially loosely arranged but the cells destined to become nephrons condense around the bud tips and undergo phenotypic transformation into epithelial renal vesicles. Each vesicle elongates to form a comma shape which folds back on itself to become an S-shaped body. The distal portion of the S-shape elongates and differentiates into proximal convoluted tubule, the descending and ascending limbs of the loop of Henle and the distal convoluted tubule, which fuses with the adjacent branch of the ureteric bud to form a continuous functional unit. The proximal S-shape is destined to become the glomerular epithelium and capillaries develop in the glomerular crevice, probably by a process known as vasculogenesis rather than angiogenesis (see below; Woolf and Loughna 1998). The primitive multilayered visceral glomerular epithelium subsequently forms a monolayer of podocytes, separated from glomerular capillary loops by glomerular basement membrane, which is synthesized by both the endothelium and epithelium (Abrahamson and Leardkamolkarn 1991). The first glomeruli are formed by 9 weeks of human gestation, nephron formation ceases around 34 weeks of gestation. Basic mechanisms of development Nephrogenesis involves a balance between key events at a cellular level including proliferation, death, differentiation, and morphogenesis (Table 3.2). Disruption of this balance has been implicated in the pathogenesis of kidney malformations, cystic diseases, and tumours (Winyard et al. 1996a,b; Woolf and Winyard 1998).

Table 3.2 Proliferation, apoptosis, PAX2, WT1, and BCL2 expression in normal developing human kidneys

Undifferentiated mesenchyme Mesenchymal condensates and vesicles S-shaped bodies Glomerular podocytes Tips of ureteric bud (ampullae) Immature collecting ducts Mature collecting ducts

Proliferation

Apoptosis

PAX2

WT1

BCL2

rare

rare

rare rare

rare

This describes the extent of proliferation and apoptosis as assessed by PCNA immunohistochemistry and propidium iodide staining respectively, and the distribution of PAX2, WT1, and BCL2 proteins using immunohistochemistry. ‘’ Indicates not detected/no staining, ‘rare ’ indicates that rare cells were apoptotic/less than 10 per cent of cells showed positive immunostaining and ‘’ to ‘’ indicates increasing levels of apoptosis/increasing percentage of positive cells or intensity of staining.

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Cell proliferation The adult mammalian kidney develops from less than a thousand cells at its inception to many millions in the mature organ and it is self evident that this process requires extensive cell proliferation. The most actively proliferating areas of the metanephros are the tips of the ureteric bud branches and the adjacent mesenchymal cells in the nephrogenic cortex (Fig. 3.4A) (Winyard et al. 1996b). Stem cells are also thought to reside in the undifferentiated renal mesenchyme and it is postulated that these divide to generate a copy of themselves and another cell which differentiates into either nephron epithelia or interstitial cells (Burrow and Wilson 1993). These cells are potentially important in the context of renal malformations since, if they are still present, it may be possible to stimulate elements of normal nephron formation if the appropriate inductive signals can be defined. True stem cells are not considered to be present in the mature adult kidney, although some cells can dedifferentiate after insults such as acute tubular necrosis to regenerate some parts of the nephron (Kawaida et al. 1994).

Fig. 3.4 Proliferation, apoptosis, and protein distribution in normal human renal development. Sections of the nephrogenic cortex from early to mid gestation, counterstained with methyl green or haematoxylin. A and C–E show immunohistochemistry. A: proliferation marker PCNA; B: in situ end labelling; C and D: transcription factors PAX2 and WTI; E: survival factor BCL2. A: Active proliferation in cells at the tips of the ureteric bud (u), renal vesicles (v) and S-shaped bodies (s). B: Apoptosis (arrows) in early nephron precursors such as comma shaped bodies (c). C: Strong PAX2 protein expression in the tips of the ureteric bud (b) and in mesenchymal condensates and vesicles (v); PAX2 expression is not detected in the uninduced mesenchyme. D: Weak WT1 expression in condensates and renal vesicles. WT1 expression is increased as glomerular podocytes are formed (not shown). E: Marked BCL2 expression in condensed mesenchyme, rapidly downregulated by the S-shaped body stage of nephron formation. Bar (m) corresponds to 60 in A–C and 100 in D and E. (See Plate 4 of the Colour Plate Section at the centre of this book.)

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Cell death/apoptosis Kampmeier (1926) reported ‘vestigial’ and ‘provisional’ zones in the human metanephros in which the first three generations of nephrons are destined to die before birth. Potter (1972) also suggested that early nephrons may die unless they moved their attachments to later branches of the ureteric bud. It is only recently, however, that the extent and mechanism of cell death has been investigated: In the rat, Coles et al. (1993) estimated that as many as 50 per cent of the cells which are born in the developing kidney will die by a process known as apoptosis, or programmed cell death. Prominent apoptosis also occurs in human renal development (Winyard et al. 1996a). The major sites of apoptosis are early nephron precursors (comma and S-shaped bodies; Fig. 3.4B) and the medulla, locations in which cell death may be important for morphogenesis and collecting duct remodelling, respectively (see above). Interestingly, therapy with some growth factors, including epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2), reduces renal cell death in both the whole animal and organ culture (Koseki et al. 1992; Coles et al. 1993; Perantoni et al. 1995). This finding is consistent with the possibility that the cells normally die during development because they lack sufficient survival factors. Excess apoptosis has also been reported in congenital renal malformations (Winyard et al. 1996a) which raises the tantalizing possibility that normal development could be rescued, at least in part, by blocking apoptosis. Differentiation The process by which cells acquire a specialized phenotype is known as differentiation. A classic example occurs in the renal mesenchyme where some cells undergo epithelial differentiation to form nephrons while others differentiate into stromal cells, or interstitial fibroblasts. Terminal differentiation occurs at a later stage and includes the specialization of cells within nephron precursors into glomerular parietal and visceral epithelia, proximal tubule, and loop of Henle cells. The term ‘lineage’ describes the series of phenotypes as a precursor differentiates into a mature cell. Morphogenesis The processes outlined above occur in individual cells whereas ‘morphogenesis’ describes the developmental process by which groups of cells acquire complex three-dimensional shapes. Examples include the formation of nephron tubules from renal mesenchymal cells, serial branching of the ureteric bud to form collecting ducts, and capillary formation. Molecular biology of nephrogenesis The molecular biology of nephrogenesis can be reviewed in several ways. One approach is to define critical stages of development and then consider which molecules control these stages. Using this scheme, crucial stages should include, for example, outgrowth of the ureteric bud, initiation of bud branching, elongation of bud derivatives, formation of the renal pelvis, mesenchymal condensation, nephron formation, and terminal differentiation. Several excellent recent reviews have used this format (Davies and Davey 1999; Woolf 1999). An alternative approach is to consider the role of individual molecules over the whole of nephrogenesis. This method is preferred here because it integrates the roles of critical molecules expressed at different stages and it is more accessible to readers with a genetic research background.

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Several classes of molecules are expressed during nephrogenesis, including transcription factors, growth factors, survival factors, and adhesion molecules. These categories are not mutually exclusive since certain molecules fall into two categories, an example being epidermal growth factor which appears to act as both a survival factor for renal mesenchyme (see above) and a growth and differentiation factor. Transcription factors Transcription factors regulate the expression of other genes, acting as ‘master regulators’ to set up basic embryonic pattern formation. They contain DNA binding domains which recognize specific sequences and hence modulate the transcription of mRNA from the target gene. The specific genetic targets of many of the transcription factors are, however, currently unknown. PAX2, WT1, EYA1, HOX, MYC, and BF2 transcription factors are reviewed below, whilst other important factors include EMX2 (Miyamoto et al. 1997), LIM1 (Shawlot and Behringer 1995), SOX9 (Bell et al. 1997), POD1 (Quaggin et al. 1998), and LMX1B (Chen et al. 1998). PAX2 PAX genes are a family of transcription factors with roles in control of embryonic patterning and cell specification in a number of organisms including Drosophila, zebrafish, frog, turtle, chick, mouse, and human (Halder et al. 1995). Only PAX2 and PAX8 are expressed in the developing kidney and there is increasing evidence that PAX2 has a critical role in normal and abnormal renal development particularly during mesenchymal condensation and epithelial transformation (Rothenpieler and Dressler 1993). Little is known about the role of PAX8, in contrast, although it is unlikely to have a major role in nephrogenesis since lack of this molecule perturbs thyroid, rather than kidney development (Mansouri et al. 1998). In the mouse, PAX2 mRNA is expressed in the mesonephric duct, the tips of the ureteric bud and the condensing mesenchyme (Dressler et al. 1990). This pattern is repeated in the outer cortex throughout nephrogenesis. Expression of the protein persists in nephron precursors such as the comma and S-shaped bodies but then decreases as these epithelia mature (Dressler and Douglas 1992). In contrast, there is little expression in ureteric bud derivatives aside from low levels in the collecting ducts. In early development of the human excretory system, PAX2 protein is observed in the mesonephric duct and distal-type mesonephric tubules, and in both the central ureteric bud stalk and peripheral bud tips (Winyard et al. 1996a). Later in human development, the distribution pattern is similar to the mouse, with PAX2-positive cells confined to ureteric bud tips, and condensing mesenchyme, comma, and S-shaped bodies (Fig. 3.4C). A series of experiments in mice and mutation analysis in humans have highlighted the critical importance of PAX2 in renal development and, taken collectively, suggest that this transcription factor has roles in mesenchyme to epithelial transformation and cell proliferation. Mice with decreased levels of PAX2 have aberrant kidney development: heterozygous mutations causes hypoplastic kidneys with reduced branching of the ureteric bud, reduced numbers of nephrons and cortical thinning whilst homozygous null mutants lack mesonephric tubules and the metanephroi fail to form because the ureteric buds are absent (Torres et al. 1995). In organ culture, addition of antisense PAX2 DNA-oligonucleotides leads to repression of PAX2 protein (Dressler et al. 1993) which causes inhibition of mesenchymal condensation and blocks the transition of mesenchyme to epithelium. Interestingly, the mesonephric tubules

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are also formed by mesenchyme to epithelium conversion which implicates PAX2 as an essential molecule in this process (Torres et al. 1995). PAX2 mutations involving a single nucleotide deletion within the conserved octapeptide sequence in exon 5 have been described in the human ‘renal-coloboma’ syndrome, which consists of optic nerve colobomas, renal anomalies, and vesicoureteral reflux (Sanyanusin et al. 1995). A similar mouse mutation (PAX2 (1Neu)) has also been described (Favor et al. 1996). Overexpression of PAX2 causes kidney abnormalities including cystic tubular changes, proteinuria and renal failure in mice (Dressler et al. 1993) and PAX2 is also overexpressed in some highly proliferative human cystic epithelia (Winyard et al. 1996a). Several other lines of evidence linking PAX gene expression and proliferation, including: high levels of PAX2 in renal clear cell carcinoma lines and the decline in rates of proliferation when these cells are treated with PAX2 antisense (Gnarra and Dressler 1995), cell transformation by overexpression of PAX (Maulbecker and Gruss 1993), and persistent expression of PAX genes in human Wilms tumours (Dressler and Douglas 1992). One of the downstream genetic targets of PAX2 during nephrogenesis may be the WT1 transcription factor (see below) since human PAX2 binds to two sites in the WT1 promoter sequence and causes up to a 35-fold increase in expression, as assessed with a reporter gene fused to the PAX2 binding sites on WT1 (McConnell et al. 1997). Interestingly, there is also evidence that WT1 downregulates PAX2, which raises the possibility that PAX2-mediated upregulation of WT1 during nephrogenesis leads to negative feedback and decreased PAX2 expression (Ryan et al. 1995). WT1 WT1 gene, which is mutated in a small percentage of Wilms tumours, encodes a transcription factor protein containing four zinc-finger DNA binding motifs (Pritchard-Jones and Hawkins 1997). Several studies have documented expression of both WT1 mRNA and protein during general mammalian development (Armstrong et al. 1993) and specifically in the urogenital system and Wilms tumours (Pelletier et al. 1991; Winyard et al. 1996a). During early human nephrogenesis, WT1 mRNA is expressed in the mesonephric glomeruli and at low levels in condensing metanephric mesenchyme. As the comma and S-shaped bodies develop, levels of WT1 increase and become restricted to the visceral glomerular epithelia (Winyard et al. 1996a) (Fig. 3.4D). Expression is restricted to podocytes in the adult kidney. Increased expression of WT1 has also been reported in Wilms tumours (Pritchard-Jones et al. 1990). The metanephros does not form in WT1 null mutant mice, and animals die around embryonic day 14 from defects in mesothelial-derived components in the heart and lungs. Small numbers of normal appearing mesonephric tubules form earlier in development but the ureteric bud fails to branch from the Wolffian duct and the intermediate mesoderm, which would normally give rise to the nephrons, dies by apoptosis (Kreidberg et al. 1993). Initial reports suggested that explants of mutant blastema do not form nephrons following heterologous culture with spinal cord and that PAX2 is not expressed in this mesenchyme (Kreidberg et al. 1993). Recently, however, Donovan et al. (1999) have demonstrated that mRNA for PAX2, and several other early markers of induction, are present in mutant WT1 / mesenchyme. This suggests that WT1 is essential for ureteric bud outgrowth and mesenchyme survival during early development, but is not required for initial mesenchymal differentiation. Several human syndromes are associated with WT1 mutations. Denys–Drash syndrome consists of genitourinary abnormalities, including ambiguous genitalia in 46 XY males,

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nephrotic syndrome with mesangial sclerosis leading to renal failure, and a predisposition to Wilms tumour (Little and Wells 1997). This is caused by point mutations of WT1, predominantly affecting the zinc finger DNA-binding domains and there is now a mouse model of this syndrome (Patek et al. 1999). The WAGR syndrome consists of Wilms tumour, aniridia, genitourinary abnormalities including gonadoblastoma, and mental retardation. Frasier syndrome is characterized by focal glomerular sclerosis with progressive renal failure and gonadal dysgenesis. This is caused by intronic point mutations of WT1, which affect the balance between different WT1 splice isoforms (Klamt et al. 1998). WT1 acts as a transcriptional repressor during nephrogenesis and several regulatory targets have been proposed including the insulin-like growth factor axis, early growth response gene 1 (EGR1) (Rackley et al. 1995). Ryan et al. (1995) moreover, demonstrated high-affinity WT1 binding sites in the 5 untranslated sequence of PAX2 and that WT1 represses expression of a reporter gene fused to a fragment of the PAX2 gene including these potential regulatory sequences. This experiment, taken together with the overlapping metanephric expression patterns of PAX2 and WT1, suggests that WT1 may downregulate PAX2 during nephrogenesis. In addition, WT1 may act as a powerful autorepressor since there are multiple WT1 binding sites in its own promoter (Rupprecht et al. 1994). EYA1 EYA1 is the mammalian homologue of the transcriptional co-activator ‘eyes absent’ gene, which is required for normal eye specification in Drosophila. In mice, EYA1 is widely expressed in the ear, branchial arches, and metanephric mesenchyme during development. Homozygote EYA1 null mutant mice have a correspondingly wide variety of abnormalities including craniofacial and skeletal defects, and absent ears (Xu et al. 1999). Affected mice, which die at birth, also lack kidneys because of defective ureteric bud outgrowth leading to failure of metanephric induction and increased mesenchymal apoptosis. Rare, straindependant, renal defects occur in EYA1 / heterozygotes, including hypoplasia and unilateral agenesis. Mutations of the human EYA1 gene occur in 20–25 per cent of patients with branchio-oto-renal syndrome (Abdelhak et al. 1997). This is characterized by a combination of hearing loss, preauricular pits, branchial fistulae, and variable renal anomalies including agenesis, hypoplasia, and dysplasia. HOX Vertebrate HOX genes encode homeodomain transcription factors which specify positional information along the anterior posterior axis. HOXA11 mRNA is normally expressed in metanephric mesenchyme (Hsieh-Li et al. 1995) but individual null mutations of these genes do not have kidney abnormalities. In contrast, double knockouts generated by interbreeding of HOXA11 and HOXD11 mutants have renal agenesis or hypoplasia (Davis et al. 1995). MYC C-myc RNA is expressed in uninduced mesenchyme and in very early epithelial structures in the mouse. Its expression is downregulated as the kidney matures and its expression pattern appears to correlate with areas of cellular proliferation during nephrogenesis (Schmid et al. 1989). N-myc is upregulated during the mesenchyme to epithelium

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conversion, where it appears to be a marker of induction, and expression is confined to the condensates, comma, and S-shaped bodies (Mugrauer and Ekblom 1991). Homozygous nullmutant mice for N-myc have mesonephroi which are hypoplastic, with increased apoptotic cells and the embryos die at around embryonic day 11 (Stanton and Parada 1992). The L-myc is restricted to the ureter of the mature kidney and it is not expressed by proliferating or embryonic cells (Mugrauer and Ekblom 1991). BF2 BF2 is one of the few genes which have been implicated in formation of interstitial cells from the mesenchyme lineage. It is a member of the winged helix family of transcription factors which are related to the Drosophila fork head gene (Lai et al. 1993). In mice, BF2 is expressed in the cells immediately surrounding condensed mesenchyme cells which express PAX2. Mice with null mutations have rudimentary, fused kidneys and die soon after birth (Hatini et al. 1996). Interestingly, the mesenchyme condenses in the null mutant mice but does not develop any further and neither comma nor S-shaped bodies are formed. The ureteric bud also fails to branch normally and RET (see below) is widely distributed in the bud epithelium, rather than confined to the bud tips. It therefore seems likely that BF2 modulates expression of a factor, or factors, from the ‘uninduced’ cells which is essential for both ureteric bud growth and maturation of pretubular aggregates. This factor has not yet been identified. Growth factors/growth factor receptors Growth factors and their cell surface receptors play an important role in nephrogenesis. There are three modes of action: paracrine factors are secreted by one cell and act on neighbouring cells, autocrine factors act on the producing cell, and juxtacrine factors become inserted into the plasma membrane of the producing cell in order to interact with receptors on adjoining cells. The growth factors bind to specific cell surface receptors, mainly receptor tyrosine kinases, which dimerize and become autophosphorylated and transduce signals into the cell. These signals may stimulate many different processes including cell division, cell survival, apoptosis, differentiation, and morphogenesis. The glial cell line-derived neurotrophic factor (GDNF), hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming growth factor (TGF) beta, vascular endothelial growth factor (VEGF), and WNT signalling systems are reviewed below. Readers are referred to alternative sources for accounts of fibroblast growth factors (FGF) (Perantoni et al. 1995; Qiao et al. 1996), insulin-like growth factors (IGF) (Rogers et al. 1991), platelet-derived growth factor (PDGF) (Leveen et al. 1994), TGF alpha (Rogers et al. 1992), and vascular endothelial growth factor (Tufro et al. 1999). GDNF/RET GDNF is the ligand for the receptor tyrosine kinase RET (Vega et al. 1996), although binding also requires the adapter molecule, GDNF receptor (GDNFR). RET is expressed in the mesonephric duct and the ureteric bud arising from it in early development, and then at the branching tips of the ureteric bud throughout metanephrogenesis (Schuchardt et al. 1994). GDNF is expressed by condensing renal mesenchyme adjacent to these bud tips, while GDNFR is expressed in the same cells as RET and, at lower levels, in renal mesenchyme (Towers et al. 1998).

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GDNF and RET are absolutely essential for normal renal development since transgenic mice with homozygous null mutations of either factor fail to develop kidneys or have severe dysplasia (Schuchardt et al. 1994; Pichel et al. 1996). The major postulated role of this signalling system is in the initial outgrowth of the ureteric bud from the mesonephric duct and its subsequent branching; levels of GDNF appear to be critical for this process. Excess GDNF, for example, promotes formation of primary ureteric buds from the mesonephric duct and increases ureteric bud branching (Sainio et al. 1997) and exogenous GDNF stimulates branching morphogenesis of a ureteric bud-derived cell line (Qiao et al. 1999). Neutralizing antibodies against GDNF, in contrast, inhibit branching morphogenesis and decreased ureteric bud branches are observed in GDNF/ heterozygous mice (Vega et al. 1996). A further possible role for GDNF may be modulation of cell survival since GDNF prevents apoptosis of ureteric bud cultured as a monolayer (Towers et al. 1998). Other members of the GDNF family have recently been implicated in collecting duct morphogenesis including persephin (Milbrandt et al. 1998) and neurturin (Davies et al. 1999). HGF/MET Hepatocyte growth factor, or scatter factor as it is also known, is the ligand for the MET receptor tyrosine kinase (Bottaro et al. 1991). Expression patterns of HGF and MET are similar in human and murine nephrogenesis: HGF is expressed in the renal mesenchyme, particularly in the cortex as the kidney matures, whilst MET mainly localizes to developing epithelia (Kolatsi-Joannou et al. 1997; Sonnenberg et al. 1993). Mice with homozygous HGF or MET null mutations die around embryonic day 13–14 with placental, liver, and muscle abnormalities (Schmidt et al. 1995). Early nephrogenesis appears grossly normal in these animals, which is surprising since blockade of this signalling system perturbs metanephric development in organ culture (Santos et al. 1994; Woolf et al. 1995). Exogenous HGF, moreover, causes branching morphogenesis of the Madin Darby Canine Kidney (MDCK) collecting duct-derived cell line in culture (Montesano et al. 1991) and overexpression leads to prominent tubular cystic disease and progressive glomerulosclerosis (Takayama et al. 1997). This system is therefore likely to be important, but not critical, for stimulating cell proliferation and organ growth during nephrogenesis. EGF Epidermal growth factor (EGF) and its embryonic homologue transforming growth factor alpha (TGF) both bind to the epidermal growth factor receptor (EGFR). Rogers et al. (1992), showed that E13 rat metanephroi produce tgf and nephrogenesis is perturbed by blocking antibodies against tgf. EGF is a potent inhibitor of cell death within the developing kidney in vivo (Coles et al. 1993) and rescues isolated renal mesenchyme from apoptosis in vitro (Koseki et al. 1992). EGF has also been reported to increase PAX2 levels in rabbit proximal tubule cells (Liu et al. 1997). Interestingly, mice with null mutations of egf receptors have different renal phenotypes depending on their genetic background (Threadgill et al. 1995). TGF Transforming growth factor beta 1 (TGF1) is the prototypic molecule of a large family of growth factors, and signalling is transduced via cell surface Type I and Type II receptors

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(TGFR1 and TGFR2) (Wrana 1998). In early mouse kidney development, TGF1 mRNA is expressed in the metanephric mesenchyme (Lehnert and Akhurst 1988; Schmid et al. 1991) and the protein is distributed in both the mesenchyme and ureteric bud (Rogers et al. 1993). At similar stages, receptor transcripts are expressed in both renal mesenchyme and immature epithelia (Mariano et al. 1998). Both the ligand and receptors are also expressed in the developing vasculature. Homozygous null mutants of TGF1 have normal kidneys, which is presumed to result from transplacental transfer of maternal circulating growth factor (Letterio et al. 1994), but perturbation of TGF1 in organ culture markedly disrupts nephrogenesis: exogenous TGF1 inhibits tubulogenesis whereas blocking antibodies enhance nephron formation (Rogers et al. 1993). Tubulogenesis of MDCK cells in tissue culture is also inhibited by TGF1 (Sakurai et al. 1997). This suggests a role for TGF1 in controlling nephron number, which is intriguing for several reasons. First, urinary tract obstruction disrupts the programme of nephrogenesis (Attar et al. 1998) and this could potentially be modulated by the upregulation of TGF1 reported in this condition (Medjebeur et al. 1997). Second, this cytokine may modulate PAX2 levels, which is critical for nephrogenesis as described above, since TGF1 decreases PAX2 levels in cultured rabbit proximal tubule cells by decreasing PAX2 mRNA stability (Liu et al. 1997). And finally, our group have recently implicated TGF1 in the pathogenesis of human dysplastic kidneys, which characteristically lack normal nephrons (Yang et al. 2000). Knockout of the closely related TGF2 ligand also causes urogenital abnormalities, although only a limited number of animals were described in the initial report (Sanford et al. 1997). Other members of the TGF superfamily, such as the bone morphogenetic proteins (BMPs) are also important during nephrogenesis. The BMP7 is expressed by the branches of the ureteric bud and is upregulated in primitive nephrons, whilst nephrogenesis is markedly impaired in BMP7 null-mutant mice, which form only a few nephrons and ureteric bud branches (Dudley et al. 1995; Luo et al. 1995). In early development, BMP4 expression is essential for nephric duct formation from intermediate mesoderm (Obara-Ishihara et al. 1999). Later in nephrogenesis, BMP4 is expressed in the non-condensed mesenchyme around the stalk of the ureteric bud, heterozygote BMP4/ knockouts develop hypoplastic or dysplastic kidneys and up- or downregulation of the BMP4 axis modulates ureteric bud elongation/growth in organ culture (Miyazaki et al. 1999).

WNT The WNT gene family, which consists of around 20 members, encodes secreted signalling molecules and several of these have been implicated in nephrogenesis. WNT4 is upregulated in renal mesenchymal cells as they differentiate and nephrogenesis is ‘frozen’ at the condensate stage in WNT4 null mutant mice (Stark et al. 1994). WNT4 is, moreover, sufficient to trigger tubulogenesis in isolated metanephric mesenchyme, in a process which is dependent on cell contact and sulphated glycosaminoglycans (Kispert et al. 1998). Isolated dorsal spinal cord also induces tubulogenesis and it may act by mimicking the normal mesenchymal action of WNT4 since spinal cord mediated induction still occurs with WNT4 mutant mesenchyme (Kispert et al. 1998). The WNT11 is expressed at the tips of the ureteric bud (Lako et al. 1998), but is not sufficient to induce tubulogenesis (Kispert et al. 1998). The WNT1-transfected

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fibroblasts induce nephron formation when co-cultured with isolated renal mesenchyme, although WNT1 is not an endogenous metanephric molecule (Herzlinger et al. 1994). Survival/proliferation factors Survival and proliferation are key cellular events and a number of genes have been identified in renal development which are linked to these processes. BCL2 The BCL2 gene, originally discovered in human follicular B-cell lymphomas, is the prototypic member of an evolutionarily conserved gene family involved in the control of apoptotic cell death (Knudson and Korsmeyer 1997). BCL2 is gene expressed in a wide variety of tissues during both murine and human development including the nervous system, immunological tissues, and epithelia (LeBrun et al. 1993). In the kidney, BCL2 protein is upregulated in the mesenchyme as it condenses around the ureteric bud tips (Fig. 3.4E) but is then rapidly down regulated in the comma and S-shaped bodies and is barely detectable in the adult organ (LeBrun et al. 1993; Winyard et al. 1996a). Homozygous BCL2 null mutants have a number of defects in haematopoiesis, hair, and kidney development. BCL2/ kidneys were initially reported to resemble polycystic kidneys with epithelial hyperproliferation and dilation of proximal and distal tubular segments (Veis et al. 1994). Further analysis, however, suggests that the abnormalities are more complex: prior to birth ‘fulminant’ apoptosis leads to small hypoplastic kidneys, with fewer nephrons and smaller nephrogenic zones (Sorenson et al. 1995), whilst cysts develop postnatally accompanied by increased proliferation in both cortex and medulla and apoptosis of interstitial cells (Sorenson et al. 1996). Hence, primary deregulation of cell survival in the BCL2 null mutant mice appears to cause a secondary upregulation of proliferation in the kidney. p53 Mutations of p53 lead to tumours in humans and it is therefore thought to act as a tumour suppresser gene. The p53 mRNA is normally expressed in comma and S-shaped bodies in the mouse but homozygous deletion of p53 has no affect on murine renal development (Donehower et al. 1995). In contrast, overexpression of p53 leads to smaller kidneys with fewer nephrons which is thought to be caused by incomplete mesenchymal differentiation (Godley et al. 1996). p57-KIP2 (CDKN1c) Cyclin-dependent kinases are essential for regulation of the cell cycle and proliferation. Cyclin kinase inhibitors such as p57-KIP2 block proliferation by binding to the kinases in G1/S phase. The p57-kip2 gene is expressed in podocytes in glomeruli and stromal cells between the renal tubules during renal development and null mutants have fewer renal tubules and small inner medullary pyramids (Zhang et al. 1997). Poorly formed medullary pyramids are also seen in human Beckwith–Wiedemann syndrome which is caused by loss of the imprinted, expressed maternal allele on chromosome 11p15.5. This site is close to the p57KIP2 gene and heterozygous mutations have been reported in some Beckwith–Wiedemann patients. The mechanism for aberrant development in the null mutant mice has not been

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reported but it seems logical that cell proliferation will be more prominent in null mutants and this may perturb apoptotic remodelling of the medulla.

Cell adhesion molecules Adhesion molecules mediate two forms of attachment, namely cell–cell and cell–matrix adhesion. Examples of cell–cell adhesion proteins are the calcium independent neural cell adhesion molecule (NCAM) (Klein et al. 1988a; Bellairs et al. 1995) and calcium-dependent E-cadherin (also known as uvomorulin) (Vestweber and Kemler 1985). Molecules involved in cell–matrix adhesion include the collagens, fibronectin (Bellairs et al. 1995), galectin 3 (Bao and Hughes 1995; Winyard et al. 1997), KAL (Duke et al. 1995; Soussi-Yanicostas et al. 1996), laminins (Klein et al. 1988b), nidogen (Ekblom et al. 1994), tenascin (Aufderheide et al. 1987), and integrin cell surface receptors (Kreidberg et al. 1996; Muller et al. 1997). Adhesion molecules may also have additional roles since some molecules, particularly proteoglycans, are able to sequester growth factors, hence modulating binding of growth factors to their receptors (Vainio et al. 1989a). It is difficult to separate the specific roles of most individual adhesion molecules so a general overview is given here, followed by a more detailed description of integrins, galectin 3 and KAL. Wallner et al. (1998) have also recently reviewed this topic. Overview Uninduced metanephrogenic mesenchyme expresses collagen I, III, and fibronectin in the mouse (Ekblom et al. 1981). The cell surface neural cell adhesion molecule (NCAM) and the proteoglycan syndecan, which acts as a receptor for interstitial matrix molecules are also expressed by the uninduced mesenchyme (Klein et al. 1988a; Vainio et al. 1989b). Following condensation, the mesenchymal cells begin their transition to polarized epithelia. Levels of syndecan increase transiently but then decrease, NCAM decreases and cells stop expressing interstitial collagens and fibronectin and begin to express uvomorulin and collagen type IV, and laminin A chain (Ekblom 1981; Ekblom et al. 1981; Vestweber and Kemler 1985; Klein et al. 1988a; Vainio et al. 1989b). Laminin is a large multidomain cruciform glycoprotein found in basement membranes, consisting of three different polypeptide chains: A, B1, and B2. Uninduced metanephric mesenchyme expresses B1 and B2 whilst the laminin A chain is expressed at the onset of epithelial polarization. Mesenchymal cells that do not undergo epithelial transformation continue to express interstitial collagens and fibronectin and upregulate tenascin as the cells around them are condensing (Aufderheide et al. 1987). Tubule formation is perturbed in mouse metanephric organ culture by antibodies to fragments E3 and E8 of the laminin A chain, because the mesenchymal cells are unable to convert to polarized epithelial cells (Klein et al. 1988b). The cell-surface receptor for the E8 fragment of laminin is the 61integrin, described below. The laminin E3 fragment binds the dystroglycan complex and antibodies that interfere with this binding also inhibit the conversion of mesenchyme to epithelia (Durbeej et al. 1995). Another basement membrane glycoprotein known as either nidogen or entactin is produced by mesenchymal cells and binds to domain III on the laminin B2 chain. This laminin B2/nidogen binding is critical for the production of epithelial basement membranes of epithelial structures formed during early nephrogenesis (Ekblom et al. 1994).

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Proteoglycans, such as heparan and chondroitin sulphate, bind/sequester diverse factors and are crucial for normal nephrogenesis (Davies et al. 1995). Proteoglycans, for example, are postulated to bind the N-glycosylated KAL protein mutated in X-linked Kallmann syndrome (Soussi-Yanicostas et al. 1996). KAL is expressed in the cortex during nephrogenesis (Duke et al. 1995) and up to 40 per cent of Kallmann syndrome patients have renal agenesis (Kirk et al. 1994). Heparan sulfate binds fibroblast growth factors, hence preventing their degradation and facilitating receptor binding (Kiefer et al. 1990), syndecan binds basic fibrobast growth factor (FGF2) (Elenius et al. 1992), and betaglycan binds TGF (Ruoslahti and Yamaguchi 1991). Integrins Integrins are transmembrane heterodimeric glycoprotein complexes consisting of and chains with diverse roles in cell–cell and cell–matrix adhesion, polarity, migration, and angiogenesis (Wallner et al. 1998). Specific ligand binding is determined by the particular combination of and chains. Integrin subunits expressed during nephrogenesis include: 1 in uninduced mesenchyme (Korhonen et al. 1990a), 2 in glomerular endothelium and distal tubules (Korhonen et al. 1990a), 3 in maturing podocytes (Ekblom 1996), 6 and 8 in the mesenchyme undergoing condensation and epithelial transformation adjacent to the ureteric bud (Falk et al. 1996; Muller et al. 1997), 1 in undifferentiated mesenchyme and glomerular endothelial cells, 3 in Bowman’s capsule and 4 in fetal collecting ducts (Korhonen et al. 1990b). Co-expression of different subunits includes 11 and 41 in uninduced mesenchyme, 21 in endothelia, and 61 in epithelia. Homozygous integrin mutant mice have a variety of defects but 3 and 8 knockouts have marked disruption of kidney development. Mice with mutations in the 3 subunit have very abnormal glomeruli containing wide capillaries, disorganized basement membrane and aberrant podocytes foot processes, plus microcystic proximal tubules and decreased branching of the medullary collecting ducts, hence suggesting roles for 3 in basement membrane organization and branching morphogenesis (Kreidberg et al. 1996). Other integrins have also been functionally implicated in tubulogenesis: blocking antibodies to the 61integrin, which is the receptor for the E8 fragment of laminin, perturb tubule formation in vitro (Sorokin et al. 1990; Falk et al. 1996) and antisense to the 2 subunit of the 21 integrin perturb cystogenesis and HGF-induced tubulogenesis of MDCK cells cultured in three dimensional collagen type I gels (Saelman et al. 1995). Mice lacking the 8 subunit exhibit profound kidney deficits with aberrant branching of the ureteric bud and formation of nephrons, which suggests, in conjunction with the expression data (Muller et al. 1997), that this molecule is essential for mesenchymal–epithelial transformation (Kreidberg et al. 1996). Aberrant expression of 1, 2, and 6 integrin subunits has also been described in human dysplastic kidneys (Daikha-Dahmane et al. 1997) Galectin 3 Galectin 3, or Mac 2 as it was formerly known, is a calcium-independent water soluble -galactoside binding lectin with a number of postulated roles in pre-mRNA splicing, oncogenic transformation, and prevention of apoptosis (Hughes 1994). Its major role, however, is likely to be in cell–matrix interactions: it binds embryonic glycoforms of laminin and fibronectin which have polylactosamine side chains and modulates laminin/integrin interactions (Sato and Hughes 1992).

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Galectin 3 is expressed in the ureteric bud lineage during human nephrogenesis (Winyard et al. 1997), but subcellular localization changes from an apical position in the actively branching tips of the ureteric bud to the basolateral domain in maturing collecting ducts. Two pieces of evidence suggest that galectin 3 may have an important role in the latter location, possibly via its interaction with laminin. First, in human cystic kidney diseases, galectin 3 is mislocalized to the apical domain (Winyard et al. 1997). The protein is unable to interact with the extracellular matrix in this location which, along with other potential roles in proliferation and in preventing apoptosis, may contribute to cyst expansion. And second, in three dimensional cell culture, MDCK cyst growth is influenced by galectin 3 expression: blocking antibodies increase cyst growth and exogenous galectin-3 slows cyst growth (Bao and Hughes 1995). These results are intriguing, but it should be noted that the kidneys appear normal in the galectin 3 knockout (Colnot et al. 1998), which suggests that other molecules must be able to compensate for lack of galectin 3 in vivo. Other molecules There are a number of functionally important metanephric molecules which do not fit comfortably into any of the categories outlined above. These include the renin–angiotensin system, described below; cyclo-oxygenase2 (cox2) an enzyme involved in synthesis of prostaglandins (Morham et al. 1995); formins, molecules of uncertain function without which the embryonic kidneys fail to form (Maas et al. 1994); mpv17, a peroxisomal protein implicated in the integrity of the glomerular filtration barrier; retinoic-acid receptors (RAR), which are important in morphogenesis (Mendelsohn et al. 1994). Renin–angiotensin system The renin–angiotensin system has an important role in renal development. Renin, which converts angiotensinogen to angiotensin I, is widely expressed in perivascular cells in the arterial system during early nephrogenesis, but is restricted to the juxtaglomerular apparatus in the mature kidney (Gomez et al. 1988). Angiotensin II, generated from angiotensin I by angiotensin converting enzyme, binds to two types of G protein-coupled receptors, AT1 and AT2. AT1 mediates the majority of the traditionally recognized functions of angiotensin II such as vasoconstriction and stimulation of cell growth, while AT2 antagonizes some of these actions and has postulated roles in the control of apoptosis. Both receptors are expressed in the developing kidney: AT1 is expressed in S-shaped bodies, developing tubules and mature glomeruli whereas AT2 is restricted to mesenchymal cells, initially around the stalk of the ureteric bud but then extending to just outside the nephrogenic cortex, and cells between collecting ducts (Kakuchi et al. 1995). This distribution of AT2 receptors corresponds to areas with high levels of apoptosis. The wide spectrum of renal/urological abnormalities that result from targeted mutation of different components of the renin–angiotensin system are discussed in Chapter 4 by Woolf (Kidney and Lower urinary tract malformations) and were recently reviewed by Pope et al. (1999). New issues Cell lineage In conventional descriptions of nephrogenesis, there is a very clear distinction between nephrons, which are said to be derived from the metanephric mesenchyme, and collecting

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ducts, which reportedly arise from the ureteric bud. Several reports of in vitro lineage tracing experiments by Herzlinger, Qiao and colleagues suggest, however, that renal cell lineages may be more complex (Herzlinger et al. 1993; Qiao et al. 1995). They isolated pure populations of mesenchymal cells or ureteric bud epithelia from embryonic day 13 rat embryos, tagged them with lacZ and co-cultured these with non-tagged ureteric buds and mesenchyme respectively. Most cells differentiated along conventional lines but some mesenchymal cells were recruited into collecting system epithelia (Qiao et al. 1995) and a small proportion of ureteric bud cells underwent an epithelial–mesenchymal transition to form part of the metanephric blastema (Herzlinger et al. 1993). There are several other examples of epithelial to mesenchymal transformations during general development, including neural crest and endocardial cushion formation from neuroepithelium and cardiac endothelia respectively (Gilbert 1997), but these were the first reports of this process in renal development. This may become particularly relevant in the future because there is a growing body of evidence that epithelial to mesenchymal transdifferentiation is a critical event in both human kidney malformations (Yang et al. 2000) and the pathogenesis of end stage renal disease (Okada et al. 1996). Vascular development Embryonic blood vessels arise by either vasculogenesis, in which mesenchyme differentiates in situ to form capillary endothelia, or angiogenesis, which involves ingrowth of existing capillaries (Woolf and Loughna 1998). Renal capillaries were initially hypothesized to arise by angiogenesis, based on experiments showing that the glomeruli that develop in organ culture are avascular (Bernstein et al. 1981) and capillary loops formed when mouse metanephroi were grafted onto avian chorioallantoic membranes are of host origin (Sariola et al. 1983). This hypothesis has been challenged, however, by grafting experiments into the anterior eye chamber and under the capsule of neonatal mouse kidneys where glomerular endothelia are derived from the donor (Hyink et al. 1996; Loughna et al. 1997). Further support for vasculogenesis comes from recent reports that molecules which characterize endothelial precursors, such as the receptors for VEGF, are expressed by non vascularized uninduced metanephric mesenchyme and cell lines derived from this tissue (Loughna et al. 1997) and the formation of cellular masses with molecular characteristics of vessels, albeit with poorly defined lumina, in hypoxic organ culture (Loughna et al. 1998). These results suggest that vasculogenesis definitely occurs during capillary formation in the metanephros, although they do not completely exclude formation of some vascular components by angiogenesis.

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McConnell, M. J., Cunliffe, H. E., Chua, L. J., Ward, T. A., and Eccles, M. R. (1997). Differential regulation of the human Wilms tumour suppressor gene (WT1) promoter by two isoforms of PAX2. Oncogene, 14(22), 2689–700. Medjebeur, A. A., Bussieres, L., Gasser, B., Gimonet, V., and Laborde, K. (1997). Experimental bilateral urinary obstruction in foetal sheep: transforming growth factor-beta 1 expression. American Journal of Physiology, 273(3, Pt 2), F372–F379. Mendelsohn, C., Lohnes, D., Decimo, D., Lufkin, T., LeMeur, M., Chambon, P., et al. (1994). Function of the retinoic acid receptors (RAR) during development. Development, 120, 2749–71. Milbrandt, J., de Sauvage, F. J., Fahrner, T. J., Baloh, R. H., Leitner, M. L., Tansey, M. G., et al. (1998). Persephin, a novel neurotrophic factor related to GDNF and neurturin. Neuron, 20(2), 245–53. Miyamoto, N., Yoshida, M., Kuratani, S., Matsuo, I., and Aizawa, S. (1997). Defects of urogenital development in mice lacking Emx2. Development, 124(9), 1653–64. Miyazaki, Y., Oshima, K., Kume, T., Dunn, R., Fogo, A., Hogan, B. L. M., et al. (1999). Ontogeny of the congenital anomalies of the kidney and urinary tract (CAKUT): Incomplete elongation of the branching ureter leads to hypo-dysplastic kidney in heterozygous Bmp4 knockouts (Bmp4/). American Journal of Nephrology, 10(408A), (Abstract). Montesano, R., Schaller, G., and Orci, L. (1991). Induction of epithelial tubular morphogenesis in vitro by fibroblast-derived soluble factors. Cell, 66, 697–711. Moore, K. L. and Persaud, T. V. N. (1998). The developing human. Clinically oriented embryology. WB Saunders, Philadelphia, Pennsylvania. Morham, S. G., Lanhenbach, R., Loftin, C. D., Tiano, H. F., Vouloumanos, N., Jennette, J. C., et al. (1995). Prostaglandin synthase 2 gene disruption cuases severe renal pathology in the mouse. Cell, 83, 473–82. Mugrauer, G. and Ekblom, P. (1991). Contrasting expression patterns of three members of the myc family of protooncogenes in the developing and adult mouse kidney. Journal of Cell Biology, 112(1), 13–25. Muller, U., Wang, D., Denda, S., Meneses, J. J., Pedersen, R. A., and Reichardt, L. F. (1997). Integrin alpha8beta1 is critically important for epithelial-mesenchymal interactions during kidney morphogenesis. Cell, 88(5), 603–13. Obara-Ishihara, T., Kuhlman, J., Niswander, L., and Herzlinger, D. (1999). The surface ectoderm is essential for nephric duct formation in intermediate mesoderm. Development, 126(6), 1103–8. Okada, H., Strutz, F., Danoff, T. M., and Neilson, E. G. (1996). Possible pathogenesis of renal fibrosis. Kidney International Supplement, 54, S37–S38. Patek, C. E., Little, M. H., Fleming, S., Miles, C., Charlieu, J. P., Clarke, A. R., et al. (1999). A zinc finger truncation of murine WT1 results in the characteristic urogenital abnormalities of Denys–Drash syndrome. Proceedings of the National Academy of Sciences, USA, 96(6), 2931–6. Pelletier, J., Bruening, W., Kashtan, C. E., Mauer, S. M., Manivel, J. C., Striegel, J. E., et al. (1991). Germline mutations in the Wilms’ tumor suppressor gene are associated with abnormal urogenital development in Denys–Drash syndrome. Cell, 67(2), 437–47. Perantoni, A. O., Dove, L. F., and Karavanova, I. (1995). Basic fibroblast growth factor can mediate the early inductive events in renal development. Proceedings of the National Academy of Sciences, USA, 92(10), 4696–700. Pichel, J. G., Shen, L., Sheng, H. Z., Granholm, A. C., Drago, J., Grinberg, A., et al. (1996). GDNF is required for kidney development and enteric innervation. Cold Spring Harbor Symposia on Quantitative Biology, 61, 445–57. Pope, J. C., Brock, J. W., III, Adams, M. C., Stephens, F. D., and Ichikawa, I. (1999). How they begin and how they end: classic and new theories for the development and deterioration of congenital anomalies of the kidney and urinary tract, CAKUT. Journal of the American Society of Nephrology, 10(9), 2018–28.

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4 Kidney and lower urinary tract malformations Adrian S. Woolf

Introduction Congenital malformations of the kidney and lower urinary tract are common causes of chronic renal failure in infants and young children. In animal experiments, the main causes of such malformations are mutations, chemical and pharmaceutical teratogens, physical obstruction of fetal urinary flow and alterations of maternal nutrition. The focus of this chapter is to consider how mutations alter gene expression during development to cause human urinary tract malformations. Some of these disorders are associated with congenital anomalies in multiple organ systems and two such syndromes are considered in detail: first, the renalcoloboma syndrome where mutations of the PAX2 transcription factor cause partial failure of urinary tract growth; second, Kallmann syndrome where mutations of the KAL gene coding for a cell signaling molecule called anosmin-1 are associated with absence of the urinary tract. In most patients, however, kidney and lower urinary tract malformations occur in isolation and in some of these individuals a genetic pathogenesis is strongly suggested by a positive family history supported by positive linkage studies: one common example is primary vesicoureteric reflux which is said to affect 1–2 per cent of very young children. Apparently sporadic kidney and lower urinary tract malformations have recently been shown to be associated with polymorphisms of genes expressed during development. In the long-term, an understanding of the genetic aspects of human urinary tract malformations could help to unravel the pathogenesis of these disorders and may facilitate the design of genetic screening tests with a view to early diagnosis. Normal development of the kidney and lower urinary tract The mammalian kidney derives from two tissue compartments of the embryonic metanephros: the ureteric bud, an epithelium which branches recurrently to form the collecting ducts, and the renal mesenchyme, which undergoes an epithelial transformation to form nephron components including glomerular and proximal tubule epithelia (Woolf 1999a; Risdon and Woolf 1998). The human metanephros appears at 5 weeks gestation and the first layer of glomeruli form by 9 weeks. Branching and nephrogenesis continue to occur in the outer rim of the kidney, the nephrogenic cortex, until 34 weeks whereas further maturation, in the form of growth and differentiation, continues postnatally. Recent histological studies using detailed measurements suggest that about two-thirds of nephrons are generated in the last third of human gestation, and that the range of nephrons found in a healthy kidney is

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rather large, about 0.5 106–1.5 106 (Hinchliffe et al. 1991, 1992b, 1993). The ureteric bud also gives rise to the epithelium of the renal pelvis, the ureter and the bladder trigone, also called the urothelium. The remainder of the bladder epithelium is derived from endoderm and both ureter and bladder become enveloped in mesodermal-derived smooth muscle. A more detailed description of these events is provided in Chapter 3. During the second half of human gestation fetal urine is the main component of amniotic fluid and is thought to be important for fetal lung growth. However, the production of urine before birth is not essential for fetal survival since the placenta effectively performs both hemodialysis and gas exchange during gestation. The spectrum of human kidney and urinary tract malformations As might be expected for such a complex pattern of development, these processes are often perturbed (Bernstein et al. 1997; Risdon and Woolf 1998), making kidney and lower urinary tract malformations relatively common (Table 4.1). Furthermore, since the development of the ureteric bud and renal mesenchyme depend on mutually inductive interactions (Woolf 1999a; Vainio and Muller 1997), it is not surprising that a primary defect of either component will affect the development of its partner. This provides one explanation for the clinical observation that an abnormal insertion of the ureter into the bladder is associated with congenital renal parenchymal anomalies (Vermillion and Heale 1973; Schwartz et al. 1981). Human urinary tract malformations are phenotypically diverse but have in common a failure of normal development before birth (Table 4.1). For instance, in agenesis the kidney and ureter are absent, whereas dysplastic kidneys contain undifferentiated tissues and are often Table 4.1 The spectrum of kidney and lower urinary tract malformations Upper urinary tract Renal agenesis: kidney is absent Renal dysplasia: kidney contains undifferentiated tissues and may be tiny (aplasia) or distended by cysts (multicystic and cystic dysplastic kidneys) Renal hypoplasia: kidney contains formed nephrons but significantly fewer than normal—when nephrons are large the condition is called oligomeganephronia Duplex kidney: the organ is separated into an upper part, which is often dysplastic and attached to an obstructed ureter, and a lower part, which is attached to a refluxing ureter Horseshoe kidney: both kidneys have become fused during development and may include dysplastic components Lower urinary tract Agenesis: the ureter and bladder trigone is absent Hydronephrosis: the renal pelvis is distended and the parenchyma may be hypoplastic or dysplastic—the ureter may be refluxing or obstructed at the level of the pelvi-ureteric or uretero-vesical junction Duplication of the ureter: may occur in association with a duplex kidney Vesicoureteric reflux: urine flows retrogradely from the bladder into the ureter, pelvis and medullary collecting ducts of the kidney Posterior urethral valves: the outflow to the urinary bladder is anatomically obstructed Some forms of upper and lower urinary tract malformations. The following incidences have been quoted: duplex ureter, 1/100; vesicoureteric reflux, 1/100; horseshoe kidney, 1/200; unilateral renal agenesis, 1/500–1/1000; unilateral multicystic kidney, 1/5000. Bilateral agenesis/dysplasia occurs in 1/5000–1/10,000 births.

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attached to structurally abnormal lower urinary tracts. Other variations are listed in Table 4.1. Any bilateral renal malformation can be associated with oligohydramnios, oligoanuria and lung hypoplasia, and this is sometimes called the ‘Potter sequence’ after the fetal pathologist, Edith Potter. The definition of malformation can be extended to microscopic abnormalities such as ‘tubular dysgenesis’ where proximal tubules form abnormally. Although some types of polycystic kidney diseases can affect infants and children (Bernstein et al. 1997; Risdon and Woolf 1998), cysts in these disorders generally arise after the period of nephron formation and these diseases are considered in full elsewhere in this book. The clinical impact of human kidney and lower urinary tract malformations With the increasing use of fetal ultrasound scanning, more individuals with congenital urinary tract malformation are being diagnosed (Anderson et al. 1997; Hiraoka et al. 1997; Yeung et al. 1997; Jaswon et al. 1999). In fact, these disorders account for about 30 per cent of all prenatally diagnosed anomalies (Noia et al. 1996). Although many appear to have minimal clinical significance, for the more serious anomalies such as bilateral renal agenesis or dysplasia, early diagnosis allows consideration of termination or active therapeutic intervention. The latter includes fetal decompression of obstructed urinary tracts as well as planning for the need for dialysis soon after birth (Noia et al. 1996). In these severe cases, accompanying lung hypoplasia can also be life threatening after birth. Severe kidney and urinary tract malformations are the main cause of chronic renal failure in young children (Ehrich et al. 1992; Drozdz et al. 1998). For example, in our own clinical service at Great Ormond Street Hospital for Children in London, UK, malformations account for over 80 per cent of children under 5 years of age with end-stage renal failure. With advances in technology, babies with minimal renal function can be dialysed from birth and infants can receive kidney transplants from the age of 1 year. These strategies, together with general improvements in the care of children with chronic renal failure will ultimately mean an increase in long-term survival into the adult period for these individuals. Clues from animal studies of kidney development The explosion of knowledge regarding the mechanisms of murine kidney development is relevant to our understanding of human malformations (Vainio and Muller 1997; Woolf and Winyard 1998). Clifford Grobstein (1967) was the first to demonstrate that mouse metanephric mesenchyme and ureteric bud failed to differentiate when cultured separately but they formed nephrons and collecting ducts when recombined. Using genetically engineered mice and organ culture, it is now established that nephrogenesis is controlled by diverse molecules, some of which may act as inductive signals postulated by Grobstein. These molecules include transcription factors, growth factors, and adhesion molecules which are essential for control of cell survival, proliferation, differentiation, and morphogenesis during normal mammalian development. ●

Transcription factors include BF2 (Hatini et al. 1996), EMX2 (Miyamoto et al. 1997), EYA1 (Johnson et al. 1999; Xu et al. 1999), hepatocyte nuclear factors (HNFs) (Lazzaro et al. 1992), HOXa11 and HOXd11 (Davis et al. 1995), LIM1 (Shawlot and Behringer 1995), LMX1B (Chen et al. 1998), N-MYC (Stanton et al. 1992), PAX2 (Torres et al. 1995), POD-1 (Quaggin et al. 1998) and WT1 (Kreidberg et al. 1993).

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Growth factors with positive effects include epidermal growth factor, bone morphogenetic protein 7 (Dudley et al. 1995), fibroblast growth factors (FGFs) 2 (Perantoni et al. 1995) and FGF 7 (Qiao et al. 1996), glial cell line-derived neurotrophic factor (Schuchardt et al. 1994; Sanchez et al. 1996; Towers et al. 1998), hepatocyte growth factor (Woolf et al. 1995), insulin-like growth factors (IGFs) I and II (Rogers et al. 1991), platelet-derived growth factor (Leveen et al. 1994), transforming growth factor (TGF) (Rogers et al. 1992), vascular endothelial growth factor (Loughna et al. 1997) and WNT4 (Stark et al. 1994). Inhibitory ‘growth’ factors include activin (Rivtos et al. 1995), leukaemia inhibition factor (Bard and Ross 1991), tumor necrosis factor (Cale et al. 1998) and TGF (Rogers et al. 1993). Cell adhesion molecules include laminins (Klein et al. 1988; Noakes et al. 1995) as well as integrins (Muller et al. 1997; Kreidberg et al. 1996). Diverse other molecules include survival factors (e.g. BCL2) (Veis et al. 1994; Sorenson et al. 1995), retinoic acid receptors (RARs) (Mendelsohn et al. 1994), enzymes (e.g. COX2) (Morham et al. 1995; Dinchuk et al. 1995) and molecules with unknown specific functions (e.g. formins; Maas et al. 1994).

Table 4.2 Molecules involved in nephrogenesis in null mutant mice Transcription factors BF2: small, fused and undifferentiated kidneys (Hatini et al. 1996) EYA11: absent kidneys (Johnson et al. 1999; Xu et al. 1999) EMX2: absent kidneys (Miyamoto et al. 1997) HOXa11 / HOXd112: small or absent kidneys (Davis et al. 1995) LIM1: absent kidneys (Shawlot and Behringer 1995) LMX1B1: malformed glomeruli (Chen et al. 1998) N-MYC: poorly developed mesonephric kidneys (Stanton et al. 1992) PAX21,3: small or absent kidneys (Torres et al. 1995) WT11: absent kidneys (Kreidberg et al. 1993) Growth factors and receptors EGF receptor: cystic collecting ducts (Threadgill et al. 1995) BMP7: undifferentiated kidneys (Dudley et al. 1995) GDNF3 and its receptor, RET: small or absent kidneys (Schuchardt et al. 1994; Sanchez et al. 1996) PDGF B: absent mesangial cells (Leveen et al. 1994) WNT4: undifferentiated kidneys (Stark et al. 1994) Adhesion molecules and receptors 3 integrin: decreased collecting duct branching (Kreidberg et al. 1996) 8 integrin: impaired ureteric bud branching and nephron formation (Muller et al. 1997) s-laminin/laminin 2: nephrotic syndrome (Noakes et al. 1995) Miscellaneous molecules BCL2: small kidneys (Veis et al. 1994; Sorenson et al. 1995) COX2: small kidneys (Dinchuk et al. 1995; Morham et al. 1995) Formin: absent kidneys (Maas et al. 1994) Neuronal nitric oxide synthase: bladder outflow obstruction (Burnett et al. 1997) RAR/22: small or absent kidneys (Mendelsohn et al. 1994) Unless otherwise stated, malformations only occur in homozygous null mutants. 1Mutations of these genes have also been implicated in malformations of the human kidney or lower urinary tract (see Table 4.4). 2Null mutation of two homologous genes is required to cause malformation. 3Heterozygous mutations also produce kidney or lower urinary tract malformations.

Table 4.2 summarizes some of the genes known to affect nephrogenesis based on analyses of null mutant mice. As with human disease, the resulting malformations are agenesis, dysplasia, and hypoplasia. These respective phenotypes represent defects in regulation of metanephric formation, epithelial morphogenesis, and nephron numbers. In addition, overexpression of


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either PAX2 (Dressler et al. 1993) or FGF7 (Nguyen et al. 1997) leads to cyst formation suggesting a role for these genes in epithelial overgrowth. These experiments show that mouse embryonic kidneys have a limited range of responses to gene inactivation or overactivity. Mutant mice also demonstrate that: ii(i) the urinary tract malformation can result from mutation of different genes (e.g. null mutation of either WTI or PAX2 transcription factor genes causes renal agenesis) (Kreidberg et al. 1993; Torres et al. 1995); i(ii) the presence and severity of the urinary tract malformation is often dependent on the genetic background of the animal (Threadgill et al. 1995); (iii) in some instances, more than one related gene must be mutated before development is perturbed (Mendelsohn et al. 1994; Davis et al. 1995). (iv) mouse ‘nephrogenesis genes’ are often also expressed in other organs where they are also critical for development for example PAX2 mutants have renal and ocular defects while WT1 / mice have a syndrome of kidney, gonadal, and cardiothoracic anomalies. Teratogenic and physical causes of kidney and lower urinary tract malformations Based in animal experiments, the major influences which perturb kidney and lower urinary tract development comprise mutations, chemical and pharmacological teratogens, urinary tract obstruction, and maternal undernutrition. The focus of this chapter is on how mutations alter gene activity and the normal program of development to generate disease. The reader is referred to recent reviews, which cover in detail the biology of the other areas (MerletBenichou et al. 1997; Chevalier 1999). Several teratogens implicated in the pathogenesis of kidney and lower urinary tract malformations are listed in Table 4.3. It is important to realize that teratogens may often act by altering the expression of genes during development (Brown 1997), as demonstrated by these three examples. 1. The use of angiotensin converting enzyme inhibitors to treat hypertension during pregnancy can cause neonatal renal failure which may have a hemodynamic basis or be associated with a lesion which resembles renal tubular dysgenesis: this can be associated with a skull malformation called hypocalvaria (Pryde et al. 1993; Barr 1994; Sedman et al. 1995). Of note, a strikingly similar disorder occurs in the absence of teratogen exposure (McFadden et al. 1997; Milunsku et al. 1997), and perhaps these individuals harbor mutations which affect angiotensin action. Certainly, animal experiments point to the importance of angiotensin action during normal kidney and urinary tract development (see below). 2. Evidence of teratogens affecting gene expression is provided by the increased incidence of kidney and lower urinary tract malformations in the offspring of mothers with diabetes (Novak and Robinson 1994; Lynch and Wright 1997). There is experimental evidence that exposure of murine embryonic kidneys to glucose alters the expression of a basement membrane component called s-laminin (Abrass et al. 1997). 3. Vitamin A and its retinoic acid derivatives provide another example. In humans and animals, high doses have been asscoaited with severe malformations such as renal agenesis,

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Adrian S. Woolf sometimes associated with ‘caudal regression’, the reduction of distal structures such as the sacrum and hindlimbs (Von Lennep et al. 1985; Rothman et al. 1995; Padmanabhan 1998). It has been reported that retinoic acid, a vitamin A derivative, can induce renal malformations in mice accompanied by fulminant apoptosis of renal mesenchyme and a downregulation of WT1 transcription factor gene expression (Tse 1998). The vitamin A story is further complicated because there is convincing animal evidence that too little may also result in failure of fetal kidney growth, perhaps mediated by impaired ureteric bud branching and downregulation of glial cell line derived neurotrophic factor receptor expression (Vilar et al. 1996; Lelievre-Pegorier et al. 1998).

Although it is unusual to elicit a history of teratogen exposure, it remains possible that occult or low-grade exposure is important. For example, the incidence of diverse malformations increases with a daily intake of vitamin A above levels as low as 10,000 IU; this includes a weak association with urogenital defects (Rothman et al. 1995). Another example of a relatively subtle environmental change which can have significant effects on development of the kidney is the effect of a low protein diet during pregnancy which in animals can lead to reduced numbers of nephrons (renal hypoplasia) and hypertension postnatally (Gilbert et al. 1990; Lucas et al. 1997). Similar effects have been postulated to occur in humans (Hinchliffe et al. 1992b; Rossing et al. 1995). In fact, it is interesting to speculate that dietary influences may explain part of the normal variation of nephron numbers found in healthy kidneys (Hinchliffe et al. 1991, 1992b, 1993). A significant minority of kidney malformations in girls, and about half of malformations in boys, are associated with obstructed urinary tracts (Peters 1997). Kidneys associated with obstruction in early gestation are usually dysplastic whereas obstruction in the last third of gestation is associated with hydronephrosis and subcortical cysts. Below, we describe how fetal urinary tract obstruction in humans and experimental animals can alter cell proliferation in developing kidney epithelia accompanied by alteration of the expression of a transcription factor called PAX2. Human malformation syndromes affecting the kidney and lower urinary tract In some cases, the kidney and lower urinary tract disorder occurs in association with a multi-organ malformation syndrome that commonly affects the central nervous, cardiovascular and skeletal systems. A brief list is shown in Table 4.3 and reader is also referred to other Table 4.3 Some teratogens of the kidney and lower urinary tract Angiotensin converting enzyme inhibitors (Fogo et al. 19902; Pryde et al. 19931; Barr 19941; Sedman et al. 19951; Tufro-McReddie et al. 19952; McCausland et al. 19972) Cocaine (Battin et al. 19951) Corticosteroids (Hulton and Kaplan 19951; Celsi et al. 19982) Ethanol (Gage and Sulik 19912; Taylor et al. 19941; Boehm et al. 19972; Moore et al. 19971) Gentamicin (Gilbert et al. 19912; Hulton and Kaplan 19951) Glucose (Novak and Robinson 19941; Abrass et al. 19972; Lynch and Wright 19971) Non-steriodal anti-inflammatory drugs (Voyer et al. 19941) Vitamin A and its derivatives (Von Lennep et al. 19851; Rothman et al. 19951; Padmanabhan 19982) 1Teratogen 2Teratogen

demonstrated in humans. demonstrated in animal experiments.


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references (McKusick 1992; Woolf and Winyard 1998) and to the constantly updated version of Online Mendelian Inheritance in Man (http:www4.ncbi.nlm.nih.gov/Omim/). Although malformation syndromes are individually rare they collectively account for considerable morbidity. Some are associated with gross chromosomal anomalies such as trisomies, 4p-syndrome and dup (10p)/del (10q) (Egli and Stalder 1973; Kulharya et al. 1993; Benjamin and al-Harbi 1997; Gilbert-Barness 1998), but cytogenetic aberrations are absent in most cases of urinary tract malformation syndromes. Other renal malformation syndromes are inherited in Mendelian patterns and in several, of them disease loci and mutations have been defined (Table 4.4). Table 4.4 Genes and human kidney and lower urinary tract malformation syndromes Apert syndrome (FGFR2 mutation—growth factor receptor) hydronephrosis and duplicated renal pelvis (Cohen and Kreiborg 1993; Wilkie et al. 1996) Bardet–Biedl syndrome1 (at least 5 loci: 11q13, 16q22, 3p13, 15q21 and 2q31): renal dysplasia and calyceal malformation (Harnett et al. 1988; Beales et al. 1999) Beckwith–Wiedemann syndrome1 (in a minority of patients, 557KIP2 mutation—cell cycle gene) renal overgrowth, cysts and dyplasia) (Choyke et al. 1999; Lam et al. 1999) Branchio-oto-renal syndrome (EYA12 mutation—transcription factor: renal agenesis and dysplasia (Konig et al. 1994; Abdelhak et al. 1997) Campomelic dysplasia (SOX9 mutation—transcription factor): diverse renal malformations (Houston et al. 1983; Wagner et al. 1994) Carnitine palmitoyltransferase II deficiency3: renal dysplasia (North et al. 1995) CHARGE association3: diverse urinary tract anomalies (Regan et al. 1999) Denys–Drash syndrome (WT12 mutation—transcription/splicing factor): calyceal defects and mesangial cell sclerosis (Jadresic et al. 1990; Coppes et al. 1993) Di George syndrome1 (locus at 22q11): renal agenesis, dysplasia and vesicoureteric reflux (Budarf et al. 1995; Czarnecki et al. 1998; Stewart et al. 1999) Glutaric aciduria type II3: cystic and dysplastic disease (Wilson et al. 1989) Fanconi anaemia (FAA mutation—DNA repair): renal agenesis, ectopic/horseshoe kidney (Lo Ten Foe et al. 1996) Kallmann Syndrome (KAL mutation—cell signaling molecule): renal agenesis (Franco et al. 1991; Legouis et al. 1991; Duke et al. 1998) Maturity onset diabetes of the young/renal malformation syndrome (HNF1 mutation—transcription factor): renal dysplasia and cysts (Bingham et al. 2000) Meckel syndrome1 (locus at 17q21–q24): cystic renal dysplasia (Salonen 1984; Paavola et al. 1995) Nail-patella syndrome (LMX1B2 mutation—transcription factor): malformation of the glomerulus and renal agenesis (Gubler and Levy 1993; Haga et al. 1997; Dreyer et al. 1998) Renal-coloboma syndrome (PAX22 mutation—transcription factor): renal hypoplasia and vesicoureteric reflux (Sanyanusin et al. 1995a,b) Simpson–Golabi–Behmel syndrome (GPC3 mutation—proteoglycan): renal overgrowth, cysts and dysplasia (Hughes-Benzie et al. 1992; Pilia et al. 1996) Smith–Lemli–Opitz syndrome1 (locus at 7q32—defect in cholesterol biosynthesis): cystic dysplastic kidneys (Akl et al. 1977; Wallace et al. 1994) VACTERL/VATER association3 (one patient with mitochondial gene mutation): diverse urinary tract anomalies (Damian et al. 1996) Zellweger syndrome (peroxisomal protein mutation): cystic dysplastic kidneys (Powers and Moser 1998; Shimozawa et al. 1992) 1Locus

known but gene undefined. of these genes have also been implicated in malformations of the murine kidney or lower urinary tract (see Table 4.4). 3Putative genetic basis in some cases. 2Mutations

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In the remainder of this chapter we firstly detail two malformation syndromes which act as striking examples where advances in genetics have illuminated the pathogenesis of abnormal urinary tract development. Secondly, we consider whether non-syndromic human urinary tract anomalies may have a genetic basis.

The renal coloboma syndrome—mutation of the PAX2 transcription factor gene As renal mesenchymal cells begin to differentiate into nephron tubules, they aggregate into condensates and undergo a burst of proliferation with increased expression of PAX2 transcription factor (Eccles et al. 1992; Winyard et al. 1996a). At the same time, the termini of the adjacent ureteric bud branches express the same gene and are highly proliferative. Later in nephrogenesis both proliferation and PAX2 expression are downregulated. Urinary tract growth is impaired in mice with inactivating PAX2 mutations (Keller et al. 1994; Torres et al. 1995; Favor et al. 1996). Null-mutants lack kidneys and ureters because the ureteric bud fails to branch from the mesonephric duct, while an associated absence of Fallopian tubes in female embryos is explained by the expression of PAX2 in Mullerian duct development. Haploinsufficiency (a partial lack of functional protein) in heterozygous PAX2 / mice is associated with renal hypoplasia and, in some cases, hydronephrosis and hydroureter, consistent with the presence of vesicoureteric reflux. The human renal-coloboma syndrome has a striking phenotypic similarity to PAX2 / mice. It comprises blindness due to an optic nerve malformation, the coloboma, with vesicoureteric reflux and hypoplastic kidneys. Sanyanusin et al. (1995a) described heterozygous mutations of PAX2 (chromosome 10q24–q25) in one kindred, and reports of other affected individuals followed (Sanyanusin et al. 1995a,b; Narahara et al. 1997; Schimmenti et al. 1997). The eye disease is explained by PAX2 expression during ocular development and it is notable that PAX2 / mutant mice also have eye disease (Keller et al. 1994; Favor et al. 1996). The first-reported human mutation was found to have arisen de novo with subsequent inheritance in a dominant manner (Sanyanusin et al. 1995a). It most likely caused haploinsufficiency because the mutated gene coded for a protein with an intact DNA-binding paired box, an interrupted octapepide domain, and a novel, truncated, carboxy terminus which was postulated to perturb the transactivation of target genes. Homozygous PAX2 mutations have yet to be described in humans, although kindreds with kidney and oviduct and uterus malformations (Battin et al. 1993; Woolf 1999b) superficially resemble mouse null-mutants (Torres et al. 1995). Of interest, in humans, mutations of other members of the PAX gene family cause Waardenburg syndromes (PAX3) and aniridia (PAX6) (Read 1995), although these do not have renal components, reflecting the organ-specific activities of the respective genes. The search for mutations of PAX2 in human, non-syndromic vesicoureteric reflux is discussed below. Thus, in mice and humans, PAX2 deficiency causes impairment of urinary tract growth, but what are the effects of PAX2 overexpression? PAX2 is expressed in human and experimental murine Wilms tumors (Dressler and Douglas 1992; Eccles et al. 1992; Sharma et al. 1994) and also in human renal carcinoma (Gnarra and Dressler 1995) where experimental ablation of PAX2 activity by antisense oligonucleotides reduced cell proliferation of renal carcinoma lines in vitro (Gnarra and Dressler 1995). Indeed, PAX2 transforms murine cells and inhibits


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the promoter of p53, a tumor suppressor (Maulbecker and Gruss 1993; Stuart et al. 1995) and transgenic overexpression of PAX2 generates renal cysts in mice (Dressler et al. 1993). Microdissection studies by Edith Potter revealed that most human dysplastic kidney tubules are malformed branching structures, which often terminate in cysts (Bernstein et al. 1997). Human dysplastic epithelia in organs from terminations express PAX2 as well as BCL2, a cell survival molecule (Veis et al. 1994; Sorenson et al. 1995), and galectin-3, a molecule implicated in epithelial growth (Winyard et al. 1996a, 1997). Strikingly, dysplastic organs harvested postnatally show persistent patterns of fetal epithelial gene expression whereas normal organs downregulate proliferation, PAX2 and BCL2 in mature epithelia (Winyard et al. 1996a, 1997). Cystic dysplastic epithelia have a high rate of proliferation, as assessed by expression of proliferating cell nuclear antigen, hence explaining why some of these renal malformations grow to distend the abdomen of infants. It is therefore notable that tumors occasionally arise in multicystic kidneys (Barrett and Winel 1980; Cromie et al. 1980; Homsy et al. 1997) and these dysplastic organs may harbor nephrogenic blastema and perilobular rests, the latter being considered a Wilms tumor precursor which also express PAX2 (Eccles et al. 1992). Conversely, apoptosis is dominant in mesenchyme around dysplastic epithelia (Winyard et al. 1996b), a compartment which has low PAX2 and BCL2 expression (Winyard et al. 1996a; Granata et al. 1997). Since multicystic dysplastic kidneys are usually associated with an ‘atretic’ ureter, obstruction early in gestation has been invoked as a cause of the malformation and, in this context, it is interesting to note that renal cyst formation associated with epithelial hyperprolifation and PAX2 overexpression can be induced by 10 days of mid-gestation experimental ureteric obstruction in sheep (Attar et al. 1998). One explanation for this effect is that increased hydrostatic pressure caused by urinary flow impairment may stretch the metanephric epithelia, hence triggering PAX2 expression and hyperproliferation. In fact, it can be demonstrated that increased pressure is associated with proliferation in vitro in cysts from Madin Darby Canine Kidney cells, a line derived from the ureteric bud/collecting duct lineage (Tanner et al. 1995).

Kallmann syndrome—mutation of KAL, a gene coding for a cell signaling molecule X-linked Kallmann syndrome is caused by mutations of KAL (also called KALIG1 and AMDXL), a gene located on the short arm of the X chromosome (Xp22.3) (Franco et al. 1991; Legouis et al. 1991). Anosmia and hypogonadotrophic hypogonadism occur in affected males because of defective prenatal migration of olfactory and gonadotrophin-releasing hormone neurons from the nasal placode into the forebrain: moreover, the olfactory bulb fails to grow and is hypoplastic (Schwanzel-Fukuda et al. 1989). It has been estimated that the syndrome affects 2 per cent of hypogonadal males (Pawlowitzki et al. 1987). Other, less constant, features include mirror movements of the hands, pes cavus, high arched palate, and cerebellar ataxia. The significance of this syndrome to our current discussion is that it constitutes a human genetic model of renal agenesis: this absent kidney is usually unilateral, resulting in a solitary functioning kidney. Although renal malformations were not noted in the ‘definitive’ report of the syndrome (Kallmann et al. 1944), Hardelin et al. (1992) documented renal agenesis in association

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with a stop mutation in the KAL gene and Kirk et al. (1994) subsequently established that 37 per cent of 17 patients had a solitary kidney as assessed by 99mTechnetium– dimercaptosuccinic acid scanning to delineate renal parenchyma. This study also documented a male infant with the Potter sequence compatible with bilateral renal agenesis, giving a total incidence of 40 per cent for renal anomalies. On occasion, other urinary tract anomalies occur including duplex systems, hydronephrosis, vesicoureteric reflux and multicystic dysplastic kidneys (Wegnenke et al. 1975; Zenteno et al. 1999; Woolf AS personal observation). Autopsy studies of Kallmann syndrome kidneys are rare although Ishida (1980) confirmed renal agenesis in one report. Patients with urinary tract agenesis also lack the vas deferens, a structure derived from the mesonephric duct which also gives rise to the ureter and collecting ducts. Female KAL mutation carriers have partial or complete anosmia but renal anomalies have not been reported in these individuals. However, urinary tract anomalies do occur in females with the rare autosomal form of the syndrome (Levy and Knudtzon 1993). We studied the seven X-linked Kallmann syndrome patients aged 22–35 years who were known to have solitary kidneys (Duke et al. 1998). Two had proteinuria with arterial hypertension and one of these patients developed chronic renal failure in the second decade. The five remaining patients had normal plasma creatinine concentrations without excess microalbuminuria although four had borderline hypertension. In one kindred, an uncle with Kallmann syndrome had chronic renal failure, but the details of his nephropathy were unavailable for review. In two sets of patients with single kidneys from the same kindreds, there was striking temporal discordance for occurrence of proteinuria and renal impairment. Perhaps these patients with solitary kidney and renal impairment have ‘hyperfiltration damage’, a decline in function of too few glomeruli which have hypertrophied and sclerosed (Brenner et al. 1997). Although renal histology in Kallmann syndrome is unknown, glomerulosclerosis can occur in other individuals with non-syndromic congenital solitary kidneys, a condition affecting 1/1000 of the general population (Kiprov et al. 1982; Argueso et al. 1992; Woolf 1997) and in oligomeganephronic renal hypoplasia (Nomura and Osawa 1990; Broyer et al. 1997). Alternatively, renal damage might be caused by exposure to an abnormal lower urinary tract because a Kallmann syndrome patient reported by Duke et al. (1998) had focal renal scarring compatible with vesicoureteric reflux and Wegenke et al. (1975) reported an X-linked case with a solitary but duplex renal tract: the upper renal moiety was attached to a dilated ureter, while the lower part connected to a separate ureter with vesicoureteric reflux. When KAL is expressed in vitro, the protein (also called anosmin-1) appears on the cell surface and in the culture medium: hence KAL may be a secreted signaling molecule (SoussiYanicostas et al. 1996). The predicted protein has four fibronectin type III repeats and homology to neural cell adhesion molecule, suggesting adhesive roles, and also a ‘four disulfide core motif’ with homology to antiproteinases. Recent evidence confirms an adhesive role for neurites in culture (Soussi-Yanicostas et al. 1998), although similar experiments have yet to be reported for epithelia from the urinary tract. In addition, the KAL gene is expressed in the developing central nervous and excretory systems (Duke et al. 1995). At 11 weeks of human gestation KAL mRNA localized to the olfactory bulb, supporting the hypothesis that KAL enables migrating neurons to enter the brain and synapse at this site. KAL transcripts were detected in the human mesonephros and metanephros at 6 weeks gestation and in the metanephros at 11 weeks, supporting the contention that KAL is directly involved in nephrogenesis. More recently, Hardelin et al. (1999) reported that KAL protein immunolocalized to


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basement membranes of the mesonephric duct and its collecting duct branch derivatives. Collectively, the data are consistent with a role for KAL protein in mediating cell adhesion during growth of the ureteric bud lineage. Certainly, other adhesion molecules have been considered critical for normal nephrogenesis in murine models (Klein et al. 1988; Noakes et al. 1995; Kreidberg et al. 1996; Muller et al. 1997). Presently, there are no published data to establish whether the gene is expressed in the adult kidney but we speculate that, if KAL was also expressed in mature kidney, mutations might cause progressive renal disease as well as kidney malformations. In this context, the WT1 transcription factor is expressed in mature podocytes and in renal mesenchyme: heterozygous WT1 mutations cause glomerulosclerosis in the Denys Drash (Coppes et al. 1993) and Frasier syndromes (Barbaux et al. 1997) whereas homozygous null-mutations cause renal agenesis in mice (Kreidberg et al. 1993). Numerous questions remain concerning renal disease in Kallmann syndrome. First, why are only some patients affected by a malformation, even in one kindred? Here, modifying genes can be invoked, as well-described in mutant mice with renal malformations (Mendelsohn et al. 1994; Davis et al. 1995; Threadgill et al. 1995). A parallel situation is established in the added effect of PKD1 and TSC2 mutations which determine the severity of polycystic kidney disease (Brook-Carter et al. 1994). With respect to the hypothesis that interacting mutations may be required for the generation of urinary tract agenesis, Colquhoun-Kerr et al. (1999) reported a family with X-linked Kallmann syndrome confirmed by mutation analysis, some of whom also had urinary tract agenesis: conversely, other members of the kindred had renal agenesis but did not have classical features of the Kallmann syndrome nor did they harbor KAL mutations. Second, why is the urinary tract malformation in Kallmann syndrome usually unilateral? Perhaps, in order to adversely affect nephrogenesis, there needs to be a second perturbation in nephrogenic precursor cells: this ‘second hit’ could be a somatic mutation in another nephrogenesis gene. Certainly, renal somatic mutations do contribute to cystogenesis in autosomal dominant polycystic kidney disease (Qian et al. 1996) and renal tumor formation (Maher 1996). Finally, yet other factors must determine renal morbidity in patients with a single kidney, based on the temporal discordance of proteinuria and renal impairment in individuals from a single kindred. Here, again there are parallel examples from animal experiments such as the Os mouse in which the kidney has significantly fewer nephrons than normal: however, the subsequent development of glomerulosclerosis is affected by the genetic background of particular strains (He et al. 1996).

Genetics of non-syndromal human urinary tract malformations The examples of the renal coloboma and Kallmann syndromes, as well as other congenital syndromes with a genetic basis (Woolf and Winyard 1998), establish beyond doubt that urinary tract malformations can be caused by mutations. Is there any evidence that mutations have a pathogenetic role in the non-syndromic urinary tract anomalies more commonly encountered by nephrologists and urologists? It is common clinical experience that most cases of renal agenesis and dysplasia appear to be sporadic. However, in 1974 Cain et al. (1974) cited 12 reports of familial renal malformations and described a kindred with two siblings: the first with bilateral renal agenesis and the second with unilateral agenesis and contralateral dysplasia. Others reported that first degree relatives of patients with bilateral urinary tract

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agenesis had a tenfold higher incidence of solitary kidney compared with controls (Roodhooft et al. 1984). In fact, there is considerable anecdotal evidence which establishes the possibility of the genetic basis of diverse other urinary tract malformations which are not associated with multiorgan syndromes. These include kindreds with renal agenesis (Rizza and Downing 1971; Carter et al. 1979; Arfeen et al. 1993), renal aplasia and dysplasia (McPherson et al. 1987; Murugasu et al. 1991), multicystic dysplastic kidney (Moazin et al. 1997; Murakami et al. 1992), oligomeganephronic renal hypoplasia (Moerman et al. 1984), renal tubular dysgenesis (McFadden et al. 1997), pelviureteric junction obstruction (Izquierdo et al. 1992), vesicoureteric reflux (Eccles et al. 1996; Feather et al. 1996), and posterior urethral valves (Farrar and Pryor 1976). Is there a kidney and lower urinary tract malformation locus on chromosome 6? An observation made by numerous investigators has been that different types of urinary tract malformation can occur in the same kindred (Atwell 1985; Squiers et al. 1987; Kobayashi et al. 1995). This is compatible with the hypothesis that specific mutations can potentially affect the development of the whole urinary tract but that the final phenotype depends on modifying factors, either genetic or environmental, which vary from individual to individual. In support of such a contention, Devriendt and Fryns (1995) proposed that there existed a genetic locus on the short arm of chromosome 6 for multicystic renal dysplasia, pelviureteral juction stenosis, and vesicoureteric reflux. Their evidence was twofold. First, Izquierdo et al. (1992) and Macintosh et al. (1989) had respectively provided linkage of hydronephrosis and vesicoureteric reflux families to HLA markers on 6p. Second, a fetal termination with bilateral multicystic dysplastic kidneys was found to have a t(6 : 19)(p21;q13.1), a translocation which Groenen et al. (1996 and 1998) determined to interrupt the CDC5L gene on chromosome 6 and the upstream stimulator factor 2 gene on chromosome 19. Transcripts for both genes were widely expressed in adult tissues including kidney, while other evidence implicates both genes in cell cycle control. However, CDC5L mutations could not be identified in 10 fetuses with severe bilateral urinary tract malformations and there was no linkage of polymorphic markers near the CDC5L gene in a large family with vesicoureteric reflux (Groenen 1999). Hence, while there is some evidence that 6p harbors a urinary tract malformation locus, the jury is out regarding the genes involved. Angiotensin II receptor 2 gene and kidney and lower urinary tract malformations Considerable evidence from animal experiments suggests that angiotensin is important for urinary tract development. For example, mice engineered to lack either angiotensin II type 1A and 1B (AT1) receptor genes or the angiotensinogen gene fail to develop a normal renal medulla and have delayed glomerular maturation (Tsuchida et al. 1998). Similar effects can be elicited by pharmacological inhibition of angiotensin II activity in rodents during early postnatal life (Fogo et al. 1990; Tufro-McReddie et al. 1995; McCausland et al. 1997). In addition, null mutations of the angiotensin II Type II receptor gene (AT2) located on the


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X chromosome, lead to a spectrum of urinary tract malformations in male mice including renal dysplasia, hypoplasia, hydronephrosis and vesicoureteric reflux (Nishimura et al. 1999). The biological basis for the AT2 phenotype is thought to be delayed apoptosis of mesenchymal cells in the fetal kidney and lower urinary tract (Yamada et al. 1996; Li et al. 1998; Nishimura et al. 1999). However, the fact that the malformation phenotype was variable in mice with the null mutation, together with the fact that other null mutants have no urinary tract malformation at all (Iichiki et al. 1995) again argues that the final phenotype is strongly dependent on modifying genes or non-genetic factors. Nishimura et al. (1999) reported an association of a polymorphism of intron one of the AT2 gene (the A–1332G transition) which perturbed AT2 mRNA splicing efficiency in USA and European patients with multicystic dysplastic kidneys and/or pelviureteric obstruction: the polymorphism occurred in 19–42 per cent of normal subjects and 74–77 per cent of patients. Subsequently, Hohenfellner et al. (1999a) reported an association of this polymorphism with a malformation called obstructive megaureter. Apart from a direct biological effect of this genetic change, an alternative explanation for the association with urinary tract malformations would be that the AT2 polymorphism may be in linkage dysequilibrium with a separate, causative, mutation of a nearby gene. A final explanation for the apparent association is that the analysis was confounded by patients and controls being drawn from separate populations with different genotypes. Also of note are the human malformations associated with use of angiotensin converting enzyme inhibitors (see above). In addition, human renal dysplasia can on occasion be associated with high plasma renin activity and systemic hypertension (Worck et al. 1995; Webb et al. 1997) and Yang et al. (1999) immunolocalized renin to dysplastic mesenchyme, while AT1 protein was detected in undifferentiated mesenchyme and dysplastic epithelia of dysplastic kidneys, and low levels of AT2 mRNA could be detected by RT-PCR of whole organs.

Genetic studies in primary, non-syndromic vesicoureteric reflux Vesicoureteric reflux describes the passage of urine from the bladder into the upper urinary tract. It can be secondary to bladder outlet obstruction and can occur as part of a multiorgan syndrome such as that associated with PAX2 mutation. Much more commonly, however, it is primary and non-syndromic and systematic screening with cystograms suggests that the disorder affects 1–2 per cent of the Caucasian population (Woolf, 1999). Although there is no experimental evidence that reflux in the absence of obstruction is harmful to the kidney (Ransley et al. 1984), associated renal parenchymal disease, or ‘reflux nephropathy’, causes up to 15 per cent of chronic renal failure in children and adults (Bailey 1993). The nephropathy is likely to have two main aetiologies. First, the passage of infected urine into the renal parenchyma via compound papillae can lead to acute pyelonephritis in children followed by scarring: this is especially common in girls. Second, it is increasingly recognized that primary vesicoureteric reflux can be associated with renal dysplasia and hypoplasia (Hinchliffe et al. 1992a; Risdon et al. 1993; Hiraoka et al. 1997). These patients are usually boys with severe grades of reflux: this population has only been defined relatively recently with the advent of routine fetal ultrasound scanning.

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Genetics may play a role in the pathogenesis of primary vescioureteric reflux because of a 30–50-fold increased risk in immediate relatives of probands vs the general population (Noe 1992; Noe et al. 1992; Scott et al. 1997) with the inheritance in many kindreds appearing to be dominant with variable penetrance and expression (Chapman et al. 1984; Eccles et al. 1996). Feather et al. (2000) recently reported the results of the first genome-wide search of vesicoureteric reflux in seven European families with up to seven affecteds who had an apparent dominant inheritance pattern. Patients were only classified as ‘affected’ if reflux had been diagnosed by cystogram: other individuals with only ‘soft’ signs such as urinary tract infection or hypertension were classified as ‘unknown’. We used 350 polymorphic markers spaced at 10 cM through the genome and used both parametric and non-parametric linkage analyses with the GENEHUNTER program in order to consider the possibilities that the disease might be either monogenic or polygenic (Lander and Kruglyak 1995). A locus on the short arm of chromosome 1 spanning 20 cM between markers GATA176C01 and D1S1653 had a parametric lod score of 3.2 (3.0 is accepted as significant for evidence of autosomal linkage). Although the data are suggestive that an important vecisoureteric reflux locus exists on chromosome 1p, specific mutations remain to be defined. Furthermore, the lod score for two of the seven kindreds were incompatible with linkage to this locus and, using a nonparametric analysis, we identified several loci with a p 0.05. Both observations are compatible with the hypotheses that (i) mutations of diverse genes may give rise to the same type of malformation (phenocopies) and (ii) that the interaction of more than one mutation may be required to generate disease. As discussed above, both concepts are well established in animal studies. Of note, no linkage was found to chromosome 6p, where a urinary tract malformation locus was previously reported (Devriendt and Fryns 1995), or to chromosome 10q, where PAX2 mutations cause vesicoureteric reflux in the renal coloboma syndrome (Sanyanusin et al. 1995a). There was however a minor locus on the long arm of the X chromosome, consistent with a minor influence of AT2. Hohenfellner et al. (1999b) were unable to find an association of the AT2A–1332G transition in 23 patients primary with vesicoureteric reflux versus 19 controls and Choi et al. (1998) excluded PAX2 mutations in kindreds with non-syndromic vesicoureteric reflux. Finally, it is also possible that genetic variation affects renal inflammation and interstitial fibrosis after acute pyelonephritis. There is indeed evidence of an association between scar formation in children with vesicoureteric reflux and polymorphisms of the angiotensin converting enzyme gene. Ozen et al. (1997) reported an association with an insertion/deletion polymorphism in intron 16 of this gene: the particular genotype (DD) is associated with increased ACE levels. A similar observation was made by Horikawa et al. (1997).

Nocturnal enuresis Nocturnal enuresis, or bed wetting at night, affects up to 10 per cent of children aged six. It can occur in families, sometimes in an autosomal dominant pattern. Eiberg et al. (1995) assigned a locus for this disorder to chromosome 13q while other loci have been suggested on 12q (Arnell et al. 1997) and 22q (Eiberg 1998). The genes remain undefined and are likely to affect bladder function rather than cause structural alterations in growth.


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Conclusions Malformations of the urinary tract are common causes of chronic renal failure in infants and young children and we have outlined emerging evidence on how mutations alter gene expression during development to cause some of these disorders. The most convincing studies come from the multiorgan malformation syndromes in which specific mutations have been defined. However, these syndromes are relatively rare and most urinary tract malformations appear to occur in isolation. In some of these individuals a genetic pathogenesis is strongly suggested by a positive family history and genetic linkage studies: one common example is primary vesicoureteric reflux. Furthermore, sporadic malformations have been shown to be associated with polymorphisms of genes expressed during construction of the urinary tract. In the longterm, an understanding of the genetic aspects of human urinary tract malformations will help to unravel the pathogenesis of these disorders and may facilitate the design of genetic screening tests with a view to early diagnosis and appropriate genetic counseling.

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Wallace, M., Zori, R. T., Alley, T., Whidden, E., Gray B. A., and Williams, C. A. (1994). SmithLemli-Opitz syndrome in a female with a de novo, balanced translocation involving 7q32: probable disruption of a SLOS gene. American Journal of Medical Genetics, 50, 368–74. Webb, N. J. A., Lewis, M. A., Bruce, J., Gough, D. C. S., Ladusans, E. J., Thompson, A. P. J., et al. (1997). Unilateral multicystic dysplastic kidney: the case for nephrectomy. Archives of Diseases of the Child, 76, 31–4. Wegnenke, J. D., Uehling, D. T., Wear Jr, J. B., Gordon, E. S., Bargman, J. G., Deacon, J. S. R., et al. (1975). Familial Kallmann syndrome with unilateral renal aplasia. Clinical Genetics, 7, 368–81. Wilkie, A. O. M., Slaney, S. F., Oldridge, M., Poole, M. D., Ashworth, G. J., Hockley, A. D., et al. (1996). Apert syndrome results from localised mutations of FGFR2 and is allelic with Crouzon syndrome. Nature Genetics, 9, 165–72. Wilson, G. N., de Chadarevian, J. P., Kaplan, P., Loehr, J. P., Frerman, F. E., and Goodman, S. I. (1989). Glutaric aciduria type II: review of the phenotype and report of an unusual glomerulopathy. American Journal of Medical Genetics, 32, 395–401. Winyard, P. J. D., Risdon, R. A., Sams, V. R., Dressler, G. R., and Woolf, A. S. (1996a). The PAX2 transcription factor is expressed in cystic and hyperproliferative dysplastic epithelia in human kidney malformations. Journal of Clinical Investigations, 98, 451–9. Winyard, P. J. D., Nauta, J., Lirenman, D. S., Hardman, P., Sams, V. R., Risdon, A. R., et al. (1996b). Deregulation of cell survival in cystic and dysplastic renal development. Kidney International, 49, 135–46. Winyard, P. J. D., Bao, Q., Hughes, R. C., and Woolf, A. S. (1997). Epithelial galectin-3 during human nephrogenesis and childhood cystic diseases. Journal of the American Society of Nephrology, 8, 1647–57. Woolf, A. S. (1997). The single kidney. In Pediatric surgery and urology: long-term outcomes (ed. M. D. Stringer, P. D. E. Mouriquand, K. T. Oldham, and E. R. Howard), pp. 625–31. WB Saunders Company Ltd, London. Woolf, A. S. (1999a). Embryology of the kidney. In Pediatric Nephrology (ed. T. M. Barratt, A. Avner, W. Harmon, 4th edn), pp. 1–19. Williams and Wilkins, Baltimore. Woolf, A. S. (1999b). Solitary kidney and bicornuate uterus in mother and child. Nephrology Dialysis and Transplantation, 14, 960–1. Woolf, A. S. (1999c). Primary vesicoureteric reflux and associated nephropathies. In Horizons in Medicine, Volume 11. Royal College of Physicians, pp. 325–33. Woolf, A. S., Kolatsi-Joannou, M., Hardman, P., Andermarcher, E., Moorby, C., Fine, L. G., et al. (1995). Roles of hepatocyte growth factor/scatter factor and the met receptor in early development of the metanephros. Journal of Cell Biology, 128, 171–84. Woolf, A. S. and Winyard, P. J. D. (1998). Advances in the cell biology and genetics of human kidney malformations. Journal of the American Society of Nephrology, 9, 1114–25. Worck, R. H., Ibsen, H., Andersen, C. B., Ibsen, K. K., and Rasmussen, F. (1995). The etiology of hypertension in nonrenovascular unilateral renal disease—two cases of renin induced hypertension in congenital renal dysplasia. Blood Pressure, 4, 113–16. Xu, P. X., Adama, J., Peters, H., Brown, M. C., Heaney, S., and Maas. R. (1999). Eya1-deficient mice lack ears and kidneys and show abnormal apoptosis of organ primordia. Nature Genetics, 23, 113–17. Yamada, T., Horiuchi, M., and Dzau, V. J. (1996). Angiotensin II type 2 receptor mediates programmed cell death. Proceedings of the National Academy of Sciences, USA, 93, 156–60. Yang, S. P., Risdon, R. A., Scott, R., O’Hare, M. J., Woolf, A. S., and Winyard, P. J. D. (1999). Role for the renin-angiotensin system and transforming growth factor in human renal dysplasia. Journal of the American Society of Nephrology, 10, 445A.


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Yeung, C. K., Godley, M. L., Dhillon, H. K., Gordon, I., Duffy, P. G., and Ransley, P. G. (1997). The characteristics of primary vesico-ureteric reflux in male and female infants with pre-natal hydronephrosis. British Journal of Urology, 80, 319–27. Zenteno, J. C., Mendez, J. P., Maya-Nunez, G., Ulloa-Aguirre, A., and Kofman-Alfaro, S. (1999). Renal abnormalities in patients with Kallmann syndrome. British Journal of Urology International, 83, 383–6. Zerres, K., Mucher, G., Bachner, L., Deschennes, G., Eggerman, T., Kaariainen, H., et al. (1994). Mapping of the gene for autosomal recessive polycystic kidney disease to chromosome 6p21-cen. Nature Genetics, 7, 429–32.


5 Urinary tract defects and chromosomal disorders David J. Amor, Lachlan de Crespigny, and R. J. McKinlay Gardner

Introduction Chromosomal abnormality may be constitutional or acquired. A constitutional abnormality is present at conception (or at least from embryogenesis), and typically affects the morphogenesis of organs. An acquired abnormality arises somatically, in a person whose constitutional karyotype may be normal or (more rarely) abnormal, and cancer is the classical example. In this chapter, we focus largely on the constitutional case, but do not ignore acquired abnormalities. At the more or less mechanical level of documentation, we record certain constitutional chromosome defects and note their association with congenital defects of the kidney and the urinary tract. We attempt a synthesis of these data in the generation of a ‘renal chromosome map’; such a map may be helpful to gene hunters. One very practical application of this karyotypic–phenotypic knowledge lies in prenatal diagnosis, and we discuss the chromosomal implications of certain ultrasonographic observations. The genetics of renal cancer are dealt with in Chapter 22, and here we note only those aspects of cancer with a particular cytogenetic relevance. We consider four categories of chromosome disorder, as follows: Chromosome disorder

Example

1 2 3 4

Trisomy Unbalanced translocation, deletion Uniparental disomy, imprinting changes Balanced translocation

Numerical abnormalities Unbalanced structural rearrangement Epigenetic phenomena Balanced structural rearrangement

For the most part, this chapter is concerned with structural unbalanced chromosome states (1 and 2 in the table above). These take various forms, including unbalanced translocations, trisomy for whole chromosomes, deletions, and duplications, but the endpoint is always a perturbation in the dosage of genes. Presumably, this mechanism is the basis of the damaging effect. That is, there is too much (duplication), or too little (deletion), of a particular chromosomal segment. Typically, the contiguous genes on these chromosome segments will be present in 150 or 50 per cent of the normal amount, and in consequence the protein products will be overproduced or underproduced by the same ratios. For many genes, including most enzymes and many structural proteins, this does not matter: one functional copy suffices to ensure normality. For some genes, however, the correct dosage of gene product is essential.

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Genes that are particularly dosage-sensitive include those whose products are part of a quantitative signalling process, and those whose products compete with each other to determine a developmental switch. In general, chromosome imbalance leads to a variable combination of developmental abnormalities, characterized by intellectual disability, growth retardation, dysmorphic facial appearance, and malformation of internal organs. Renal abnormalities comprise one of the most common categories of organ defect. An unbalanced state may also exist at the functional level, and in this we refer to the concept of parent-of-origin imprinting of chromosomes. In a chromosomal segment subject to imprinting, only one of each homologue, either the maternal or paternal, is genetically active, and a ‘50 per cent’ output from that locus is normal. If both of these chromosomal segments originate from the one parent, which is known as uniparental disomy (UPD), the balance is disturbed. There will either be a 0 or a 100 per cent output, so to speak, and this underproduction or overproduction may have a deleterious phenotypic effect. UPD for a chromosome that is not subject to imprinting has no untoward consequence. Balanced chromosome rearrangements, by definition, do not involve a net loss or gain of genetic material, and have no phenotypic effect, other things being equal. Occasionally, an apparently balanced rearrangement disrupts a gene that happens to be located at the breakpoint of the rearrangement, and this, of itself, causes phenotypic defect. Such cases can be providential in mapping genes, and one ‘nephrogenesis gene’ has been found this way (the branchio-oto-renal gene, see below). One unique role of the balanced rearrangement in renal disease is the constitutional 3p translocation which sets the stage for the development of renal cancer (see below).

The effects of constitutional chromosomal disorders on the kidney Normal kidney development is a dynamic process of proliferation, cell death, differentiation, and morphogenesis, controlled by the temporal and spatial expression of hundreds of different genes (Davies and Brandli 1997). These include transcription factors, growth factors, and adhesion molecules. While the functions of most of these genes are yet to be determined, it may safely be presumed that many will be dosage-sensitive. In nephrogenesis, a fine balance exists between cell proliferation and cell death. Excessive proliferation may lead, for example, to cystic changes, or to Wilms tumour; whereas excessive apoptosis could lead to renal hypoplasia. The embryology of normal and abnormal renal development is dealt with more fully in Chapters 3 and 4, but Table 5.1 summarizes the patterns of renal malformation seen in chromosomal disorders, along with their postulated developmental bases. Renal abnormalities are a surprisingly consistent feature of chromosomal aneuploidy; and there are few common chromosome abnormalities in which renal malformations have not been described. Table 5.2 illustrates the particular contribution of the three major autosomal trisomies. This suggests that there are many dosage-dependent genes for nephrogenesis, and that these genes are spread throughout the genome. Alternatively, there may be a nonspecific effect of aneuploidy on cell growth and differentiation operating at a chromosomal level. The other notable feature of renal changes in chromosome disorders is that the phenotypic effects are limited to a relatively small number of malformations. The fact that the same phenotypic consequence can result from a variety of different chromosome imbalances is testament to the developing kidney’s limited repertoire of responses to aneuploidy.

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Table 5.1 Embryological basis of renal malformations in chromosomal disorders Renal agenesis Failure of formation of metanephros Failure of ureteric bud formation Failure of ureteric bud to reach, or interact with nephrogenic blastema Interruption of vasculature Regression of cystic dysplastic kidney Ectopic kidney Error of ascent Horseshoe kidney Migration of nephrogenic cells across the primitive streak (before 5 weeks gestation) Mechanical fusion of ascending kidneys (after 5 weeks gestation) Dysplastic/polycystic kidney Error of mutual induction between ureteric bud and metanephric blastema Failure of normal differentiation of renal parenchyma Changes secondary to urinary tract obstruction Obstruction/hydronephrosis Stenosis or atresia of pelvoureteric junction, ureter, or urethra Poorly functioning bladder/vesico-ureteric reflux Extrinsic compression of ureter Failure of recanalization of the ureter (not patent at mid-gestation) Duplications Early duplication of ureteric bud leading to duplicated kidney Later duplication of ureteric bud leading to duplicated ureter

Table 5.2 Renal malformations in common trisomies (%) Feature

Trisomy 13

Trisomy 18

Trisomy 21

Any structural abnormality Hydronephrosis Horseshoe kidney Duplex kidney/collecting system Cortical cysts/cystic dysplasia Glomerular microcysts Renal hypoplasia

61 18 1 12 34 NR NR

62 17 26 23 14 NR NR

6 4 1 1 3 26 21

Based on combined data of Ariel et al. (1991), Egli and Stalder (1973), Lo et al. (1998), Taylor (1968), Warkany et al. (1966), and Zellweger et al. (1977). NR not recorded.

Renal anomalies in chromosome disorders can be divided into five main groups. 1. Developmental error or growth failure during embryogenesis of the renal tract. This includes renal agenesis and hypoplasia, horseshoe kidney, duplication defects, migration defects, and lower urinary tract malformation. 2. Obstructive uropathy, whereby developmental error leads to secondary abnormalities proximal to the obstruction. When the obstruction occurs early in development, the result is cystic dysplasia, possibly secondary to the induction of aberrant expression of various

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renal genes. Later obstruction leads to hydronephrosis, hydroureter, poor renal parenchymal growth, and cortical cysts, secondary to the hydrostatic effect of obstruction. 3. Renal dysplasia describes a kidney comprising undifferentiated and metaplastic cells surrounding poorly branched ureteric bud derivatives. It may be unilateral or bilateral, focal or diffuse, and the kidney may be enlarged or small. Dysplasia may be secondary to obstruction, or may occur as a primary process; in the case of chromosome disorders, the root cause is likely to be the aberrant expression of nephrogenesis genes. 4. ‘Acquired’ tubular disorders and glomerular lesions, including glomerulonephritis. 5. Cancer is primarily represented by Wilms tumour. This usually occurs in the context of so-called ‘nephrogenic rests’, that is, the abnormal persistence of embryonal cells into postnatal life. Renal cell adenocarcinoma associated with constitutional 3p translocation is a very rare case. These various urinary tract abnormalities may range in their clinical implications from in utero lethality through to being inconsequential. Between these extremes, clinical consequences include vulnerability to urinary tract infection, hydronephrosis, hypertension, cancer, and chronic renal failure. Urinary tract abnormalities in specific chromosome disorders In this section, we provide a correlation between certain chromosome abnormalities and the urinary abnormalities that have been described with each condition. In certain of them, the information is rather academic (which is not to say uninteresting), and the renal defect is merely one of an aggregation of defects that are, in sum, of lethal effect. In others, survival is the rule, and these data may serve as a useful reference for clinicians dealing with patients with particular chromosome abnormalities. We have concentrated mainly on two areas: firstly, those chromosome abnormalities that are relatively common, or comprise well-recognized syndromes; and secondly, those chromosome abnormalities that are particularly associated with urinary tract abnormalities. These data are summarized in a ‘renal chromosome map’ (Fig. 5.1), in which we document karyotypic segments apparently linked, in the unbalanced state, with urinary tract defects. To this end, we have reviewed a substantial fraction of the cytogenetic literature in which an abnormality of the urinary system has been recorded. Our three main sources of information have been the computerized medical publication databases, the POSSUM syndrome identification system (Bankier et al. 1999), and the Oxford Cytogenetic Database (Schinzel 1994). For the purposes of this exercise, the urinary tract starts at the level of the renal parenchyma, and ends at the external urinary meatus. There are potential confounders with this method of data acquisition. First, reporting of cases is necessarily incomplete, particularly in the case of renal abnormalities, which are often asymptomatic. Second, there may be bias in that report of an abnormality in one case of a particular syndrome will encourage seeking out the same abnormality in other patients. This is especially true for asymptomatic internal malformations and may exaggerate a supposed association. Third, data on urinary abnormalities are frequently clustered under the nonspecific heading of ‘genitourinary malformations’, making it impossible to separate structural renal malformations from common genital abnormalities such as cryptorchidism and hypospadias.


Del

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Dup

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

Fig. 5.1 A renal chromosomal map. Lines indicate those chromosomal segments associated with urinary tract abnormality in approximately 20 per cent of reported cases. Sources given in text. Unbroken lines deletion, dotted lines duplication.

Selected autosomal trisomies Trisomy 8 mosaicism Mosaic trisomy 8 is a well-recognized syndrome, the features of which include mental retardation, dysmorphic facies, skeletal anomalies, congenital cardiac defects, and malformations of the kidney. The great majority of cases are due to mitotic (postzygotic) duplication (Karadima et al. 1998). As is typical in mosaicism, there is a wide range of phenotype. Ureteral or renal anomalies are present in 75 per cent, with the predominant category being obstructive uropathy with vesico-ureteric reflux and hydronephrosis (Riccardi 1977; Schinzel 1994). Renal agenesis, cystic dysplasia, urethral valves, and horseshoe kidney are also reported. An association between constitutional mosaic trisomy 8 and malignancy has been suggested (Seghezzi et al. 1996), and two cases of Wilms tumour are on record (Niss and Passarge 1976; Nakamura et al. 1985). Trisomy 9 Trisomy 9 is rare, and typically seen in the mosaic state. Neonatal mortality is high, and survivors are severely retarded. The phenotype includes facial dysmorphism and eye defects, joint dislocations, and variable organ defects, particularly cardiac and renal malformations. Renal defects occur in 60 per cent of non-mosaic cases and in 39 per cent of mosaic cases (Arnold et al. 1995), with cystic dysplasia and hydronephrosis being the predominant

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contributors. Horseshoe kidney has been reported in five cases of non-mosaic trisomy 9 (Roberts et al. 1993a; Chitayat et al. 1995; Saura et al. 1995; Sandoval et al. 1999). Micropenis and cryptorchidism are common. Trisomy 13 Trisomy 13 is typically lethal in the first few days. There is a distinctive facies, frequently with cleft lip and palate, severe brain malformations, polydactyly, and cardiac and genital abnormalities. Renal abnormalities are common (see Table 5.2). One-third of cases have renal cortical cysts, and duplication of kidneys or ureters and hydronephrosis are also common. Unilateral renal agenesis is an infrequent observation. As an historical point, one of the original cases of ‘Patau syndrome’ published in 1960 had a persistent urachus (Patau et al. 1960). Horseshoe kidney is rare (in contradistinction to trisomy 18, see below). Nephrogenic rests are a frequent finding at autopsy, and given their property of being a Wilms precursor lesion (Beckwith 1998), it is not surprising that Wilms tumour has been reported in two surviving children with trisomy 13 (Sweeney and Pelegano 2000). Trisomy 18 Trisomy 18 is characterized in particular by growth retardation, severe mental defect, craniofacial abnormalities, cardiac and genital defects, and digital anomalies. The birth frequency is about 1 in 6000; most die within the first year. Renal defects are common, but the spectrum differs from that of trisomy 13 (see Table 5.2). Horseshoe kidney is the most common, present in about a quarter of cases. Other abnormalities include cystic kidneys (14 per cent), renal or ureteric duplication (23 per cent), cystic dysplasia (14 per cent), and renal ectopy (10 per cent). As with trisomy 13, nephrogenic rests occur frequently, and a number of surviving children with trisomy 18 have had Wilms tumour (Olson et al. 1995). Trisomy 21 Down syndrome is common, and most children survive. We therefore pay detailed attention to the range of urinary tract anomaly and dysfunction. Numerous structural abnormalities and acquired renal diseases have been described, most appearing as case reports rather than as population surveys. Two recent autopsy series provide a useful review: Ariel et al. (1991) with 124 cases, and Lo et al. (1998) with 43 cases along with a control group. Gross structural abnormalities of the kidneys and urinary system are infrequent in Down syndrome compared with other trisomies. Obstructive uropathy is an occasional concomitant and can be associated with dysplastic change. Bladder neck stenosis with neonatal death due to Potter syndrome is also recorded, as are urethral valves. Abnormalities at the level of the parenchyma are more common. Renal hypoplasia is present in about 20 per cent, and the glomeruli are often immaturely formed and reduced in number. Glomerular microcysts are seen in 24–30 per cent (cf. 3.5 per cent in controls), and appear to reflect an inherent morphological characteristic of trisomy 21, rather than being secondary to obstruction. Focal dilatation of the tubules occurs in 8 per cent, and simple cysts in 5.6 per cent (compared with 0.1 per cent in a general paediatric population). Acquired glomerular disease is also seen, and this is less simple to ascribe to the chromosomal defect; accompanying complications such as cyanotic heart disease and chronic infection could be contributory. Recorded diseases include focal segmental glomerulosclerosis, minimal change disease, early glomerulonephritis with crescents and vasculitis, membranous nephropathy, renal amyloidosis, haemolytic–uraemic syndrome, and hypercalcaemia with secondary medullary nephrocalcinosis. Unlike trisomy 13


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and trisomy 18, there is no association with Wilms tumour, this having been reported on only one occasion (Kusumakumary et al. 1995). The kidneys function less well in Down syndrome, and reduced clearance of creatinine and uric acid has been described (Coburn et al. 1967). It is likely that renal failure occurs more commonly in individuals with Down syndrome compared with the general population, although accurate data demonstrating this are not available. Renal dialysis and transplantation have become therapeutic options for patients with Down syndrome and their families. The utilization of living family members as kidney donors has increased the availability of kidneys for transplantation, but the decision of whether to pursue such an option is up to the individual family. Baqi et al. (1998) identified 14 Down syndrome patients who underwent renal transplantation in the USA between 1987 and 1995. All but one were in the age group 7–17 years, and 10 had been on dialysis prior to transplantation. The indications for transplant were obstructive uropathy (4), hypoplastic/dysplastic renal disease (3), focal segmental glomerulosclerosis (2), membranous nephropathy (1), chronic glomerulonephritis (2) and unspecified (2). Living related donors were used in six cases, and cadaver donors in the remaining eight cases. At the time of writing, there had been three patient deaths, two grafts had been rejected, and the remaining nine kidneys were functioning normally. Trisomy 22 Trisomy 22, either mosaic or non-mosaic, is rare, and the clinical picture is severe (Bacino et al. 1995). Few cytogenetic conditions manifest renal malformation more frequently than this one: 82 per cent of full trisomy 22 cases show renal defects, of which dysplasia and hypoplasia are the typical (Schinzel 1994; Bacino et al. 1995). The proportion is less in mosaic cases (Crowe et al. 1997). With respect to functional renal compromise Wertelecki et al. (1986) reported a female with trisomy 22 mosaicism who, at age 4 years, developed nephrotic syndrome unresponsive to steroid therapy. Renal ultrasound and intravenous pyelogram were normal. Renal biopsy at 12 years of age showed mild focal mesangial proliferation consistent with minimal change glomerulopathy, and remission was finally achieved with a short course of cyclophosphamide. One infant with multiple malformations and full trisomy 22 had proteinuria and haematuria, with the typical observation (on ultrasonography) of small dysplastic kidneys; death occurred at 2 12 months of age (Bacino et al. 1995). Triploidy Triploidy (69 chromosomes) occurs in about 1 per cent of all recognized pregnancies. It is due to a double chromosomal contribution from either parent, either fertilization of a normal oocyte by two sperms (or by a single diploid sperm), or fertilization of a diploid oocyte by a single sperm. Almost all (more than 99 per cent) are lost as first trimester miscarriage or second trimester fetal death in utero. Triploidy with a double paternal contribution shows the classical placental phenotype of partial hydatidiform mole, and the fetuses are microcephalic and of relatively normal size. Triploidy due to a double maternal contribution is more likely to survive into the second trimester, and develop as a severely growth-retarded fetus with relatively enlarged head and an abnormally small and non-molar placenta (McFadden and Kalousek 1991). These parent-of-origin differences likely reflect genomic imprinting. In the very rare cases of survival until birth, death occurs in the perinatal period. There is a wide variety of associated malformations and dysmorphogenesis. Of the renal malformations, the most common are cystic dysplasia, renal hypoplasia, hydronephrosis, and horseshoe kidney (Schinzel 1994). Mittal et al. (1998) detected renal

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abnormalities in three of 20 fetuses diagnosed with triploidy on the basis of antenatal ultrasound findings; and at pathology examination, renal abnormalities were identified in an additional six cases, including renal hypoplasia, cystic dysplasia, renal agenesis, and horseshoe kidney. Sex chromosome disorders Turner syndrome (monosomy X) The invariable features of 45,XO Turner syndrome are short stature and ovarian dysgenesis, and the condition occurs in 1 in 2500 females. Renal defects are high on the list of associated malformations, being seen in about one-third of cases (Lippe et al. 1988). The most common of these are horseshoe kidney (7 per cent), duplex collecting system (8 per cent), unilateral renal agenesis (3 per cent), and pelvo-ureteric junction obstruction (2 per cent). Other abnormalities include vesico-ureteric junction obstruction, pelvic kidney, rotational abnormalities of kidney position, and aberrant renal vessels. Simple cysts and cystic dysplasia have been reported (Herman and Siegel 1994) but are uncommon. Renal defects are seen less often with mosaic karyotypes (Flynn et al. 1996). Thus, each newly diagnosed child (or adult) should proceed to renal ultrasonography, and those found to have an abnormal urinary tract should be monitored for urinary infections and progressive renal impairment (Elsheikh et al. 1999). Whether or not structural renal abnormalities are present, there is a risk for hypertension (Nathwani et al. 2000). This aneuploidy is another that may predispose to the development of a Wilms tumour. In the review of Olson et al. (1995), with reference to the National Wilms Tumor Study in the United States, there were four children with Turner syndrome (two of whom had a horseshoe kidney, a malformation which itself predisposes to Wilms tumour). The expectation, according to which comparative baseline data were used, was either 0.3 or 1.6 cases. XXX and Polysomy X About 1 in 1000 females has a 47,XXX karyotype. The important phenotypic concomitant is that of a mild intellectual deficit (Ratcliffe 1999). Organ malformations are infrequent. Lin et al. (1993) reviewed eight cases of 47,XXX in whom urinary malformations had been reported, and hypothesized that development of the ‘urogenital field’ may be affected by X chromosome trisomy. The abnormalities were unilateral or bilateral renal agenesis (3), renal hypoplasia (1), hydronephrosis (1), cystic kidneys (1), extrophy of the cloaca (1), and ureteral stricture (1). If this association is truly causal (an as yet unsettled question), it is nevertheless apparent that the increased risk would be a small one. The addition of each supernumerary X chromosome is associated with a progressively more severe phenotype. In a review of Penta-X (49,XXXXX) syndrome, Kassai et al. (1991) noted urinary tract abnormalities in four out of six girls who had renal investigation. The abnormalities included cystic kidney, renal hypoplasia, horseshoe kidney, and urolithiasis. XXY Klinefelter syndrome Klinefelter syndrome is the most common sex chromosome disorder, affecting about 1 in 600 boys. The main features are testicular failure with androgen deficiency and failure of spermatogenesis, and a typical body habitus, and there is a minor neuropsychological component to the phenotype (Ratcliffe 1999). Gross malformations of the kidney do not seem to occur more frequently in Klinefelter syndrome than in the general population. Egli and Stalder (1973) found only one renal abnormality, a cystic kidney, in a series of 276 cases with


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47,XXY. Urinary abnormalities were, however, present in two out of 17 men with a 49,XXXXY karyotype: both had hydronephrosis, which in one case was associated with ureterocele and hydroureter. As with poly-X females, the presence of one supernumerary X chromosome appears to be well tolerated by the urinary tract, but each additional X chromosome carries an increasing risk of abnormality. XYY syndrome About 1 in 1000 males has a 47,XYY karyotype, although most are never diagnosed. Typical features include increased linear growth, mild learning difficulty, and behavioural problems (Ratcliffe 1999). Although sex chromosome studies on large populations have failed to detect a higher incidence of organ malformation, the observations from several case reports suggest a specific association with renal agenesis. Rudnik-Schöneborn et al. (1996) reviewed 10 reported cases of renal malformations in XYY males and added three cases (each with bilateral agenesis) of their own. Unilateral or bilateral renal agenesis was recorded in 11 of the 13 cases, with polycystic kidneys in one case and renal hypoplasia in another. Based on an expected frequency of bilateral renal agenesis of 1 in 6000 births (Bankier et al. 1985), the two conditions should occur together by chance once in 6,000,000 male births. Rudnik-Schöneborn et al. (1996) calculated an expectation of only 0.1 cases in the period of time during which their three cases were observed. Duplication (Dup), Deletion (Del), Triplication (Trp), and Rearrangement (Rea) syndromes The Trp (2q) A single case of trp(2)(q11.2q21) is nevertheless of interest in its association with a severe and extensive renal phenotype (Wang et al. 1999). A 32-week gestation infant with Potter syndrome had very large polycystic kidneys (combined weight 69.8 g, five times the expected 12.6 g), atretic ureters, and a hypoplastic bladder. Renal defects have not been noted in cases of duplication for this region, suggesting that a ‘renal malformation threshold’ exists between a 150 and a 200 per cent dosage for certain genes within the 2q11.2q21 segment. The Dup (3q) The dup(3q) syndrome has a distinctive phenotype, in which the facies is reminiscent of Brachmann–de Lange syndrome (Steinbach et al. 1981). The trisomy involves a variable segment of 3q, usually within the region 3q21-qter. Renal abnormalities are a prominent feature. Wilson et al. (1985) reviewed 23 cases of dup(3q) in which the information was available, and recorded the high fraction of 48 per cent with renal abnormalities. Urinary malformations are typically severe, with multicystic dysplastic kidney a frequent observation, often with concomitant ureteric duplication, and ureteric and renal pelvis dilatation. Double renal artery (Wilson et al. 1985) and horseshoe kidney (Falek et al. 1966) have also been described. The Del (4p), Wolf–Hirschhorn syndrome (WHS) Both deletion and duplication of 4p have a renal phenotype, suggesting that there may be renal genes in this region sensitive to dosage effects in either direction. Partial deletion of the distal short arm of chromosome 4 (4p) causes a well-known clinical entity, the WHS. The main features are characteristic facial appearance, growth retardation, mental defect, and skeletal and cardiac abnormalities. Urinary malformations are now recognized to be more common in WHS than previously thought. Renal hypoplasia is the most common abnormality

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(Schinzel 1994), and a variety of other abnormalities has been described including hydronephrosis, cystic dysplasia, vesico-ureteric reflux, cortical cysts, bilateral and unilateral renal agenesis (Lazjuk et al. 1980; Lurie et al. 1980; Grisaru et al. 2000). In a series of five fetuses with antenatally diagnosed del(4p), Tachdjian et al. (1992) observed major renal hypoplasia with lack of lobation in all five. Premature regression of metanephric blastema in the youngest fetuses and oligonephronia were the main histological findings. Cortical dysplasia was observed in one case. Katz and Smith (1991) described a boy with WHS and a structurally normal urinary tract, who underwent renal biopsy at age 12 for proteinuria. Histology showed focal segmental sclerosis and unclassified ultrastructural abnormalities of the basal lamina and tubular interstitium. The Dup (4p) Trisomy 4p is a distinct clinical entity, although it is seldom recognized prior to cytogenetic evaluation (Patel et al. 1995). There is a distinctive facies with unusual nasal and chin anatomy and distal limb anomalies; psychomotor retardation is universal. The frequency of renal malformation has not been formally studied, but a number of case reports refer to renal defects. These include hydronephrosis, unilateral renal agenesis, hypoplastic and ectopic kidneys, and horseshoe kidney (Schinzel 1994). In one small group of 12 cases who had renal imaging, Gonzalez et al. (1977) reported defects in two. The genitalia are abnormal in 81 per cent of males (Patel et al. 1995). The Del (5p), Cri-du-chat syndrome The cri-du-chat syndrome is due to variable degrees of deletion of the short arm of chromosome 5, and is characterized in particular by mental deficiency and the unusual ‘mewing’ cry in infancy which gave it its name. Renal anomalies appear to be fairly unusual, although hypoplasia, agenesis, renal duplication, and horseshoe kidney have all been described (Egli and Stalder 1973; Rethoré 1977). The Dup (6p) In partial 6p trisomy there is a characteristic facies which includes blepharophimosis and hypotelorism, and mental defect is invariable. There is a rather distinctive and consistent renal phenotype. In the great majority (85 per cent) there is hypoplasia, and most (64 per cent) show proteinuria (Rudnik-Schöneborn et al. 1997), the mechanism of which is undetermined. Histopathology is available for six cases. Subchronic glomerulonephritis is recorded at autopsy in one case (Breuning et al. 1977). In a brother and sister with dup(6p) (and a concomitant 4p deletion), renal biopsy in one sib showed focal segmental mesangial sclerosis with focal segmental glomerulosclerosis, whereas in the other there was only small focal areas of mesangial hypercellularity and interstitial fibrosis accompanied by fine mesangial staining for IgM (Pierpont et al. 2000). In three other cases renal histopathology was normal (Therkelsen et al. 1971). There is a single report of horseshoe kidney (Smith and Pettersen 1985). The Del (7q11.2), Williams syndrome Williams syndrome is one of the more readily recognized of the contiguous gene syndromes, and the facies and the behaviour are distinctive. Renal defects are occasionally (18 per cent) noted (Pankau et al. 1996), and include agenesis, duplication, vesico-ureteric reflux, and renal artery stenosis. Renal function is not usually compromised, and creatinine clearance


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was reduced in only 2 out of the 130 patients in one series (Pankau et al. 1996). High blood pressure is common (about 40 per cent of patients), but this may more usually reflect a primary vascular defect, rather than a secondary renal manifestation (Broder et al. 1999). The Rea (8q13.3), Branchio–Oto–Renal (BOR) syndrome The BOR syndrome is actually a Mendelian disorder, in which the renal component can comprise rather a range: unilateral or bilateral agenesis or hypoplasia, severe cystic dysplasia, crossed ectopia, and horseshoe malformation, bifid renal pelves, and duplication of the collecting system. The ‘branchio’ and ‘oto’ elements refer to neck (branchial cleft) fistulas, and to ear defects. An inherited chromosomal abnormality was the entrée to mapping the gene for the BOR syndrome, which justifies its mention here. Haan et al. (1989) studied a BOR family segregating an 8q insertion, dir ins(8)(q24.11q13.3q21.13). Presumably, the BOR gene on the insertion chromosome was deleted or disrupted. In due course, the gene itself proved to be a Drosophila homologue, the EYA1 gene (Abdelhak et al. 1997). This gene is expressed in metanephric cells as the ureteric branches form; it is likely that its half-functioning state in the ins(8q) family was the underlying cause of the renal maldevelopment. The Del (9p) Deletion of the short arm of chromosome 9 produces a syndrome with a rather distinctive craniofacial dysmorphology along with mental retardation (Huret et al. 1988). The Del(9p) is associated with XY sex reversal and gonadal dysgenesis (McDonald et al. 1997), but other urological abnormalities are not a feature. Tetrasomy 9p Tetrasomy 9p (triplication) is a rare syndrome due to a supernumerary isochromosome 9p. Mosaic and non-mosaic forms exist, and severity varies accordingly from minor anomalies to neonatal death. Numerous malformations are described (Tonk 1997), and renal abnormalities are frequent. These include hypodysplasia, ectopia, fusion, and hydronephrosis. Interestingly, renal defects are uncommonly reported in 9p trisomic state (Schinzel 1994), rather like the situation with trp(2q) and dup(2q) (see above). The Del (11p13), Wilms–Aniridia–Genital–Retardation (WAGR) syndrome This syndrome is of historic importance: it was the first deletion syndrome in which a renal component figured as a prominent observation, and it pointed the way to the discovery of a gene with a critical role in kidney development, the WT1 gene. The very specific features of Wilms tumour, aniridia (absence of the iris), and genitourinary abnormalities suggested the agency of particular genes, and indeed it transpired that loss of WT1 and PAX6 on one chromosome 11 was responsible for three of the four manifestations of WAGR syndrome. Other loci in 11p13 presumably cause the mental retardation. The loss of one WT1 allele first impairs the process of genitourinary tract formation, and second comprises a first hit in the generation of Wilms tumour (see Chapter 13); the PAX6 loss causes the eye defect. Children with del(11p13) have a high (but yet unquantified) risk for Wilms tumour, and should have ultrasonographic surveillance on a 3-monthly schedule for the first 7 years; the maximum risk for cancer is in the second year (Beckwith 1998; and see Chapter 13). Sporadic aniridia also implies a risk (in the region of 30 per cent) for Wilms tumour. Those cases due to PAX6 deletion might also have a defect in the neighbouring WT1 gene, and Gupta et al. (1998) outline a PCR-based methodology to check for this possibility.

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The Dup/Rea (11p) Chromosome 11p has two important renal roles. It carries the WT1 gene in 11p13 mentioned above, and it contains the critical region for Beckwith–Wiedemann syndrome (BWS) in 11p15.5. Duplications of 11p can include either region. Curiously, duplication of 11p13 does not have a particular renal implication, and of the dozen or so recorded cases, only one had a urinary tract anomaly, a urethral stenosis with bilateral hydronephrosis. This was seen in a stillborn male, who was also trisomic for the BWS region at 11p15.5 and monosomic for part of 7q (Ogur et al. 1988). Thus the proposition of Aalfs et al. (1997) that ultrasonographic surveillance be done in children with dup(11p13) is based more on a suspicion about WT1 than on empirical evidence. Beckwith–Wiedemann syndrome is a remarkable condition, and one of the archetypical disorders of genomic imprinting (Li et al. 1998). It presents a phenotype of intra-uterine and postnatal overgrowth, in which the renal manifestations include nephromegaly, medullary and calyceal cyst formation, hydronephrosis, and a predisposition to Wilms tumour (Elliott et al. 1994). Various mechanisms lead to a perturbation of the function of a cluster of ‘BWS candidate genes’ mapping to 11p15.5, these including the imprinted loci IGF2, H19, CDKN1C (p57KIP2), and KCNQ1 (KVLQT1). The growth-promoting gene IGF2 is expressed from the paternal chromosome, whereas H19 and CDKN1C activity arise from the maternally originating chromosome. A functional balance of reciprocal growth promoting and suppressing genes maintains a normal growth rate. In BWS, this balance is disturbed. About 2 per cent of sporadic BWS is due to structural abnormalities involving chromosome 11p15.5, falling into two main groups (see Fig. 5.2). (i) In paternally inherited duplications (de novo or inherited), there will be two functioning IGF2 copies, and overgrowth results. These children frequently have a concomitant monosomy for some other chromosomal segment, and thus display additional phenotypic defects; this was, for example, the case in 11 of 17 patients in the series of Slavotinek et al. (1997). (ii) Maternally inherited translocations demonstrate a different modus operandi: the translocation breakpoint at 11p15.5 appears to disrupt the KCNQ1 locus, possibly resulting in a secondary inhibition of the growth inhibitory effect of CDKN1C. BWS due to uniparental disomy is noted below. Mosaic tetrasomy 12p, Pallister–Killian syndrome (PKS) The PKS is a malformation/retardation syndrome with the interesting feature that the distribution of the abnormal chromosome, a 12p isochromosome, is tissue-limited. The distribution apparently includes the kidney, since renal defects are frequent. Among 24 cases from the literature, 9 (37 per cent) had anomalous urinary tract development. The defects most commonly noted are hydronephrosis and cystic dysplasia (McLean et al. 1992; Rodriguez et al. 1994; Los et al. 1995; Chiesa et al. 1998). Large kidneys (Bernert et al. 1992), persistence of the urogenital sinus (Reynolds et al. 1987), and renal duplication (Steinbach and Rehder 1987) are also on record. In severely affected PKS cases, with neonatal death, renal malformations are more often seen, presumably reflecting a higher mosaic load. The Del (15q11–q13), Prader–Willi syndrome (PWS) and Angelman syndrome (AS) Unlike the renal interest of Beckwith–Wiedemann syndrome, the two other classical cytogenetic imprinting disorders, PWS and AS, do not manifest a prominent renal phenotype. A very few cases of pelvic kidney (Pauli et al. 1983) and hydronephrosis (Schwartz et al. 1985; Galan et al. 1991) in PWS may or may not be causally associated. The same reservation

Urinary tract defects and chromosomal disorders pat mat (11) (11)

pat dup mat (11) (11)

pat der (11) mat (11)

Other chromosome

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Pat UPD for 11p or whole chromosome 11 pat mat pat pat (11) (11) (11) (11)

BWS critical region 11p15.5

Normal chromosome 11’s

Paternal duplication

Maternal translocation disrupting BWS critical region

Mosaic paternal UPD (11)

Fig. 5.2 The different chromosomal mechanisms in the aetiology of (BWS) Chromosomal abnormalities are responsible for approximately 2 per cent of BWS, and an additional 20 per cent is due to paternal uniparental disomy. The lightly shaded chromosome indicates the paternally derived chromosome 11 and the unshaded chromosome indicates the maternally derived chromosome 11. Diagonal lines indicate the BWS critical region. The dark shaded chromosome is a different chromosome involved in a translocation with chromosome 11.

applies to the single case of an 8-year-old PWS girl with membranoproliferative glomerulonephritis (Robson and Leung 1992). The Del (17p13.3), Miller–Dieker syndrome (MDS) The very specific feature of MDS is lissencephaly (smooth brain), due to deletion of the LIS1 gene from one chromosome 17. This and other defects confer early mortality. From the first reports, which noted associated renal defects, it might have seemed that the contiguous genes lost in the deletion would have included, among others, a renal gene or genes. Miller’s original cases (two siblings) had unilateral renal agenesis, and one of Dieker’s four patients showed fetal lobulation and cystic changes. But there has been little mention of the kidney in subsequent reports. Van Allen and Clarren (1983) noted duplex collecting system and hydronephrosis in two MDS cases. Is there a candidate locus in 17p13.3 that might have a renal role? HIC is telomeric to LIS1, and codes for a putative transcription factor that is expressed in the developing kidney (Grimm et al. 1999). It would be speculative, yet, to imagine its agency, in the haplo-insufficient state, in the causation of MDS renal defects. The Del (17p11.2), Smith–Magenis syndrome (SMS) Urinary tract defects are common (about 30 per cent) in this syndrome of congenital malformation and functional neurological compromise (including the particular observations of sleep disturbance and self-injurious behaviour such as onychotillomania, that is, pulling out fingernails and toenails). The abnormalities include unilateral renal agenesis, hydronephrosis and ectopic kidney, and duplication of the collecting system (Smith et al. 1986; Greenberg

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et al. 1996). This being so, renal imaging may be warranted. One patient with vesico-vaginal fistula and urachal diverticulum is on record (Lockwood et al. 1988). The Del (18q) This is one of the first deletion syndromes to have been delineated, generally presenting a severe phenotype of dysmorphology and mental defect, although the severity may relate to the extent of the deletion. Estimates of the frequency of structural renal abnormalities in 18q deletions vary from 10 per cent (Cody et al. 1999) to 43 per cent (Egli and Stalder 1973). Fused or horseshoe kidney, small or single kidney and hydronephrosis appear to comprise the majority of abnormalities. One del(18q) child had Wilms tumour, which may or may not have been causally related (Gilbert and Opitz 1979). The Dup (20p) Renal abnormalities are not infrequently seen in this rare cytogenetic abnormality, and include renal duplication, cystic kidneys, and renal hypoplasia (Grammatico et al. 1992; Schinzel 1994). The Del (22q11) (22q deletion syndrome/velocardiofacial syndrome/DiGeorge syndrome) This is probably the most common of all the deletion syndromes, and presents a variable clinical picture, as its different names attest. Features that may be present include conotruncal heart defects, cleft palate, facial dysmorphism, learning difficulties, hypoparathyroidism, and immune abnormalities. A large proportion (36 per cent) has urinary tract defects. Multicystic/dysplastic kidneys and obstructive abnormalities are the most common, but renal agenesis, duplex kidney, nephrocalcinosis, and vesico-ureteric reflux have all been reported (Ryan et al. 1997; Stewart et al. 1999). Directed investigation is certainly appropriate in individuals having this cytogenetic diagnosis. (Devriendt et al. 1996). Renal anomalies on obstetric ultrasound may lead to prenatal diagnosis (Goodship et al. 1997). At the other end of the urinary tract, hypospadius, and small penis may accompany other genital defects in the male (undescended testes, shawl scrotum) (Ryan et al. 1997). Tetrasomy 22pter–q11 (Cat-eye syndrome) This triplication syndrome takes its syndromal name from the iris colobomas that are one feature of the phenotype. Numerous other dysmorphic signs characterize the condition, with anal and auricular anomalies being among the more specific. The cytogenetic basis is an inverted duplication of the short arm and proximal long arm of chromosome 22, existing as a supernumerary marker (Schinzel et al. 1981). Urinary abnormalities are frequent, and include renal agenesis and hypoplasia, horseshoe kidney, ectopic kidney, cystic dysplasia, urethral stenosis, and vesico-ureteric reflux (Egli and Stalder 1973; Schinzel 1994). Reduced glomerular filtration rate has been documented in a single patient (Bellinghieri et al. 1994). Uniparental disomy Uniparental disomy (UPD) exists when both members of a pair of chromosomes (or in some cases a part of the pair of chromosomes) come from one parent. The UPD causes abnormality when a chromosome segment is subject to genomic imprinting. In this case, a segment of a chromosome, or perhaps just a single locus, is genetically active, or not active (silent), according to whether it has been transmitted from the mother or from the father. The delineation of syndromes associated with uniparental disomy has been complicated by three


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factors. First, the UPD cell line may coexist in the mosaic state with a trisomic cell line, and it may be trisomic cells that are responsible, in part or in whole, for the clinical phenotype. Second, phenotypic effects may be due to homozygosity for a monogenic recessive gene located on the ‘UPD chromosome’. Third, UPD may involve only part of a chromosome rather than a whole chromosome (segmental UPD). In general, the observation of UPD for a particular chromosome in an individual of normal phenotype argues against its being subject to imprinting. The reported associations between UPDs and urinary tract abnormalities are listed hereunder. Maternal UPD 2 Maternal UPD 2 with confined placental mosaicism may cause severe fetal growth retardation (Webb et al. 1996). Of three infants reported, one developed acute renal failure in the neonatal period associated with small kidneys and bilateral vesico-ureteric reflux. The other two did not, and the nature of the renal association remains to be determined. Maternal UPD 7 Maternal UPD 7 is responsible for about 7 per cent of cases of Russell–Silver syndrome (Preece et al. 1999), a condition characterized by pre- and post-natal growth restriction, a characteristic facies, and body asymmetry. Observed urinary tract abnormalities in Russell–Silver syndrome include a functional defect at the level of the renal parenchyma (renal tubular acidosis), and functional or structural defects of the urine conduit system (pelvo-ureteric junction obstruction, vesico-ureteric reflux, posterior urethral valves) (Haslam et al. 1973; Ortiz et al. 1991; Alvarenga et al. 1995). It is yet to be determined whether these observations apply to the subgroup of Russell–Silver syndrome with maternal UPD 7. Paternal UPD 11 The renal phenotype in BWS is of the greatest cytogenetic interest, as we discuss above under the heading dup(11p). Some 10–20 per cent of cases are due to paternal UPD for distal 11p; to be precise, a mosaic segmental paternal isodisomy for chromosome 11p. Here the mechanism is postzygotic: the two No. 11 homologues recombine, the point of exchange being in the short arm, proximal to 11p15.5. A cell line is thus generated in which both distal 11p segments are of paternal origin, and inappropriate overactivity of the growth-promoting genes in 11p15.5 results. Since this UPD cell line may be distributed asymmetrically, the renal enlargement may be unilateral. A routine cytogenetic test would show, of course, a normal karyotype. As for UPD of the whole chromosome, there is a single case report of mosaic paternal isodisomy 11, in which the clinical picture did not differ from typical BWS (Dutly et al. 1998), and one example of non-mosaicism associated with intra-uterine death (Webb et al. 1995), with no report of renal abnormalities. UPD 14 Uniparental disomy 14 is associated with syndromes according to the parental origin of the chromosomes, with 18 cases of maternal (Kotzot 1999) and four of paternal UPD on record (Cotter et al. 1997). In neither are renal defects noted. UPD 15 Maternal UPD 15 and paternal UPD 15 result in PWS and AS respectively. These syndromes are dealt with under chromosome deletions above; neither claims a prominent renal phenotype.

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Maternal UPD 16 This is one of the more commonly recognized UPDs, typically presenting as intra-uterine growth retardation (Kotzot 1999). A single case with renal agenesis is on record (Woo et al. 1997). Chromosomal implications of fetal renal anomalies detected at ultrasonography Clearly, as outlined above, structural defects of the urinary tract are common in many chromosomal disorders. Thus, discovery of a urinary tract defect at fetal ultrasonography raises a concern about the possibility of chromosomal abnormality. The immediate question is this: should fetal karyotyping be offered? How, in this context, is the renal defect to be assessed? In many countries, ultrasound examination is performed at least once in nearly every pregnancy. A ‘fetal anomaly’ scan is usually done between 18 and 20 weeks of gestation; ultrasound examination may also be done for various reasons at other gestational ages. If there are multiple defects, which may or may not include renal defects, the likelihood of a chromosomal abnormality is high, and cytogenetic analysis becomes a matter of urgency. If a renal abnormality is detected as an isolated observation, an underlying chromosomal abnormality is much less likely, and in some cases the risk may not be significantly greater than that of the background population. We here review the chromosomal associations of various categories of fetal renal defect, and discuss these associations in relation to the indications for prenatal karyotype analysis. Isolated renal pyelectasis Pyelectasis is dilatation of the fetal renal pelvis, and can be readily identified on ultrasound in the second and third trimester. It is observed in about 1 per cent of all pregnancies, although the prevalence varies greatly according to the definition used. It has been variously described as a minimum anteroposterior pelvic diameter of 3, 4, or 5 mm (Cockell and Chitty 1998). If 3 mm or greater is taken as the cut-off, the prevalence of pyelectasis may be as high as 18 per cent (Hoddick et al. 1985). Snijders et al. (1995) defined pyelectasis as a minimum anteroposterior pelvic diameter of 4 mm before 20 weeks gestation, and this is the most commonly used cut-off in the mid trimester. The renal pelvis varies somewhat in size during the pregnancy, and Corteville et al. (1992) used a higher cut-off of 7 mm after 33 weeks gestation. Snijders et al. (1995) assessed 1177 fetuses with pyelectasis 4 mm, with no calyceal dilatation and normal renal echogenicity, at 16–24 weeks gestation. When pyelectasis was the only abnormality (805 cases), a chromosome abnormality was present in nine (1.1 per cent), of which five (0.6 per cent) were trisomy 21. This incidence of trisomy 21 (0.6 per cent) was greater, but not significantly so, than that expected on the basis of maternal age alone (0.4 per cent) (Figs 5.3 and 5.4). These workers calculate that at least 1,000,000 pregnancies would be required to demonstrate a statistical difference! On the one hand, they suggest that couples might be counselled that mild hydronephrosis does not significantly increase the risk for fetal trisomy 21; but alternatively, they comment that the risk is 1.6 times higher than the maternal age and gestational age-related risk. Pooled data from other studies indicate that trisomy 21 occurs in about 1 per cent of fetuses with pyelectasis (Cockell and Chitty 1998), although individual studies show a range from 0.3 per cent (Corteville et al. 1992; Chudleigh et al. 1999) to as high as 3.7 per cent


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Fig. 5.3 Bilateral renal pyelectasis: appearance at antenatal ultrasound.

Risk of trisomy 21 (%)

100

10

1 Age related Isolated One Two Three

0.1

0.01 15

20

25

30 35 40 Maternal age (years)

45

50

Fig. 5.4 Maternal age-related risk for trisomy 21 at 20 weeks gestation (bottom line) and the risks in the presence of isolated mild hydronephrosis, and with one, two, and three or more additional abnormalities. From Snijders et al. (1995), courtesy Prof. K. Nicolaides, and with the permission of S. Karger AG.

(Wickstrom et al. 1996). This range may reflect differences in the definition of pyelectasis (see above), the timing of ultrasound, and important differences in the populations studied. In some studies the incidence may have been inflated by high-risk populations, such as those presenting for genetic amniocentesis, or by the inclusion of cases with other (non-renal)

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markers of aneuploidy. Conversely, it is not surprising that no case of trisomy 21 was found in 423 fetuses with isolated pyelectasis when nuchal translucency and second trimester maternal serum screen had previously been normal (Thompson and Thilaganathan 1998). The Down syndrome risk may also be influenced by the sex of the fetus: although pyelectasis is more common in the male, chromosome defects are almost twice as frequent in the female fetus with pyelectasis (Nicolaides et al. 1992). A definitive interpretation is yet to be drawn. It seems likely that isolated renal pyelectasis does represent a real risk factor, but a rather small one, for trisomy 21. How should couples be advised? Taking the data at face value, a case could be made for discussing the risk, and offering fetal karyotyping. The difficulty with this approach is that renal pyelectasis is only one of a number of different ultrasound markers of trisomy 21. If all of these markers are considered individually, we could find that most pregnancies present one or more ‘markers of trisomy 21’ at mid-trimester. When other screening tests for aneuploidy such as nuchal translucency and serum screening are added to this scenario, the problem is, potentially, compounded, and the false-positive rate would be quite unacceptable. A logical solution is to consider the risk factors together rather than separately. An overall risk assessment can be generated which incorporates the pregnant woman’s age-related risk, the results from serum screening and/or nuchal translucency, and ultrasound findings such as renal pyelectasis: the whole is then greater than the sum of the parts. A single risk figure would allow a greater sensitivity for the detection of trisomy 21, and avoid the problem of excessive false-positives. Some workers have addressed this task by combining renal pelvic size with maternal age (Snijders et al. 1995; Wickstrom et al. 1996) (Fig. 5.4), and with serum screening results (Verdin and Economides 1998). The calculation of a single risk figure is facilitated by the use of a likelihood ratio (defined as frequency of the abnormal test result in the population with disease over the frequency of the test result in the population without disease). In a meta-analysis, Smith-Bindman et al. (2001) calculated the likelihood ratio for renal pyelectasis in the detection of Down syndrome to be 1.9. Renal pyelectasis in the presence of other ultrasound abnormalities When pyelectasis exists in the presence of other markers of aneuploidy, the situation is much more straightforward. The risk of an underlying chromosome abnormality is undoubtedly higher, and karyotype analysis should be offered. In the study of Snijders et al. (1995) the risk of aneuploidy increased from 1.1 per cent when pyelectasis was present in isolation, to 5.4 per cent when with one other ultrasound marker of aneuploidy, and to 22.9 and 63.3 per cent when associated with two or three other markers respectively (Fig. 5.5). Fetal obstructive uropathy Urinary tract obstruction (partial or complete) is presumed when there is dilatation of the renal pelvis of 10 mm. Dilatation of the surrounding calyces may also be noted. The presence or absence of dilatation of the ureters and/or bladder is used to determine the level of the obstruction. In the second and third trimesters of pregnancy, complete urinary obstruction is usually associated with severe oligohydramnios or anhydramnios, a partial obstruction being suspected when amniotic fluid is present. Reasonable data are available for the group with lower tract obstruction, for which treatment is potentially available in the form of ultrasoundguided vesico-amniotic shunt. Investigating 30 such fetuses with a view to inserting a shunt,


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Fig. 5.5 Fetal obstructive uropathy: appearance at antenatal ultrasound. Megacystis and bilateral hydronephrosis are present.

Nicolaides et al. (1986) found nine (30 per cent) with a chromosome abnormality, including five cases of trisomy 18 and two cases of trisomy 13. In a retrospective analysis of obstructive uropathy due to upper or lower tract obstruction, Brumfield et al. (1991) identified chromosome abnormality in a similar proportion (23 per cent; seven out of 30 fetuses). There were two trisomy 13s, two trisomy 18s, and isolated cases of 45,X, unbalanced translocation, and an (apparently balanced) inversion. In all seven chromosomally abnormal fetuses, the obstruction was bilateral or at the level of the urethra, and in two of the seven, other malformations (nervous system, cardiac) were detected. Favre et al. (1999) found an abnormal karyotype in four of 16 cases of early fetal megacystis (two with trisomy 13, and single cases of trisomy 18 and trisomy 21). Multicystic kidneys Nicolaides et al. (1992) reviewed their experience in London over a 6-year period during which they assessed 173 cases of multicystic dysplasia, 109 bilateral and 64 unilateral. A chromosome abnormality was found in 12 per cent of all cases. This figure varied according to the presence or absence of other abnormalities. When associated with other abnormalities on ultrasound, the proportion with a chromosomal defect was 33 per cent for bilateral multicystic kidneys and 44 per cent for unilateral disease; the respective figures were 6 and 2 per cent when the kidney defect occurred in isolation. Trisomies 13, 18, 21, triploidy, and various deletions accounted for most of the abnormal karyotypes. Lazebnik et al. (1999) report a similar experience from Pittsburgh. Out of 102 cases of prenatally diagnosed

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Table 5.3 Estimated likelihood to detect a chromosomal abnormality according to nature of the ultrasonographic fetal renal defect (%) Renal defect

Isolated

With other defects

Unilateral Pyelectasis Obstructive uropathy Multicystic kidneys Renal agenesis

Bilateral 1

Low 1 Low

23 3 5

5; 23; 63* 40 29 38

Drawn from data referenced in the text; some figures rounded. *With 1, 2 3 other defects respectively.

multicystic dysplastic kidneys, 11 (11 per cent) were chromosomally abnormal, including four cases of trisomy 18. A chromosomal defect was more likely if the defect was bilateral, if there were extrarenal abnormalities, and if the fetus was female. On the other hand, unilateral isolated involvement was not associated with chromosomal defect, and the outcome was favourable. Renal agenesis Nicolaides et al. (1992) assessed 27 cases of fetal renal agenesis. Of 19 cases in which bilateral renal agenesis was the only abnormality, only one (5 per cent) was chromosomally abnormal. This was 47,XYY, and it is possible that this sex chromosome aneuploidy may be causally related (see above). In contrast, of the eight cases with extrarenal defects in addition, three (38 per cent) showed an abnormal karyotype, of which two were trisomies 13 and 18 (Table 5.3). Urinary tract tumours Cancer is, in a sense, a chromosomal disease. This being so, we shall make brief mention of cytogenetic aspects of tumours of the urinary tract. From the viewpoint of the clinical geneticist, it is the familial and constitutional abnormalities that are of predominant interest; and, since examples are so very few, a review is easily and swiftly accomplished. In contrast, the cytogenetics of sporadic urinary tract cancer could (but will not) command most of the chapter. A more comprehensive molecular review of cancer is to be found in Chapter 22. Renal cell carcinoma (RCC) RCC is the most common kidney cancer, and papillary and non-papillary forms account for most cases (Motzer et al. 1996; Kovacs et al. 1997). The VHL (Von Hippel–Lindau) gene is involved in most non-papillary renal cell carcinomas, while MET gene mutation typically underlies papillary RCC. Familial forms of RCC are rare (Zbar and Lerman 1998). Chromosomal mechanisms are central in two categories of familial RCC, as follows. 1. In a handful of recorded families, hereditary RCC has been associated with a familial constitutional translocation, in each involving chromosome 3. For the record, these include t(3;8)(p14.2;q24.1), t(3;6)(p13;q25), t(2;3)(q33;q21), t(1;3)(q32;q13.3), and


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t(3;6)(q12;q15) (Cohen et al. 1979; Gemmill et al. 1998; Druck et al. 2001; Eleveld et al. 2001; Kanayama et al. 2001). Translocation carriers have a high risk for non-papillary (clear cell) RCC, which may be bilateral. A three-hit scenario is envisaged. The first step is the inheritance of the translocation, and the second is loss of heterozygosity in a renal cell, due to somatic loss of the derivative chromosome that carries the distal 3p segment. The third hit is then somatic mutation in the VHL gene, located on distal 3p on the normal homologue (Bodmer et al. 1998; Kanayama et al. 2001). 2. Hereditary papillary RCC is due to germline mutation in the MET gene on chromosome 7q31. Tumours typically show trisomy 7, due to somatic duplication (which could thus be considered as a second hit) of the chromosome carrying the mutant gene (Zhuang et al. 1998). Presumably, duplication of output from the mutant locus is instrumental in generating the cancer. As for cancer which arises sporadically and somatically in individuals with a 46,XX or 46,XY constitutional karyotype, a wide range of tumour karyotypes is seen, and some patterns are emerging (Verdorfer et al. 1999). Loss of heterozygosity on the short arm of chromosome 3 typifies non-papillary RCC, and is presumed to involve the VHL locus and other tumour suppressor genes (Ogawa et al. 1991; Zbar and Lerman 1998; Martinez et al. 2000). Other genomic imbalances identified in non-papillary RCC lines include losses of 8p, 4q, and 14q, and gains of 5q, 7q, 8q, and 1q (Yang et al. 2000). One particular translocation occupies a unique niche: the X-autosome translocation 46,X,t(X;1)(p11.2;q21). This karyotype characterizes childhood papillary RCC. The translocation fuses a chromosome 1-borne and an X-borne gene, and this leads to the production of a hybrid protein, which initiates the process of carcinogenesis (Sidhar et al. 1996; Zbar and Lerman 1998). Renal oncocytoma This is regarded as being a benign neoplasm, and may occasionally be familial (Weirich et al. 1998). Cytogenetically there are three distinct subgroups: those with loss of the Y chromosome and chromosome 1; those with rearrangements affecting band 11q13; and those with involvement of 12q12–q13 (Fuzesi et al. 1998; Dal Cin et al. 1999). Wilms tumour and mesoblastic nephroma Constitutional karyotypic abnormalities which convey a risk for Wilms tumour include deletion of 11p13 (see above), and the following aneuploidies: trisomy 18 and 45,X, and possibly trisomy 13 and trisomy 8 mosaicism (Niss and Passarge 1976; Nakamura et al. 1985; Olson et al. 1995). The increased risk in Turner syndrome may relate to the association of this karyotype with horseshoe kidney, the latter being a risk factor for Wilms tumour. In the review of Olson et al. (1995), Wilms tumour was diagnosed between the ages of 5 and 13 in the four children with trisomy 18. Since survival to this age is so rare in trisomy 18, it may be that the Wilms association is a rather strong one. As for isolated sporadic Wilms tumour, cytogenetic evolution includes rearrangement at segments 11p13 and 11p15, 1p, 7p, iso7q, 11q, 12q, 16q, 17p, and monosomy 21 and 22 (Kaneko et al. 1991; Coppes and Egeler 1999). ‘Mesoblastic nephroma’ is pathologically heterogeneous; cytogenetic data are accumulating, and trisomy 11 may characterize one subtype (Roberts et al. 1993b; Lowery et al. 1995; see also Chapter 20).

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Renal angiomyolipoma This hamartomatous tumour can occur sporadically, but is best known to geneticists as a typical concomitant of tuberous sclerosis (TSC) (and see Chapter 15). Chromosomal studies show that most sporadic angiomyolipomas, but not TSC-related tumours, carry acquired abnormalities, with a predilection for involvement of chromosome 5q33–q34 (Kattar et al. 1999). Renal pelvis, ureteric, bladder, and urethral cancer (urothelial cancer) An initial discovery of a constitutional t(5;20)(p15;q11) associated with familial urothelial cancer suggested that familial translocations might be an important contributor to this disease, but in fact a formal study of 30 Dutch families thereafter revealed no other one with a chromosomal aberration (Aben et al. 2001). As for sporadic bladder cancer, complex rearrangements are common, with certain chromosomal segments preferentially involved (Koo et al. 1999).

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6 Dysmorphic syndromes with renal involvement Ian D. Young

Introduction The human kidney is an extremely sophisticated organ which represents the end-point of a long and complicated evolutionary process. Its formation requires the successful coordinated orchestration of a complex series of developmental steps including cellular migration, proliferation, differentiation, and polarization. All of these events are under genetic control and although the processes involved are still poorly understood it is reasonable to conclude that the demands imposed upon the human genome for successful nephrogenesis are second only to those required for maturation of the cerebral cortex in terms of their degree of complexity. Given this significant genetic requirement for successful renal development and function it is not surprising that the underlying processes are highly sensitive to any disturbance of normal morphogenesis. This is clearly illustrated by the high incidence of renal abnormalities in almost every recognized chromosomal syndrome involving visible autosomal imbalance as discussed in Chapter 5. Further support is provided by the accumulating evidence that normal renal structure and/or function are compromised in numerous dysmorphic syndromes, many of which show single-gene inheritance. For example, a recent edition of the London Dysmorphology Database (Winter and Baraitser 1999) reveals that renal abnormalities have been described in more than 400 syndromes as outlined in Table 6.1. Those syndromes which are most commonly associated with specific abnormalities are summarized in Table 6.2.

Table 6.1 Specific renal abnormalities and the numbers of syndromes in which they have been documented Abnormality

Number of syndromes

Renal agenesis Multiple cysts Dysplasia Ectopic/supernumerary kidney Horseshoe kidneys Hydronephrosis Large kidneys Small kidneys Nephritis and nephropathy

88 91 54 49 27 110 24 68 74

Data adapted from Winter and Baraitser (1999).

Table 6.2 Renal abnormalities and their more common syndrome associations Renal agenesis

Multiple cysts/dysplasia

Ectopic/supernumerary kidneys

Nephritis/nephropathy

Hydronephrosis

Enlarged kidneys

Branchio-oculo-facial syndrome Branchio-oto-renal syndrome DiGeorge/Shprintzen syndrome Fanconi anaemia Fraser syndrome Fryns syndrome Goldenhar syndrome IDDM embryopathy Kallmann syndrome Lenz microphthalmos syndrome MURCS assocation Nager acrofacial dysostosis Pallister–Hall syndrome Sirenomelia Smith–Lemli–Opitz syndrome Thalidomide embryopathy Townes–Brocks syndrome VATER association

Alagille syndrome Bardet–Biedl syndrome Beckwith–Wiedemann syndrome Branchio-oto-renal syndrome Glutaric aciduria type II Ivemark (asplenia/polysplenia) syndrome Jeune syndrome Kaufman–McKusick syndrome Marden–Walker syndrome Meckel syndrome Oral-facial-digital syndrome type I Roberts syndrome Short rib polydactyly syndromes type I–III Simpson–Golabi–Behmel syndrome Smith–Lemli–Opitz syndrome Townes–Brocks syndrome VATER association Zellweger syndrome

Baller–Gerold syndrome Branchio-oto-renal syndrome CHARGE association Floating-Harbor syndrome Fronto-metaphyseal dysplasia Hydrocephalus/VATER association MURCS association Pallister–Hall syndrome Peters plus syndrome Schinzel–Giedion syndrome VATER association Williams syndrome

Alport syndrome Alstrom syndrome Bardet–Biedl syndrome Drash syndrome Frasier syndrome Glycogen storage disease type I Lowe syndrome Nail-patella syndrome Nezelof syndrome Perlman syndrome Rothmund–Thomson syndrome Sialidosis

Apert syndrome Bardet–Biedl syndrome Branchio-oto-renal syndrome CHARGE association EEC syndrome Fronto-metaphyseal dysplasia Goldenhar syndrome Johanson–Blizzard syndrome Kabuki make-up syndrome Kaufman–McKusick syndrome Marshall–Smith syndrome Megacystis-microcolon-intestinal Hypoperistalsis syndrome Nijmegen breakage syndrome Ochoa syndrome Perlman syndrome Prune-belly anomaly Schinzel–Giedion syndrome Simpson–Golabi-Behmel syndrome Smith–Lemli–Opitz syndrome VATER assocation Weyer oligodactyly syndrome

Beckwith–Wiedemann syndrome Galactosialidosis Glutaric aciduria type II Glycogen storage disease type I Leprechaunism Perlman syndrome Proteus syndrome Simpson–Golabi–Behmel syndrome

The information in this table has been derived from the London Dysmorphology Database (Winter and Baraitser 1999).

Dysmorphic syndromes with renal involvement

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In this chapter, rather than attempt to provide a totally comprehensive account of every dysmorphic syndrome in which a renal abnormality has been documented, attention shall be focussed instead on the relatively small number of more commonly encountered disorders in which renal complications can make a significant contribution to morbidity and mortality. Note that the branchio-oto-renal syndrome and the chromosome microdeletion syndromes, including the Williams and DiGeorge syndromes, are discussed in Chapter 5. The Alstrom and Bardet–Biedl syndrome, are considered in Chapter 17. Alagille syndrome Clinical features The five cardinal features of Alagille syndrome are paucity of intrahepatic bile ducts, peripheral pulmonary stenosis, posterior embryotoxon, butterfly vertebrae, and a characteristic facial appearance with prominent forehead, deep set eyes, and long straight nose. Expression is very variable, however, and many asymptomatic family members show only mild involvement on careful examination. Symptomatic patients present most commonly with jaundice and other features of cholestasis, such as pruritus, which can ultimately progress to hepatic failure. Other cardiac abnormalities can include Fallot’s tetralogy, atrial and ventricular septal defects, and aortic stenosis. Whilst the commonest ocular manifestation is posterior embryotoxon, which is also found in around 10 per cent of the general population, careful eye examination can also reveal retinal pigmentary changes, ectopic pupils, Axenfeld anomaly, and a macular dystrophy. In the absence of severe liver or cardiac involvement the long-term prognosis is usually good with normal intellectual development (Krantz et al. 1997). Renal involvement Mesangiolipidosis was noted in 17 out of 23 patients in one study (Alagille et al. 1987). This is generally thought to be secondary to the hyperlipidaemia that is associated with cholestasis. Structural abnormalities have also been described but are much rarer. These include unilateral agenesis, hypoplasia, and multicystic dysplasia (Martin et al. 1996). Although renal lesions are not usually a major cause of morbidity in Alagille syndrome, there has been a report of an affected 2-month-old infant dying in renal failure as a result of subcortical cysts, interstitial fibrosis, and nephronophthisis (Tolia et al. 1987). In their report of three children with cystic renal disease Martin et al. (1996) concluded that all patients with Alagille syndrome should be evaluated by renal ultrasound. Molecular genetics Inheritance is autosomal dominant with variable expression and close to complete penetrance if potential gene carriers are subjected to an investigative protocol which includes liver function tests, echocardiography, ophthalmoscopy, and spinal radiography. Early reports of occasional patients with deletions involving chromosome 20p11.2 led to isolation of the causative gene, Jagged 1, in which frameshift, nonsense, and splice site mutations have been identified (Yuan et al. 1998). Less than 10 per cent of cases have an identifiable 20p11.2 deletion.

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CHARGE association Clinical features The term ‘CHARGE association’ was introduced by Pagon et al. (1981) to describe the spectrum of abnormalities in a group of children with variable combinations of Coloboma of the iris, Heart abnormalities, Atresia of the choanae, Retardation of growth and development, Genital hypoplasia, and Ear anomalies with or without hearing loss. To make the diagnosis, there should be at least four of these features including either an ocular coloboma and/or choanal atresia. Numerous other abnormalities have been documented including central nervous system (CNS) malformations, cranial nerve palsies, cleft lip/palate, exomphalos, and anal atresia. Almost 50 per cent of patients die before the age of 6 months and feeding problems in infancy can be very severe (Tellier et al. 1998). Intellectual capacity in survivors can vary from normal to severe retardation (Harvey et al. 1991). Renal involvement Renal abnormalities are present in 25–30 per cent of cases. These are variable in nature and include hypoplasia, dysplasia, hydronephrosis, malrotation, horseshoe kidney, and crossed renal ectopia (Blake et al. 1990; Oley et al. 1988). Bladder outflow obstruction due to either urethral atresia or posterior urethral valves has also been reported. Molecular genetics The cause of the CHARGE association is unknown. There have been occasional reports of parent to child transmission and various chromosome abnormalities, including 22q11 deletion, have been reported in isolated cases; however, no clear or consistent pattern has emerged and most cases represent isolated events within a family. Cockayne syndrome Clinical features The natural history of Cockayne syndrome (CS) is dominated by severe postnatal growth failure and progressive multisystem involvement with mental retardation, microcephaly, sensorineural deafness, pigmentary retinopathy, optic atrophy, cataracts, and photosensitivity (Fig. 6.1). Older children show striking short stature with sunken eyes, hollow cheeks, and a thin beaked nose. Neuropathological findings include reduced myelination throughout the nervous system with calcification in the cerebral cortex and basal ganglia. The mean age at death in a series of 37 cases was 12 years although this sample may have been biased by the inclusion of some children with a severe phenotype which shows overlap with the cerebrooculo-facial-skeletal (COFS) syndrome (Nance and Berry 1992). Renal involvement Renal complications are reported to occur in approximately 10 per cent of all cases with the commonest presenting feature being severe hypertension. Findings at renal biopsy include deposition of immunoglobulin and complement in the glomeruli, interstitial fibrosis, tubular


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Fig. 6.1 A young man at 16 years of age with the characteristic facial features of CS with sunken eyes, ‘bird-like nose’, and aged appearance (from Ellaway et al. 2000).

atrophy, and thickening of the glomerular basement membrane (GBH) (Higginbottom et al. 1979; Nance and Berry 1992; Saito et al. 1988). Death due to renal failure has been reported. Regular monitoring of blood pressure and urinalysis to detect proteinuria are indicated. Molecular genetics Cockayne syndrome shows autosomal recessive inheritance with two complementation groups, CSA and CSB, in both of which there is defective repair of UV-induced damage in transcriptionally active DNA. The genes for CSA and CSB have both been cloned and are on chromosomes 5 and 10, respectively (Cleaver et al. 1999). Prenatal diagnosis can be achieved by studying the effects of UV light on cultured cells or, when available, by direct mutation analysis. Frasier syndrome Clinical features This condition is also known as the cryptophthalmos syndrome although this feature is not always present. The other characteristic findings are syndactyly, colobomata of the alae nasi, malformed low set ears, and genital abnormalities, which can include a small phallus, cryptorchidism, vaginal atresia, and ambiguity. Less common findings are cleft lip/palate, laryngeal

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atresia, and CNS malformations. Approximately 50 per cent of cases die before birth or within the first year of life. Renal involvement Two literature reviews yielded figures of 37 and 84 per cent for the incidence of renal agenesis or severe hypoplasia (Thomas et al. 1986; Gattuso et al. 1987), this being the commonest cause of death in the neonatal period (Boyd et al. 1988). Cystic dysplasia secondary to bladder outflow tract obstruction has also been noted. It is important that Fraser syndrome, with its associated recurrence risk of one in four, be considered in the differential diagnosis of any child with bilateral renal agenesis (Potter ‘syndrome’), which in isolation conveys a much lower risk to siblings of around 3 per cent. Molecular genetics Inheritance is autosomal recessive and the relevant gene has not been mapped. Prenatal diagnosis can be achieved only by ultrasonography. Goldenhar syndrome (hemifacial microsomia, oculoauriculovertebral dysplasia) Clinical features Most of the features seen in this group of disorders represent the consequences of abnormal branchial arch development although other organs, including the kidneys, can be affected. Typically, there is unilateral hypoplasia of the lower half of one side of the face with the ear on the same side being malformed. The other hallmark features are ocular epibulbar dermoid tumours and errors of segmentation in the upper cervical vertebrae. The manifestations can be very variable ranging from quite subtle facial asymmetry to severe unilateral hypoplasia with anotia. Preauricular tags and ipsilateral macrostomia are often seen along the line of fusion of the maxillary and mandibular facial processes. CNS malformations and variable mental retardation occur in approximately 10 per cent of cases. Other findings can include pulmonary hypoplasia, cardiac malformations such as Fallot’s tetralogy, and an imperforate anus. Renal involvement Abnormalities of the genitourinary system have been reported in approximately 5 per cent of cases (Rollnick et al. 1987). These include renal agenesis or hypoplasia, crossed renal ectopia, double kidney, and hydronephrosis. Although the documented incidence of renal involvement is relatively low this could be the result of underinvestigation so that renal ultrasonography would be a prudent precaution in all cases. Molecular genetics Most cases are sporadic but there have been sufficient reports of parent–child transmission, albeit with very variable expression, to suggest that a small proportion of cases show autosomal dominant inheritance. If parental examination reveals no abnormality the empirical sibling recurrence risk is between 2 and 6 per cent (Rollnick and Kaye 1983). Confirmation that


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genetic factors can play a role comes from studies in a transgenic mouse line in which a mutation in mouse chromosome 10 causes a hemifacial microsomia phenotype (Naora et al. 1994). Jeune syndrome (asphyxiating thoracic dystrophy) Clinical features Presentation is usually in the newborn period with short limbs, small thorax, and occasional post-axial polydactyly. Radiologically the ribs are short and horizontal and the pelvis shows squaring of the iliac wings with flattening of the acetabular roofs. These show medical spiky protrusions giving a trident-shaped appearance (Fig. 6.2). The combination of pulmonary hypoplasia and restricted chest movement often proves lethal in early infancy (Oberklaid et al. 1977). Survival beyond infancy is usually associated with the development of renal failure in childhood. Other potential complications include tapetoretinal degeneration and progressive hepatic fibrosis leading to cirrhosis. Renal involvement Almost all aspects of renal structure and function can be affected. Tiny glomerular and tubular cysts are present at birth and contribute to the development of the typical changes of cystic dysplasia. Both the glomeruli and the renal tubules can be affected, with biopsy findings of mesangial sclerosis and clinical evidence of renal tubular acidosis. The renal findings in older children can be almost identical to those seen in juvenile nephronophthisis (Donaldson et al. 1985; Turkel et al. 1985). The very high incidence of renal complications in survivors beyond early infancy indicates that all children with Jeune syndrome should be under the care of a paediatric nephrologist.

A

B

Fig. 6.2 X-ray photograph of an infant with Jeune syndrome. A: chest, B: pelvis.

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Molecular genetics Inheritance is autosomal recessive. The disease locus has not been mapped. It has been suggested that the locus could be on chromosome 12 based on a single report of an affected child with a de novo deletion of 12p11.21–12.2 (Nagai et al. 1995). Lowe oculocerebrorenal syndrome (OCRL) Clinical features The three cardinal features of Lowe syndrome are congenital ocular abnormalities, mental retardation and renal tubular dysfunction. The ocular findings include cataract, glaucoma, megalocornea, and microphthalmos; vision is usually severely impaired in affected males. Carrier females show subtle lens opacities (Lin et al. 1999). Affected males show a wide range of intellectual impairment often in association with severe behavioural disturbance characterized by negativism, stubbornness, temper tanrums and obsessional preoccupations (Kenworthy and Charnas 1995). The typical phenotype in Lowe syndrome is that of a severely retarded boy with gross megalocornea, corneal clouding or microphthalmia, hyperactivity, and progressive renal rickets (Abbassi et al. 1968). Renal involvement Tubular dysfunction is usually present from birth whereas glomerular involvement shows a later onset and is more slowly progressive. Proteinuria, generalized aminoaciduria, and phosphaturia occur in the first year of life. Renal biopsy shows thickening in the GBM loops, Bowman’s capsules and the renal tubules, with an increase in glomerular cellularity, focal glomerular fibrosis, and diffuse interstitial fibrosis (Witzleben et al. 1968). In the past, death in childhood was common but with appropriate metabolic therapy survival well into adult life is possible. Renal failure usually occurs in the third or fourth decade (Charnas et al. 1991). Molecular genetics Inheritance is X-linked recessive. Almost all heterozygous adult females show small, nonrefractile, peripheral cortical lens opacities, an observation which can be utilized for carrier detection (Lin et al. 1999). The gene which causes Lowe syndrome (OCRL1) has been cloned and shown to code for a Golgi complex protein with phosphatidylinositol (4,5) biphosphate 5-phosphatase activity. It is located at Xq26.1. Many different loss-of-function mutations have been identified in OCRL1 and tightly linked highly polymorphic mirosatellites have also been identified for use in carrier detection and prenatal diagnosis (Lin et al. 1997; Satre et al. 1999). Meckel syndrome Clinical features This is a lethal malformation syndrome characterized by the classical triad of occipital encephalocele, polycystic kidneys, and postaxial polydactyly. Other common findings include microcephaly, cleft lip/palate, hepatic fibrosis, and genital ambiguity. More rarely there may be other malformations such as holoprosencephaly, Dandy–Walker cyst, microphthalmia,


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Fig. 6.3 Post-mortem view of the abdomen showing enlarged cystic kidneys in a fetus with Meckel syndrome.

a congenital heart defect, or bowing of the long bones. Survival beyond the neonatal period is very unusual. Disorders which feature in the differential diagnosis include the hydrolethalus syndrome and the severe form of the Smith–Lemli–Opitz syndrome. Renal involvement The kidneys are usually very enlarged and cause abdominal distension (Fig. 6.3). Histologically, they show multiple large cysts surrounded by fibromuscular tissue with few nephrons and glomeruli and poor corticomedullary differentiation (Bernstein et al. 1974). Often there is complete anuria resulting in the oligohydramnios sequence phenotype with squashed facies, pulmonary hypoplasia, and talipes. Molecular genetics Inheritance is autosomal recessive with confirmed locus heterogeneity. In Finland the gene has been mapped to chromosome 17q21–24 (Paavola et al. 1995), whereas in Middle Eastern and North African families autozygosity mapping points to a locus at chromosome 11q13 (Roume et al. 1998). In these latter families the CNS involvement was particularly severe with holoprosencephaly, occipital encephalocele, and rhombic roof dysgenesis giving an appearance similar to that seen in the Dandy–Walker malformation (Roume et al. 1997). MURCS association Clinical features The acronym MURCS was derived to describe the association of Müllerian duct, Renal and Cervicothoracic Somite abnormalities. The main features are absence of the vagina and uterus (also known as the Rokitansky malformation), renal aplasia, and errors of segmentation and

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fusion in the cervical and upper thoracic vertebrae (Duncan et al. 1979). Presentation is usually in early adult life with primary amenorrhoea. Rare associations include hearing loss, facial asymmetry, and an encephalocele (Lin et al. 1996). Renal involvement Renal abnormalities were identified in 22 of 25 patients by Duncan et al. (1979). These consisted of either agenesis, usually unilateral, or ectopy or both, with 13 patients having a single pelvic kidney. Renal dysplasia has also been noted. Molecular genetics The cause is unknown and no genetic factors have been implicated. The pathogenesis is thought to involve a defect in the development of the lower cervical–upper thoracic somites and pronephric ducts at around 25–28 days gestation. Oral–facial–digital syndrome, type I (OFDI) Clinical features The oral–facial–digital syndromes constitute a heterogeneous group of disorders consisting of at least nine discrete entities with overlapping clinical features (Toriello 1993). In type I there is often a prominent forehead with hypertelorism, a broad nasal bridge and alar hypoplasia. Oral findings can include a mid-line cleft lip, sometimes in the form of a ‘pseudocleft’, highly arched or cleft palate, tongue clefts or nodules, and multiple oral frenulae (Fig. 6.4A). The digital findings in the hands can take the form of brachydactyly, clinodactyly, and syndactyly (Fig. 6.4B), whereas in the feet preaxial polysyndactyly is the most characteristic finding. Mild mental retardation is said to occur in as many as 40 per cent of all cases, possibly in association with a CNS malformation such as hydrocephalus or partial agenesis of the corpus callosum (Feather et al. 1997a).

A

B

Fig. 6.4 A: View of the mouth. B: View of the hand in a child with the oral–facial–digital syndrome type I.


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Renal involvement The characteristic lesion is that of adult onset polycystic disease, and although the overall incidence is unknown, there have been several reports of families with multiple affected members (Feather et al. 1997a; Donnai et al. 1987). OFDI differs from typical autosomal dominant polycystic kidney disease in that the kidneys are usually of normal size and shape and the renal histology shows that the cysts are glomerular in origin (glomerulocystic kidneys are also found in Jeune syndrome, the short-rib polydactyly syndromes, tuberous sclerosis, and Zellweger syndrome). The polycystic kidney disease in OFDI can culminate in renal failure so that all cases should be monitored for this complication from late childhood onwards. Molecular genetics Inheritance is X-linked dominant with lethality in affected male embryos. Thus, for an affected female there is one chance in two that each of her daughters will be affected whereas sons who survive to term will be unaffected. The disease gene has been isolated having been previously mapped to a relatively small region of the X chromosome at Xp22.2–Xp22.3 (Feather et al. 1997b; Gedeon et al. 1999). It has been designated as CXORFS and the function of its protein product is unknown. Smith–Lemli–Opitz (SLO) syndrome Clinical features These fall within a spectrum ranging from variable mental retardation with characteristic dysmorphic features (SLO type I) to a very severe presentation with death in the neonatal period (SLO type II). Patients with classical type I SLO syndrome (Fig. 6.5) show microcephaly, ptosis, anteverted nares, micrognathia, postaxial polydactyly, 2/3 toe syndactyly, and a genital anomaly such as hypospadias, cryptorchidism, or ambiguity (Ryan et al. 1998). Congenital heart defects, most commonly an atrioventricular septal defect / coarctation, occur in approximately one-third of cases. Most affected children show moderate to severe mental retardation. In the severe lethal type II SLO syndrome, there is a high incidence of severe internal malformations such as congenital heart defects, abnormal pulmonary lobulation, and renal abnormalities. Almost all affected babies have a cleft palate, small tongue, postaxial polydactyly, and short limbs. Most 46, XY cases show either genital ambiguity or complete sex reversal (Curry et al. 1987). Renal involvement A large survey undertaken in the USA, based on cases investigated locally plus published reports, indicated that 57 per cent of affected children had an upper urinary tract abnormality (Joseph et al. 1987). A similar review of biochemically proven cases in the UK gave an incidence of renal abnormality of 27 per cent (Ryan et al. 1998). Combining both of these studies the renal abnormalities included agenesis, hypoplasia, cystic dysplasia, duplication, and hydronephrosis. In a series of 19 type II SLO cases, five had unilateral renal agenesis, one had unilateral cystic dysplasia and one had a single fused ectopic kidney (Curry et al. 1987). Molecular genetics Inheritance is autosomal recessive and the basic defect lies in cholesterol biosynthesis. The diagnosis can be confirmed by demonstrating an elevated level of 7-dehydrocholesterol and a

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C

B

D

Fig. 6.5 Clinical features of Smith–Lemli–Opitz syndrome (from Kelley and Hennekam 2000). A: Syndactyly of the second, third and fourth toes. B: Hand abnormalities—postaxial polydactyly along with duplication/ulnar deviation of the distal phalanx of the thumb. C: Ambiguous genitalia. D: Ear is small.

reduced level of cholesterol in serum. Recently, mutations have been identified in the 7-dehydrocholesterol reductase gene on chromosome 11q12–13 in several patients (Wassif et al. 1998). Confirmation of this diagnosis is important not only for genetic counselling but also because there is some evidence that cholesterol therapy has a beneficial effect on growth and behaviour in some children and adults (Kelley 1998). A full evaluation of the proposed benefits of cholesterol therapy is awaited. Townes–Brocks syndrome Clinical features These overlap with those seen in the VATER/VACTERL association from which distinction is important because of the fundamental difference in genetic aetiology. The classical findings


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as recorded in the original report by Townes and Brocks (1972) are auricular anomalies, hearing loss, triphalangeal or supernumerary thumbs, and imperforate anus. Typically, the ears are small and cup shaped and hearing loss can vary from mild to severe. Hand abnormalities can include preaxial polydactyly along with duplication and/or ulnar deviation of the distal phalanx of the thumb (Fig. 6.6). The most common finding in the feet is overlapping of the second, third and fourth toes. The anus is imperforate in approximately 50 per cent of cases, often in association with a rectovaginal or rectourethral fistula. Cardiac abnormalities have been noted in a few cases. Most individuals with this condition are of normal intelligence (Powell and Michaelis 1999). Renal involvement This is common and can include unilateral agenesis, uni- or bilateral hypoplasia, multicystic changes and hydronephrosis secondary to obstruction caused by posterior urethral valves and meatal stenosis. Hypoplasia is the commonest manifestation of renal involvement and has been reported in association with end-stage renal failure both in infancy and in adult life (Newman et al. 1997). Several patients have undergone renal transplantation (Powell and Michaelis 1999). Renal ultrasonography is indicated in all patients supplemented by regular monitoring of renal function when an abnormality is identified. Molecular genetics Inheritance is autosomal dominant with marked intrafamilial variation. Reports of several affected individuals with balanced chromosome rearrangements involving a breakpoint at 16q12.1 led to isolation of the causative gene known as SALL1. This is a developmental transcription regulator gene which is conserved throughout evolution. Mutations in SALL1 have been identified in both familial and isolated cases (Kohlhase et al. 1998). VATER association Clinical features The concept of the VATER association was proposed to account for the tendency of Vertebral, Anorectal, Tracheo-Esophageal, Radial, and Renal abnormalities to occur together more often than would be expected by chance. It is generally accepted that at least three of these five cardinal features are necessary for this diagnosis to be accepted. The phenotype was subsequently expanded to include cardiac abnormalities, hence the ‘VACTERL’ association, in which C and L represent cardiac and limb abnormalities, respectively. Other recognized associated findings include single umbilical artery, cleft lip-palate, and diaphragmatic hernia. The combination of VATER anomalies and hydrocephalus is thought to represent a separate discrete entity. Renal involvement Estimates of the incidence of renal abnormalities in children with the VATER association vary from 41 per cent (Khoury et al. 1983) to 61 per cent (Weaver and Mapstone 1986) to 80 per cent (Botto et al. 1997). These can take the form of unilateral or bilateral agenesis, hypoplasia, cystic dysplasia, hydronephrosis, crossed renal ectopia, or a horseshoe kidney. Bladder outflow obstruction due to urethral atresia can also occur. This high incidence and protean nature

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B

C

Fig. 6.6 A: Dysplastic ear with preauricular tag and cheek tag in a child with Townes–Brocks syndrome. B: Ulnar deviation of distal phalanges of the thumbs in a patient with Townes–Brocks syndrome. C: Second and fourth toes overlapping third, and fifth toe clinodactyly in a patient with Townes–Brocks syndrome. (From Powell and Michaelis 1999.)


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of renal involvement fully justify renal ultrasonography in all children with more than one VATER association abnormality. Molecular genetics Almost all cases represent isolated events within a family and there is little if any evidence to support a significant genetic role. A possible link with underlying tissue mosaicism for a mitochondrial mutation has not been confirmed (Stone and Biesecker 1997). There is some evidence that the association of VATER anomalies with hydrocephalus may show autosomal recessive inheritance (Evans et al. 1989). Zellweger syndrome Clinical features This is the most severe condition caused by a disorder of peroxisomal biogenesis. Presentation is usually in the neonatal period with severe hypotonia, poor feeding, and convulsions. Affected babies have a high forehead, large anterior fontanelle, shallow supraorbital ridges, puffy eyelids, and flat nasal bridge. Other findings can include corneal clouding, cataracts, cardiac defects such as a VSD, hepatomegaly, and camptodactyly. The average length of survival is 3 months (Wilson et al. 1986). Developmental progress is very limited and at autopsy the brain shows changes of abnormal neuronal migration with microgyria, pachygyria, lissencephaly, and hypoplasia of the olfactory bulbs. White matter changes consistent with a sudanophilic leukodystrophy are also present (Volpe and Adams 1972). Renal involvement The kidneys can be either large or small. Almost invariably, they contain numerous small cortical cysts which are mainly glomerular in origin. Histologically, these cortical microcysts are associated with focal sclerotic and involutional changes with evidence of deficient metanephric differentiation (Bernstein et al. 1974). Both albuminuria and aminoaciduria are often present. The potential impact of these renal findings is usually totally overshadowed by the profound neurological sequelae of the CNS involvement. Molecular genetics Inheritance is autosomal recessive. The Zellweger syndrome phenotype can be caused by mutations in several different genes with at least 10 recognized complementation groups (Moser et al. 1995). The diagnosis is confirmed by measurement of very long chain fatty acids, pipecolic acid, phytanic acid, and plasmalogens. Histology confirms the absence of peroxisomes in liver and kidney. Prenatal diagnosis can be achieved by assay of the levels of very long chain fatty acids in cultured chorionic villus samples (CVS) or amniocytes. Conclusion There is increasing recognition that children and adults with dysmorphic syndromes and other multisystem genetic disorders should benefit from multidisciplinary assessment and

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management. This is particularly relevant for most of the disorders discussed in this chapter, especially those such as the Jeune and OFDI syndromes in which renal involvement is the main cause of morbidity in late childhood and adult life. This also applies to the Williams and DiGeorge/Shprintzen microdeletion syndromes as discussed in Chapter 5. Furthermore, many children with one of the unexplained sporadic associations (e.g. CHARGE, MURCS, and VATER) will also be found to have a renal malformation if appropriately investigated. Given that renal malformations are often aysmptomatic until associated complications reach an advanced stage, a strong case can be made for including renal imaging as an integral component of the standard investigative protocol for all children with a multisystem dysmorphic syndrome. The distress caused by the non-invasive procedure of renal ultrasonography is minimal. In contrast the potential for preventing morbidity and mortality is enormous. Acknowledgements The author is grateful to Dr David Semeraro for the permission to reproduce Fig. 6.3 and to Dr Alan Watson for critical review of the manuscript. References Abbassi, V., Lowe, C. U., and Calcagno, P. L. (1968). Oculo-cerebro-renal syndrome. A review. American Journal of Diseases of Children, 115, 145–67. Alagille, D., Estrada, A., Hadchouel, M., Gautier, M., Odièvre, M., and Dommergues, J. P. (1987). Syndromic paucity of interlobular bile ducts (Alagille syndrome or arteriohepatic dysplasia): review of 80 cases. Journal of Pediatrics, 110, 195–200. Bernstein, J., Brough, A. J., and McAdams, A. J. (1974). The renal lesions in syndromes of multiple congenital malformations. Birth Defects: Original Article Series, X(4), 35–43. Blake, K. D., Russell-Eggitt, I. M., Morgan, D. W., Ratcliffe, J. M., and Wyse, R. K. H. (1990). Who’s in CHARGE? Multidisciplinary management of patients with CHARGE assocation. Archives of Disease in Childhood, 65, 217–23. Botto, L. D., Khoury, M. J., Mastroiacovo, P., Castilla, E. E., Moore, C. A., Skjaerven, R., et al. (1997). The spectrum of congenital anomalies of the VATER association: an international study. American Journal of Medical Genetics, 71, 8–15. Boyd, P. A., Keeling, J. W., and Lindenbaum, R. H. (1988). Fraser syndrome (cryptophthalmossyndactyly syndrome): a review of eleven cases with postmortem findings. American Journal of Medical Genetics, 31, 159–68. Charnas, L. R., Bernardini, I., Rader, D., Hoeg, J. M., and Gahl, W. A. (1991). Clinical and laboratory findings in the oculocerebrorenal syndrome of Lowe with special reference to growth and renal function. New England Journal of Medcine, 324, 1318–25. Cleaver, J. E., Thompson, L. H., Richardson, A. S., and States, J. C. (1999). A summary of mutations in the UV-sensitive disorders: xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. Human Mutation, 14, 9–22. Curry, C. J. R., Carey, J. C., Holland, J. S., Chopra, D., Fineman, R., Golabi, M., et al. (1987). Smith–Lemli–Opitz syndrome–type II: multiple congenital anomalies with male pseudohermaphroditism and frequent early lethality. American Journal of Medical Genetics, 26, 45–57. Donaldson, M. D. C., Warner, A. A., Trompeter, R. S., Haycock, G. B., and Chantler, C. (1985). Familial juvenile nephronophthisii, Jeune’s syndrome and associated disorders. Archives of Disease in Childhood, 60, 426–34.


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Donnai, D., Kerzin-Storrar, L., and Harris, R. (1987). Familial orofaciodigital syndrome type I presenting as adult polycystic kidney disease. Journal of Medical Genetics, 24, 84–7. Duncan, P. A., Shapiro, L. R., Stangel, J. J., Klein, R. M., and Addonizio, J. C. (1979). The MURCS association: Mullerian duct aplasia, renal aplasia, and cervicothoracic somite dysplasia. Journal of Pediatrics, 95, 399–402. Ellaway, C. J., Duggins, A., Fung, V. S., Earl, J. W., Kamath, R., Parsons, P. G., et al. (2000). Cockayne syndrome associated with low CSF 5-hydroxyindole acetic acid levels. Journal of Medical Genetics, 37, 553–7. Evans, J. A., Stranc, L. C., Kaplan, P., and Hunter, A. G. W. (1989). VACTERL with hydrocephalus: further delineation of the syndrome(s). American Journal of Medical Genetics, 34, 177–82. Feather, S. A., Winyard, P. J. D., Dodd, S., and Woolf, A. S. (1997a). Oral–facial–digital syndrome type I is another dominant polycystic kidney disease: clinical, radiological and histopathological features of a new kindred. Nephrology, Dialysis, Transplantation, 12, 1354–61. Feather, S. A., Woof, A. S., Donnai, D., Malcom, S., and Winter, R. M. (1997b). The oral–facial– digital syndrome type 1 (OFD1), a cause of polycystic kidney disease and associated malformations maps to Xp22.2-Xp22.3. Human Molecular Genetics, 6, 1163–67. Gattuso, J., Patton, M. A., and Baraitser, M. (1987). The clinical spectrum of the Fraser syndrome: report of three new cases and review. Journal of Medical Genetics, 24, 549–55. Gedeon, A. K., Oley, C., Nelson, J., Turner, G., and Mulley, J. C. (1999). Gene localization for oral–facial–digital syndrome type 1 (OFD1 : MIM 311200) proximal to DXS85. American Journal of Medical Genetics, 82, 352–4. Harvey, A. S., Leaper, P. M., and Bankier, A. (1991). CHARGE association: clinical manifestations and developmental outcome. American Journal of Medical Genetics, 39, 48–55. Higginbottom, M. C., Griswold, W. R., Jones, K. L., Vasquez, M. D., Mendoza, S. A., and Wilson, C. B. (1979). The Cockayne syndrome: an evaluation of hypertension and studies of renal pathology. Pediatrics, 64, 929–34. Joseph, D. B., Uehling, D. T., Gilbert, E., and Laxova, R. (1987). Genitourinary abnormalities associated with the Smith–Lemli–Opitz syndrome. Journal of Urology, 137, 719–21. Kelley, R. I. (1998). RSH/Smith–Lemli–Opitz syndrome: mutations and metabolic morphogenesis. American Journal of Human Genetics, 63, 322–6. Kelley, R. I. and Hennekam, R. C. (2000). The Smith–Lemli–Opitz syndrome. Journal of Medical Genetics, 37, 321–35. Kenworthy, L. and Charnas, L. (1995). Evidence for a discrete behavioural phenotype in the oculocerebrorenal syndrome of Lowe. American Journal of Medical Genetics, 59, 283–90. Khoury, M. J., Cordero, J. F., Greenberg, F., James, L. M., and Erickson, J. D. (1983). A population study of the VACTERL association: evidence for its etiologic heterogeneity. Pediatrics, 71, 815–20. Kohlhase, J., Wischermann, A., Reichenbach, H., Froster, U., and Engel, W. (1998). Mutations in the SALL1 putative transcription factor gene cause Townes–Brocks syndrome. Nature Genetics, 18, 81–3. Krantz, I. D., Piccoli, D. A., and Spinner, N. B. (1997). Alagille syndrome. Journal of Medical Genetics, 34, 152–7. Lin, H. J., Comford, M. E., Hu, B., Rutgers, J., K. L., Beall, M. H., and Lachman, R. S. (1996). Occipital encephalocele and MURCS association: case report and review of central nervous system anomalies in MURCS patients. American Journal of Medical Genetics, 61, 59–62. Lin, T., Lewis, R. A., and Nussbaum, R. L. (1999). Molecular confirmation of carriers for Lowe syndrome. Ophthalmology, 106, 119–22. Lin, T., Orrison, B. M., Leahey, A. M., Suchy, S. F., Bernard, D. J., Lewis, R A., et al. (1997). Spectrum of mutations in the OCRL1 gene in the Lowe oculocerebrorenal syndrome. American Journal of Human Genetics, 60, 1384–8.

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Martin, S. R., Garel, L., and Alvarez, F. (1996). Alagille’s syndrome associated with cystic renal disease. Archives of Disease in Childhood, 74, 232–5. Moser, A. B., Rasmussen, M., Naidu, S., Watkins, P. A., McGuinness, M., Hajra, A. K., et al. (1995). Phenotype of patients with peroxisomal disorders subdivided into sixteen complementation groups. Journal of Pediatrics, 127, 13–22. Nagai, T., Nishimura, G., Kato, R., Hasegawa, T., Ohashi, H., and Fukushima, Y. (1995). Del(12)(p11.21p12.2) associated with an asphyxiating thoracic dystrophy or chondroectodermal dysplasia-like syndrome. American Journal of Medical Genetics, 55, 16–18. Nance, M. A. and Berry, S. A. (1992). Cockayne syndrome: review of 140 cases. American Journal of Medical Genetics, 42, 68–84. Naora, H., Kimura, M., Otani, H., Yokoyama, M., Koizumi, T., Katsuki, M., et al. (1994). Transgenic mouse model of hemifacial microsomia: cloning and characterization of insertional mutation region on chromosome 10. Genomics, 23, 515–19. Newman, W. G., Brunet, M. D., and Donnai, D. (1997). Townes–Brocks syndrome presenting as end stage renal failure. Clinical Dysmorphology, 6, 57–60. Oberklaid, F., Danks, D. M., Mayne, V., and Campbell, P. (1997). Asphyxiating thoracic dystrophy. Archives of Disease in Childhood, 52, 758–65. Oley, C. A., Baraitser, M., and Grant, D. B. (1988). A reappraisal of the CHARGE association. Journal of Medical Genetics, 25, 147–56. Paavola, P., Salonen, R., Weissenbach, J., and Peltonen, L. (1995). The locus for Meckel syndrome with multiple congenital anomalies maps to chromosome 17q21-q24. Nature Genetics, 11, 213–15. Pagon, R. A., Graham, J. M., Zonana, J., and Yong, S. L. (1981). Coloboma, congenital heart disease and choanal atresia with multiple anomalies: CHARGE association. Journal of Pediatrics, 99, 223–7. Powell, C. M. and Michaelis, R. C. (1999). Townes–Brocks syndrome. Journal of Medical Genetics, 36, 89–93. Rollnick, B. R. and Kaye, C. I. (1983). Hemifacial microsomia and variants. American Journal of Medical Genetics, 15, 233–53. Rollnick, B. R., Kaye, C. I., Nagatoshi, K., Hauck, W., and Martin, A. O. (1987). Oculoauriculovertebral dysplasia and variants: phenotypic characteristics of 294 patients. American Journal of Medical Genetics, 26, 361–75. Roume, J., Genin, E., Cormier-Daire, V., Ma, H. W., Mehaye, B., Attie, T., et al. (1998). A gene for Meckel syndrome maps to chromosome 11q13. American Journal of Human Genetics, 63, 1095–101. Roume, J., Ma, H. W., Le Merrer, M., Cormier-Daire, V., Girlich, D., Genin, E., et al. (1997). Genetic heterogeneity of Meckel syndrome. Journal of Medical Genetics, 34, 1003–6. Ryan, A. K., Bartlett, K., Clayton, P., Eaton, S., Mills, L., Donnai, D., et al. (1998). Smith–Lemli–Opitz syndrome: a variable clinical and biochemical phenotype. Journal of Medical Genetics, 35, 558–65. Saito, H., Saito, T., Kurosawa, K., Ootaka, T., Furuyama, T., and Yoshinaga, K. (1988). Renal lesions in Cockayne syndrome. Clinical Nephrology, 29, 206–9. Satre, V., Monnier, N., Berthorn, F., Ayuso, C., Joannard, A., Jouk, P. S., et al. (1999). Characterization of a germline mosaicism in families with Lowe syndrome, and identification of seven novel mutations in the OCRL1 gene. American Journal of Human Genetics, 65, 68–76. Stone, D. L. and Biesecker, L. G. (1997). Mitochondrial NP3243 point mutation is not a common cause of VACTERL association. American Journal of Medical Genetics, 72, 237–8. Tellier, A. L., Cormier-Daire, V., Abadie, V., Amiel, J., Sigandy, S., Bonnet, D., et al. (1998). CHARGE syndrome: report of 47 cases and review. American Journal of Medical Genetics, 76, 402–9. Thomas, I. T., Frias, J. L., Felix, V., Sanchez de Leon, L., Hernandez, R. A., and Jones, M. C. (1986). Isolated and syndromic cryptophthalmos. American Journal of Medical Genetics, 25, 85–98. Tolia, V., Dubois, R. S., Watts, F. B., and Perrin, E. (1987). Renal abnormalities in paucity of interlobular bile ducts. Journal of Pediatric Gastoenterology and Nutrition, 6, 971–6. Toriello, H. V. (1992). Oral–facial–digital syndromes, 1992. Clinical Dysmorphology, 2, 95–105.


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Townes, P. L. and Brocks, E. R. (1972). Hereditary syndrome of imperforate anus with hand, foot and ear anomalies. Journal of Pediatrics, 81, 321–6. Turkel, S. B., Diehl, E. J., and Richmond, J. A. (1985). Necropsy findings in neonatal asphyxiating thoracic dystrophy. Journal of Medical Genetics, 22, 112–18. Volpe, J. J. and Adams, R. D. (1972). Cerebro-hepato-renal syndrome of Zellweger: an inherited disorder of neuronal migration. Acta Neuropathologica, 20, 175–98. Wassif, C. A., Maslen, C., Kachilele-Linjewile, S., Lin, D., Linck, L. M., Connor W. E., et al. (1998). Mutations in the human sterol 7-reductase gene at 11q12-13 cause Smith–Lemli–Opitz syndrome. American Journal of Human Genetics, 63, 55–62. Weaver, D. D. and Mapstone, C. L. (1986). The VATER association. American Journal of Diseases of Children, 140, 225–9. Wilson, G. N., Holmes, R. G., Custer, J., Lipkowitz, J. L., Stover, J., Datta, N., et al. (1986). Zellweger syndrome: diagnostic assays, syndrome delineation and potential therapy. American Journal of Medical Genetics, 24, 69–82. Winter, R. M. and Baraitser, M. (1999). The London Dysmorphology Database. Oxford University Press, Oxford. Witzleben, C. L., Schoen, E. J., Tu, W. H., and McDonald, L. W. (1968). Progressive morphologic renal changes in the oculo-cerebro-renal syndrome of Lowe. American Journal of Medicine, 44, 319–24. Yuan, Z. R., Kohsaka, T., Ikegaya, T., et al. (1998). Mutational analysis of the Jagged 1 gene in Alagille syndrome families. Human Molecular Genetics, 7, 1363–9.

Recommended further reading Bergsma, D. (ed.) (1974). The clinical delineation of birth defects. Part XVI. Urinary system and others. Birth defects: Original Article Series, X(4). Gorlin, R. J., Cohen, M. M., and Hennekam, R. C. M. (2001). Syndromes of the head and neck, 4th edn., Oxford University Press, New York, NY. Jones, K. L. (1996). Smith’s recognizable patterns of human malformation, 5th edn., Saunders, Philadelphia, PA. Limwongse, C., Clarren, S. K., and Cassidy, S. B. (1999). Syndromes and malformations of the urinary tract. In Paediatric nephrology (ed. T. M. Barratt, E. D. Avner, and W. E. Harman, 4th edn), pp. 427–52. Lippincott Williams & Wilkins, Baltimore, MD. Stevenson, R. E., Hall, J. G., and Goodman, R. M. (1993). Human malformations and related anomalies. Oxford University Press, New York, NY.


7 Primary hereditary nephropathies Karl Tryggvason

Introduction Congenital nephrotic syndrome (CNS) is defined as proteinuria leading to clinical symptoms such as edema soon after birth, especially at the age limit of 3 months (Rapola et al. 1992; Mauch et al. 1994). Most CNS have a genetic basis and poor outcome. Recent extensive progress in molecular genetics and biology has given new insight into the molecular basis of these diseases. In many cases, precise diagnosis of CNS or other hereditary nephropathies can now be based on gene analysis, in addition to clinical, laboratory, and histological criteria. Since the gene for nephrin, a key component of the slit diaphragm (SD) was identified as the disease gene in Finnish type CNS in 1998, several proteins of the SD complex have been found. Alterations or lack of any of the proteins of this SD complex will lead to an abnormal glomerular filter structure and proteinuria of variable onset and severity. In this chapter, the various hereditary conditions that may be associated with nephrotic syndrome at birth or even later in life are reviewed. Congenital nephrotic syndrome of the Finnish type Clinical features The most common type of CNS is congenital nephrotic syndrome of the Finnish type (CNF or NPHS1), a disorder particularly frequent in Finland, where the incidence is 1 : 10,000 births (Huttunen 1976). It was first described by Hallman et al. (1956). The children are often born prematurely, one third of the patients have low birth weight. The placenta is larger than normal and weighs more than 25 per cent of the newborn’s birth weight. The basic abnormality in CNF is massive nonselective proteinuria, beginning in utero (Huttunen 1976). Edema and abdominal distension, that are present immediately after birth or within the following next few weeks, are secondary to the protein deficiency. Without nutritional support and albumin supplementation, the classic picture of CNF develops: widened cranial sutures and fontanelles, a small nose, wide-set eyes and low ears, abdominal distension, ascites, umbilical hernias, a tendency to assume an opisthotonic position, and generalized edema (Hallman et al. 1956; Rapola et al. 1992; Mauch et al. 1994). Laboratory findings In CNF, the first urine analysis already shows proteinuria, microscopic hematuria, and often some leukocytes. The level of proteinuria exceeds 20 g/l only when the serum albumin concentration is above 15 g/l (Holmberg et al. 1995). In addition to albumin, many large proteins, such as immunoglobulin G, transferrin, apoproteins, lipoprotein lipase (LPL),

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antithrombin III (ATIII), ceruloplasmin, vitamin D binding protein, and thyroid binding globulin are lost into urine (Antikainen et al. 1992; Mauch et al. 1994). The loss of these proteins, leads to secondary metabolic disturbances. Urinary excretion of plasminogen and ATIII leads to compensatory protein synthesis, resulting in increased levels of macroglobulins, fibrinogen, thromboplastin, and factors II, V, VII, X, and XII, contributing to coagulopathy (Panicucci et al. 1983; Rapola et al. 1992). Low serum albumin and postheparin plasma LPL activities and high free fatty acid concentrations lead to hypertriglyceridemia, may increase the risk of arteriosclerosis (Antikainen et al. 1994). There are no pathognomonic pathologic features in CNF, the most typical histological finding is dilation of the proximal tubules (Huttunen et al. 1980). The kidneys are also large and have been found to contain a larger number of nephrons than age-matched controls (Tryggvason and Kouvalainen 1975). Electron microscopy reveals no abnormal features of the glomerular basement membrane (GBM) itself, although there is a loss of foot processes of the glomerular epithelial cells, a finding characteristic of nephrotic syndrome of any cause (Huttunen 1976; Huttunen et al. 1980). Chemical analyses carried out on the composition of the GBM of CNF patients in the 1970s did not reveal any typical changes (Tryggvason 1977), and later studies on GBM proteins, such as type IV collagen, laminin, and heparan sulfate proteoglycan (HSPG), or their genes did not reveal any association with CNF (Ljungberg et al. 1993; Kestila et al. 1994). There is no evidence of an immune complex cause of CNF, as shown by immunofluorescence studies and lack of dense deposits in electron microscopy. Genetics The CNF is inherited as an autosomal recessive trait (Norio 1966). The gene was identified by positional cloning using DNA from 17 Finnish CNF families, each having one or two affected individuals (Kestila¨ et al. 1994, 1998; Lenkkeri et al. 1999). Through linkage analysis, a novel gene on chromosome 19 was shown to be mutated in the CNF patients (Kestila¨ et al. 1998) (Fig. 7.1). The gene, termed NPHS1, was shown to be expressed in the kidney specifically in glomeruli, and to encode a novel protein termed nephrin. The NPHS1 gene is 26-kb long, with 29 exons (Kestila¨ et al. 1998). Two mutations in the NPHS1 gene accounting for 90 per cent of mutations in Finland are a 2-bp deletion in exon 2 (Fin-major) and a nonsense mutation in exon 26 (Fin-minor). Of these patients, 65 per cent were homozygous for the 2-bp deletion in exon 2 (Fin-major), 8 per cent were homozygous for a nonsense mutation in exon 26 (Fin-minor), and 16 per cent were compound heterozygotes. These mutations, that lead to complete absence of nephrin and podocyte SD, are responsible for a severe, therapyresistant form of nephrotic syndrome (NS). Mutations in the NPHS1 gene have also been found in many non-Finnish cases. Altogether, over 50 mutations have been detected in NPHS1 in patients worldwide. These mutations include insertions, deletions, and nonsense and missense mutations that are scattered throughout the entire gene (Lenkkeri et al. 1999; Aya et al. 2000; Patrakka et al. 2000; Beltcheva et al. 2001). This emphasizes the importance of being able to provide diagnosis of NPHS1 mutations, especially in Finland where an assay for the two most common mutations enables specific prenatal or neonatal diagnosis of about 90 per cent of CNF cases. The two most common mutations found in the Finnish population result in premature stop codons and truncated nephrin molecules. In contrast to insertions, deletions, and nonsense mutations, that lead to synthesis of a truncated polypeptide, missense mutations result in full size proteins with amino acid substitutions. A recent study on the subcellular

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Chromosome 19 q13.1 A

D19S208 D19S191

D19S609 MAG

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F 19399 F 15549 R 28051

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APLPI

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C

E 3⬘

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5⬘

Fig. 7.1 Physical map of the NPHS1 locus at 19q13.1 and genomic organization of the NPHS1 gene. A: Physical map of the 920-kb region between D19S208 and Results D19S224. B: Overlapping cosmid clones spanning the 150-kb critical region containing the NPHS1 gene. Location of polymorphic markers are characterization of genes indicated by arrows. at the NPHS1 locus. C: Location of five genes, NPHS1, APLP1, A, B, and C, characterized following localization of the NPHS1 gene to 19q13.1, and analysed for mutations in this study, overlapping cosmid clones from the interval of interest. D: Schematic structure of the NPHS1 gene. (Adapted from Kestilä, M., Lenkkeri, U., Lamerdin, J., et al. (1998). Positionally cloned gene for a novel glomerular protein—nephrin is mutated in congenital nephrotic syndrome. Molecular Cell, 1, 575–82.)

localization of 21 missense mutants from CNF patients in transfected human embryonic kidney cells showed that in the majority of cases, the mutant proteins are retained in the endoplasmic reticulum and do not appear on the plasma membrane (Liu et al. 2001). This suggests that misfolding and defective intracelluar transport with consequent absence of the mutant nephrin on the plasma membrane is the most common cause in development of CNF carrying missense mutations. Interestingly, some of the NPHS1 mutations lead to a slower progression to end-stage renal disease (ESRD) than characteristic for patients with the more severe Finnish type of CNS. For example, one patient with a Fin-major mutation in one allele and a missense mutation (a change of arginine 743 to cysteine in the extracellular Ig5 domain) in the other, had partially

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defective nephrin but still a normal looking SD and he responded to drug therapy (Patrakka et al. 2000). This emphasizes the importance of understanding the nature and effect of the mutations as it can help physicians choose treatment and evaluate the prognosis of the disease. Diagnosis and treatment Knowing that mutations in the nephrin gene are the cause of CNF prompted physicians to perform a systemic analysis of the clinical manifestations and renal pathology of Finnish patients with CNF. Prenatally, a high maternal serum fetoprotein (AFP) concentration (more than 2.5 multiples of the median) suggests fetal abnormalities such as CNF, neural tube defects, or defects of abdominal wall closure (Seppälä et al. 1979). In Finland, elevated levels of AFP in amniotic fluid was previously used to guide prenatal CNF diagnosis in families at risk. However, a carrier with the most common Finnish haplotype (the Fin-major mutation) may have an elevated amniotic fluid AFP concentration (up to 100,000 g/l) in early pregnancy, which later decreases and leads to an unaffected child at birth (Ma¨nnikko¨ et al. 1997). Retrospective studies indicate that a fetus heterozygous for NPHS1 mutations shows temporary proteinuria and renal lesions resembling those of the affected fetuses. AFP concentration in both maternal serum and amniotic fluid could be elevated. Thus, the use of AFP measurements for the prenatal diagnosis of congenital nephrosis is unreliable and easily leads to unjustified abortions. Therefore, genetic analysis of NPHS1 should be done when CNS is suspected (Jaakko Patrakka et al. 2002). High urinary protein with intrauterine onset is always seen in CNF patients. Serum albumin is less than 10 g/l before treatment. When the serum albumin is corrected to 15 g/l, the urinary protein is 20 g/l. An enlarged placenta indicates massive intrauterine proteinuria. Positive family history and exclusion of other types of CNS are also criteria of diagnosis. Now, analysis of the NPHS1 gene is the method of choice for precise diagnosis of CNF. The CNF is a progressive disease, usually leading to death during the first 2 years of life, with kidney transplantation being the only life-saving treatment (Holmberg et al. 1995). Immunosuppressive therapy with steroids and cyclophosphamide does not bring CNF into remission, but may cause severe side effects (McDonald et al. 1971; Rapola et al. 1992; Mauch et al. 1994). Thus, all therapeutic decisions should aim at renal transplantation (Mahan et al. 1984; Holmberg et al. 1995). The goals are to provide good nutrition, control edema, and prevent thrombosis and infections, allowing the child to reach a weight and body size consistent with successful kidney transplantation. In order to achieve this goal, albumin substitution is usually required. One group has used a more aggressive approach for 12 years (Holmberg et al. 1999). Regular, parenteral albumin infusions are started at birth with a dosage of 3–4 g/kg per day. Indwelling deep-vein catheters are used from the age of 3–4 weeks. Albumin is given together with intravenous furosemide (0.5 mg/kg) as a 20 per cent solution. With this substitutions the serum albumin concentration remains around 15 g/l and the patients do not have substantial edema (Rapola et al. 1992). If the albumin dosage is increased, it leads only to more urinary loss and a vicious cycle, with increased edema, fluid overload, and cardiac insufficiency. Finnish patients receive enterally 130 kcal/kg of energy and 4 g/kg of protein per day (in addition to parenteral albumin). The energy intake is 10– 14 per cent protein, 40–50 per cent fat, and 40–50 per cent carbohydrate. Rapeseed oil (10–15 ml) and fish oil (2 ml) are added. Other protein is given as a casein-based protein product and additional energy as glucose polymers. Additional medications including thyroxine, low-dose aspirin and dipyridamole or warfarin can be used according to the situation


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(Holmberg et al. 1999). Infections are a major problem in infants with CNF. Antibiotic therapy should be started promptly on suspicion and should cover the major hospital strains of bacteria. Although normal growth can be achieved with nutritional support and protein supplementation, the patients are still malnourished and hypoproteinemic (Antikainen et al. 1992; Holmberg et al. 1995). To optimize treatment one group perform bilateral nephrectomy and commence dialysis before kidney transplantation (Holmberg et al. 1999). The first report of successful kidney transplantation in CNF dates back to 1973 (Hoyer et al. 1973). Since 1987, 43 patients with CNF have received a new kidney in Helsinki, all except one after bilateral nephrectomy. The patient survival at 5 years is 98 per cent and graft survival 82 per cent, with a mean glomerular filtration rate (GFR) of 67 ml/min per 1.73 m2. The main risk after kidney transplantation is recurrence of NS, that has been reported in nine patients with CNF (Sigström et al. 1989; Lane et al. 1991; Flynn et al. 1992; Laine et al. 1993). Twenty per cent of patients that have undergone renal transplantation, show recurrence of nephrotic syndrome because of neoantigen–nephrin-induced auto-antibodies that may be pathogenic by perturbing the glomerular filtration barrier (Wang et al. 2001). The nephrin zipper The discovery of nephrin, the protein mutated in CNF, was the first in a chain of recent advances in kidney research that have considerably elucidated the SD structure. This has provided new understanding of the pathomechanism of proteinuria. The cDNA-deduced amino acid sequence predicted that nephrin is a type I transmembrane protein of the Ig superfamily (Kestila¨ et al. 1998). Nephrin has eight extracellular Ig-like and one fibronectin type III-like modules (Fig. 7.2). The Ig-like modules are of the so-called type C2 that is found predominantly in proteins participating in cell–cell or cell–matrix interactions (Chothia 1997; Kestila¨ et al. 1998). High homology of nephrin between species suggests that nephrin has an important role for maintaining cell function (Ahola et al. 1999; Kawachi et al. 2000; Putaala et al. 2000). By using in situ hybridization, the NPHS1 gene product was shown in the kidney to be expressed specifically in podocytes of developing human glomeruli (Kestila¨ et al. 1998) and immunostaining with anti-nephrin displayed strong staining in the GBM region, while the cell bodies of podocytes appeared to be negative (Ruotsalainen et al. 1999). Immunoelectron microscopic analysis of human glomeruli with an antibody reacting with the N-terminal Ig-like modules demonstrated that the epitope is located exclusively in the SD region (Ruotsalainen et al. 1999). This was an important observation, because it was the first demonstration of a known protein to be located in the SD. The fact that nephrin appears to be specific for the slit indicates a crucial role of nephrin for the glomerular filter structure. Structurally, nephrin is the predominant component of the SD, which is thought to form an isoporous, zipper-like structure, that constitutes the ultimate size-selective barrier. Physiologically, the intracellular domain of nephrin is rich in serine and tyrosine residues with potential phosphorylation sites, pointing to the possibility that nephrin is involved in outside-in signaling. Recent studies have demonstrated that nephrin is tyrosine-phosphorylated suggesting that it is a signaling molecule activating a canonical mitogen activated protein kinase (MAPK) cascade (Huber et al. 2001). Diffuse mesangial sclerosis The term diffuse mesangial sclerosis (DMS) denotes nephrotic syndrome associated with the histologic change of DMS of the renal glomeruli. The entity was described by Habib and

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C

C C–C

C–C

C–C

C–C

C–C

C–C

C–C

C–C

N

C C

B

Plasma membrane

Plasma membrane

Podocyte

Slit membrane

Podocyte

Fig. 7.2 Hypothetical model of nephrin assembly to form the isoporous filter of the podocyte slit diaphragm. A: Schematic domain structure of nephrin. The Ig repeats are shown by incomplete circles connected by disulfide bridges (C–C). The locations of free cysteine residues are indicated by a –C. B: Possible mode of interdigitating association of four nephrin molecules in the slit between two foot processes. For the sake of clarity, nephrin molecules from opposite foot processes are illustrated in different colors. In this model, it is assumed that Ig repeats 1–6 of a nephrin molecule of one foot process associate in an interdititating fashion with Ig repeats 1–6 in a neighboring molecule from the opposite foot process. Cysteine residues are depicted by black lines and two potential disulfide bridges crosslinking four nephrin molecules in the center of the slit are illustrated. The remaining single free cysteine present in the fibronectin domain may react with another nephrin molecule, or some other as yet unknown molecule, which may connect with the plasma membrane or cytoskeleton. (Adapted from Ruotsalainen, V., Ljungberg, P., Wartiovaara, J., and Tryggvason, K. (1999). Nephrin is specifically located at the slit diaphragm of glomerular podocytes. Proceedings of the National Academy of Sciences, USA, 96, 7962–7.)

Bois (1973). It may become manifest in the first 3 months of life, but it is more often detected later in infancy, often in the second and third years of life (Barakat et al. 1982; Mendelsohn et al. 1982; Habib 1993; Habib et al. 1993). Pregnancy and delivery are usually normal. Clinical features at onset are typical of NS, with proteinuria, hypoalbuminemia, and edema. Decline of GFR ensures rapidly and all children develop ESRD within a few months to years. A large proportion of patients develop hypertension. Laboratory findings are milder but similar to those in CNF. The pathological changes of DMS are characteristic (Habib et al. 1993). In the early phase, the glomeruli show expansion of the mesangium by fibrillar increase of the mesangial matrix, podocyte hypertrophy is pronounced. In the next diagnostic phase, the GBM is thickened and the expanded mesangium shows a delicate periodic-acid-Schiff (PAS)-positive mesh with embedded mesangial cells. The mesangial expansion causes obliteration of the capillary lumens. In the advanced phase, the mesangial sclerosis causes contraction of the glomerular tufts, which appear as solidified masses within somewhat expanded urinary space. Dilated tubules are also seen, but not as obvious as in CNF. In electron microscopy, the abundant mesangial matrix often contains filaments and collagenlike fibrils; GBM is thickened, often multilayered and corrugated (Habib et al. 1993; Rumpelt and Bachmann 1980). There is no indication of an


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immune complex cause of DMS, although some studies have shown variable amounts of IgM, C1q, and C3 in the mesangium or in the periphery of the glomeruli (Barakat et al. 1982; Mendelsohn et al. 1982; Habib 1993). The presence of affected siblings of both genders in several families suggest DMS is autosomal recessive inheritance (Barakat et al. 1982; Mendelsohn et al. 1982; Kristal and Lichtig 1983; Habib 1993; Ozen and Tinaztepe 1996). The interesting feature of DMS is that it is not confined to primary NS but is also associated with some syndromic types of NS, most notably the Denys–Drash syndrome (DDS) (Habib et al. 1985). The DNA analysis can yield mutations of the WT1 gene in isolated DMS and DMS associated with DDS, suggesting that the WT1 gene is involved in the pathogenesis of the disease (Schumacher et al. 1998). However the question of removing both kidneys at the time of transplantation is raised by the theoretical risk of developing a Wilms tumor. The WT1 gene, located at chromosome 11p13, encodes a transcription factor that has a critical role in kidney and gonad development, as demonstrated in WT1 knockout mice lacking both kidneys and gonads (Pitchard-Jones et al. 1990). Steroids and other immunosuppressive drugs are ineffective in DMS. Renal transplantation appears to be the only effective treatment of this disease (Habib 1993). Pretransplant treatment is similar to that which has been outlined for CNF, but with less aggressive protein substitution.

Other hereditary diseases leading to nephrotic syndrome Familial steroid-resistant nephrotic syndrome Familial idiopathic NS in childhood is mostly inherited as an autosomal recessive trait (White 1973; Tejani et al. 1983; Fuchshuber et al. 1995), whereas in adulthood, autosomal dominant forms are more frequently observed (Conlon et al. 1995; Mathis et al. 1998; Winn et al. 1999). A subgroup of nephrosis—the familial steroid resistant NS (SRN) was used to isolate the disease-causing gene. It has characteristic features as below: ● ● ● ● ● ●

familial occurrence early onset between 3 months and 5 years resistance to steroid therapy rapid progression to ESRD (within a few years) absence of recurrence after renal transplantation absence of extra-renal disorders.

Pathological examination shows minimal glomerular changes on early biopsy samples and focal segmental glomerulosclerosis at later stages (Fuchshuber et al. 1995). Positional cloning of the gene, NPHS2, causing familial steroid-resistant nephrotic syndrome yielded the existence of a novel podocyte-specific protein called podocin (Boute et al. 2000). The NPHS2 gene, that was mapped to chromosome 1q25–q32 (Fuchshuber et al. 1995), is 2 kb long and has 8 exons. The initially identified mutations included premature protein termination as well as missense mutations. The latter are predicted to cause either nonconservative substitutions of replacement of residues that are highly conserved among the stomatin-like protein family members and are probably crucial for podocin function

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(Boute et al. 2000). Podocin is located specifically in the SD region (Schwarz et al. 2001). It is an integral plasma membrane phosphoprotein with both ends located intracellularly (Boute et al. 2000). It has been proposed to have two closely related functions, that is, recruitment and/or stabilization of nephrin and CD2-associated protein (CD2AP) at the podocyte foot process, and augmentation of nephrin signaling. Signaling mediated by the nephrin–podocin complex has been reported to trigger additional cellular signaling cascades and programs to preserve the structural integrity of the podocyte SD (Huber et al. 2001). Thus, podocin may be involved in the glomerular filtration barrier, mainly by associating nephrin with the cytoskeleton (Boute et al. 2000; Huber et al. 2001). Recently podocin, nephrin, and CD2AP were reported to be associated with lipid rafts of the SD (Schwarz et al. 2001). Podocin has also been shown to bind to both CD2AP and nephrin via its COOH terminus (Schwarz et al. 2001), so it may thereby serve as a scaffolding protein in the organization of the SD complex. Homozygous or composite heterozygous mutations in the NPHS2 gene have also been found in Italian patients with nonfamilial steroid resistant focal segmental glomerulosclerosis (Caridi et al. 2001). In addition to the previously described defects, two novel mutations at exon 4 were identified, four single nucleotide polymorphisms (SNPs) and one dinucleotide repeat were also identified. Familial focal segmental glomerulosclerosis Focal segmental glomerulosclerosis (FSGS) is defined by the presence of segmental sclerosis in some, but not all glomeruli, and is seen in all ethnic groups, although it is particularly common in individuals of African descent. It may occur as a primary process that is thought to result at least in some instances from a defect in glomerular podocyte function (Ichikawa and Fogo 1996), may also be inherited as a mendelian trait. Three large families with similar features were used to isolate the causative gene on chromosome 19q. They have a mild increase in urine protein excretion starting in the teenage years or later, slowly progressive renal dysfunction, and the development of ESRD in some affected individuals (Kaplan et al. 2000). Finally, ACTN4, which encodes -actinin-4, an actin-filament crosslinking protein, was found being mutated in this disease (Kaplan et al. 2000). Three different missense mutations were found. The A682G and C695T nucleotide changes cause a lysine-to-glutamate substitution at residue 228 and a threonine-to-isolecucine substitution at residue 232. The third mutation is T703C, causing a serine-to-proline substitution at residue 235. The ACTN4 gene has widespread expression, but immunostaining of human kidney sections has revealed glomerular podocyte location (Kaplan et al. 2000). Although -actinin-4 is less likely to be a component of the podocyte SD protein complex, it undoubtedly plays an important role in the integrity of podocyte foot processes and glomerular filtration barrier. Glomerular filtration barrier The central role of the kidney is to filter plasma during the formation of primary urine. Ultrafiltration of plasma occurs in the glomerulus, a tuft of anastomasing capillaries surrounded by the Bowman’s capsule. The glomerular filtration barrier consists of three layers: a fenestrated endothelium, the GBM and the outermost podocyte foot processes with their interconnecting SD. The plasma first traverses the fenestrae of the endothelial cells, allowing direct contact of the plasma with the GBM and hardly restricting the passage of


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macromolecules. Then, the filtrate passes through the GBM, that is thought to function as a pre-filter of large plasma proteins, and, finally, it passes through the SD, the ultimate sizeselective macromolecular filter, that prevents proteins of the size of albumin or greater from penetrating into the urinary space (Karnovsky and Ainsworth 1972). During the last decade, studies on genetic disease and distinct proteins have unraveled a picture of glomerular filtration barrier. Glomerular basement membrane (GBM) The GBM has been regarded as the primary size- and charge-selective molecular sieve of the glomerulus. Similar to other basement membranes in the body, the GBM is a highly organized network of extracellular proteins, such as type IV collagen, laminin, nidogen/entactin, and heparin sulphate proteoglycan (HSPG). Type IV collagen is a basement-membrane-specific protein that belongs to the large family of collagens (Hudson et al. 1993). These are the main structural proteins of the connective tissue in multicellular organisms. All collagens consist of three -chains, which assemble intracellularly into a triple-helical molecule. Once secreted, the triple-helical type IV collagen molecules assemble by end-to-end aggregation and lateral alignment into a tightly crosslinked network structure. Six genetically distinct type IV collagen -chains, termed 1–6, have been identified (Hudson et al. 1993). Among those, the 1 and 2 chains are found in practically all basement membranes in an 1:1:2 composition. In contrast, the 3,4,5, and 6 chains are minor component chains with a more restricted tissue distribution and, thus, probably specialized functions. The embryonic GBM contains 1:1:2 type of collagen IV, but postnatally this is replaced by adult collagen IV trimers containing 3:4:5 chains (Miner and Sanes 1994). Because of its higher cysteine content, the adult form contains more intermolecular disulfide crosslinks that are thought to provide more strength to the GBM. Defects in the adult type IV collagen form lead to a distorted GBM and Alport syndrome (Barker et al. 1990; Hudson et al. 1993; Mochizuki et al. 1994; Miner and Sanes 1996; Chapter 8, this volume). Laminin is another large trimeric basement membrane component. This protein is considered particularly important for cellular differentiation and adhesion, but it also contributes to the basement membrane structure by forming its own structural meshwork. Embryonic GBM contains a laminin-10 isoform (5 : 1 : 1), but this is replaced by laminin-11 (5 : 2 : 1) after birth (Miner et al. 1997). The actual roles of these two laminin isoforms in the GBM is unknown, but mice deficient for the laminin 2 chain develop nephrotic syndrome (Noakes et al. 1995). Kidney development in laminin 5 chain knock out mice is arrested at a very early stage, a proper GBM does not form and podocyte do not assemble in a correct pattern (Miner and Li 2000). This indicates an important role for laminin-10 and 11 isoforms in the maintenance of a normal glomerular filtration barrier. Perlecan is the predominant HSPG in basement membranes, but agrin is an additional major proteoglycan in the GBM (Hassel et al. 1980; Groffen et al. 1998). Both are rich in negatively charged heparan sulfate moieties that are thought to contribute to the anionic electric barrier for the negatively charged macromolecules of the plasma. Decrease in GBM heparan sulfate has previously been reported to induce proteinuria (Caulfield and Farquhar 1978; Van Den Born et al. 1992), which points to the relevance of HSPG for charge-dependent permeability. Perlecan-deficient mouse embryos die at embryonic days 10–12 due to

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rupture of basement membranes in the heart (Arikawa-Hirasawa et al. 1999; Costell et al. 1999). However, mice containing heparan sulfate deficient perlecan do not develop proteinuria (Rossi et al. 2003). This indicates that at least heparan sulfate in perlecan is not important for the filtration barrier. Agrin-deficient mutant mice show aberrations in the development of neuromuscular junctions and they die shortly before birth (Gautam et al. 1996). In both cases, no prominent kidney malformations have been reported. Nidogen/entactin, a 150-kDa sulfated glycoprotein, is a major component of basement membranes. It forms a highly stable noncovalent complex with laminin, probably providing a link between the laminin and type IV collagen meshworks. Nidogen also has a perlecanbinding site and, thus, appears to have a crucial role for the assembly of the GBM (Yurchenco and Schittny 1990; Paulsson 1992). Slit diaphragm It is well established that the glomerular filtration barrier behaves both as a size- and chargeselective filter that restricts the passage of plasma macromolecules based on their size, shape, and charge. Although it is generally acknowledged that the GBM can restrict the movement of large plasma proteins, there is also ample evidence showing that the ultimate size-selective barrier for proteins resides in the SD (Latta 1970; Karnovsky and Ainsworth 1972). The SD is a unique structure that appears to be distinct from any other type of intercellular junctions between polarized epithelial cells. The 40-nm wide intercellular space distinguishes the SD from tight junctions where the distance between the cells is only 15 nm. There is no apparent accumulation of actin microfilaments or intermediate-sized filaments to the SD which also distinguishes it from adherens junctions and desmosomes. Until recently, knowledge about the structure and molecular properties of this membrane was very limited. However, Rodewald and Karnovsky showed already in 1974 that the SD has an isoporous, zipper-like pattern based on electron microscopy (Rodewald and Karnovsky 1974). According to their controversial hypothesis, the 4 14 nm2 around large pores are small enough to prevent the passage of albumin into the urinary space. The molecular nature of the SD has hitherto been a mystery, but characterization of glomerular proteins associated with this structure has progressed rapidly during the last 3 years. Before nephrin was discovered, Orikasa et al. (1988) generated monoclonal antibodies against glomeruli, one of which, 5-1-6, recognized a 51-kDa protein, that was localized exclusively to the SD (Orikasa et al. 1988). However, attempts to identify this SD-specific protein were unsuccessful. Later, Farquhar and coworkers localized the -isoform of the intracellular tight junction protein zonula occludens-1 (ZO-1) in the glomerulus primarily to the sites where the SD is inserted into the plasma membrane of the foot processes (Schnabel et al. 1990; Kurihara et al. 1992). ZO-1 has been proposed to connect components of the SD to the cytoskeleton. The renal glomerular epithelial cell, or podocyte, is a highly differentiated cell with characteristic interdigitating foot processes covering the outer aspect of the GBM. The foot processes are covered on their apical and lateral surfaces with a polyanionic glycocalyx. This contains podocalyxin, a sialoglycoprotein, that has been proposed to help maintain the spacing between the interdigitating foot processes by charge repulsion (Schnabel et al. 1989). The recent discoveries of specific proteins located at the SD region are evolving into a precise picture of the molecular structure of this size-selective filter. It appears that the SD is a


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highly organized porous filter made by proteins in the SD itself, as well as by some intracellular proteins, all these proteins forming a specific SD complex. Nephrin appears to be a key component of the SD proper. Its necessity for this structure is demonstrated by the lack of a SD and development of fatal proteinuria and nephrotic syndrome in the human NPHS1 disease and in nephrin knockout mice (Kestila¨ et al. 1998; Lenkkeri et al. 1999; Putaala et al. 2001). The intracellular integral membrane protein podocin is also an essential component of the SD complex, as its absence or abnormal function leads to steroid-resistant congenital nephrotic syndrome in early childhood (Boute et al. 2000). Podocin interacts with the intracellular domain of nephrin and somehow connects it to the actin cytoskeleton together with other intracellular proteins of the SD complex such as CD2AP. The latter, in turn, has been shown to react with both nephrin and podocin on the one hand and with actin on the other. Altogether, current data suggest that a functional SD complex requires the interplay of specific proteins that build up the filter itself, as well as intracellular membrane-bound and cytosolic proteins that connect the extracellular proteins to the actin cytoskeleton of the foot process. A hypothetical model of the SD complex is depicted in Fig. 7.3. Alterations or lack of any of the proteins of this SD complex will lead to an abnormal filter structure and proteinuria of variable onset and severity (Table 7.1) as reviewed above.

Urinary space Podocyte foot process Direction of filtration P-cadherin

Actin ZO-1 Collagen IV Laminin GBM Nidogen Proteoglycan

Podocin

CD2AP

α-Actinin-4

Endothelial cell

Slit diaphragm made of nephrin molecules from two adjacent foot processes

Capillary lumen Fig. 7.3 Schematic model of the glomerular filtration barrier. The three layers of glomerular filtration barrier are the fenestrated endothial cells lining the inside of the glomerular capillary, the GBM and the podocyte with their intervening slit diaphragm. The GBM is a highly organized network of extracellular proteins, mainly composed of type IV collagen, laminin, nidogen and heparan sulfate proteoglycans. The slit diaphragm is built up of a SD protein complex. Intercellularly, nephrin molecules of adjacent foot processes form the zipper-like filter by homophilic interaction. CD2AP, podocin and ZO-1 are intracellular proteins of the SD complex that may connect the nephrin molecules to the actin cytoskeleton. (See Plate 5 of the Colour Plate Section at the centre of this book.)

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Table 7.1 Diseases related with the mutations of the protein in SD complex Disease name

Hereditary trade

Gene locus

Gene/protein

Gene structure

CNS of Finnish type (CNF) Diffuse mesangial sclerosis (DMS) Familial steroid-resistant nephrotic syndrome (SRNS) Familal focal segmental glomerular sclerosis (FSGS)

AR AR AR

19q12–13 11p13 1q25–32

NPHS1/nephrin WT1/WT1 NPHS2/podocin

26 kb, 29 exons 50 kb, 10 exons 2 kb, 8 exons

AD

19q13

ACTN4/a-acinin-4

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Gautam, M., Noakes, P. G., and Moscoso, L. (1996). Defective neuromuscular synaptogenesis in agrindeficient mutant mice. Cell, 85, 525–35. Groffen, A. J., Ruegg, M. A., Dijkman, H., van de Velden, T. J., Buskens, C. A., van den Born, J., et al. (1998). Agrin is a major heparan sulfate proteoglycan in the human glomerular basement membrane. Journal of Histochemisty and Cytochemistry, 46, 19–27. Habib, R. (1993). Nephrotic syndrome in the 1st year of life. Pediatric Nephrology, 7, 347–53. Habib, R. and Bois, E. (1973). Heterogeneite des syndromes nephrotiques a debut precoce du nourrison (syndrome nephrotique ‘infantil’). Helvetica Pediatric Acta, 28, 91–107. Habib, R., Gubler, M. C., Antignac, C., and Gagnadoux, M. F. (1993). Diffuse mesangial sclerosis: a congenital glomerulopathy with nephrotic syndrome. Advances in Nephrology from the Necker Hospital, 22, 43–57. Habib, R., Loirat, C., Gubler, M., Niaudet, P., Bensman, A., Levy, M., et al. (1985). The nephropathy associated with male pseudo-hermaphroditism and Wilms’ tumor (Drash syndrome): a distinctive glomerular lesion–report of 10 cases. Clinical Nephrology, 24, 269–78. Hallman, N., Hjelt, L., and Ahvenainen, E. K. (1956). Nephrotic syndrome in newborn and young infants. Annales Paediatriae Fenniae, 2, 227–41. Hassel, J. R., Robey, P. G., Barrach, H. J., Wilczedk, J., Rennard, S. I., and Martin, G. R. (1980). Isolation of a heparan sulfate-containing proteoglycan from basement membrane. Proceedings of the National Academy of Sciences, USA, 77, 4494–8. Holmberg, C., Antikainen, M., Rönnholm, K., Ala Houhala, M., and Jalenko, H. (1995). Management of congenital nephrotic syndrome of the Finnish type (Review). Pediatric Nephrology, 9, 87–93. Holmberg, C., Jalanko, H., Tryggvason, K., and Rapola, J. (1999). Congenital nephrotic syndrome. In Pediatric nephrology (ed. T. M. Barratt, E. D. Avner and W. E. Harmon), pp. 765–77. Lippincott Williams & Wilkins, Baltimore, MD. Hoyer, J. R., Kjellstrand, C. M., Simmons, R. L., Naharian, J. S., Mauer, S. M., Buselmeier, T. J., et al. (1973). Successful renal transplantation in three children with congenital nephrotic syndrome. Lancet, 1, 1410–12. Huber, T. B., Köttgen, M., Schilling, B., Walz, G., and Benzing, T. (2001). Interaction with podocin facilitates nephrin signaling. Journal of Biological Chemistry, 45, 41,543–6. Hudson, B. G., Reeders, S., and Tryggvason, K. (1993). Type IV collagen: structure, gene organization, and role in human disease: molecular basis of Goodpasture and Alport syndromes and diffuse leiomyomatosis. Minireview, Journal of Biological Chemistry, 268, 26,033–6. Huttunen, N. P. (1976). Congenital nephrotic syndrome of Finnish type. Archives of Diseases in Childhood, 51, 344–8. Huttunen, N. P., Vehaskari, M., Viikari, M., and Laipio, M. L. (1980). Proteinuria in congenital nephrotic syndrome of the Finnish type. Clinical Nephrology, 13, 12–19. Ichikawa, I. and Fogo, A. (1996). Focal segmental glomerulosclerosis. Pediatric Nephrology, 10, 374–91. Kaplan, J. M., Kim, S. H., and North, K. N. (2000). Mutations in ACTN4, encoding alpha-actinin-4, cause familial focal segmental glomerulosclerosis. Nature Genetics, 24, 251–6. Karnovsky, M. J. and Ainsworth, S. K. (1972). The structural basis of glomerular filtration. Advances in Nephrology from the Necker Hospital, 2, 35–60. Kawachi, H., Koike, H., and Kurihara, H. (2000). Cloning of rat nephrin: expression in developing glomeruli and in proteinuric states. Kidney International, 57, 1949–61. Kestilä, M., Lenkkeri, U., Männikkö, M., Lamerdin, J., McCready, P., Putaala, H., et al. (1998). Positionally cloned gene form a novel glomerular protein-nephrin-is mutated in congenital nephrotic syndrome. Molecular Cell, 1, 575–82. Kestilä, M., Männikkö, M., Holmberg, C., Gyapay, G., Weissenbach, J., Savolainen, E. R., et al. (1994). Congenital nephrotic syndrome of the Finnish type maps to the long arm of chromosome 19. American Journal of Human Genetics, 54, 757–64.

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Kestilä. M., Männikkö, M., Holmberg, C., Korpela, K., Savolainen, E. R. Peltonen, L., et al. (1994). Exclusion of eight genes as mutated loci in congenital nephrotic syndrome of the Finnish type. Kidney International, 45, 986–90. Kristal, H. and Lichtig, C. (1983). Infantile nephrotic syndrome clinicopathological study of 11 cases. Israel Journal of Medical Sciences, 19, 626–30. Kurihara, H., Anderson, J. M., and Farquhar, M. G. (1992). Diversity among tight junctions in rat kidney: Glomerular slit diaphragms and endothelial junctions express only one isoform of the tight junction protein ZO-1. Proceedings of the National Academy of Sciences, USA, 89, 7075–79. Laine, J., Jalanko, H., Holthöfer, H., Krogerus, L., Rapola, J., von Willebrand, E., et al. (1993). Posttransplantation nephrosis in congenital nephrotic syndrome of the Finnish type. Kidney International, 44, 867–74. Lane, P. H., Schnaper, H. W., Vernier, R. L., and Bunchman, T. E. (1991). Steroid-dependent nephrotic syndrome following renal transplantation for congenital nephrotic syndrome. Pediatric Nephrology, 5, 300–3. Latta, H. (1970). The glomerular capillary wall. Journal of Ultrastructural Research, 32, 526–44. Lenkkeri, U., Männikkö, M., McCready, P., Lamerdin, J., Gribouval, O., Niaudet, P. M., et al. (1999). Structure of the gene for congenital nephrotic syndrome of the Finnish type (NPHS1) and characterization of mutations. American Journal of Human Genetics, 64, 51–61. Liu, L., Doné, S. C., Khoshnoodi, J., Bertorello, A., Wartiovaara, J., Berggren, P. O., et al. (2001). Defective nephrin trafficking caused by missense mutations in the NPHS1 gene: insight into the mechanisms of congenital nephrotic syndrome. Human Molecular Genetics, 23, 2637–44. Ljungberg, P., Jalanko, H., Holmberg, C., and Holthöfer, H. (1993). Congenital nephrosis of the Finnish type (CNF): matrix components of the glomerular basement membranes and of cultured mesangial cells. Histochemical Journal, 25, 606–12. Mahan, J., Mauer, S., Sibley, R., and Vernier, R. L. (1984). Congenital nephrotic syndrome: evolution of medical management and results of renal transplantation. Journal of Pediatrics, 105, 549–57. Männikkö, M., Kestilä, M., Lenkkeri, U., Alakurtti, H., Holmberg, C., Leioti, J., et al. (1997). Improved prenatal diagnosis of the congenital nephrotic syndrome of the Finnish type based on DNA analysis. Kidney International, 51, 868–72. Mathis, B. J., Kim, S. H., Calabrese, K., Haas, M., Seidman, J. G., Seidman, C. E., et al. (1998). A locus for inherited focal segmental glomerulosclerosis maps to chromosome 19q13. Kidney International, 53, 282–6. Mauch, T. J., Vernier, R., Burke, B. A., and Nevins, T. E. (1994). Nephrotic syndrome in the first year of life. In Pediatric nephrology (ed. M. A. Holliday, T. M. Barratt, and E. D. Avner, 2nd edn), pp. 788–802. Williams & Wilkins, Baltimore, MD. McDonald, R., Wiggelinkhuitzen, J., and Kaschula, O. C. (1971). The nephrotic syndrome in very young infants. American Journal of Diseases in Children, 122, 507–12. Mendelsohn, H., Krauss, M., Berant, M., and Lichtig, C. (1982). Familial early-onset nephrotic syndrome: diffuse mesangial sclerosis. Acta Paediatrica Scandinavica, 71, 753–8. Miner, J. H. and Li, C. (2000). Defective glomerulogenesis in the absence of laminin 5 demonstrates a developmental role for the kidney glomerular basement membrane. Developmental Biology, 217, 278–89. Miner, J. H., Patton, B. L., Lentz, S. I., Gilbert, D. J., Snider, W. D., Jenkins, N. A., et al. (1997). The laminin alpha chains: expression, developmental transitions, and chromosomal locations of 1–5, identification of heterotrimeric laminins 8–11, and cloning of a novel 3 isoform. Journal of Cell Biology, 137, 685–701. Miner, J. H. and Sanes, J. R. (1994). Collagen IV 3, 4, and 5 chains in rodent basal laminae: sequence, distribution, and association with laminins, and developmental switches. Journal of Cell Biology, 127, 879–91. Miner, J. H. and Sanes, J. R. (1996). Molecular and functional defects in kidneys of mice lacking collagen 3 (IV): implications for Alport syndrome. Journal of Cell Biology, 135, 1403–13.


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Mochizuki, T., Lemmink, H. H., Maryiyama, M., et al. (1994). Identification of mutations in the (IV) and (IV) collagen genes in autosomal recessive Alport syndrome. Nature Genetics, 8, 77–8. Noakes, P. G., Miner, J. H., Gautam, M., Cunningham, J. M., Sanes, J. R., and Merlie, J. P. (1995). The renal glomerulus of mice lacking s-laminin/laminin beta-2: nephrosis despite molecular compensation by laminin beta-1. Nature Genetics, 10, 400–6. Norio, R. (1966). Heredity in congenital nephrotic syndrome. Annales Paediatriae Fenniae, 12 (Suppl. 27), 1–94. Orikasa, M., Matsui, K., Oite, T., and Shimizu, F. (1988). Massive Proteinuria induced in rats by a single intravenous injection of a monoclonal antibody. Journal of Immunology, 141, 807–14. Ozen, S. and Tinaztepe, K. (1996). Diffuse mesangial sclerosis: a unique type of congenital and infantile nephrotic syndrome. Nephron, 72, 288–91. Panicucci, F., Sagripanti, A., Vispi, M., Puiori, E., Lecchini, L., Barsotti, G., et al. (1983). Comprehensive study of haemostasis in nephrotic syndrome. Nephron, 33, 9–13. Patrakka, J., Kestilä, M., and Wartiovaara, J. (2000). Congenital nephrotic syndrome (NPHS1): Features resulting from different mutations in Finnish patients. Kidney International, 58, 972–80. Patrakka, J., Martin, P., Salonen, R., Ruotsalainen, V., Kestila-, M., Mannikko- , M., et al. (2002). Proteinuria and prenatal diagnosis of congenital nephrosis in fetal carriers of nephrin gene mutations. Lancet, 359, 1575–7. Paulsson, M. (1992). Basement membrane proteins: structure, assembly, and cellular interactions. Critical Reviews in Biochemistry and Molecular Biology, 27, 93–127. Pitchard-Jones, K., Fleming, S., Davidson, D., Bichmore, W., Porteous, D., Gosden, C., et al. (1990). The candidate Wilms’ tumour gene is involved in genitourinary development. Nature, 346, 194–7. Putaala, H., Sainio, K., Sariola, H., and Tryggvason, K. (2000). Primary structure of mouse and rat Nephrin cDNA and structure and expression of the mouse gene. Journal of the American Society of Nephrology, 11, 991–1001. Putaala, H., Soininen, R., Kilpeläinen, P., Wartiovaara, J., and Tryggvason, K. (2001). The murine nephrin gene is specifically expressed in kidney, brain and pancreas: inactivation of the gene leads to massive proteinuria and neonatal death. Human Molecular Genetics, 10, 1–8. Rapola, J., Huttunen, N.-P., and Hallman, N. (1992). Congenital and infantile nephrotic syndrome. In Pediatric kidney disease (ed. C. M. Edelman, J. Bernstein, A. Meadow, et al., 2nd edn), pp. 1291–305. Little, Brown, Boston, MA. Rodewald, R. and Karnovsky, M. J. (1974). Porous substructure of the glomerular slit diaphragm in the rat and mouse. Journal of Cell Biology, 60, 423–33. Rossi, M., Morita, H., Sormunen, R., Airenne, S., Kreivi, M., Wang, L., et al. (2003). Heparan sulfate chains of perlecan are indespensible in the lens but not in the kidney. EMBO Journal, 22, 236–45. Rumpelt, H. and Bachmann, H. (1980). Infantile nephrotic syndrome with diffuse mesangial sclerosis: a disturbance of glomerular basement membrane development? Clinical Nephrology, 13, 146–50. Ruotsalainen, V., Ljungberg, P., and Wartiovaara, J. (1999). Nephrin is specifically located at the slit diaphragm of glomerular podocytes. Proceedings of the National Academy of Sciences, USA, 96, 7962–7. Schnabel, E., Anderson, J. M., and Farquhar, M. G. (1990). The tight junction protein ZO-1 is concentrated along slit diaphragms of the glomerular epithelium. Journal of Cell Biology, 111, 1255–63. Schnabel, E., Dekan, G., Miettinen, A., and Farquhar, G. (1989). Biogenesis of podocalyxin. The major glomerular sialoglycoprotein in the new born rat kidney. European Journal of Cell Biology, 48, 313–26. Schumacher, V., Schärer, K., Wühl, E., Attrogge, H., Bonzel, K. E., Guschmann, M., et al. (1998). Spectrum of early onset nephrotic syndrome associated with WT1 missense mutations. Kidney International, 53, 1594–600. Schwarz, K., Simons, M., and Reiser, J. (2001). Podocin, a raft-associated component of the glomerular slit diaphragm, interacts with CD2AP and nephrin. Journal of Clinical Investigations, 108, 1621–9. Seppälä, M., Ranta, T., Aula, P., et al. (1979). Alpha-fetoprotein in normal and abnormal pregnancy. In Carcino-embryonic proteins: chemistry, biology, clinical applications (ed. F. G. Lehmann), pp. 191–7. Elsevier, Amsterdam.

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8 Alport syndrome Frances Flinter

Introduction Alport syndrome (AS) is the most common hereditary nephropathy, with a gene frequency of 1 in 5000. It causes 0.6 per cent of cases of chronic renal failure in Europe (Rigden et al. 1996). The majority of cases are inherited in an X-linked fashion, with a very variable phenotype in women. During the last decade, the cloning of several genes involved in the assembly of type IV collagen, which is abnormal in patients with AS, has increased our understanding of the pathogenesis of this progressive glomerulonephritis considerably. Mutation detection studies are enabling genotype/phenotype correlations to be made, and facilitating more accurate carrier detection and prenatal diagnosis. History The family reported by Alport (1927) had been described previously by others. Alport made two important additional observations however, commenting that the occurrence of ‘nerve’ deafness in most of the patients with haematuria probably represented a specific clinical syndrome, and noting that males were usually affected more severely than females. The eponym was adopted by Williamson (1961), but as there were no specific diagnostic criteria for the diagnosis of AS until recently, a variety of clinically heterogeneous inherited nephritides was included for many years (Crawfurd 1988). Classic (X-linked) Alport syndrome In 1988 a set of four clinical diagnostic criteria was described that enables the identification of patients who are affected with the same disease as Alport’s original family (Flinter et al. 1988). In order to diagnose classic AS in a family, examination of the proband and other relatives must reveal evidence of at least three out of four of the following criteria (different features may occur in different family members): 1. 2. 3. 4.

Positive family history of macro/microscopic haematuria, chronic renal failure (CRF), or both. Electron microscopic evidence of AS on renal biopsy. Characteristic ophthalmic signs (i.e. anterior lenticonus/macular flecks). High-tone sensorineural deafness.

In some cases, previous molecular analysis within an affected family may mean that a specific mutation has already been defined, and a relative presenting with isolated haematuria can

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be tested for the mutation with no necessity for further clinical tests. A family which does fulfil these criteria is most likely to have an X-linked disease because autosomal forms of AS are less common (15 per cent of all cases at most). Autosomal dominant forms are usually not associated with the characteristic eye signs (Crawfurd 1988; Flinter 1997; Colville et al. 2000), although autosomal recessive forms may be (Colville and Savige 1997). Positive family history Individuals are often unaware of the presence, or significance, of renal disease in relatives. Rambausek et al. (1987) found that only 20 per cent of patients with some form of familial glomerulonephritis knew of their positive family history, and 50 per cent of patients in that study had AS. In earlier generations, suspicions should be raised by deaths from ‘Bright disease’ and deaths during pregnancy or delivery ascribed to pre-eclampsia. It will often be informative to perform a urinalysis, an ophthalmological examination and an audiogram on first-degree relatives (particularly parents) of people with unexplained haematuria. Renal biopsy It is essential that a renal biopsy from a patient suspected of having AS be examined under an electron microscope (EM). Light microscopy (LM) results are usually normal in children under 10 years, and in adults the findings are nonspecific. An experienced pathologist may report thickening of the glomerular capillary walls, but generally LM will reveal only segmental sclerosis and obsolescence, tubular atrophy, interstitial fibrosis, and infiltration by lymphocytes and plasma cells with clusters of foam cells. Under EM, there are various characteristic ultrastructural lesions of the glomerular basement membrane (GBM) (Hinglais et al. 1972; Spear and Slusser 1972). In childhood, the GBM may appear predominantly thin, especially in females, because of a reduction in the thickness of the lamina densa (Grünfeld 1985). This has often led to the erroneous conclusion that the patient has ‘thin basement membrane disease’ with the prediction that the condition will follow a benign course (so-called ‘benign familial haematuria’—see below). Subsequent biopsies, however, reveal increasing areas of thickening and ultimately splitting of the GBM, with electron-lucent areas containing dense granules (Hinglais et al. 1972; Rumpelt 1980; Cangiotti et al. 1996). It is probably wise to be extremely cautious about the long-term renal prognosis of any child whose renal biopsy reveals even small areas of GBM thickening. Immunohistochemical findings Human anti-GBM (Goodpasture) autoantibodies fail to bind to the GBM of renal biopsy material from patients with AS (McCoy et al. 1976; Olson et al. 1980), suggesting absence of a normal GBM constituent in these patients. In 1984, Wieslander et al. localized the Goodpasture epitope to type IV collagen, and it was identified subsequently as the carboxy terminal non-collagenous (NCI) domain of a novel type IV collagen chain, 3(IV) (Saus et al. 1988). A monoclonal antibody directed against the Goodpasture antigen confirmed the absence of this epitope from the GBM of many males with AS (Savage et al. 1986; Gubler

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et al. 1988), and the final evidence implicating type IV collagen in the aetiology of AS came with the demonstration that the antibodies which AS patients generated against transplanted kidneys were directed against epitopes of type IV collagen (Kashtan et al. 1986; Brainwood et al. 1998). In 1990, Hostikka et al. cloned COL4A5, the gene encoding the 5 chain of type IV collagen, and Barker et al. (1990) then identified pathogenic mutations within this gene that co-segregated with the disease in affected families (Table 8.1). The failure of anti-GBM antibodies to bind to Alport GBM can be useful diagnostically, but has limited sensitivity and specificity. In females there may be a mosaic pattern of Goodpasture epitope expression (Jenis et al. 1981). In the majority of cases of AS that are X-linked, studies of the binding of antibodies to the 5(IV) chain may be the most relevant (Table 8.2). Table 8.1 Type IV collagen genes Chain

Gene

Chromosome

1(IV) 2(IV) 3(IV) 4(IV) 5(IV) 6(IV)

COL4A1 COL4A2 COL4A3 COL4A4 COL4A5 COL4A6

13 13 2 2 X X

Renal disease

Autosomal recessive AS Autosomal recessive AS X-linked AS X-linked AS with leiomyomatosis (contiguous deletion involving COL4A5 and COL4A6)

Table 8.2 Typical distribution of the six (IV) collagen chains in selected renal and skin basement membranes from normal individuals and AS patients (hemizygotes and autosomal recessive homozygotes)

Normal GBM Bowman’s capsule Distal tubule Collecting duct EBM Hemizygote GBM Bowman’s capsule Distal tubule Collecting duct EBM AR homozygote GBM Bowman’s capsule Distal tubule Collecting duct EBM

1

2

3

4

5

6

/ /

/ /

ND

GBM: glomerular basement membrane; EBM: epidermal basement membrane; AR: autosomal recessive; ND: not determined. (Reproduced with permission from Pirson, Y. (1999). Kidney International, 56, 766.)

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In X-linked AS, the absence of 5(IV) is predictable, but often absence of 3(IV) and 4(IV) is found simultaneously. Presumably, a mutation in COL4A5 prevents the incorporation or expression of the other two collagen chains. Immunohistochemical studies may be particularly helpful when standard EM examination of the GBM has given equivocal results. In XL heterozygotes, random X-chromosome inactivation leads to a mosaic expression of 3 and 5 (Pirson 1999), and the degree of 5 expression correlates negatively with the severity of renal disease (Nakanishi et al. 1998). In autosomal recessive AS, there is absence of 3(IV) and 4(IV) but some 5(IV) expression (Pirson 1999). Overall, the combination of EM and histochemical studies enabled a diagnosis of AS in 92 per cent of a cohort of 108 Italian patients (Mazzucco et al. 1998).

Skin immunohistochemistry The 5(IV) and 6(IV) chains are expressed in the epidermal basement membrane (EBM), but not the 3(IV) and 4(IV) chains, so skin biopsies may be helpful diagnostically (Kashtan and Michael 1996; van der Loop et al. 1999). Generally, the pattern of expression of 5(IV) in the EBM mirrors that found in the GBM in patients with X-linked AS, and skin biopsy may be a helpful diagnostic test. Absence of 5(IV) expression in a male, or clearly mosaic 5(IV) expression in a female suggests a diagnosis of X-linked AS with a COL4A5 mutation. Normal 5(IV) expression in the skin could arise because (i) the patient has a COL4A5 mutation which allows skin expression of 5(IV) (van der Loop et al. 1999) or (ii) the patient has autosomal recessive AS or some other disease. Therefore, if skin expression of 5(IV) is normal, it may be appropriate to proceed to renal biopsy (Kashtan 1999); however, a discordant pattern with 5(IV) present in the EBM and absent in the GBM has been reported by Naito et al. (1997). In autosomal recessive homozygotes 5(IV) expression in the EBM is normal (Gubler et al. 1995).

Ophthalmic signs Sohar first reported the characteristic eye signs found in AS in 1954. 3, 4, and 5(IV) are all distributed in the lens capsule, Descemet’s membrane, Bruch’s membrane, and the internal limiting membrane of the eye. It is necessary to examine the eye with a slit lamp ophthalmoscope to reveal the three characteristic features: anterior lenticonus (which is pathognomic of AS) (Fig. 8.1), macular flecks (Fig. 8.2), and peripheral coalescing flecks (Fig. 8.3) (Govan 1983; Colville and Savige 1997). Recurrent, non-traumatic corneal erosions have been described in 23 per cent of 44 patients with severe AS (Rhys et al. 1997). Anterior lenticonus is a conical protrusion of the central portion of the lens, usually into the anterior chamber of the eye, and it may cause a progressive axial myopia, anterior capsular cataract, or spontaneous rupture of the anterior capsule. Occasionally the lens requires surgical removal. Anterior lenticonus is not present at birth or during early childhood, and the youngest male in whom the author has observed it was 13 years. There does

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Fig. 8.1 Ophthalmic sign of AS. Anterior lenticonus revealed by eye examination under slit lamp ophthalmoscope. (See Plate 6 of the Colour Plate Section at the centre of this book.)

Fig. 8.2 Ophthalmic sign of AS. Macular flecks revealed by eye examination under slit lamp ophthalmoscope. (See Plate 7 of the Colour Plate Section at the centre of this book.)

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Fig. 8.3 Ophthalmic sign of AS. Peripheral coalescing flecks revealed by eye examination under slit lamp ophthalmoscope. (See Plate 8 of the Colour Plate Section at the centre of this book.)

seem to be a correlation with the severity of the renal disease, as anterior lenticonus is frequently first noted around the time the kidneys fail. It has been recorded in 22 per cent of 94 AS patients under 25 years (Pirson 1999). Macular flecks are faint, pale (often white), dot-like lesions in the perifoveal area or mid-periphery of the retina. They were present in 37 per cent of 94 patients less than 25 years old (Pirson 1999). They are not present in infancy or early childhood, and progress with time in parallel with the decline in renal function. The flecks do not affect vision and fluorescein angiography of the macula is normal. Peripheral white flecks are less common. At least one ophthalmic feature of AS has been reported in up to 72 per cent of males and 38 per cent of females with AS (Flinter 1990; Jais et al. 2000; Dagher et al. 2001). High-tone sensorineural deafness An audiogram should be performed in any patient with unexplained haematuria, as significant high-tone sensorineural hearing loss may be detected in apparently asymptomatic patients. The deafness in AS is never congenital, and generally has an insidious onset and slowly progressive course during later childhood (Gregg and Becker 1963; Flinter 1990). The loss may be asymmetrical, but it is usually bilateral, and occasionally awareness of hearing problems may precede the recognition of haematuria or other evidence of renal disease. The deterioration in hearing may be rapid during the teens (especially in males) but often reaches a plateau subsequently (Grünfeld 1985); patients do not usually become completely deaf

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6000 4000 8000

Hearing loss (dB) (British standard)

10 20 30 40 50 60 70 80 90 100 = Right ear

= Air conduction

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Fig. 8.4 High-tone sensorineural deafness.

(Iverson 1974). Occasionally, there is some improvement in hearing post-transplant (McDonald et al. 1978) but this may be secondary to the treatment of uraemia (Gubler et al. 1981). The hearing loss affects the higher tones first and most significantly; later there is a reduction at lower frequencies as well (Fig. 8.4). Patients may be prescribed hearing aids, but often do not wear them because of their limited effectiveness. Eighty three per cent of males with X-linked AS are deaf, having presented clinically at an average age of 11 years and with an average deficit of 66 dB (Flinter 1989; Jais et al. 2000). Between 4 and 57 per cent of females are deaf, with an average loss of 50 dB, but they generally present two or three decades later than males (Flinter 1989; Jais et al. 2000; Dagher et al. 2001). Patients who are affected with autosomal recessive AS have a two in three chance of demonstrating sensorineural deafness by 20 years (Lemmink et al. 1997). Histologically, EM studies have revealed a multilayered basement membrane in the vas spirale, and it is known that 3, 4, and 5(IV) are localized in the inner/outer sulci, spiral to the tectorial membrane and basilar membrane of the ear (Kalluri et al. 1998). Typical clinical course of a male with X-linked AS Sixty seven per cent of males present with macroscopic haematuria, usually during an intercurrent infection at an average age of 3 years (Flinter and Chantler 1990). Once the macroscopic haematuria clears, there is residual microscopic haematuria on careful testing. A male at 50 per cent risk of inheriting AS who has consistently normal urinalysis at the age of

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5 years is very unlikely to develop AS. All affected males develop proteinuria subsequently (never the other way round, as is the case in the Samoyed dog animal model) and up to 30 per cent become frankly nephrotic (Grünfeld 1985; Kashtan and Michael 1996). Deafness becomes apparent at an average age of 11 years, and hypertension develops during the teens. By 20 years renal function is deteriorating, and renal replacement therapy is required, on average, 16 months after the serum creatinine reaches 200 mol/l. Ninety-four per cent of males have abnormal renal function by 25 years, and the average age of reaching end-stage CRF for males with X-linked AS is 21 years (Flinter 1997). The rate of progression of renal disease tends to run true to some extent within families (Grünfeld and Knebelmann 1998), and it is unusual for males to retain normal renal function beyond 30 years (Flinter and Chantler 1990). There are a few notable families, however, where the age at CRF in affected males can vary by up to 20 years in a single pedigree (Renieri et al. 1994; Barker et al. 1996). Renal transplant is the treatment of choice for patients with end-stage CRF secondary to AS, and graft survival is good (Kashtan et al. 1995). If a living related kidney donor is being considered, it is important to remember that apparently asymptomatic female relatives may also carry the Alport gene and therefore be at risk of developing impaired renal function themselves in due course (Kursat et al. 1998). The long-term effect of a nephrectomy on an asymptomatic carrier is unknown, but kidneys from first-degree relatives have been used in the absence of other suitable living donors (Sessa et al. 1995). About 5 per cent of transplanted patients develop post-transplantation anti-GBM antibody nephritis which may lead to graft loss, usually within a year of transplant (Turner and Rees 1996). The risk of recurrence in subsequent allografts is very high. Patients who have a large deletion in COL4A5 appear to be at highest risk (15 per cent risk), and they are also likely to have had an earlier onset of deafness and renal failure.

Typical clinical course of a female with X-linked AS This is very variable, presumably because of random or non-random X-inactivation. At the most severe end of the spectrum a small minority (approximately 1–2 per cent) of females follows a similar clinical course to affected males. At the other extreme, healthy women in their eighties with a family history of AS may be found to have previously-undetected microscopic haematuria with no other clinical signs. The majority of women carrying X-linked AS develop microscopic haematuria during childhood (which may be intermittently macroscopic). Thirty six per cent present with macroscopic haematuria at an average age of 9 years, and a further 40 per cent are detected when they are found to have microscopic haematuria on routine urinalysis. The penetrance of the gene in adult female carriers probably approaches 100 per cent (Ferguson and Rance 1972; Flinter and Chantler 1990), although some reports have suggested it might be only 85 per cent (Hasstedt and Atkin 1983). Recently it has become apparent that the incidence of somatic and germinal mosaicism in X-linked AS may be considerable, and this could account for some cases of apparent non-penetrance in female carriers (Bruttini et al. 2000; Plant et al. 2000). One-third develop hypertension (usually in middle age), and the lifetime risk of CRF is between 8 and 15 per cent (Flinter 1990; Dagher et al. 2001).

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Autosomal recessive AS Parental consanguinity, absence of a positive family history, and a phenotype in which males and females are affected with equal severity at a young age are clinical features that may suggest autosomal recessive inheritance; however, this mode of inheritance probably accounts for only about 10–15 per cent of all cases of AS (Torra et al. 1999). Affected individuals develop CRF between 5 and 15 years of age, and their parents often have isolated microscopic haematuria but no deafness or ophthalmological signs (Dagher et al. 2001). Immunohistochemical studies reveal absent 3(IV), 4(IV), and 5(IV) staining in the GBM (Nomura et al. 1998), but the presence of 5(IV) in extra-glomerular basement membranes including capsular, collecting duct, and EBMs, a combination never observed in X-linked AS (Gubler et al. 1995). It has been suggested that heterozygote carriers, who often have microscopic haematuria, may have what has sometimes been called benign familial haematuria, an apparently dominantly-inherited condition in which non-progressive haematuria is sometimes associated with thin GBM on renal biopsy (Smeets et al. 1996; Piccini et al. 1999; Buzza et al. 2001). Autosomal dominant AS There are more than 30 published pedigrees with apparent male-to-male transmission of an hereditary nephritis, but generally they do not fulfil the strict clinical criteria listed above, and autosomal dominant AS is probably very rare (Flinter 1997). Typically renal failure does not develop until middle age, and the characteristic eye signs are not found. In one large pedigree demonstrating autosomal dominant inheritance, the renal biopsy findings were characteristic, and one member with mild sensorineural deafness developed CRF at 35 years. Linkage to COL4A5 was demonstrated, and it was postulated that a missense mutation in exon 5 of the adjacent COL4A3 gene might lead to a more severe phenotype in family members who inherited it (Jefferson et al. 1997). Haematological abnormalities, including macrothrombocytopenia, have been reported in association with autosomal dominant AS (Epstein et al. 1972; Kashtan and Michael 1996), and this association is now explained at the molecular level by the demonstration of linkage to markers on chromosome 22q11–13 (Toren et al. 1999). Extra-renal abnormalities Apart from the ocular and audiological involvement described earlier, the most significant extrarenal abnormality is oesophageal, tracheobronchial, and genital leiomyomatosis (Johnston et al. 1953; Grünfeld et al. 1987). The underlying cause is a contiguous deletion involving a variable amount of COL4A5 plus the first two exons of COL4A6 which lie adjacent on the X chromosome (Antignac and Heidet 1996). Patients whose deletion extends beyond the second intron of COL4A6 do not have leiomyomatosis, but may have congenital cataracts. A variety of haematological abnormalities have been reported, mostly in association with autosomal dominant AS (e.g. Epstein’s anomaly and Fechtner syndrome; Toren et al. 1999),

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but there is also a single case report of a contiguous deletion syndrome on Xq22 which causes elliptocytosis (Vitelli et al. 1999). Knebelmann et al. (2001) suggest that hereditary nephritis with macrothrombocytopenia should no longer be regarded as an Alport syndrome variant. Other reported associations, including antithyroid antibodies and hyperprolinaemia/ hyperaminoaciduria are probably coincidental.

Molecular biology of AS Type IV collagen is the main structural component of GBM, and the two major polypeptide chains, 1(IV), and 2(IV) are present in a 2 : 1 ratio, forming a triple helical heterotrimeric molecule. Other associated glycoproteins include laminin, nidogen (entactin), and heparin sulphate proteoglycan. The genes coding for these two chains (COL4A1 and COL4A2) lie adjacent to each other on chromosome 13. The rarer 3(IV) and 4(IV) chains are coded for by a similar pair of genes on chromosome 2, and the 5(IV) and 6(IV) chains are encoded by COL4A5 and COL4A6 which lie on Xq22. The majority (80–90 per cent) of cases of AS are X-linked and caused by mutations in COL4A5. More than 300 mutations have been reported to date (Heiskari et al. 1996; Kawai et al. 1996; Knebelmann et al. 1996; Renieri et al. 1996; Tryggvason 1996; Lemmink et al. 1997; Martin et al. 1998; Inoue et al. 1999; Plant et al. 1999; Jais et al. 2000; Barker et al. 2001), and the mutation detection rate has ranged from 55 to 90 per cent (Flinter and Plant 1998). The COL4A5 gene is large, containing 51 exons, covering 240–310 kb of genomic DNA which produce a 6.5 kb transcript, making it one of the longest collagen genes characterized to date. The 5 chain contains 1685 amino acids, divided into a 26-residue signal peptide, a 1430 residue collagenous domain containing short non-collagenous interruptions, and a 229-residue carboxy terminal non-collagenous (NC) domain (Zhou et al. 1992). Most mutations described have been unique to the pedigree, with the small number of shared mutations probably explained by a common ancestor (Barker et al. 1996). About 10–15 per cent of patients have a large deletion which is detectable on Southern blotting. The largest deletion identified to date involves the loss of 450 kb of DNA, only about 10 kb of which lie within the gene, the rest of the deletion extending beyond the 3 end (Boye et al. 1991; Vetrie et al. 1992). As this patient appears to have a classic phenotype, it suggests the absence of other important genes adjacent to the 3 end of COL4A5. Other mutations identified have been distributed throughout the gene, with no particular mutation hotspots. A variety of smaller deletions, insertions, duplications, inversions, and missense, nonsense, and splice-site mutations has also been reported. The most common abnormality appears to be a missense mutation involving a guanine substitution in the first or second position of glycine codons which leads to glycine substitution in the collagenous domain (Jais et al. 2000). The new mutation rate is 15 per cent (Flinter 1997). Linkage studies have not suggested a second locus for X-linked AS, so the remaining mutations either lie within COL4A5 (as suggested by Martin et al. 1998, who have achieved a mutation detection rate of 84 per cent using PCR and direct sequencing), in non-coding segments of COL4A5, or in another gene nearby. The analysis of mRNA extracted from hair roots enables the detection of some mutations not picked up on analysis of DNA extracted from blood (King et al. 2001).

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Mutations in COL4A3 and COL4A4 have been found in patients with autosomal recessive AS (Lemmink et al. 1994; Mochizuki et al. 1994; Cosgrove et al. 1996; Boye et al. 1998; Heidet et al. 2001). COL4A4 has been implicated by linkage studies in one large autosomal dominant pedigree (Jefferson et al. 1997) and a splice site mutation in COL4A3 is the cause in another (van der Loop 2000).

Mosaicism in AS Three cases of somatic mosaicism have been demonstrated at a molecular level, with the mosaics having a milder phenotype (Plant et al. 2000), and one case of gonadal mosaicism has been reported (Bruttini et al. 2000). Genotype–phenotype correlation More than 90 per cent of male patients with a large rearrangement of COL4A5 or with small mutations leading to premature stop codons develop CRF before 30 years. These patients generally also have early-onset deafness and eye signs are present, and there is an increased risk of developing anti-GBM antibody nephritis following renal transplantation. The rate of deterioration of renal function is slower in patients with splice site or missense mutations. The majority of families, particularly those with a large rearrangement or frameshift mutation, show intrafamilial correlation for the age of CRF in affected males (Renieri et al. 1994; Jais et al. 2000), but not in females where there can be very striking differences in the severity of renal disease within families, presumably because of differential X-inactivation. Contiguous deletions Deletions involving a variable length of COL4A5 plus the first two exons of COL4A6 have been demonstrated in several patients with AS and leiomyomatosis. The breakpoint in intron 2 of COL4A6 appears to be significant because deletions extending beyond this point are not associated with leiomyomatosis (Heidet et al. 1995, 1997; Antignac and Heidet 1996). The exon size pattern of COL4A6 is highly homologous with that of COL4A2. A novel contiguous deletion syndrome, AMME (Alport syndrome, mental retardation, midface hypoplasia, and elliptocytosis) has been reported (Vitelli et al. 1999). Progressive nephropathy associated with mitochondrial mutations Progressive non-diabetic renal disease, diabetes mellitus, and sensorineural hearing loss have been reported in several families with a 3243A6G mutation in mitochondrial DNA demonstrating maternal transmission. The combination of renal failure and deafness had been misdiagnosed previously as AS (Jansen et al. 1997; Cheong et al. 1999). This mutation is also known to be associated with MELAS syndrome (myopathy, encephalopathy, lactic acidosis, and stroke-like episodes).

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Gene therapy Genetic renal diseases (see Chapter 24, this volume) which are not associated with life-threatening extra-renal complications will be attractive targets for gene therapy for several reasons. The therapy can be targeted solely at the kidneys, which have their own circulatory system; and the turnover of type IV collagen is fairly slow, with a half-life of more than a year. The GBM will be the prime target, and a transfer efficiency of 15 per cent has been reported using intra-arterial injection of liposomes together with Sendai virus (Tomita et al. 1992). More recently, using a kidney perfusion method, an 85 per cent transfer efficiency was obtained into pig glomeruli with an adenovirus vector containing the ß-galactosidase reporter gene (Heikkilä et al. 1996). The adenovirus expression continued for only 6–8 weeks however, as it is not integrated into the genome, so treatment would need to be repeated periodically throughout life. There are hopes that gene therapy will become a viable option eventually, but this will depend on the development of better gene transfer vectors, and on greater knowledge of the regulation of the genes requiring treatment (Tryggvason et al. 1997; Heikkilä et al. 2001). Animal models X-linked hereditary nephritis in Samoyed dogs is a model for AS, although proteinuria precedes haematuria in the dogs, and their vision and hearing are normal. Treatment with angiotensin-converting enzyme (ACE) inhibitors has been shown to delay the decline in the glomerular filtration rate, and to reduce proteinuria. There was also a transient reduction in GBM splitting, and treated dogs survived 1.36 times longer than untreated dogs (Grodecki et al. 1997). Bull terrier hereditary nephritis is inherited as an autosomal dominant disease causing haematuria, renal failure, and anterior lenticonus, but not deafness. The renal histology in affected bull terriers shows an identical ultrastructural appearance to that seen in humans with X-linked AS (Hood et al. 1995). English cocker spaniels provide a model of autosomal recessive AS, with proteinuria and juvenile-onset CRF in association with typical renal histology (Lees et al. 1998), and there is also a COL4A3 knockout mouse model (Cosgrove et al. 1996). Benign familial haematuria Benign familial haematuria (BFH) describes a clinical phenotype that is often associated with a renal biopsy finding of a thin GBM. Neither term is entirely satisfactory. Isolated glomerular haematuria may occur as a familial or a sporadic condition—but familial haematuria is not always benign, and benign haematuria not always familial (Kashtan 1998). Thin GBM is a histological finding which may be found in a variety of different conditions, including BFH, IgA nephropathy, minimal change nephrotic syndrome, AS (particularly in females and in childhood) and even as a normal variant. GBM thickness is age- and gender-dependent, and various normal values exist; however, intraglomerular variability in GBM width is small in thin GBM disease, and marked variability suggests an alternate diagnosis of AS (Kashtan 1998; Marquez et al. 1999). Patients with a thin GBM are at increased risk of premature glomerular obsolescence, hypertension, and occasionally late-onset renal insufficiency

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(Nieuwhof et al. 1997). Immunohistochemical studies in patients with BFH are usually normal, and ocular abnormalities are not seen (Colville et al. 1997). Benign familial haematuria is usually transmitted as an autosomal dominant condition, and in some families linkage to COL4A3 and COL4A4 on chromosome 2 has been demonstrated. Affected individuals in a large Dutch pedigree carry a heterozygous missense mutation in COL4A4 (Lemmink et al. 1996); however, linkage to the collagen genes on chromosome 2 has been excluded in other BFH families (Piccini et al. 1999), suggesting that the condition is clinically heterogeneous (Saito et al. 1997; Piccini et al. 1999). Internet sites AS home page: http://www.cc.utah.edu/~cla6202/ASHP.htm The Hereditary Nephritis Foundation website: http://www.cc.utah.edu/~cla6202/HNF.htm

References Alport, A. C. (1927). Hereditary familial congenital haemorrhagic nephritis. British Medical Journal, 1, 504–6. Antignac, C. and Heidet, L. (1996). Mutations in Alport syndrome associated with diffuse esophageal leiomyomatosis. Contributions to Nephrology, 117, 172–82. Barker, D. F., Hostikka, S. L., Zhou, J., Chow, L. T., Oliphant, A. R., Gerken, S. C., et al. (1990). Identification of mutations in the COL4A5 collagen gene in Alport syndrome. Science, 248, 1224–7. Barker, D. F., Pruchno, C. J., Jiang, X., Atkin, C. L., Stone, E. M., Denison, J. C., et al. (1996). A mutation causing Alport syndrome with tardive hearing loss is common in the western United States. American Journal of Human Genetics, 58, 1157–65. Barker, D. F., Denison, J. C., Atkin, C. L., and Gregory, M. C. (2001). Efficient detection of Alport syndrome COL4A5 mutations with multiplex genomic PCR-SSCP. American Journal of Human Genetics, 98, 148–60. Boye, E., Vetrie, D., Flinter, F. A., Buckle, B., Pihlajaniemi, T., Hamalainen, E.-R., et al. (1991). Major rearrangements in the 5(IV) collagen gene in three patients with Alport’s syndrome. Genomics, 11, 1125–32. Boye, E., Mollet, G., Forestier, L., Cohen-Solal, L., Heidet, L., Cohat, P., et al. (1998). Determination of the genomic structure of the COL4A4 gene and of novel mutations causing autosomal recessive Alport syndrome. American Journal of Human Genetics, 63, 1329–40. Brainwood, D., Kashtan, C., Gubler, M. C., and Turner, A. N. (1998). Targets of alloantibodies in Alport anti-glomerular basement membrane disease after renal transplantation. Kidney International, 53, 762–6. Bruttini, M., Vitelli, F., Meloni, I., Rizzari, G., Volpe, M. D., Mazzucco, G., et al. (2000). Mosaicism in Alport syndrome and genetic counselling. Journal of Medical Genetics, 37, 717–9. Buzza, M., Wilson, D., and Savige, J. (2001). Segregation of haematuria in thin basement membrane disease with haplotypes at the loci for Alport syndrome. Kidney International, 59, 1670–6. Cangiotti, A. M., Sessa, A., Meroni, M., Montironi, R., Ragaiolo, M., Mambelli, V., et al. (1996). Evolution of glomerular basement membrane lesions in a male patient with Alport syndrome: ultrastructural and morphometric study. Nephrology, Dialysis, Transplantation, 11, 1829–34. Cheong, H. I., Chae, J. H., Kim, J. S., Park, H. W., Ha, I. S., Hwang, Y. S., et al. (1999). Hereditary glomerulopathy associated with a mitochondrial tRNA(Leu) gene mutation. Pediatric Nephrology, 13, 477–80.

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Colville, D. J. and Savige, J. (1997). Alport syndrome. A review of the ocular manifestations. Ophthalmic Genetics, 18, 161–73. Colville, D., Savige, J., Branley, P., and Wilson, D. (1997). Ocular abnormalities in thin basement membrane disease. British Journal of Ophthalmology, 81, 373–7. Colville, D., Wang, Y. Y., Jamieson, R., Collins, F., Hood, J., and Savige, J. (2000). Absence of ocular manifestations in autosomal dominant Alport syndrome associated with haematological abnormalities. Ophthalmic Genetics, 21, 217–25. Cosgrove, D., Meehan, D. T., Grunkemeyer, J. A., Kornak, J. M., Sayers, R., Hunter, W. J., et al. (1996). Collagen COL4A3 knockout: a mouse model for autosomal Alport syndrome. Genes and Development, 10, 2981–92. Crawfurd, M. d’A. (1988). Hereditary nephritis with deafness (Alport’s disease). In The genetics of renal tract disorders. Oxford Monographs on Medical Genetics No. 14, p. 351. Oxford University Press, Oxford. Dagher, H., Buzza, M., Colville, D., Jones, C., Powell, H., Fassett, R., et al. (2001). A comparison of the clinical, histopathologic, and ultrastructural phenotypes in carriers of X-linked and autosomal recessive Alport’s syndrome. American Journal of Kidney Diseases, 38, 1217–28. Epstein, C. J., Sahud, M. A., Piel, C. F., Goodman, J. R., Bernfield, M. R., Kushner, J. H., et al. (1972). Hereditary macrothrombocytopathia, nephritis and deafness. American Journal of Medicine, 52, 299–310. Ferguson, A. C. and Rance, C. P. (1972). Hereditary nephropathy with nerve deafness (Alport’s syndrome). American Journal of Diseases of Children, 124, 84–8. Flinter, F. A. (1989). A clinical and genetic study of Alport’s syndrome. MD thesis. University of London. Flinter, F. A. (1990). Alport’s syndrome. A clinical and genetic study. In Recent advances in hereditary nephritis (ed. A. Sessa and G. Battini), Contributions to Nephrology Series 80, pp. 9–16. Karger, Basel, Switzerland. Flinter, F. (1997). Alport’s syndrome. Journal of Medical Genetics, 34, 326–30. Flinter, F. A. and Chantler, C. (1990). The inheritance of Alport’s syndrome. In Inheritance of kidney and urinary tract diseases (ed. A. Spitzer and E. D. Avner), pp. 107–20. Kluwer Academic Publications, London and Boston, MA. Flinter, F. and Plant, K. (1998). Why are mutations in COL4A5 not detectable in all patients with Alport’s syndrome? Nephrology, Dialysis, Transplantation, 13, 1348–51. Flinter, F. A., Cameron, J. S., Chantler, C., Houston, I., and Bobrow, M. (1988). Genetics of classic Alport’s syndrome. Lancet, ii, 1005–7. Govan, J. A. A. (1983). Ocular manifestations of Alport’s syndrome: a hereditary disorder of basement membrane? British Journal of Ophthalmology, 67, 493–503. Gregg, J. B. and Becker, S. F. (1963). Concomitant progressive deafness, chronic nephritis and ocular lens disease. Archives of Ophthalmology, 69, 293–9. Grodecki, K. M., Gains, M. J., Baumal, R., Osmond, D. H., Cotter, B., Valli, V. E., et al. (1997). Treatment of X-linked hereditary nephritis in Samoyed dogs with angiotensin converting enzyme (ACE) inhibitor. Journal of Comparative Pathology, 117, 209–25. Grünfeld, J. P. (1985). The clinical spectrum of hereditary nephritis. Kidney International, 27, 83–92. Grünfeld, J. P. and Knebelmann, B. (1998). Alport’s syndrome. In Oxford textbook of clinical nephrology (ed. A. M. Davison, J. S. Cameron, J. P. Grünfeld, D. N. S. Kerr, E. Ritz, and C. G. Winearls), pp. 2427–37. Oxford University Press, Oxford. Grünfeld, J. P., Grateau, G., Noel, L.-H., Charbonneau, R., Gubler, M. C., Savage, G. O. S., et al. (1987). Variants of Alport’s syndrome. Pediatric Nephrology, 1, 419–21. Gubler, M., Levy, M., Broyer, M., Naizot, C., Gonzales, G., Perrin, D., et al. (1981). Alport’s syndrome: a report of 58 cases and a review of the literature. American Journal of Medicine, 70, 493–505. Gubler, M. C., Mounier, F., Gros, F., Wieslander, J., and Beziau, A. (1988). Glomerular distribution of subunits M1 and M2 of the globular domain of basement membrane collagen in Alport’s syndrome.

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In Progress in basement membrane research: renal and related aspects in health and disease (ed. M. C. Gubler and M. Sternberg), pp. 177–82. Libbey Eurotext, London. Gubler, M. C., Knebelmann, B., Beziau, A., Broyer, M., Pirson, Y., Haddoum, F., et al. (1995). Autosomal recessive Alport syndrome: immunohistochemical study of type IV collagen chain distribution. Kidney International, 47, 1142–7. Hasstedt, S. J. and Atkin, C. L. (1983). X-linked inheritance of Alport syndrome: family P revisited. American Journal of Human Genetics, 35, 1241–51. Heidet, L., Dahan, K., Zhou, J., Xu, Z., Cochat, P., Gould, J. D., et al. (1995). Deletions of both alpha 5(IV) and alpha 6(IV) collagen genes in Alport syndrome and in Alport syndrome associated with smooth muscle tumours. Human Molecular Genetics, 4, 99–108. Heidet, L., Cohen-Solal, L., Boye, E., Thorner, P., Kemper, M. J., David, A., et al. (1997). Novel COL4A5/COL4A6 deletions and further characterization of the diffuse leiomyomatosis-Alportsyndrome (DL-AS) locus define the DL critical region. Cytogenetics and Cell Genetics, 78, 240–6. Heidet, L., Arrondel C., Forestier, L., Cohen-Solal, L., Mollet, G., Gutierrez, B., et al. (2001). Structure of the human type IV collagen gene COL4A3 and mutations in autosomal Alport syndrome. Journal of the American Society of Nephrology, 12, 97–106. Heikkilä, P., Parpala, T., Lukkarinen, O., Weber, M., and Tryggvason, K. (1996). Adenovirus-mediated gene transfer into kidney glomeruli using an ex vivo and in vivo kidney perfusion system—first steps towards gene therapy of Alport syndrome. Gene Therapy, 3, 21–7. Heikkilä, P., Tibell, A., Morita, T., Chen, Y., Wu, G., Sado, Y., et al. (2001). Adenovirus-mediated transfer of type IV collagen alpha 5 chain cDNA into swine kidney in vivo: deposition of the protein into the glomerular basement membrane. Gene Therapy, 8, 882–90. Heiskari, N., Zhang, X., Zhou, J., Leinonen, A., Barker, D., Gregory, M., et al. (1996). Identification of 17 mutations in ten exons in the COL4A5 collagen gene, but no mutations found in four exons in COL4A6: a study of 250 patients with hematuria and suspected of having Alport’s syndrome. Journal of the American Society of Nephrology, 7, 702–9. Hinglais, N., Grünfeld, J.-P., and Bois, E. (1972). Characteristic ultrastructural lesion of the glomerular basement membrane in progressive nephritis (Alport’s syndrome). Laboratory Investigations, 27, 473–87. Hood, J. C., Savige, J., Hendtlass, A., Kleppel, M. M., Huxtable, C. R., and Robinson, W. F. (1995). Bull terrier hereditary nephritis: a model for autosomal dominant Alport syndrome. Kidney International, 47, 758–65. Hostikkä, S. L., Eddy, R. L., Byers, M. G., Höyhtyä, M., Shows, T. B., and Tryggvason, K. (1990). Identification of a distinct type IV collagen chain with restricted kidney distribution and assignment of its gene to the locus of X chromosome-linked Alport syndrome. Proceedings of the National Academy of Sciences, USA, 87, 1606–10. Inoue, Y., Nishio, H., Shirakawa, T., Nakanishi, K., Nakamura, H., Sumino, K., et al. (1999). Detection of mutations in the COL4A5 gene in over 90% of male patients with X-linked Alport’s syndrome by RT-PCR and direct sequencing. American Journal of Kidney Disease, 34, 854–62. Iverson, U. M. (1974). Hereditary nephropathy with hearing loss: ‘Alport’s syndrome’. Acta Paediatrica Scandinavica Supplement, 245, 1–23. Jais, J. P., Knebelmann, B., Giatras, I., de Marchi, M., Rizzoni, G., Renieri, A., et al. (2000). X-linked Alport syndrome: natural history in 195 families and genotype-phenotype correlations in males. Journal of the American Society of Nephrology, 11, 649–57. Jansen, J. J., Maassen, J. A., van der Woude, F. J., Lemmink, H. A., van der Ouweland, J. M., t’Hart, L. M., et al. (1997). Mutation in mitochondrial tRNA(Leu(UUR)) gene associated with progressive kidney disease. Journal of the American Society of Nephrology, 8, 1118–24. Jefferson, J. A., Lemmink, H. H., Hughes, A. E., Hill, C. M., Smeets, H. J., Doherty, C. C., et al. (1997). Autosomal dominant Alport syndrome linked to the type IV collagen alpha 3 and alpha 4 genes (COL4A3 and COL4A4). Nephrology Dialysis Transplantation, 12, 1595–9.

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Jenis, E. H., Valeski, J. E., and Calcagno, P. L. (1981). Variability of anti-glomerular basement membrane binding in hereditary nephritis. Clinical Nephrology, 15, 111–4. Johnston, J. B., Clagett, O. T., and McDonald, J. R. (1953). Smooth-muscle tumours of the oesophagus. Thorax, 8, 251–65. Julian, B. A., Waldo, F. B., Rifai, A., and Mestecky, J. (1988). IgA nephropathy, the most common glomerulonephritis worldwide: a neglected disease in the United States? American Journal of Medicine, 84, 129–32. Kalluri, R., Gattone, V. H., and Hudson, B. G. (1998). Identification and localization of type IV collagen chains in the inner ear cochlea. Connective Tissue Research, 37, 143–50. Kashtan, C. E. (1998). Alport syndrome and thin glomerular basement membrane disease. Journal of the American Society of Nephrology, 9, 1736–50. Kashtan, C. E. (1999). Alport syndrome: is diagnosis only skin-deep? Kidney International, 55, 1575–76. Kashtan, C. E. and Michael, A. (1996). Alport syndrome. Kidney International, 50, 1445–63. Kashtan, C., Fish, A. J., Klepel, M., Yoshioka, K., and Michael, A. F. (1986). Nephritogenic antigen determinants in epidermal and renal basement membranes of kindreds with Alport-type familial nephritis. Journal of Clinical Investigation, 78, 1035–44. Kashtan, C. E., McEnery, P. T., Tejani, A., and Stablein, D. M. (1995). Renal allograft survival according to primary diagnosis: a report of the North American Pediatric Renal Transplant Cooperative Study. Pediatric Nephrology, 9, 679–84. Kawai, S., Nomura, S., Harano, T., Harano, K., Fukushima, T., and Osawa, G. (1996). The COL4A5 gene in Japanese Alport syndrome patients: spectrum of mutations in all exons. Kidney International, 49, 814–22. King, K., Flinter, F. A., and Green, P. M. (2001). Hair roots as the ideal source of mRNA for genetic testing. Journal of Medical Genetics, 38, E20. Knebelmann, B., Breillat, C., Forestier, L., Arrondel, C., Jacassier, D., Giatras, I., et al. (1996). Spectrum of mutations in the COL4A5 collagen gene in X-linked Alport syndrome. American Journal of Human Genetics, 59, 1221–32. Knebelmann, B., Fakhouri, F., and Grunfeld, J. P. (2001). Hereditary nephritis with macrothrombocytopenia: no longer an Alport syndrome variant. Nephrology, Dialysis, Transplantation, 16, 1101–3. Kursat, S., Erkin, E., and Kiliccioglu, B. (1998). The simultaneous appearance of Alport syndrome in a renal transplant donor and the recipient as primary disease recurrence. Nephron, 79, 364. Lees, G. E., Helman, R. G., Kashtan, C. E., Michael, A. F., Homco, L. D., Millichamp, N. J., et al. (1998). A model of autosomal recessive Alport syndrome in English cocker spaniel dogs. Kidney International, 54, 706–19. Lemmink, H. H., Mochizuki, T., van den Heuvel, L. P., Schröder, C. H., Barrientos, A., Monnens, L. A. H., et al. (1994). Mutations in the type IV collagen 3 (COL4A3) gene in autosomal recessive Alport syndrome. Human Molecular Genetics, 3, 1269–73. Lemmink, H. H., Nillesen, W. N., Mochizuki, T., Schroder, C. H., Brunner, H. G., van Oost, B. A., et al. (1996). Benign familial hematuria due to mutation of the type IV collagen alpha4 gene. Journal of Clinical Investigation, 98, 1114–8. Lemmink, H. H., Schroder, C. H., Monnens, L. A., and Smeets, H. J. (1997). The clinical spectrum of type IV collagen mutations. Human Mutation, 9, 477–99. Marquez, B., Stavrou, F., Zouvani, I., Anastasiades, E., Patsias, C., Pierides, A., et al. (1999). Thin glomerular basement membranes in patients with hematuria and minimal change disease. Ultrastructural Pathology, 23, 149–56. Martin, P., Heiskari, N., Zhou, J., Leinonen, A., Tumelius, T., Hertz, J. M., et al. (1998). High mutation detection rate in the COL4A5 collagen gene in suspected Alport syndrome using PCR and direct DNA sequencing. Journal of the American Society of Nephrology, 9, 2291–301.

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Mazzucco, G., Barsotti, P., Muda, A. O., Fortunato, M., Mihatsch, M., Torri-Tarelli, L., et al. (1998). Ultrastructural and immunohistochemical findings in Alport’s syndrome: a study of 108 patients from 97 Italian families with particular emphasis on COL4A5 gene mutation correlations. Journal of the American Society of Nephrology, 9, 1023–31. McCoy, R. C., Johnson, H. K., Stone, W. J., and Wilson, C. B. (1976). Variation in glomerular basement membrane antigen in hereditary nephritis. Laboratory Investigations, 34, 325–6. McDonald, T. J., Zincke, H., Anderson, C. F., and Ott, N. T. (1978). Reversal of deafness after renal transplantation in Alport’s syndrome. Laryngoscope, 88, 38–42. Mochizuki, T., Lemmink, H. H., Mariyama, M., Antignac, C., Gubler, M. C., Pirson, Y., et al. (1994). Identification of mutations in the alpha 3 (IV) and alpha 4 (IV) collagen genes in autosomal recessive Alport syndrome. Nature Genetics, 8, 77–81. Naito, I., Nomura, S., Inoue, S., Kagawa, M., Matsubara, T., Araki, T., et al. (1997). X-linked Alport syndrome with normal distribution of collagen IV alpha chains in epidermal basement membrane. Contributions to Nephrology, 122, 134–9. Nakanishi, K., Iijima, K., Kuroda, N., Inoie, Y., Sado, Y., Nakamura, H., et al. (1998). Comparison of alpha5(IV) collagen chain expression in skin with disease severity in women with X-linked Alport syndrome. Journal of the American Society of Nephrology, 9, 1433–40. Nieuwhof, C. M., de Heer, F., de Leeuw, P., and van Breda Vriesman, P. J. (1997). Thin GBM nephropathy: premature glomerular obsolescence is associated with hypertension and late onset renal failure. Kidney International, 51, 1596–601. Nomura, S., Naito, I., Fukushima, T., Tokura, T., Kataoka, N., Tanaka, I., et al. (1998). Molecular genetic and immunohistochemical study of autosomal recessive Alport’s syndrome. American Journal of Kidney Disease, 31, E4. Olson, D. L., Anand, S. K., Landin, B. H., Heuser, E., Grushkin, C. M., and Lieberman, E. (1980). Diagnosis of hereditary nephritis by failure of glomeruli to bind anti-glomerular basement membrane antibodies. Journal of Pediatrics, 96, 697–9. Piccini, M., Casari, G., Zhou, J., Bruttini, M., Volti, S. L., Ballabio, A., et al. (1999). Evidence for genetic heterogeneity in benign familial hematuria. American Journal of Nephrology, 19, 464–7. Pirson, Y. (1999). Making the diagnosis of Alport’s syndrome. Kidney International, 56, 760–75. Plant, K. E., Green, P. M., Vetrie, D., and Flinter, F. A. (1999). Detection of mutations in COL4A5 in patients with Alport syndrome. Human Mutation, 13, 124–32. Plant, K. E., Boye, E., Green, P. M., Vetrie, D., and Flinter, F. A. (2000). Somatic mosaicism associated with a mild Alport syndrome phenotype. Journal of Medical Genetics, 37, 238–9. Rambausek, M., Hartz, G., Waldherr, R., Andrassy, K., and Ritz, E. (1987). Familial glomerulonephritis. Pediatric Nephrology, 1, 416–8. Renieri, A., Meroni, M., Sessa, A., Battini, G., Servelloni. P., Tarelli, L. T., et al. (1994). Variability of clinical phenotype in a large Alport family with gly1143ser change of collagen 5(IV)-chain. Nephron, 67, 444–9. Renieri, A., Bruttini, M., Galli, L., Zanelli, P., Neri, T., Rossetti, S., et al. (1996). X-linked Alport syndrome: an SSCP-based mutation survey over all 51 exons of the COL4A5 gene. American Journal of Human Genetics, 58, 1192–204. Rhys, C., Snyers, B., and Pirson, Y. (1997). Recurrent corneal erosion associated with Alport’s syndrome. Kidney International, 52, 208–11. Rigden, S. P., Mehls, O., Jones, E. H., and Valderrabano, F. (1996). Report on management of renal failure in Europe, XXVI, 1995. The child-adult interface: a report on Alport’s syndrome, 1975–1993. The ERA-EDTA Registry. Nephrology Dialysis Transplantation, 11, S21–7. Rumpelt, H. J. (1980). Hereditary nephropathy (Alport’s syndrome): correlation of clinical data with glomerular basement membrane alterations. Clinical Nephrology, 13, 203–7. Saito, A., Yamazaki, H., Nakagawa, Y., and Arakawa, M. (1997). Molecular genetics of renal diseases. Internal Medicine, 36, 81–6.

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Saus, J., Wieslander, J., Langeveld, J. P. M., Quinones, S., and Hudson, B. G. (1988). Identification of the Goodpasture antigen as the 3 chain of collagen IV. Journal of Biological Chemistry, 263, 13,374–80. Savage, C. O. S., Pusey, C. D., Kershaw, M. J., Cashman, S. J., Harrison, P., Hartley, B., et al. (1986). The Goodpasture antigen in Alport’s syndrome: studies with a monoclonal antibody. Kidney International, 30, 107–12. Sessa, A., Pietrucci, A., Carozzi, S., Torri Tarelli, L., Tazzarri, S., and Giordano, F. (1995). Renal transplantation from living donor parents in two brothers with Alport syndrome. Can asymptomatic female carriers of the Alport gene be accepted as kidney donors? Nephron, 70, 106–9. Smeets, H. J. M., Knoers, V. V. A. M., van de Heuvel, L. P. W. J., Lemmink, H. H., Schröder, C. H., and Monnens, L. A. H. (1996). Hereditary disorders of the glomerular basement membrane. Pediatric Nephrology, 10, 779–88. Sohar, E. (1954). A heredo-familial syndrome characterized by renal disease, inner ear deafness and ocular changes. Harefuah, 27, 161. Spear, G. S. and Slusser, R. J. (1972). Alport’s syndrome. Emphasising electron microscopic studies of the glomerulus. American Journal of Pathology, 69, 213–20. Tomita, N., Higaki, J., Morishita, R., Kato, K., Mikami, H., Kaneda, Y., et al. (1992). Direct in vivo gene introduction into rat kidney. Biochemistry and Biophysics Research Communications, 186, 129–34. Toren, A., Amariglio, N., Rozenfeld-Granot, G., Simon, A. J., Brok-Simoni, F., Pras, E., et al. (1999). Genetic linkage of autosomal dominant Alport syndrome with leukocyte inclusions and macro (Fechtner syndrome) to chromosome 22q11–13. American Journal of Human Genetics, 65, 1711–7. Torra, R., Badenas, C., Cofan, F., Callis, L., Perez-Oller, L., and Darnell, A. (1999). Autosomal recessive Alport syndrome: linkage analysis and clinical features in two families. Nephrology Dialysis Transplantation, 14, 627–30. Tryggvason, K. (1996). Mutations in type IV collagen genes and Alport phenotypes. In Molecular pathology and genetics of Alport syndrome (ed. K. Tryggvason), Vol. 177, pp. 154–71. Basel, Karger. Tryggvason, K., Heikkilä, P., Pettersson, E., Tibell, A., and Thorner, P. (1997). Can Alport syndrome be treated by gene therapy? Kidney International, 51, 1493–9. Turner, A. N. and Rees, A. J. (1996). Goodpasture’s disease and Alport’s syndrome. Annual Reviews in Medicine, 47, 377–86. van der Loop, F. T., Monnens, L. A., Schroder, C. H., Lemmink, H. H., Bruening, M. H., Timmer, E. D., et al. (1999). Identification of COL4A5 defects in Alport’s syndrome by immunohistochemistry of skin. Kidney International, 55, 1217–24. van der Loop, F. T., Heidet, L., Timmer, E. D., van den Bosch, B. J., Leinonen, A., Antignac, C., et al. (2000). Autosomal dominant Alport syndrome caused by a COL4A3 splice site mutation. Kidney International, 58, 1870–5. Vetrie, D., Boye, E., Flinter, F. A., Bobrow, M., and Harris, A. (1992). DNA rearrangements in the 5(IV) collagen gene of individuals with Alport’s syndrome (AS): further refinement using pulsed field electrophoresis. Genomics, 14, 624–33. Vitelli, F., Piccini, M., Caroli, F., Franco, B., Malandrini, A., Pober, B., et al. (1999). Identification and characterization of a highly conserved protein absent in the Alport syndrome (A), mental retardation (M), midface hypoplasia (M), and elliptocytosis (E) contiguous gene deletion syndrome (AMME). Genomics, 55, 335–40. Wieslander, J., Barr, J. F., Butkowski, R., Edwards, S. J., Bygren, P., Heinegard, D., et al. (1984). Goodpasture antigen of the glomerular basement membrane: localisation to noncollagenous regions of type IV collagen. Proceedings of the National Academy of Sciences, USA, 81, 3838–42.

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9 Autosomal dominant polycystic kidney disease Anand K. Saggar-Malik and Stefan Somlo

Autosomal dominant polycystic kidney disease (ADPKD) typically presents in the third or fourth decade with haematuria, loin (flank) pain or hypertension. It is characterized by the progressive development and enlargement of bilateral renal cysts and often leads to end-stage renal disease (ESRD) by late middle age. It is well recognized now that ADPKD is a multisystem disorder with many extra-renal changes and complications. These extrarenal manifestations include hepatic cysts, cerebral aneurysms, and cardiac valve abnormalities. With increasing awareness of potential complications, there has been a greater drive to diagnose patients pre-symptomatically such that many patients may now be asymptomatic for many years. Sudden and premature death is also a recognized problem. Rupture of intracranial aneurysms (ICA) is an important cause of sudden death in polycystic patients, but only accounts for 14 per cent of all deaths with less than 10 per cent of patients first presenting with an acute subarachnoid haemorrhage (SAH) secondary to ICA rupture (Iglessia et al. 1983). ADPKD is one of the most common single-gene disorders of humans, and is both the most frequent inherited form of cystic kidney disease and the most common inherited renal disease. The prevalence of ADPKD is estimated at 1 in 1000 to 1 in 400 (Iglesias et al. 1983; Chapman and Schrier 1991; Davies et al. 1991; Bear et al. 1992). This approximates to 4–6 million affected individuals worldwide. In a combined report of the European Dialysis and Transplantation Association (Brunner et al. 1988), ADPKD had a prevalence of 6.9 per cent in Europe with similar figures later identified by Parfery in North America and Europe (Parfrey et al. 1990) and in Australasia (Disney and Correll 1981). However in Japan there appears to be a low rate of prevalence, approximating 3 per cent at most (Teraoka et al. 1995; Higashihara et al. 1998). Enlarged kidneys with bilateral cysts were first described Bartolomeo Eustachi in Rome (1520–1574) while early thoughts on the pathophysiology mechanisms of cystogenesis were made by Alexis Littre in Paris (1700). In the 1880s several clinical studies were published in Europe with subsequent contributions by Brash in 1916 and later Dalgaard in 1957. For an excellent and more complete account of the historical aspects of ADPKD the reader is referred to Watson et al. (1996). Table 9.1 shows a brief timeline of discovery in ADPKD. Cysts in this disease vary considerably in size and appearance, from a few millimetres to many centimetres and can produce an enlarged kidney as big as 40 cm in length and weighing as much as 8 kg (Gabow 1993). Rarely cysts may be unilateral (Bear 1999; Fick-Brosnahan et al. 1999) or be seen in the fetus (MacDermot et al. 1998).

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Table 9.1 Timeline of discovery in ADPKD 1500s 1880s 1957 1985 1986 1992 1993 1994 1995 1996 1996 2002

Enlarged kidneys with bilateral cysts first described by Bartolomeo Eustachi in Rome Early clinical studies published in Europe Publication of first large scale epidemiological study of ADPKD by Dalgaard PKD1 gene locus identified to alpha globin gene on chromosome 16 Heterogeneity described with second locus identified on chromosome 4 TSC2 locus discovered at chromosome 16p13.3 PKD2 gene localized at chromosome 4q13–q23 PKD1 gene isolated by the European Polycystic Kidney Disease Consortium Evidence for a third PKD locus PKD2 gene cloned Two-hit model for cystogenesis proposed Complete mutation screen of PKD1 and PKD2 by DHPLC

Historically, it has been suggested that ADPKD is less common in the non-white Caucasian population. Certainly there have been differences in numbers of reported cases, but these almost certainly reflect under-diagnosis, under-investigation or misdiagnosis. ADPKD has been described to occur in Africans and Asians and many other races (Iglesias 1997; Yersin et al. 1997; Phakdeekitcharoen 2000; Lee et al. 2001; Ding et al. 2002; Mizoguchi et al. 2002), but again underlying the lack of study in different racial groups, the prevalence of ESRD in non-European subjects has not been extensively determined. Although the disease is said to be less common in some parts of Africa, little information exists (Seedat et al. 1984). In US Blacks ESRD from ADPKD is about as frequent as in Whites, but age at ESRD is more variable and lower in Blacks than in Whites (Yium et al. 1994; Freedman et al. 2000). Furthermore ESRD in ADPKD blacks with sickle-cell trait is earlier than in those without sickle trait (Yium et al. 1994).

Cyst formation Cysts increase in size and number with time, however some patients remain asymptomatic and may only be diagnosed at autopsy (Iglesias et al. 1983). Detectable renal cysts, by ultrasound scanning, are often only first seen in the second decade of life. The cysts themselves are fluid filled structures which vary in diameter. Those below the diameter of 200 m tend to communicate with the tubules from which they arise. Above this size most cysts (70 per cent) exist as non-communicating structures (Gardner 1992). It is believed that overall only 5 per cent of tubules are affected by early cystic expansion (Grantham et al. 1987). The reasons for such a low number of tubules being affected is ill-understood but more recently a ‘two hit’ hypothesis has been postulated as one explanation (Pei 2001). In the initial stages the tubule from which the cyst derives communicates with the proximal and distal segment. Therefore, fluid from the glomerular filtrate may enter the cysts and exit to the downstream tubule. Growth of cells have been found obstructing the outflow of fluid from some cysts, but this is not a universal finding in all cysts (Grantham et al. 1987). It is believed that as a cyst enlarges and protrudes into the renal interstitium, various shear

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forces break the connections of the parent tubule in the majority of cysts, leaving it as an isolated sac. The mechanism(s) by which cysts accumulate fluid is ill-understood but remains of clinical importance. If the tubule epithelium lining the cyst functioned normally the cyst would collapse, since the fluid entering the cavity would be absorbed by the normal transepithelial transport of sodium chloride out of the lumen. However, cyst expansion continues to occur despite the lack of a glomerular source of fluid indicating that net trans-epithelial fluid secretion must be responsible for continued accumulation of fluid (Sullivan et al. 1998). Several mechanisms of fluid secretion have been suggested (Calvet and Grantham 2001). Sodium, potassium-ATPase has been found to be preferentially distributed on apical plasma membranes lining human ADPKD cysts, demonstrating that the sodium pump is mislocated (Wilson et al. 1991, 2000). This appears to be a reversion to fetal origin and may represent epithelial immaturity. A role for a chloride/bicarbonate exchanger has also been suggested (Ye and Grantham 1993). Ongoing fluid accumulation together with other secretagouges such as c-AMP (Mangoo-Karim et al. 1989) lead to continued cyst expansion and this, in part, along with other inflammatory mediators is blamed for the progressive failure of kidney function (Wilson 1996; Carone et al. 1996; Grantham et al. 1997) Cyst fluids vary in composition although sodium is the most prevalent extracellular cation and undoubtedly accounts for the osmotic activity of the cysts. It is believed that the cystic fluid originally begins as an ultrafiltrate of plasma, but there must be other modifying factors since there can be a several hundred-fold variation in non-sodium and sodium solute concentrations (Gardner and Bernstein 1990). There must either be the addition of water to, or the removal of sodium solutes from the original glomerular filtrate and/or the addition of non-sodium solutes higher than that found in normal serum. Despite genetic heterogeneity it appears that in all types of ADPKD the process of cystogenesis and progressive enlargement appears to be similar, particularly in that bilateral renal cysts originate from relatively few nephrons and collecting ducts and over several decades progressively expand. The extracellular matrix surrounding cysts is remarkably abnormal and appears to change concomitantly with cyst formation (Carone and Kanwar 1996; Carone et al. 1996). There is an abundance of Types I, III, and IV collagen, smooth muscle actin, laminin, and fibronectin amongst many other changes (Wilson 1996). Furthermore, the presence of mononuclear cells in the renal interstitium suggest that cysts produce a reaction which may be more important in disturbing renal function than the mere physical presence of the cyst. This is given weight by the clinical observation that kidneys may be grotesquely enlarged by cysts, yet function normally, while there may be others which are mildly enlarged and completely dysfunctional. The role of the interstitium and specifically inflammation and fibrotic mechanisms continues to be examined by numerous research workers and the field of knowledge is expanding rapidly.

Animal studies It is assumed that growth factors play a major role in cyst growth beside their effects of progression towards end-stage renal failure since any type of 5/6 nephrectomy for example, pole resection, pole ligation, and ligation of branches of the renal artery will induce cyst

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formation. This indicates that cyst formation is a common endpoint of the factors influencing renal tissue regeneration. Mouse models A number of autosomal recessive models exist in mice while few mouse models seem to resemble ADPKD (Schieren et al. 1996; Montagutelli 2000). Recently, gene targeting has been utilized to produce a genuine mouse model of the ADPKD complete with a propensity for acquiring second hits and chronic progressive renal failure in middle life (Wu et al. 1998, 2000). Mouse models are easier to breed, they develop renal cysts easily and die of uraemia within 16–24 months. Unfortunately, mice are small animals with a small blood volume. They are therefore not suitable for longitudinal assessment of disease which requires frequent blood sampling. Blood pressure measurement and metabolic studies are also difficult to study in mice, although technology is improving.

Rat models Several genetically determined rat models have been described (Gretz et al. 1996; Gallagher et al. 2000). The rat model most commonly used is the so-called Hanover rat (Han:SPRD cy/). This model is characterized by a slow progression of uraemia, proteinuria, and hyperlipidaemia. Characterization and documentation of the course of the disease continues in this model which promises to be the most interesting model so far of human PKD. Polycystic kidney disease has also been described in many other different animal species, including antelope, cat, ferret, pigeon, and even goldfish!

Clinical diagnosis Autosomal dominant polycystic kidney disease presents mainly in adult life. The clinical expression is variable, ranging from incidental findings at autopsy in old age to end-stage renal failure (ESRF) by the age of 30. Developing renal cysts can usually be detected well before the onset of symptoms. In one study Bear et al. (1984), cysts were visualized by ultrasound in 56 per cent of gene carriers by 10 years of age and in 90 per cent of gene carriers by the age of 20. Neonatal development of renal cysts and/or early clinical manifestation of ADPKD is known to occur with an estimated frequency of 2 per cent (Zerres et al. 1993). In these families there is a 40 per cent recurrence risk for similar early presentation in subsequent affected children. The biological basis of this marked change in clinical expression of the disease from parent to offspring is, at present, not understood and not explained by locus heterogeneity. For a more detailed review of very early presentation the reader is referred to Fick et al. (1993, 1994) and MacDermot et al. (1998). The ADPKD is most readily diagnosed using ultrasonography. A positive ultrasound diagnosis is often unambiguous, but a negative scan is complicated by the fact that cysts develop gradually over time. In the past Bear’s (Bear et al. 1984) classification of three cysts between two kidneys, in a person at 50 per cent risk was used. From this study, Bear estimated that the


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possibility of a false negative diagnosis that is, the risk that the patient may go on to develop kidney cysts later in life following a normal ultrasound (assuming 90 per cent specificity) was 0.46, 0.28, and 0.14 for persons at 50 per cent a priori risk in their first, second, and third decades, respectively. This data is now however considered to have insufficient specificity, particularly in older individuals. Certainly, using this classical definition, a false positive diagnosis by this criterion is highly unlikely before the age of 30, but recent studies have shown that two cysts in one or both kidneys at ages below 30 years are diagnostic (Ravine et al. 1994). However, from the ages of 30–59 years, observation of two cysts in each kidney may be required, and for persons aged 60 or more, where isolated kidney cysts are common in aging kidneys, there may need to be at least four cysts in each kidney to establish a reliable positive diagnosis. Table 9.2 gives the positive and negative predictive values of various ultrasound diagnostic criteria taken from Ravine et al. (1994). In PKD2, disease probability of false negative ultrasound diagnosis is higher at all ages (Bear et al. 1992; Ravine et al. 1992; Demetriou et al. 2000; Hateboer et al. 1999). An isolated kidney cyst in a child is very rare (Ravine et al. 1993) and raises a strong suspicion of ADPKD in a child with a positive family history (Fick et al. 1994; Gabow et al. 1997). Genetic diagnosis is also possible. Since ultrasound is so good, genetic testing presently is reserved for special circumstances such as an at-risk transplant donor below age 30. Linkage testing is feasible if the family structure permits; more recently, sequence-based direct gene mutation detection has become commercially available. The sensitivity and specificity of this test is complicated by the difficulties in mutation detection in large genes without any predominant mutations in the population and experience is just now beginning to accrue on how to best utilize gene-based diagnosis. Important prognostic information from knowing if one has PKD1 or PKD2 may some day be available, but current understanding does not permit application of this knowledge to the prognosis of individual patients. The ADPKD patients are successfully transplanted and in rare circumstances polycystic kidneys have even been used as donor organs (Koene 2001). In summary, using modern day ultrasound scanners which have a much higher resolution, general figures for counselling the risk of a false negative scan in a first degree relative approximate to 10 per cent at the age of 20 and much less than 1 per cent at the age of 30. These figures can be reduced further by the use of fine-cut renal CT scanning.

Table 9.2 The positive (PPV) and negative (NPV) predictive values of various ultrasound diagnostic criteria taken from Ravine et al. (1994) Criteria

1 cyst 2 cysts* 2 cysts in one kidney, 1 cyst in the other 2 cysts in each kidney 4 cysts in each kidney *Unilateral or bilateral

PPV, NPV(%) at age 20

30

40

50

60

70

100, 96.6 100, 96.6 100, 90.5

97.7, 100 99.2, 100 99.2, 100

96.9, 100 98.9, 100 98.9, 100

77.2, 100 95.6, 100 98.2, 100

73.8, 100 94.7, 100 97.9, 100

45.5, 100 61.2, 100 85.2, 100

100, 87.7 100, 85.1

100, 100 100, 89.9

100, 100 100, 100

100, 100 100, 100

100, 100 100, 100

90.7, 100 96.9, 100

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Presymptomatic and prenatal diagnosis ADPKD is relatively easy to diagnose. The disease however has a highly variable course, with approximately 50 per cent having no major complications in a normal lifetime. It is impossible to judge during the asymptomatic early period of the disease who will experience problems. This complicates prognosis counselling. It had been thought that pre-natal testing would become important with respect to ‘prevention’ of polycystic kidney disease in the population. However, there is very little demand for pre-natal, as well as predictive testing, and this is undoubtedly in part due to attitudes to adult onset diseases such as ADPKD, where there is a variable age of onset and prognosis and where there are no obvious stigmatizing external abnormalities. Issues such as insurance premium loading and career prospects also play a role. Future success in molecular biology and discovery of the protein defect in ADPKD will undoubtedly make real prevention a possibility. Disease knowledge in families that ADPKD is an inherited disease is usually poor. Several patients have been told that ADPKD is something that they have been infected with and that there is no risk to any offspring (personal observation). In a study by Sahney et al. (1982) only 5 of 22 patients knew at the time of diagnosis that their disease was hereditary and in only 4 patients was genetic counselling suggested. In a further study by Zerres and Stephan (1986) none of 79 persons at risk had sufficient knowledge of the inheritance of the disease at the time of contact. Such lack of knowledge about the possible risk causes concern since adequate understanding has important implications for life planning and early treatment of complications. In this same study by Zerres and Stephan (1986), 46 of 100 persons would not have had children, or would not have any more children. Thirty-three had no opinion and of the group of 100 patients, 91 said their marriage plans would not have been influenced by early diagnosis. Sujansky et al. (1990) found that less than 5 per cent of patients would actually have an abortion if there was prenatal testing. Macnicol et al. (1991) reported similar findings that many families asking for prenatal diagnosis refuse after discussing the different implications. This is a strong argument against early diagnosis. However, against this must be weighed the fact that although ADPKD has no cure, there are important treatable components which make a strong argument for early diagnosis. These complications include hypertension, urinary tract infection, the impact of repeated pregnancies and impaired renal function. There may also be other subtle abnormalities which may predict premature cardiovascular disease (Saggar-Malik et al. 1994). Early treatable complications appear quite common in children (Ravine et al. 1991; Zeier et al. 1993). Ravine et al. (1991) demonstrated that 25 of 68 (37 per cent) so far undiagnosed cases had one or more treatable complications at the time of diagnosis. These complications included 20 cases of hypertension, 7 cases of impaired renal function and 4 cases of bacterial urinary tract infection. Eight persons had several complications. Thus, there appears to be a clear and strong need for genetic counselling and clinical information in ADPKD families, although not prenatal testing.

Clinical aspects The clinical manifestations can be divided into renal and extrarenal (Table 9.3). There also appears to be some degree of correlation between renal size and symptoms or signs.


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Table 9.3 Frequency of renal and extrarenal manifestation of ADPKD (adapted from Gabow 1994) Manifestation Renal Cysts Cyst calcification Hypertension (adult) Hypertension (children) Haematuria Renal pain Urinary tract infection Nephrolithiasis ESRF Extrarenal Hepatic cysts Pancreatic cysts Colonic diverticulae Cardiac valve abnormalities Intracranial aneurysm Thoracic aortic aneurysm Arachnoid cysts Left ventricular hypertrophy (LVH)

Frequency 100% Common 60% (pre-ESRF) 80% (post-ESRF) 30% 50% 60% Common 30% 45% by age 60 50% 7% 80% (post-ESRF) 30% 8% 8% 8% 40%

The larger kidneys are more often associated with pain (Milutinovic et al. 1984), and are associated with hypertension (Gabow et al. 1990). Pain can be difficult to control and in some can require narcotic analgesia. An excellent review of pain management in ADPKD is provided by Bajwa et al. (2001). In a few patients where there is an easily identifiable and accessible renal or liver cyst, percutaneous drainage (Bennett et al. 1987) and alcohol sclerosis provides relief. Open surgical drainage has also been undertaken where cysts are smaller and more numerous (Elzinga et al. 1992). Neither of these procedures have been reported to enhance or prevent renal deterioration. Common clinical problems in ADPKD are summarized in (Table 9.3). Haematuria occurs in 30–50 per cent of ADPKD patients and may be the first presenting sign. The mean age at first episode is usually about 30 years of age and repeated episodes are associated with a worse renal outcome (Gabow et al. 1992a,b). Haematuria is self-limiting and usually lasts no more than a few days. Management involves bed rest, analgesia, fluids, and the exclusion of urine infection. Proteinuria also is a further poor prognostic indicator with 30 per cent of children demonstrating microalbuminuric levels of excretion, hypertensive individuals more than 40 per cent and adults approximately 23 per cent (Chapman et al. 1994). More rarely, bleeding may also occur into a renal cyst. Urinary tract infection also commonly occurs in the bladder, the kidney tissue or cyst fluid. Infection within cystic fluid often fails to respond to initial antibiotic treatment because many of the commonly used first-line antibiotics, for example, penicillins or cephalosporins fail to concentrate sufficiently within cysts (Schwab et al. 1987). Ideally non-ionized lipid soluble antibiotics should be used. There is no good data on the length of treatment for cyst infection

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but often patients have required several weeks of therapy with cyst-penetrating antibiotics for example, trimethoprim-sulphamethoxazole (Elzinga et al. 1987) or ciprofloxacin, a fluorinated quinolone particularly active against gram-negative enteric bacteria, the pathogen most frequently implicated in cyst infections (Schwab et al. 1987). Treating such infections can be difficult (Gibson and Watson 1998) and is best done under the supervision of specialist units. Nephrolithiasis occurs in about 25 per cent of ADPKD patients and is one of the differentials for patients presenting with pain or haematuria (Torres et al. 1988; Levine and Grantham 1992). CT scanning may be required to identify stones given the distortion and acoustic shadowing that can obscure a clear image on ultrasound. ADPKD patients are prone to develop calculi because of the relative intrarenal stasis and distortion produced by the cysts. Furthermore a common metabolic abnormality is hypocitraturia (Torres et al. 1988), a known risk factor for nephrolithiasis. The most important functional abnormality is renal failure. The rate of loss of renal function is therefore commonly used to assess progression. The family history cannot be used in this assessment since in affected members of the same generation, some progress to ESRD while others do not. Similarly, family history of age at ESRD does not provide information for the individual about the rate of progression. The rate of change of glomerular filtration rate (GFR) with time may help predict the age at which ESRD occurs. Approximately 45 per cent of patients will have ESRD by the age of 60 years (Churchill et al. 1984; Parfrey et al. 1990; Bear et al. 1992; Gabow et al. 1992b; Hateboer et al. 1999). Age at onset of renal failure is variable (in the range of 2–80 years), even within families. This variation is presumed due to the stochastic nature of pathogenic ‘second hits’ and to genetic modifiers within and between families (Upadhya et al. 1999; Peters and Breuning 2001). Table 9.4 lists some of the factors associated with a worse renal prognosis. There seems much hope that renal prognosis will improve further since the quoted figures are based on a previous generation, not all of whom were treated adequately or sufficiently early. In a review by Grunfeld (1998) the mean rate of decline of creatinine clearance in two groups of ADPKD patients was about 4 ml/min/year. These data by Grunfeld also agree with those collected prospectively in the US MDRD study (Klahr et al. 1995). In these and other studies (Gretz and Strauch 1992), no correlation was found between rate of progression and protein intake. In Grunfeld’s study, there was a slight positive correlation between the rate of progression and mean blood pressure recorded during follow-up, whatever the hypertensive treatment. Age distribution of ESRD in their study showed that approximately 15 per cent of the ADPKD population progressed to end-stage before the age of 40, 10 per cent after the age Table 9.4 Factors associated with a worse renal prognosis History of hypertension Repeated haematuria Proteinuria Urine infection (in males) Massive polycystic liver disease Male gender Black race Concurrence of sickle-cell trait More than 2 children (in women)


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of 65, and 75 per cent between 41 and 65. This suggested that the better management, such as blood pressure control, may not play a dramatic role in slowing the rate of renal progression. Similar disappointing results have been seen in other studies (Klahr et al. 1995; Locatelli et al. 1996; Maschio et al. 1996). However, a consistent problem with these studies, from the ADPKD point of view, has been the low level of renal function at the inception of the trial and the short duration of follow-up. A recent study by Ecder et al. (2000) found that whilst enalapril compared to amlodipine reduced proteinuria more significantly, there was no difference between the groups in the level of blood pressure reduction and both drugs equally slowed renal functional loss over a 5-year period. In this study, all patients began with a creatinine clearance greater than 50 ml/min/1.73 m2. If the rate of progression is to be modified, identification of predictive factors is vital for early intervention when therapeutic modifications become possible in the future. Hypertension in ADPKD Hypertension occurs in 60 per cent of polycystic patients before renal impairment (Gabow et al. 1984) and is an early event (Hansson 1974; Calabrese 1982; Milutinovic et al. 1984; Gabow et al. 1984, 1990). Hypertension is also a significant factor in the progression of renal disease. The association between hypertension and progressive renal impairment has been well documented in ADPKD. Despite this, hypertension remains inadequately managed in ADPKD (Ecder and Schrier 2001). Watson and colleagues (Li Kam et al. 1997) determined the 24-h blood pressure load in hypertensive ADPKD individuals compared to matched essential hypertensive subjects with normal renal function. Day to night change in blood pressure, that is, the nocturnal decline in both systolic and diastolic blood pressure (nocturnal dipping), in ADPKD individuals was diminished compared to the essential hypertensives. Similar findings are seen in children (Zeier et al. 1993). One important justification for the increased use of early ultrasound screening of at risk family members has been that early initiation of effective anti-hypertensive treatment may delay progression of renal disease, although frequent monitoring of blood pressure in at risk individuals can be used in place of early presymptomatic ultrasound diagnosis. Hypertension in children presents a particular problem. In children with ADPKD approximately 20 per cent are reported to be hypertensive (Sedman et al. 1987; Gabow et al. 1990; Fick et al. 1994), defined as a blood pressure above the 95 per cent confidence limits for age. Milder forms of hypertension are more difficult to assess in children. In Sedman’s study (Sedman et al. 1987) 13 per cent of children had abnormal renal function at the time of presentation. The pathogenesis of the hypertension remains to be elucidated however. Disturbances have been found in many mechanisms involved in the regulation of blood pressure in ADPKD. Activation of the renin–angiotensin–aldosterone system (RAAS); alterations in tubular sodium handling; changes in plasma and extracellular volume, and abnormal levels of natriuretic substances have all been the focus of investigation at some time. Throughout all of these studies it is very difficult to establish whether the observed abnormalities are primary or secondary, or unrelated epi-phenomena. Furthermore, when comparing across studies it is clear that various groups have looked at normotensive and hypertensive patients, patients with normal or decreased renal function or patients with variable salt balance states. Not all studies have compared healthy subjects as controls.

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Hypertensive patients with ADPKD and normal renal function seem to have significantly larger kidneys than those with normal blood pressure although there are many instances of extremely large kidneys in normotensive individuals. Since growth of relatively few cysts can result in compression of adjacent normal nephrons it has been hypothesized that growth of cysts relates to hypertension through progressive tubular damage. This leads to sodium retention and/or activation of the RAAS. The compressed/occluded renal microvasculature leads to focal intra-renal ischaemia (Ettinger et al. 1969; Cornell 1970) and in turn stimulates RAAS activation. Some studies have shown an increase in juxta-glomerular and vascular granules containing renin in kidneys of patients with ADPKD (Torres et al. 1992). In view of the involvement of the RAAS, there is increasing speculation that intra-renal formed angiotensin II could act as a growth factor for cysts (Torres et al. 1992). The search to elucidate the exact mechanism(s) that result in hypertension continue and will undoubtedly be helped by understanding the ADPKD gene products and their function. The most common cause of death in ADPKD patients is cardiovascular disease (Fick et al. 1995). Left ventricular hypertrophy (LVH) has been clearly established as a complication of ADPKD with or without hypertension (Harrap et al. 1991; Timio et al. 1992; Zeier et al. 1993; Saggar-Malik et al. 1994; Bardaji et al. 1998). LVH is a well-known risk factor for premature cardiovascular disease and is very likely to be a consequence of early elevation in blood pressure load. This fact supported by the observation that reducing blood pressure also reduces LVH (Ecder et al. 1999). Similarly, acceleration of atheroma formation as a consequence of hypertension and a high mortality from cardiovascular disease in ADPKD patients points to the significance of early elevations of blood pressure (Iglesias et al. 1983). Detection and early treatment of hypertension on the assumption that it will delay progression of renal disease and other types of target organ damage represents one of the best justifications for early diagnosis of ADPKD. However, the crucial and yet still unanswered question is the extent to which a higher blood pressure and at what level, is the rate of decline in renal function significantly accelerated. Adequate treatment of hypertension in ADPKD subjects is important. There is no clear consensus over the target level of blood pressure that should be achieved with treatment. Treatment of blood pressure greater than 140/95 in patients with essential hypertension is of small but significant value and therefore provides the level at which treatment is clearly indicated in ADPKD (Ecder et al. 2000). Intervention below 140/95 is speculative but American bodies conclude that control of blood pressure less than 140/90 reduces the incidence of ESRD and may confer even greater benefit if it is treated to a level less than 130/85 (National Blood Pressure Education Programme, USA, 1991). Given the high incidence of hypertension in the ADPKD population this implies that large numbers of patients will be on treatment. The choice of hypertensive agents is at present guided mostly by patient compliance. There are theoretical advantages for using angiotensin converting enzyme inhibitors early in the course of disease. They are well tolerated and they have also been shown to be reno-protective in other renal diseases independent of blood pressure control and reduce proteinuria significantly in ADPKD (Ecder et al. 2000; Torres 2000). However, in the later stages of decline in renal function, angiotensin converting enzyme inhibitors may in certain cases exacerbate renal failure (Chapman et al. 1991). A higher incidence of atypical chest pain, palpitations, and cardiac valvular defects has been described in ADPKD (Gabow et al. 1984; Harrap et al. 1991; Timio et al. 1992). There is an approximately 30 per cent incidence of valvular heart abnormalities, the mitral and then


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aortic being the commonly affected (often manifesting as mitral or aortic valvular prolapse). It is possible that some of the cardiovascular deaths seen in ADPKD result from arrhythmias secondary to this ventricular enlargement or valvular changes. Other vascular abnormalities described are thoracic, iliac, and abdominal aneurysms (Gabow 1993; Stiasny et al. 1995), arterial dolichoectasia (Stiasny et al. 1995) and intracranial arterial dissection (Schievink et al. 1997). In summary, hypertension occurs frequently and early in ADPKD adults and children prior to loss of renal function. Early renal impairment amongst this group suggests that greater awareness and earlier informed diagnosis in those adults most at risk may reduce morbidity and possibly delay renal disease progression. Clearly, factors that lower blood pressure, may also lead to a reduction in ventricular mass, and so further help reduce subsequent cardiovascular risk although further work needs to be done in this respect. Hepato-biliary Simple hepatic cysts occur in 2.5–4.6 per cent of patients referred for ultrasound examination of the abdomen. Such cysts are solitary in two-thirds of patients, but no more than three cysts are usually found in those with simple multiple lesions. Hepatic cysts are seen in a number of other conditions and are very common in ADPKD (Table 9.5). Hepatic cysts are seen in approximately 50 per cent of ADPKD patients overall (Milutinovic et al. 1980; Gabow et al. 1990). They increase in size and number as in the kidney, but rarely result in any problems or compromise hepatic function. The incidence of cysts increases with age from approximately 20 per cent in the third to 75 per cent in the seventh decade of life (Torres 1996). They are more common in women than in men, and the cysts are often more numerous and larger than in men. Women with ADPKD who have had no oestrogens or previous pregnancies are less likely to have hepatic cysts than those who have used hormones, been pregnant or both (Torres 1996). Polycystic liver disease (PLD) has been used to describe the presence of multiple cysts throughout the liver, usually found in association with ADPKD. Rarely, families with ADPKD may have PLD with very few renal cysts. Furthermore, autosomal dominant PLD without renal cysts has been documented in a small numbers of families. Isolated PLD without a family history of either PKD or PLD cannot be separated from multiple simple cysts. It is not known whether sporadic PLD reflects inadequate ascertainment of dominant PLD or PKD families or new mutations. More recently a gene locus has been isolated for autosomal dominant PLD, marking it out as genetically different from ADPKD (Reynolds et al. 2000). As in ADPKD the derivation of cysts from biliary microhamartomas has been clearly shown and as occurs in the kidney, the cyst becomes disconnected during growth from the tubular ducts from which they derive. The natural history of PLD has been described in Table 9.5 Non-ADPKD conditions associated with cystic liver disease Dilatation of the extra and/or intrahepatic bile ducts Segmental dilatation of intrahepatic bile ducts (Caroli disease) Congenital hepatic fibrosis Autosomal recessive polycystic kidney disease

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patients with ADPKD (Gabow 1990; Torres 1996). Hepatic cysts are exceptionally rare in children and liver specimens in children with ADPKD usually show no micro- or macroscopic changes. Most patients with PLD are asymptomatic and have normal liver function tests. The development of symptoms in PLD is usually due to mass effect or a massively enlarged polycystic liver with or without rare complications for example, hepatic vein thrombosis (Budd–Chiari syndrome) (Torres et al. 1994). Furthermore, haemorrhage into a cyst can cause severe acute abdominal pain, fever, elevation of liver enzymes, and imitate acute cholecystitis or hepatic abscess. A ruptured cyst causing haemo-peritoneum is exceptionally rare. The MRI imaging is the most sensitive technique to differentiate the complicated from an uncomplicated hepatic cyst. As with normal pyogenic abscesses, cysts are best aspirated and cultured and drainage is usually effective and safe. Oral antibiotics that concentrate in the biliary tree and cysts, such as trimethoprim-sulfamethoxazole or ciprofloxacin should be used. Congenital hepatic fibrosis, a hallmark of autosomal recessive polycystic kidney disease (see Chapter 10, this volume) has been reported in 22 patients from 13 families with ADPKD (Lee and Paes 1985; Tamura et al. 1994; Torres 1996). In general where treatment is required it is directed at reducing the volume of cystic tissue. The approach can be preventative or surgical which in turn can be open or closed. On the basis of epidemiological studies and anecdotal observations avoidance of exogenous factors such as oestrogens, c-AMP agonists, alcohol and possible hepatotoxins is prudent. The administration of H2 blockers or somatostatin reduces the release of secretin which theoretically may be helpful in reducing the secretory activity of the cyst walls under certain conditions (Everson et al. 1990). However, the clinical value is not proven. Cyst aspiration, alcohol sclerosis, surgical resection with or without fenestration and liver transplantation have all been used successfully to treat patients with severe symptomatic PLD. The treatment choice is dictated by the size and distribution of one or more symptomatic cysts. A full review can be obtained from Tan et al. (2002). Main complications of alcohol sclerosis are temporary local pain and an alcohol rush. Large superficial hepatic cysts can be treated by laparoscopic fenestration. In some patients with massive polycystic livers, extended surgical fenestrations where successively deeper cysts are unroofed and drained through more superficial ones, have been used to treat some patients whilst some extreme cases have had liver transplantation (He et al. 1999).

Intracranial aneurysms (ICA) The association of ICA and ADPKD has been established for many years. Aneurysm rupture is life-threatening and entails a 30–50 per cent mortality rate in the general population. Although an uncommon manifestation, ICA rupture is part of the spectrum of extrarenal features that may occur at any time in ADPKD patients. Subarachnoid haemorrhage as a cause of death is found in about 0.7 per cent of the general population and in about 6 per cent of ADPKD patients. The poor prognosis of ruptured ICA, and the issues surrounding screening for asymptomatic ICA in ADPKD patients have long been a matter for debate. In the meantime, there have been substantial advances in neuroradiology, both to screen for silent ICA and to ablate ICA through the endovascular route. The development of high-resolution computer tomography (CT) and magnetic resonance angiography (MRA) has recently allowed the non-invasive detection of


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ICA. These new techniques are especially welcome in ADPKD subjects who are at increased risk of complications from conventional cerebral angiography. Among 32 subjects undergoing this investigation, 25 per cent experienced various neurovascular complications, a rate 2.5 times higher than that observed in the general population (Chapman et al. 1999). The timing of surgery for ruptured ICA has been modified and there have been improvements in the prevention and treatment of cerebral vasospasm, a major risk factor for severe morbidity after subarachnoid haemorrhage. A substantial proportion of ICA in ADPKD patients do not rupture and remain asymptomatic. Historically, a wide variability in prevalence rates has been described for asymptomatic ICA in ADPKD, figures ranging from 0 to 60 per cent at autopsy or with conventional angiography (reviewed by Chapman et al. 1993). This most probably reflects selection bias, small size of the samples and the retrospective nature of the studies and different methods of diagnosis. In the largest, carefully conducted autopsy study, unruptured ICA were found in 4 per cent of ADPKD patients in whom the cause of death was not a ruptured ICA, that is, a rate twice as high although not significantly different, from a control-matched population (Schievink et al. 1992). The only characteristic clearly associated with the presence of ICA in ADPKD is a family history of same. In Huston’s series (Huston et al. 1993), combined ICA and ADPKD appeared to be almost 3 times more frequent in patients with a definite family history of ICA or subarachnoid haemorrhage than in those without such a history. Pathogenesis The mechanisms leading to the formation of ICA in ADPKD are largely unknown. As in nonADPKD patients, ICA mostly develop at bifurcation sites of the circle of Willis. These areas are exposed to maximum blood flow-induced sheer stress. The internal elastic lamina is thought to be the crucial structure which is first damaged before an ICA will form (YongZhong and van Alphen 1990). Disruption of this layer is a constant feature of saccular ICA. It is of note that ICA are known to develop in a variety of other inherited connective tissue disorders such as Marfan syndrome and the vascular form of Ehlers–Danlos syndrome. The unique susceptibility of intracranial arteries to develop aneurysms may in part be due to the virtual absence of a well-developed external elastic lamina (Yong-Zhong and van Alphen 1990). Similarly an increased association between thoracic aortic dissection, ICA and ADPKD has been observed. The prevalence of hypertension in polycystic kidney disease may be responsible for this increased frequency of vascular associations. More recently evidence suggests that both polycystin-1 and polycystin-2 are strongly expressed in vascular smooth muscle cells of large elastic distributive arteries. Therefore, vascular manifestations may be a direct consequence of the basic genetic defect in ADPKD (Kim et al. 2000; Torres et al. 2001). A mutational basis for ADPKD-associated ICA is supported by both the young age of some affected patients, the familial aggregation of the association (Chauveau et al. 1994) and recent mutational analysis (Rossetti et al. 2003). Epidemiology High-resolution, fine cut (1.5 mm) CT has allowed the detection of 74 (97 per cent) of 76 angiographically or autopsy-verified ICA in a series of non-ADPKD patients; the two ICA escaping CT were 8 mm in size (Schmidt et al. 1987). This method is however not specific

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enough: angiography confirmed the presence of an ICA in only two out of seven ADPKD patients with a suspicious CT result (Chapman et al. 1992). Recently, advances in MR techniques have allowed a better and probably safer method of imaging intracranial vascular structures including ICA. Magnetic resonance angiography (MRA) in non-ADPKD patients can detect 79 per cent of angiographically-proven ICA (Horikoshi et al. 1994). The specificity of the method is 92 per cent. Using MRI techniques in a large group of ADPKD subjects, Huston et al. (1993) and Ruggieri et al. (1994) found an ICA prevalence of about 10 per cent. Chapman et al. (1992) detected ICA in 4 per cent of 88 ADPKD patients using CT. These three prospective studies have provided the most accurate estimation of the prevalence (7.9 per cent) in asymptomatic patients, a rate significantly higher than the 1 per cent rate found in the general population (Atkinson et al. 1989). Most of the ICA detected in the three prospective series were of small size. Only three, that is, 1 per cent, of the examined patients had an ICA greater than 6 mm in diameter, and one patient had one greater than 7 mm. However, just as in the general population, 90 per cent of the ICA in ADPKD patients were found in the anterior circulation. Huston et al. (1993) and Ruggieri et al. (1994) tried to identify clinical features associated with the presence of occult ICA in ADPKD patients. No significant association was found between ICA and age, gender, prevalence of hypertension, or abnormal renal function. In the Huston study, there was a trend for an association between ICA and severe polycystic liver disease. The only characteristic clearly associated with the presence of ICA was a family history of ICA. In the combined three series mentioned above, the relative risk for harbouring an ICA was 2.6 times higher among patients with a definite family history of ICA or subarachnoid haemorrhage than in those without. ICA are associated with both the PKD1 and PKD2 forms of ADPKD. An ADPKD family with ICA, unlinked to PKD1 or PKD2 has also been described (Chauveau et al. 1994). It still remains unknown whether specific mutations of the ADPKD gene, as suggested by familial aggregation, accounts for the association to ICA. Besides the PKD mutation, other genetic and non-genetic factors could play a role in the development of ICA. They could explain the frequent discordance for ICA within ADPKD families, even in monozygotic twins (Chauveau et al. 1994).

Risk of rupture ICA rupture accounts for only a minority of fatal neurological events in ADPKD patients although the mortality from cerebrovascular accident is higher in ADPKD than in nonADPKD ESRD patients, 11 versus 5 per cent in the Toronto Registry (Roscoe et al. 1993) and 13 versus 10 per cent in the EDTA Registry (Ritz et al. 1994). Hypertensive haemorrhage or cerebral infarction more commonly accounts for acute neurological events than ruptured ICA. In patients admitted with ADPKD and stroke, only 5 per cent were found to have a ruptured ICA (Ryu 1990). In the Heidelberg autopsy series (Zeier et al. 1988), cerebral complications were observed in 20 per cent of ADPKD patients, but an ICA rupture was found in only 12 per cent of them. Therefore, in polycystic patients presenting with stroke there requires to be unequivocal demonstration of a rupture. The risk of suffering an ICA rupture in ADPKD patients is not well defined. On the basis of the population data of Rochester, US, this risk in ADPKD patients was calculated to be


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about 1/2000 person-year overall, and 1/1000 person-year in patients above the age of 30 years (Schievink et al. 1992). Clinical features Symptoms arising from an ICA are virtually always due to its rupture. These include nausea and vomiting, photophobia, focal neurological deficit, seizures, lethargy, and loss of consciousness. Neck stiffness may take up to 6 hours to develop. ICA rupture may also mimic an acute psychiatric illness. However as in the general population ICA rupture in polycystic patients can be preceded by herald bleeds in almost 40 per cent of cases (Leblanc 1987), that is, headaches due to a minor leaks of blood a few hours to 2 weeks before rupture. The onset of headaches should be investigated urgently when they are of sudden onset, unusual character or severity in an ADPKD patient. Recognizing such warning headaches is important, since delay in the diagnosis of ICA rupture increases morbidity and mortality. Rupture has a 35–55 per cent risk of combined mortality and morbidity (Schievink et al. 1992; Chauveau et al. 1994). This is close to the corresponding figures reported in non-ADPKD patients (Saveland et al. 1986; Sarti et al. 1991). These high rates of mortality and morbidity are attributed mainly to the consequences of damage from initial bleeding, re-bleeding, and cerebral ischaemia. Intracerebral haematomas are also not uncommon with ruptured ICA. Early diagnosis and management will hopefully improve prognosis. Rarely, ICA becomes symptomatic through local expansion. Focal findings such as cranial nerve palsy (particularly oculomotor and optic nerves) or seizures may result from compression by a large ICA. Transient ischaemic attacks may result either because of an embolus from within an ICA or from direct compression of adjacent vessels. Three studies have allowed the development of a clinical profile of ADPKD patients presenting with an ICA rupture: a critical review by Lozano and Leblanc (1992) of 79 cases gathered from the literature up to 1989, the second is a review of 41 cases at the Mayo Clinic between 1950 and 1989 (Schievink et al. 1992) and the last one, a multicentre European Concerted Action Group of Polycystic Disease study of 71 cases (Chauveau et al. 1994). The mean age of rupture is lower in ADPKD patients in these 3 studies (39, 47, and 39 years, respectively) than in the general population as a whole (51 years), but close to that found in the subset with the familial form of ICA where the mean age of rupture was 42 (Lozano and Leblanc 1987). In Chauveau’s study however, age at rupture ranged from 15 to 69 years; with, 10 per cent of the patients aged less than 21 years (Chauveau et al. 1994). Chronic hypertension is a recognized risk factor for ICA rupture in the general population. These studies also examined blood pressure before rupture and found that 25–29 per cent of patients were previously normotensive, demonstrating that established hypertension is not a prerequisite for the development of ICA (Lozano and Leblanc 1992; Schievink et al. 1992; Chauveau et al. 1994). This does not exclude a role for episodes of acute hypertension in ADPKD patients. At the time of rupture 50 per cent of patients in the European study had normal renal function while 38 per cent had CRF and 12 per cent were on renal replacement therapy. In the Mayo series, 60 per cent had normal renal function. Overall ICA rupture was the first manifestation of ADPKD in 10 per cent of patients and 50 per cent of ruptured ICA were located on the middle cerebral artery (Chauveau et al. 1994). This site appears to be more frequently involved, both in ADPKD and in non-ADPKD patients. As in non-ADPKD

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patients, multiple ICA are not uncommon in ADPKD: in the Mayo series (Schievink et al. 1992), 31 per cent of the patients with a ruptured ICA had at least an additional intact ICA versus 20 per cent in non-PKD patients. The second most frequent location was the anterior communicating artery. There are no predictive clinical features identifying ADPKD patients with a ruptured ICA. Just as for asymptomatic ICA, the only characteristic associated with ruptured ICA is a family history of ICA: such a history was found 5 times more often in patients with a ruptured ICA than in a control group of polycystic patients without previous rupture (Pirson and Chauveau 1996). There is an overall early mortality rate of at least 10 per cent. At the Mayo Clinic mortality rate within 6 months following rupture is as high as 55 per cent (Schievink et al. 1992). Morbidity is also serious. Almost 43 per cent of patients surviving beyond 3 months after rupture have severe neurological disability (Chauveau et al. 1994). In the Mayo Clinic study there was a trend for previous hypertension to be associated with severity and poor outcome after haemorrhage.

Investigation and management The principles of management in ADPKD patients are fundamentally similar to those of ruptured ICA in the general population. Management includes a medical and surgical approach and patients ideally require management in a neurosurgical unit. Although the important points are summarized, full details of the investigation, management, and follow-up of patients with ruptured ICA are beyond the scope of this chapter and the reader is referred to more standard neurosurgical texts on this subject (Meyer et al. 1995). About 30 per cent of subarachnoid haemorrhages are diagnosed incorrectly or late which markedly increases the mortality and morbidity. The reasons for late diagnosis range from failure to appreciate the spectrum of presentation of SAH and the associated symptoms; failure to appreciate the limitations of CT scan, which include not detecting small bleeding if a CT scan with contrast is not used and the possibility of false negative results in patients who have very small volume bleeds or who are anaemic. The sensitivity of CT scanning is maximal at 95 per cent in the first day after a bleed. This value drops to about 50 per cent by 5 days after the episode. Nonetheless the first-line investigation is non-contrast CT scanning. Lumbar puncture should be reserved for the 15 per cent of cases in whom the diagnosis remains questionable despite CT (Kopitinik and Sanmson 1993). Xanthochromia in the cerebrospinal fluid should also be looked for at this time. Once the diagnosis of SAH has been established, conventional 4-vessel angiography should be undertaken with the aim of localizing the ruptured ICA, delineating its size and neck, assessing the degree of vasospasm and detecting other unruptured ICA. Unfortunately in about 15 per cent of non-ADPKD patients with proven SAH, angiography will not detect the ICA (van Gijn 1997). What proportion of ADPKD have angiogramnegative SAH is unknown. Besides supportive nursing, treatment is aimed at preventing both cerebral ischaemia and rebleeding. Drug therapy includes using the lipophilic, dihydropyridine nimodipine since it can reduce by 34 per cent the incidence of cerebral infarction (Pickard et al. 1989). Whether newer therapies such magnesium sulphate will improve survival further remain to be proven (Veyna et al. 2002).


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The mainstay of surgical treatment is ICA occlusion. Surgically clipping the ruptured ICA at its neck is the standard method. Morbidity and mortality rates remain at about 10 per cent because of occasional intraoperative ICA rupture (Kopitinik and Sanmson 1993). Asymptomatic ICA accessible during the surgical exposure should also be clipped. New techniques of selective endovascular occlusion now represent valuable alternatives to surgery. Detachable balloons were first used but this technique has now been largely abandoned (Nichols et al. 1994). A detachable platinum coil system has been reported to show at 6 months follow-up evaluation that 84 per cent of patients made a good recovery; 4 per cent had residual disability and 11 per cent died. In 84 per cent of cases the ICA were completely occluded (Casasco et al. 1993). Undoubtedly endovascular therapy will continue to play a major role in the treatment of ICA. Management of unruptured ICA The key question is whether ICA in ADPKD are more likely to rupture than aneurysms in the general population. Management is based on this risk of rupture. Available evidence suggests that the majority of asymptomatic ICA do not rupture although the natural history remains largely unknown. Reference is therefore always made to current knowledge about unruptured ICA in the general population. ICA size has long been appreciated to be an important risk factor for first rupture (Juvela et al. 1993). However, where there is a previous history of rupture, risk of a further bleed is inversely correlated with age. A recent retrospective study of unruptured ICA (ISUIA 1998), calculated the annual risk for first rupture to be 0.05 per cent per year for aneurysms measuring less than 10 mm in diameter; almost 1 per cent per year for aneurysms measuring 10–24 mm in diameter, and 6 per cent in the first year for aneurysms measuring 25 mm or greater. Where there was a previous history of rupture, the risk of rupture was approximately 0.5–1 per cent per year, independent of size, suggesting that there are unique factors that increase the risk of a second bleed. In addition to size, location is also important and aneurysms in the posterior circulation are more likely to rupture than aneurysms in the anterior circulation. Other possible factors include family history of subarachnoid haemorrhage, smoking, binge drinking, sudden blood pressure elevations, the presence of daughter sacs, and possibly the distensibility of the aneurysm (Meyer et al. 1995; Juvela et al. 2000; Pirson et al. 2002). Screening for unruptured ICA In ADPKD patients successfully operated for a first ICA rupture, the annual incidence rate of another ICA rupture is between 1.4 and 4 per cent for those with multiple ICA (Chauveau et al. 1990; Pirson and Chauveau 1996). The multiplicity and the new development of ICA together with the severe outlook following ICA rupture has prompted a suggestion that, at least in a subset of ADPKD patients, regular screening for another ICA is justified. The original decision analysis using conventional angiography done by Levey et al. (1983) has been reconsidered because of recent advances in the possibility to detect and endovascularly treat ICA before rupture, the advent of non-invasive screening techniques such as MRA and new information on ICA prevalence and age at rupture. Presymptomatic screening is mostly indicated in patients who are at a high risk of having ICA, that is, patients who have a strong family history of ICA or personal history of previous

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Anand K. Saggar-Malik and Stefan Somlo Suspected ICA/at risk individual Magnetic resonance angiogram (MRA)

ICA not confirmed

ICA confirmed

If still in doubt

ICA not confirmed

Repeat MRA in 5 years

Conventional angiogram

Size ≤ 6 mm

Size > 6 mm

Repeat in 2 years

Surgical/endovascular treatment

Fig. 9.1 Summary of the investigation and management of patients with ICA.

bleed. Screening is also indicated in special cases where the patient has a high risk occupation such as private pilot or for patients planning major elective surgery. MRA has emerged as the best non-invasive screening test and has a sensitivity of 100 per cent for ICA 6 mm (Pirson and Chauveau 1996), which is appropriate since this is also the size threshold for surgical repair. The current specificity is 92 per cent (Horikoshi et al. 1994). It remains to be determined how frequently at-risk patients with negative MRA should be re-evaluated. At St George’s Hospital, London, UK and also the Mayo Clinic, Rochester, USA, an MRA is recommend every 5 years. Patients surviving a first ICA rupture warrant MRA re-screening every 2–3 years with early intervention at low ICA size. The age threshold is unknown but it seems reasonable today to screen ADPKD patients with a family history of ICA from the ages 18–35 years. Guidelines of surgical intervention should be as for the general population (Chapman et al. 1993) (Fig. 9.1) where prophylactic surgery is undertaken for asymptomatic ICA greater than 6–7 mm. ADPKD genetics The notion that polycystic kidney disease was inheritable was first suggested in 1925. It was to be another 60 years before gene linkage to the alpha globin gene locus on chromosome 16 (later called PKD1) was described (Reeders et al. 1985). Genetic heterogeneity with a second locus elsewhere, was later reported by both Kimberling et al. (1988) and Romeo et al. (1988). Kimberling et al. (1993) identified this second locus to be on chromosome 4 (PKD2). If this was not enough there have been several reports after the initial finding by Daoust et al. (1995) that there is at least one other locus, the position as yet unknown. However, the existence of this third locus has been called into question (Paterson and Pei 1998). Finally, in 1994 came the dramatic news that the PKD1 gene had been cloned by the


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European Polycystic Kidney Disease Consortium (EPKDC) led by Peter Harris and co-workers in Oxford (EPKDC 1994). Subsequently, news broke of the cloned gene sequence for PKD2 on chromosome 4 (Mochizuki et al. 1996). Present estimates indicate that ~85 per cent of ADPKD is due to PKD1 in a white European population. Mutations at the PKD2 locus on chromosome 4q3–q23, probably account for ~15 per cent of ADPKD. However, the prevalence of PKD2 disease among elderly patients with ADPKD (~40 per cent) is almost 3 times the prevalence of the disease in the general ADPKD population (Torra et al. 2000). It is also possible that the proportion of PKD2 families is greater outside of white European populations. Mutations at the PKD1 locus are suggested to produce a clinically more severe form of the disease (mean age of ESRD, ~53 years) than mutations in the PKD2 gene (mean age of ESRD, ~69 years) (Hateboer et al. 1999). The population frequency of ADPKD is relatively high compared to that of other autosomal dominant conditions, at least in part to the fact that the clinical manifestations rarely occur before those affected have passed down mutations to their offspring. The rate at which new mutations arise is difficult to interpret, but it estimated to be about 1 in 27 cases or about 1–2 per 100,000 gametes per generation (Rossetti et al. 2001). An essential insight to explain both the significant intrafamilial variability and the focal nature of the disease in the kidney and liver came with the discovery that cyst formation can be initiated by somatic acquisition of second hits (i.e. mutations) in the normal allele at the respective PKD gene (Qian et al. 1996; Watnick et al. 1998; Wu et al. 1998; Koptides et al. 1999; Pei et al. 1999; Torra et al. 1999) This model first proposed by Knudson (1971) to explain the variation in retinoblastoma has been applied to ADPKD. Wild-type inactivation of polycystin-1 and polycystin-2 has been reported by several laboratories (Qian et al. 1996; Koptides et al. 1998; Watnick et al. 2000). It has been shown that almost a quarter of cysts examined show loss of heterozygosity (LOH). Similar findings have been reported for liver cysts cells where there may be as much as 40 per cent LOH (Watnick et al. 1998). The loss of both alleles provides a loss of function model for ADPKD. Table 9.6 summarizes the salient features of PKD1 and PKD2.

Table 9.6 Comparison of polycystin 1 (PKD1) and polycystin 2 (PKD2)

Genomic size Exons Transcript Localization

Transmembrane domains COOH terminus NH2 terminus Homology

Function

PKD1

PKD2

~50 kb 46 14.8 kb Integral membrane protein:basolateral plasma membrane and cilium(?) 11 Intracellular (~210 aa) Extracellular (~3000 aa) PKD2; sea urchin receptor for egg jelly (suREJ3); nematode LOV-1 protein Cell surface receptor

~68 kb 15 5.1 kb Integral membrane protein; endoplasmic reticulum and cilium 6 Intracellular (~280 aa) Intracellular (~225 aa) TRP family of Ca2 channels; voltage activated Ca2 channels Cation channel (Ca2)

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The PKD1 gene The gene for the most common form of ADPKD, has been mapped, fully sequenced and characterized. This PKD1 gene, on chromosome 16p13.3, encodes a 14.8 kb transcript of 46 exons (EPKDC 1994; IPKDC 1995). Intron 21 contains the largest polypyrimidine tract identified in the human genome (IPKDC 1995; Germino 1997). This tract has been suggested to account for the high mutation rate seen in ADPKD (Wang et al. 1996) but recent evidence has failed to document clustering of mutations in this region of the gene (Rossetti et al. 2001). About 70 per cent of the genomic sequence encompassing exons 1–33 is reiterated at least 5 times more proximally on the same chromosome in humans. These homologous genes have approximately 95 per cent similarity to PKD1 and are transcribed into mRNA. Recent data suggest that the proximal copies are pseudogenes (Bogdanova et al. 2001). The predicted PKD1 protein, polycystin-1, is an integral membrane protein of 4302 amino acids with 7–11 transmembrane domains, a large extracellular region including leucine-rich repeats, a C-type lectin domain, 16 immunoglobulin-like repeats, and a REJ (Receptor for Egg Jelly) domain with homology to the sperm receptor for egg jelly in the sea urchin (Moy et al. 1996; Sandford et al. 1997; Hughes et al. 1999). This homology between the REJ module and human PKD1 suggests that PKD1 could be involved in ionic regulation. The extracellular tail appears to be involved in cell–cell and cell–matrix interactions and a role as homophilic associate has been proposed (Ibraghimov-Beskrovnaya et al. 2000). Evidence has emerged that polycystin-1 may be involved in G-protein coupled signalling (Delmas et al. 2002; Parnell et al. 2002) and that it may regulate epithelial proliferation directly by activating JAK/STAT signalling (Bhunia et al. 2002). In the latter model, loss of polycystin-1 as occurs in individual cells in ADPKD leads to loss of inhibition of the cell cycle resulting, in turn, in the relatively proliferative phenotype of cyst lining cells. Finally the PKD1 gene has a cytoplasmic carboxy terminus that contains motifs which may interact with the cell cytoskeleton and influence intracellular signalling (Hughes et al. 1995). In particular the COOH terminus has a coiled-coil domain which mediates interaction with PKD2 (Qian et al. 1997). Subcellular localization of polycystin-1 has been dogged by conflicting reports in the literature. This undoubtedly due to differences between individual monoclonal and polyclonal antibodies raised against various epitopes and the low abundance of polycystin-1. In general, the following conclusions can be drawn: there is a wide tissue distribution with higher levels of expression during development that decrease postnatally; there is expression in the brain, liver, heart, vascular smooth muscle, pancreas, and of course the kidneys, where developmental regulation is particularly strong. In the complete absence of polycystin-1, kidney development is disrupted with defective maturation and elongation of renal tubules resulting in in utero cyst formation (Lu et al. 1997).

The PKD2 gene The PKD2 gene on chromosome 4q21 has also been identified and characterized (Mochizuki et al. 1996) and encodes an integral membrane protein, polycystin-2, of 968 amino acids containing six transmembrane domains flanked by cytoplasmic NH2 and COOH termini. The transmembrane domains are reported to be 25 per cent identical and 50 per cent similar to the last 5–6 transmembrane domains of polycystin 1. The genomic sequence spans 68 kb and has 15 exons. Unlike PKD1 it is a single copy locus. The first exon is however very GC rich (Koptides et al. 1999), but clustering of mutations have not been observed in this gene either.


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Homology of PKD2 to the transient receptor potential (TRP) and voltage-activated Ca2 channels, suggests a related role for the PKD2 protein (Mochizuki et al. 1996). Qian et al. (1997) and Tsiokas et al. (1999) in separate experiments suggested there was heterodimer binding between polycystin-1 and polycystin-2 through interaction in their respective COOHtermini. Altogether these reports are highly suggestive of their involvement in a common signalling pathway that links extracellular adhesion events to alterations in ion transport, possibly regulating transmembrane Ca2 fluxes. More recently, genetic evidence in the nematode has provided support for the notion that the polycystins do indeed function in a common pathway since mutations in either or both orthologues of polycystin result in the identical mechano- or chemosensory defect in C. elegans (Barr et al. 2001). Co-expression of polycystin-1 and polycystin-2 in cells results in novel cation currents (Hanaoka et al. 2000) and polycystin-2 is itself a nonselective cation channel that likely conducts Ca2 ions in vivo (Gonzalez-Perret et al. 2001; Koulen et al. 2002). Recently, data has emerged that the polycystins may be expressed on luminal monocilia of renal tubular cells. This seems quite clear for polycystin-2 (Pazour et al. 2002) and is possible for polycystin-1 as well (Yoder et al. 2002). It has been proposed but not proven that cilia may be luminal flow sensors and that the polycystins may play a role as the molecular mediators of this signal. For a more detailed review of the structure, function, and regulation of polycystin the reader is referred to Koptides and Deltas (2000), Wilson (2001) and Igarashi and Somlo (2002). Progression of polycystic kidney disease results from a combination of altered tubular cell proliferation, apoptosis, secretion, and tissue fibrosis and inflammation that together lead to eventual renal failure. Despite advances in understanding the molecular pathogenesis of ADPKD, the exact mechanisms underlying these processes remain ill-defined. What is clear is that the polycystins play an important role in organizing and integrating signals that regulate and maintain the highly specialized structure of the kidney. Knowledge about the gene mutation combined with clinical studies examining specific risk factors (e.g. LVH, HPT) will allow a better understanding of the pathogenesis, pathophysiology, and progression of the disease and its associations. Such knowledge will undoubtedly help to reduce mortality and morbidity and delay progression in this common condition.

Genotype–phenotype considerations Non-genetic factors exert their effects either on the kidney itself or on modulation of gene expression. Regulation of PKD gene expression may, therefore, in part explain phenotypic variability. The role of genetic factors have been underlined by several studies (Gabow et al. 1992b; Ravine et al. 1992; Wright et al. 1993; Hateboer et al. 1999, 2000; Persu et al. 2000). From both a genetic and clinical viewpoint, the discovery of at least a second gene (PKD2) appears to be significant as this genotype appears to be less aggressive (Hateboer et al. 1999). In this study median age at death or onset of ESRD was ~53 years in individuals with PKD1 and ~69 years in those with PKD2. Women with PKD2 had a significantly longer median survival than men by 4 years but no sex influence was apparent in PKD1. Age at presentation with kidney failure was almost 20 years later in PKD2 than in PKD1 and PKD2 patients were less likely to have hypertension, a history of urinary-tract infection or haematuria. The molecular basis of the greater severity of PKD1 disease over PKD2 disease most likely has to do with the relative rate of occurrence of second hits at the respective gene loci.

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The increased size of the PKD1 transcript, the very dense arrangement of the gene, the existence of homologous loci on the same chromosome and, perhaps, unique sequence elements within the gene all may contribute to the relatively higher second hit rate when compared to PKD2. However, some PKD1 families with slow renal progression have also been described. The nature of the gene defect is stressed by the observation that large deletions which disrupt the contiguous tuberous sclerosis (TSC2) and the PKD1 genes are associated with unusually severe polycystic disease of the kidneys (Brook-Carter et al. 1994; Harris 1997; Sampson et al. 1997; Martignoni et al. 2002). Racial differences exist. ESRD has been shown to develop almost 10 years earlier in blacks with ADPKD than whites (Yium et al. 1994; Freedman et al. 2000). Among blacks with sickle cell trait, there has been an even more rapid progression of disease (Yium et al. 1994). The association of the ACE D allele with clinical severity has been described in several diseases, including IgA nephropathy, a glomerular renal disease associated with hypertension and progressive renal impairment. It has been suggested that ACE genotype may be associated with increased blood pressure and this in turn with greater renal disease severity (Baboolal et al. 1997; van Dijk et al. 1999). Similar claims had been made for angiotensinogen polymorphisms and clinical phenotype in ADPKD. Despite conflicting reports, several papers have shown no association to support such a role in ADPKD (Lee et al. 2000; SaggarMalik et al. 2000; van Dijk et al. 2000; Schiavello et al. 2001). These findings do not support the proposition that ACE genotype or angiotensinogen polymorphisms are associated with a worse prognosis in patients with ADPKD. Phenotypic variability in ADPKD is reflected in the age of onset of symptoms, signs and complications, both within and between families. It is estimated that fewer than 5 per cent of all nephrons develop cysts even though all cells carry the same germ line mutation. The interfamilial variability is not explained by genotype variation alone and is more probably explained by different somatic mutation rates; the intrafamilial variation is almost certainly largely the result of these differences. Modifying genes (Woo et al. 1997; Upadhya et al. 1999), metabolic load and environmental factors may play some part but the ‘two-hit’ model of somatic mutation seems to have the most support. The description by Grantham of ADPKD as neoplasia in disguise may now seem more apt than ever (Grantham 1990, 2000).

Mutation detection Mutation screening in ADPKD has been difficult not least of all because of the genomic reiteration of exons 1–33 more proximally of the PKD1 transcript on chromosome 16. The sequence homology of the proximal pseudogenes has complicated sequence amplification of the PKD1 gene since common mutation detection tests based on genomic polymerase chain reaction (PCR) cannot be used in this region. Mutation detection has proved to be successful in the single copy region of the PKD1 gene by SSCP analysis and it is therefore not surprising that the majority of the reported mutations initially have been in the 3 unique end even though this represents only 20 per cent of the coding sequence. More recently, a strategy using sequential PCR reactions, the first step being long range amplification using primers designed to take advantage of the 5 per cent sequence differences between PKD1 and it pseudogenes followed by more conventional PCR using the long range product as template


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has permitted mutation detection along the full length of PKD1. Genetic heterogeneity at the PKD1 and PKD2 loci with most mutations being unique within only a single family have complicated things further. Different mutations in PKD1 have been described but no clear hot spot is apparent (Peral et al. 1997; Rossetti et al. 2001). Finally, the high rate of apparent missense alterations and of polymorphism in PKD1 continues to pose problems in interpretation of the pathogenic significance of sequence variants detected. Recently, new semi-automatic methods of mutational analysis using denaturing high performance liquid chromatography (DHPLC) have been developed and the decreasing costs associated with direct sequencing have allowed this to be used as well. These methods have a high level of sensitivity and do not require conventional electrophoresis (Xiao and Oefner 2001). A mutation screen using DHPLC has very recently been reported by Rossetti where overall, 70 per cent of all mutations (across both PKD1 and PKD2) were detected (Rossetti et al. 2002). It has also been suggested that certain mutational sites may be associated with a more severe outcome (Hateboer et al. 2000; Rossetti et al. 2002) or ICA (Rossetti et al. 2003). Use of DHPLC or direct sequencing-based mutation detection should now allow the question of phenotype and mutational site to be investigated further having already allowed development of a commercial PKD gene test. Treatment prospects and the future Ultimately there requires to be some process or treatment whereby progression of renal disease in ADPKD can be modified. Human and animal studies have not always shown consistent results. With careful control of blood pressure, microalbuminuria and early treatment of complications, the rate of renal disease progression can be reduced (Ecder et al. 2000). Institution of low protein diets (Ogborn and Sareen 1995), soy modification (Ogborn et al. 1998), antioxidants (Nagao et al. 2000), anti-inflammatory agents (Gattone et al. 1995), hormonal modulators (Gattone et al. 1995) and the use of lipid lowering (HMG-CoA reductase) agents (van Dijk et al. 2001) may also be of value in ADPKD. The use of novel agents to specifically target mediators of cyst formation, for example, use of tyrosine kinase inhibitors (Sweeney et al. 2000) and gene therapy have also been considered. However, none of the data regarding these interventions are conclusive regarding use in human disease. For a full review of this rapidly changing area the reader is referred to Qian et al. (2001). References Atkinson, J. L. D., Sundt, T. M., Houser, O. W., and Whisnant, J. P. (1989). Angiographic frequency of anterior circulation intracranial aneurysms. Journal of Neurosurgery, 70, 551–5. Baboolal, K., Ravine, D., Daniels, J., Williams, N., Holmans, P., Coles, G. A., et al. (1997). Association of the angiotensin I converting enzyme gene deletion polymorphism with early onset of ESRF in PKD1 adult polycystic kidney disease. Kidney International, 52, 607–13. Bajwa, Z. H., Gupta, S., Warfield, C. A., and Steinman, T. I. (2001). Pain management in polycystic kidney disease. Kidney International, 60, 1631–44. Bardaji, A., Vea, A. M., Gutierrez, C., Ridao, C., Richart, C., and Oliver, J. A. (1998). Left ventricular mass and diastolic function in normotensive young adults with autosomal dominant polycystic kidney disease. American Journal of Kidney Diseases, 32, 970–5.

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10 Autosomal recessive polycystic kidney disease Lisa M. Guay-Woodford

Introduction Autosomal recessive polycystic kidney disease (ARPKD) is an often devastating form of polycystic kidney disease. The typical onset occurs in neonates and infants and is characterized by cystic dilatation of the renal collecting ducts and dysgenesis of the biliary ductal plate. In the past, ARPKD has been variably referred to as infantile polycystic kidney disease, infantile polycystic disease of the kidneys and liver, sponge kidneys, microcystic kidney disease, and polycystic kidneys, Potter type I (Zerres et al. 1984). While these descriptors were initially intended to highlight the predominant age of onset or the key pathologic features, they have proven to be more confusing than useful. For example, the clinical presentation of ARPKD can be delayed until adolescence or even adulthood (Neumann et al. 1988; Shaikewitz and Chapman 1993; Perez et al. 1998). Conversely, other clinically distinct renal cystic diseases can present in infancy (Guay-Woodford and Bernstein 1999). Therefore, the term ARPKD is now generally preferred, because it denotes a specific genetic disorder. Epidemiology The precise incidence of ARPKD is not known. Estimates have varied widely, ranging from 1 : 6000 to 1 : 14,000 in American reports (Bosniak and Ambos 1975), to 1 : 40,000 in the European literature (Zerres et al. 1984). An even higher incidence has been reported in the Finnish population (Kaariainen 1987) and among the Afrikaan-speaking population in South Africa (Lombard et al. 1989). These disparities most likely reflect differences in the study populations and in the ascertainment methodologies. More recent genetic analyses indicate that the incidence in the Caucasian population is 1 : 20,000 (Zerres et al. 1998). Based on this estimate figure, a heterozygosity frequency of 1 : 70 should be used for genetic counseling. Limited data are available regarding the distribution of ARPKD among different ethnic or racial groups, but it appears that ARPKD occurs rarely in non-Caucasian populations (Kaplan et al. 1989; Mattoo et al. 1994). Genetics In their seminal studies, Blythe and Ockenden (1971) recognized and characterized the phenotypic variability in ARPKD. They stratified patients into four phenotypic groups according to the age of presentation, the proportion of dilated renal collecting ducts, and the

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degree of biliary tract fibrosis. They concluded that within a given sibship the phenotypic manifestations were uniform. Based on their nosology, they hypothesized that ARPKD involved mutations in four distinct genes. However, re-evaluation of their cases cast doubt on the validity of this rigid classification (Kaplan et al. 1989). Moreover, while disease expression among affected siblings is often similar, several subsequent reports described sibships with discordance in disease onset and clinical phenotypes (Chilton and Cremin 1981; Gang and Herrin 1986; Kaplan et al. 1988; Deget et al. 1995). These observations prompted Zerres et al. (1984) to propose that ARPKD is caused by mutations in a single gene. This genetic model is appealing in that it can account both for the relatively high phenotypic concordance within sibships as well as the broad range of phenotypes that is evident among different families. With this single-gene model, the discordant disease expression observed in some sibships could be explained by other genetic modifiers, termed quantitative trait loci, that modulate the expression of the ARPKD phenotype. Positional cloning studies have confirmed that ARPKD involves a single gene, PKHD1, that maps to the short arm of chromosome 6 (6p21–p12) (Zerres et al. 1994). The full spectrum of phenotypic variants appear to involve mutations in this gene (Guay-Woodford et al. 1995). Recently, two groups have used complementary strategies to identify the PKHD1 gene (Onuchic et al. 2002; Ward et al. 2002). The genomic sequence includes at least 86 exons that are variably assembled into a number of alternatively spliced transcripts. The longest continuous open reading frame encodes a very large protein, polyductin, that is predicted to have a single transmembrane spanning domain near its carboxyl terminus. Several transcripts encode truncated products that lack the transmembrane domain and may be secreted polypeptides. The PKHD1 gene products are members of a novel protein class and appear to belong to a superfamily of proteins involved in regulation of cell proliferation as well as cellular adhesion and repulsion.

Insights into pathogenesis ARPKD typically begins in utero and the renal cystic lesion appears to be superimposed on a normal developmental sequence (Bernstein and Slovis 1992). The tubular abnormality involves fusiform dilatation of the collecting ducts. The factors involved in this cystogenic process remain unclear, but tubular obstruction has been excluded as a contributing mechanism. Interstitial, renal vascular, and glomerular abnormalities are not significant features of ARPKD. The biliary lesion appears to involve defective remodeling of the ductal plate in utero (Desmet 1998). As a result, primitive bile duct configurations persist and progressive portal fibrosis evolves. The remainder of the liver parenchyma develops normally. The defect in ductal plate remodeling is accompanied by abnormalities in the branching of the portal vein. The resulting histopathologic pattern is referred to as congenital hepatic fibrosis. A variety of animal models and in vitro cell culture systems have been used to investigate pathogenic mechanisms that cause the renal and biliary abnormalities in PKD. These studies have demonstrated (i) dysregulation of epithelial cell proliferation and differentiation; (ii) alterations of tubular basement membrane constituents and the associated extracellular matrix; (iii) abnormalities of epithelial cell polarity with apical mislocalization of key receptors and enzymes; and (iv) abnormalities in transepithelial fluid transport (reviewed by Calvet 1998). Of particular note, several laboratories have demonstrated that abnormalities in

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the epidermal growth factor (EGF)/transforming growth factor- (TGF-)/EGF receptor (EGFR) axis play a significant role in cystogenesis in both human PKD and several mouse PKD models (reviewed by Murcia et al. 1999). Among the numerous mouse PKD models described (reviewed by Schieren et al. 1996), the bpk mutation causes disease that is strikingly similar to human ARPKD (Nauta et al. 1993). Affected homozygotes develop both cystic dilatation of the renal collecting ducts as well as biliary dysgenesis. Avner and colleagues have recently demonstrated that a newly developed inhibitor of EGFR tyrosine kinase activity (EKI-785) significantly attenuates the renal and biliary abnormalities in the mice that are homozygous for the bpk mutation (Sweeney et al. 2000). These mouse studies potentially represent a seminal therapeutic breakthrough for ARPKD patients and intensive efforts to design appropriate clinical trials are in progress. Clinical manifestations The clinical manifestations of ARPKD are variable and depend on the age at presentation (Fig. 10.1). The majority of cases are identified either in utero or at birth. The most severely affected fetuses have enlarged echogenic kidneys and oligohydramnios due to poor fetal urine output. These fetuses develop the ‘Potter’s phenotype’, with pulmonary hypoplasia, a characteristic facies, and deformities of the spine and limbs. At birth, these neonates often have a critical degree of pulmonary hypoplasia that is incompatible with survival. Renal function, though frequently compromized, is rarely a cause of neonatal death (Guay-Woodford and Bernstein 1999). For those infants who survive the perinatal period, hypertension, renal failure, and portal hypertension usually evolve, but there is significant variability in the onset and severity of these manifestations. Hypertension usually develops in the first few months and ultimately affects 70–80 per cent of patients (Zerres et al. 1996; Roy et al. 1997). Without early aggressive treatment, severe hypertension may cause cardiac hypertrophy, congestive heart failure, and even death. The pathogenic mechanisms that underlie the development and progression of systemic hypertension remain undefined. Renal and hepato-biliary Renal

Hepato-biliary

Neonates Infants/children Adolescents Fig. 10.1 Correlation of clinical ARPKD phenotypes and age.

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ARPKD patients have defects in both urinary diluting capacity and concentrating capacity. Hyponatremia has been reported in affected infants and presumably results from defects in free water excretion (Kaplan et al. 1989). While net acid excretion may be reduced in these patients, metabolic acidosis is not a significant clinical feature. Abnormal urinalyses are common in both infants and older children. Microscopic or gross hematuria, proteinuria, or pyuria have all been reported (Zerres et al. 1996; Roy et al. 1997). The presence of pyuria does not always indicate urinary tract infection. The clinical presentation and appropriately obtained urine cultures should confirm the diagnosis and guide antibiotic therapy. Retrospective studies report an increased incidence of urinary tract infections in ARPKD patients, but controlled studies remain to be done (Zerres et al. 1996). In the first six months of life, ARPKD infants may have a transient improvement in their glomerular filtration rate (GFR) due to renal maturation (Cole et al. 1987). Subsequently, a progressive but variable decline in renal function occurs, with some patients not progressing to end-stage renal disease (ESRD) until adolescence or early adulthood. With advances in effective therapy for ESRD, prolonged survival is common and for many patients, the hepatic complications come to dominate the clinical picture. Portal hypertension frequently is the predominant clinical abnormality in older children and adolescents with ARPKD. These children typically present between 5 and 13 years of age with hepatosplenomegaly and bleeding esophageal or gastric varices, as well as hypersplenism with consequent thrombocytopenia, anemia, and leukopenia (Birnbaum and Suchy 1998). Hepatocellular function is usually preserved. However, ascending suppurative cholangitis is a serious complication and can cause fulminant hepatic failure (Zerres et al. 1988). Associated Caroli disease is a rare cause of chronic cholestasis and intrahepatic stones in young adults. Pathology Kidney The renal involvement is bilateral and largely symmetric. The histopathology varies depending on the age of presentation and the extent of cystic involvement. In the affected neonate, the kidneys can be 10 times normal size but retain their reniform configuration. Dilated, fusiform collecting ducts extend radially through the cortex (Fig. 10.2, panel A). In the medulla, the dilated collecting ducts are more often cut tangentially or transversely. Up to 90 per cent of the collecting ducts are involved. There is little evidence of interstitial fibrosis (Fig. 10.2, panel B). In perinatal survivors, the kidney size and extent of cystic involvement tend to be more limited. Between 10 and 60 per cent of the collecting ducts are involved. Cysts, particularly involving medullary collecting ducts, can expand up to 5 cm in diameter and assume a more spherical configuration (Bernstein and Slovis 1992). Progressive interstitial fibrosis is probably responsible for secondary tubular obstruction. In older children, medullary ductal ectasia is the predominant finding (Fig. 10.2, panel C). Cysts are lined with a single layer of nondescript cuboidal epithelium. The glomeruli and nephron segments proximal to the collecting ducts are generally normal in structure, but are often crowded between ectatic collecting ducts or displaced into subcapsular wedges. Cartilage or other dysplastic elements are not evident in ARPKD kidneys.

A

B

C

D

Fig. 10.2 Pathologic features of ARPKD. Panel A: Cut section of a kidney from an ARPKD neonate demonstrating the dilated cortical collecting ducts. Panel B: The classic renal histopathology in neonatal ARPKD. Dilated, radially arrayed cortical collecting ducts and the dilated, spheroid medullary collecting ducts are the predominant features. H&E 10. Panel C: The typical renal histopathology in later-onset ARPKD. The cortex is to the left, and the medulla is to the right of the section, with the cortico-medullary junction indicated by the arrow. Medullary ductal ectasia is the most prominent finding. H&E 10. Panel D: The hepatic ductal plate malformation lesion in an ARPKD neonate. Portal areas are enlarged and contain elongated, dilated biliary ducts that encircle the portal area. H&E 40. (Pathologic images courtesy of Dr. J Bernstein, William Beaumont Hospital, Royal Oak, MI.)

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Liver The liver in ARPKD can be either normal in size or somewhat enlarged. The hepatic parenchyma may be intersected by delicate fibrous septa that link the portal tracts. Bile ducts are dilated (biliary ectasia) and marked cystic dilatation of the entire intrahepatic biliary system (Caroli disease) has been described (Desmet 1998). In neonatal ARPKD, the bile ducts are increased in number, rather tortuous in configuration, and often located around the periphery of the portal tract (Fig. 10.2, panel D). In older children, the biliary ectasia is accompanied by increasing portal fibrosis and hypoplasia of the small portal vein branches. Fibrosis sometimes progresses to true cirrhosis, but the hepatocytes are seldom affected. Diagnosis Prenatal diagnosis Given the autosomal recessive mode of inheritance, the parents of affected children are obligate heterozygotes and each pregnancy has a 25 per cent recurrence risk. Prenatal diagnosis of ARPKD is suggested by sonographic evidence of enlarged, echogenic kidneys in the fetus (Fig. 10.3, panel A), oligohydramnios, and absence of urine in the fetal bladder. However, even with state-of-the-art technology, fetal sonography has limited reliability in second-trimester diagnoses (Zerres et al. 1988). While the increased renal size and echogenicity often become evident between 16 and 26 weeks of gestation, these abnormalities may not be detected until late in the third trimester (Luthy and Hirsch 1985; Barth et al. 1992). Oligohydramnios and absence of urine in the bladder are usually not manifest until after the 20th week (Bernstein and Slovis 1992). The genetic mapping of the PKHD1 locus and the absence of genetic heterogeneity in ARPKD have provided the basis for gene linkage or haplotype-based prenatal diagnosis in ‘at risk’ families (Zerres et al. 1998). It must be emphasized that haplotype-based testing is an indirect diagnostic method. As such, the feasibility and reliability of this test are predicated on access to the DNA from previous affected sibling(s) and accurate phenotypic diagnosis in the proposed index case(s). While the PKHD1 gene is quite large and has a complex array of transcripts, the identification of this gene should lead to the development of a direct gene-based diagnostic test. Such a test could be applied to ‘at-risk’ pregnancies as well as aid in specific diagnoses for fetuses and children with large, echogenic kidneys. Postnatal diagnosis Real-time sonography has replaced the intravenous pyelogram (IVP) in the evaluation of children with cystic kidney disease. The sonographic findings in ARPKD vary with patient age and the severity of renal involvement (Kaariainen et al. 1998). The kidney size typically peaks at 1–2 years of age, then gradually declines relative to the child’s body size, and stabilizes by 4–5 years. Sonography in affected neonates reveals symmetrically enlarged, diffusely echogenic kidneys with poor demarcation from surrounding tissues as well as among the cortex, medulla, and renal sinus (Boal and Telle 1980). With high resolution sonography, the radial array of dilated collecting ducts may be imaged (Fig. 10.3, panel B). As patients age, the sonographic

A

B

C

D

Fig. 10.3 Radiologic findings associated with ARPKD. Panel A: In utero sonogram of an ARPKD fetus at 26 weeks gestational age. Transverse view reveals enlarged, diffusely echogenic kidneys (indicated by the hatch marks) that occupy most of the abdominal cavity. Panel B: ARPKD in a neonate. High resolution renal sonography reveals radially arrayed, dilated collecting ducts (arrows). Panel C: ARPKD in a symptomatic 4-year-old girl. Contrastenhanced CT shows a striated nephrogram and prolonged corticomedullary differentiation. Panel D: ARPKD with Caroli’s disease. Sonography reveals marked biliary ductal ectasia associated with patchy hepatic echogenicity. (Radiologic images courtesy of Dr. ZH Jafri, William Beaumont Hospital, Royal Oak, MI.)

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pattern changes and reveals increased medullary echogenicity with scattered small cysts, measuring less than 2 cm in diameter. These cysts and progressive fibrosis can alter the reniform contour and ARPKD in older children may be mistaken for autosomal dominant polycystic kidney disease (ADPKD) (Gang and Herrin 1986; Kaplan et al. 1989; Zerres 1992). Contrast-enhanced computerized tomography (CT) can be useful in delineating the dilated collecting ducts in older children (Fig. 10.3, panel C). However, this streaky pattern of contrast retention can be seen in neonates with either ARPKD or ADPKD Saunders et al. 1999). The liver may be either normal in size or enlarged. It is usually less echogenic than the kidneys. Prominent intrahepatic bile duct dilatation (Fig. 10.3, panel D) suggests associated Caroli disease. With age, the portal fibrosis tends to progress and in older children, sonography typically reveals hepatosplenomegaly and a patchy increase in hepatic echogenicity. In patients with portal hypertension, Doppler flow studies of the vasculature may indicate reversal of portal flow (Premkumar et al. 1988). Promising initial reports indicate that magnetic resonance cholangiography (MRC), a new non-invasive imaging technique, may become the diagnostic modality of choice for evaluating the biliary tree in ARPKD patients (Jung et al. 1999).

Differential diagnosis In addition to ARPKD, ADPKD, glomerulocystic kidney disease (GCKD), and diffuse cystic dysplasia may also present in the perinatal period or the first few years of life with enlarged, diffusely echogenic kidneys (Table 10.1) (Guay-Woodford et al. 1998). While genetically distinct, these disorders can have overlapping clinical, radiographic, and morphologic features and are variably associated with biliary dysgenesis (Bernstein 1993). Liver biopsy evidence of biliary dysgenesis therefore must be interpreted with caution in distinguishing ARPKD from these other disorders. In 30–70 per cent of patients who present with ADPKD in utero or in the first year of life, parental sonography identifies a previously undiagnosed parent (Chapman 1996). Thus, the single most useful diagnostic test for a child with early-onset renal cystic disease is renal sonography of the parents and when the parents are less than 30 years of age, even of the grandparents (Ogborn 1994). Negative parental ultrasounds reduce the probability of ADPKD in the affected infant to the frequency of a spontaneous mutation, which according to recent estimates occurs in fewer than 10 per cent of ADPKD patients (Gabow and Grantham 1997). Glomerular cysts are the classic histopathologic manifestation of infantile ADPKD, but GCKD can also occur as an isolated, sporadic disorder. The kidneys in both forms of GCKD often contain abnormally differentiated pyramids, a type of medullary dysplasia (Bernstein and Slovis, 1992). Both forms of GCKD are associated with biliary dysgenesis in about 10 per cent of cases (Bernstein 1993). Recent reports describe a syndrome that includes a ARPKD-like polycystic kidneys and biliary tract fibrosis as well as a host of skeletal and facial dysmorphisms. In one of the four described sib-pairs, haplotype analyses excludes linkage to the PKHD1 locus (Hallermann et al. 2000). While this disorder is quite rare, it bears mention as a phenocopy of ARPKD. Cystic renal dysplasia is associated with gross cysts and typically occurs in the context of multiple malformation syndromes. Therefore, the clinical context, renal sonography and renal histopathology readily distinguish this disorder from ARPKD. Transient neonatal


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Table 10.1 Differential diagnosis of ARPKD Genetic disorders

Renal cystic lesion

Associated with CHF

ARPKD ARPKD with skeletal and facial anomalies ADPKD Glomerulocystic disease Malformation syndromes Meckel–Gruber syndrome Ivemark syndrome Chondrodysplasia syndrome of Jeune Chondrodysplasia syndrome of Saldino and Noonan Bardet–Biedl syndrome Zellweger syndrome Short-rib polydactyly syndromes Beckwith–Wiedemann syndrome

Collecting duct Collecting duct

Invariably Yes

Along entire nephron Bowman’s space

Rarea Rarea

Diffuse cystic dysplasia Cystic dysplasia Cystic dysplasia

Yesb Yesb Yesb

Cystic dysplasia

Yesb

Cystic dysplasia Cystic dysplasiac Cystic dysplasia Nephroblastoma; Cystic dysplasia

Yes Yes Yes No

Cystic dysplasia

Yesb

Cystic dysplasia

Yesb

Metabolic disorders Glutaric acidemia type II Chromosomal disorders Trisomy 9

CHF: congenital hepatic fibrosis. a Rare—evident in less than 10 per cent of cases. b Associated with renal-biliary-pancreatic dysplasia. c Cystic lesion in Zellweger syndrome primarily involves the glomerulus.

nephromegaly, neonatal contrast-induced nephropathy, nephroblastomatosis, and bilateral Wilms tumor are less common disorders that may present with renal sonographic findings similar to ARPKD (Stapleton et al. 1981; Avner et al. 1982; Kinoshita et al. 1986). Finally, the ultrasound findings of medullary ductal ectasia in adults may suggest the diagnosis of ARPKD or medullary sponge kidney. Despite the sonographic similarities, medullary sponge kidney is a generally non-heritable, isolated renal lesion that occurs primarily in adult women and therefore, should be distinguished readily from ARPKD by the clinical context (Guay-Woodford 1999). Clinical management The survival of ARPKD neonates has improved significantly in the last decade due largely to advances in mechanical ventilation and other supportive measures. Aggressive interventions such as unilateral (Bean et al. 1995), or bilateral nephrectomies (Sumfest et al. 1993) and continuous arteriovenous hemofiltration have been advocated in neonatal management, but prospective, controlled studies have not been performed.

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For those children who survive the perinatal period, careful blood pressure monitoring is required. Treatment with angiotensin converting enzyme inhibitors, calcium channel blockers, beta blockers, and diuretics, particularly loop agents, are efficacious. The management of ARPKD children with declining GFR should follow the standard guidelines established for chronic renal insufficiency in other pediatric patients (Warady et al. 1999). Renal transplantation is the treatment of choice for ARPKD patients who develop ESRD. Native nephrectomies may be warranted in patients with massively enlarged kidneys to allow allograft placement. Living-related donors are preferable for pediatric transplant candidates (Warady et al. 1997). Because ARPKD is a recessive disorder, either parent may be a suitable kidney donor. Given the relative urinary concentrating defect, ARPKD children should be monitored for dehydration during intercurrent illnesses associated with fever, tachypnea, nausea, vomiting, or diarrhea. In those infants with severe polyuria, thiazide diuretics may be effective in decreasing distal nephron solute and water delivery. While metabolic acidosis is not a prominent clinical issue, acid–base balance should be closely monitored and supplemental bicarbonate therapy initiated as needed. Close monitoring for portal hypertension is warranted in all ARPKD patients. The development of portal hypertension and its progression can be followed by serial ultrasound and Doppler flow studies. Hematemesis or melena suggests the presence of esophageal varices. Primary management of variceal bleeding may include endoscopic approaches, such as sclerotherapy or variceal banding. Alternatively, in the hands of appropriately skilled operator, the transjugular intrahepatic portosystemic shunt (TIPS) procedure has been quite effective (Karrer and Narkewicz 1999). Porta-caval or spleno-renal shunting may be indicated in some patients. Although hypersplenism occurs fairly commonly, splenectomy is seldom warranted. Unexplained fever with or without elevated transaminase levels suggests bacterial cholangitis and requires meticulous evaluation, often including a percutaneous liver biopsy, to make the diagnosis and guide aggressive antibiotic therapy (Kashtan et al. 1999).

Prognosis Perinatal mortality has been estimated to occur in 30–50 per cent of ARPKD neonates, but no definitive data are available. It is not yet possible to distinguish prospectively between potential neonatal survivors and those with severe pulmonary hypoplasia which is incompatible with extra-uterine life. The reported mortality rate for ARPKD patients during the first year of life ranges from 6–24 per cent (Cole et al. 1987; Zerres et al. 1996; Roy et al. 1997). However, for those infants who survive the first month of life, the reported mean 5-year patient survival rate was 80–95 per cent (Zerres et al. 1996; Roy et al. 1997) and survival into adulthood is not uncommon (Jamil et al. 1999). Effective management of systemic and portal hypertension, coupled with successful renal replacement therapy, appears to have contributed substantially to this extended patient survival. These data demonstrate that the prognosis in ARPKD, particularly for those children who survive the first month of life, is far less bleak than previously believed. Therefore, aggressive medical therapy is warranted. In those patients with ESRD and severe portal hypertension, combined kidney and liver transplantation may be indicated.


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Jamil, B., McMahon, L., Savige, J., Wang, Y., and Walker, R. (1999). A study of long-term morbidity associated with autosomal recessive polycystic kidney disease. Nephrology, Dialysis, Transplantation, 14, 205–9. Jung, G., Benz-Bohm, G., Kugel, H., Keller, K.-M., and Querfeld, U. (1999). MR cholangiography in children with autosomal recessive polycystic kidney disease. Pediatric Radiology, 29, 463–6. Kaariainen, H., Jaaskelainen, J., Kivisaari, L., Koskimies, O., and Norio, R. (1988). Dominant and recessive polycystic kidney disease in children: classification by intravenous pyelography, ultrasound, and computed tomography. Pediatric Radiology, 18, 45–50. Kaariainen, H. (1987). Polycystic kidney disease in children: a genetic and epidemiological study of 82 Finnish patients. Journal of Medical Genetics, 24, 474–81. Kaplan, B., Fay, J., and Shah, V. (1989). Autosomal recessive polycystic kidney disease. Pediatric Nephrology, 3, 43–9. Kaplan, B. S., Kaplan, P., de Chadarevian, J.-P., Jequier, S., O’Regan, S., and Russo, P. (1988). Variable expression of autosomal recessive polycystic kidney disease and congenital hepatic fibrosis within a family. American Journal of Medical Genetics, 29, 639–47. Karrer, F. and Narkewicz, M. (1999). Esophageal varices: current management in children. Semin Pediatric Surgery, 8, 193–204. Kashtan, C., Primack, W., Kainer, G., Rosenberg, A., McDonald, R., and Warady, B. (1999). Recurrent bacteremia with enteric pathogens in recessive polycystic kidney disease. Pediatric Nephrology, 13, 678–82. Kinoshita, T., Nakamura, Y., Kinoshita, M., Fukuda, S., Nakashima, H., and Hashimoto, T. (1986). Bilateral cystic nephroblastomas and botryoid sarcoma in a child with Dandy-Walker syndrome. Archives of Pathology and Laboratory Medicine, 110, 150–2. Lieberman, E., Salinas-Madrigal, L., Gwinn, J., Brennan, L., Fine, R., and Landing, B. (1971). Infantile polycystic disease of the kidneys and liver: clinical, pathological and radiological correlations and comparison with congenital hepatic fibrosis. Medicine (Baltimore), 50, 277–318. Lombard, E., Kromberg, J., Thomson, P., Milner, L., Biljon, Iv., and Jenkins, T. (1989). Autosomal recessive polycystic kidney disease: evidence for high frequency of the gene in Afrikaans-speaking population. South African Medical Journal, 76, 321–3. Luthy, D. A. and Hirsch, J. H. (1985). Infantile polycystic kidney disease: observations from attempts at prenatal diagnosis. American Journal of Medical Genetics, 20, 505–17. Mattoo, T. K., Khatani, Y., and Ashraf, B. (1994). Autosomal recessive polycystic kidney disease in 15 Arab children. Pediatric Nephrology, 8, 85–7. Murcia, N. W., Sweeney, J., and Avner, E. (1999). New insights into the molecular pathophysiology of polycystic kidney disease. Kidney International, 55, 1187–97. Nauta, J., Ozawa, Y., Sweeney, W., Rutledge, J., and Avner, E. (1993). Renal and biliary abnormalities in a new murine model of autosomal recessive polycystic kidney disease. Pediatric Nephrology, 7, 163–72. Neumann, H. P. H., Zerres, K., Fischer, C. L., Wolff, G., Schaefer, H. E., Gal, A., et al. (1988). Late manifestation of autosomal-recessive polycystic kidney disease in two sisters. American Journal of Nephrology, 8, 194–7. Ogborn, M. (1994). Polycystic kidney disease—a truly pediatric problem. Pediatric Nephrology, 8, 194–7. Onuchic, L., Furu, L., Nagasawa, Y., Hou, X., Eggermann, T., Ren, Z., et al. (2002). PKHD1, the polycystic kidney and hepatic disease 1 gene, encodes a novel large protein containing multiple IPT domains and PbH1 repeats. American Journal of Human Genetics, 70, 1305–17. Perez, L., Torra, R., Badenas, C., Ara, J., Coll, E., Moises, J., and Darnell, A. (1998). Autosomal recessive polycystic kidney disease presenting in adulthood. Molecular diagnosis of the family. Nephrology, Dialysis, Transplantation, 13, 1273–6.


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Premkumar, A., Berdon, W. E., Levy, J., Amodio, J., Abramson, S. J., and Newhouse, J. H. (1988). The emergence of hepatic fibrosis and portal hypertension in infants and children with autosomal recessive polycystic kidney disease. Pediatric Radiology, 18, 123–9. Roy, S., Dillon, M., Trompeter, R., and Barratt, T. (1997). Autosomal recessive polycystic kidney disease: long-term outcome of neonatal survivors. Pediatric Nephrology, 11, 302–6. Saunders, A., Denton, E., Stephens, S., and Reid, C. (1999). Cystic kidney disease presenting in infancy. Clinical Radiology, 54, 370–76. Schieren, G., Pey, R., Bach, J., Hafner, M., and Gretz, N. (1996). Murine models of polycystic kidney disease. Nephrology, Dialysis, Transplantation, 11, 38–45. Shaikewitz, S. T., and Chapman, A. (1993). Autosomal recessive polycystic kidney disease: issues regarding the variability of clinical presentation. Journal of the American Society of Nephrology, 3, 1858–62. Stapleton, F., Hilton, S., and Wilcox, J. (1981). Transient nephromegaly simulating infantile polycystic disease of the kidneys. Pediatrics, 67, 554–9. Sumfest, J. M., Burns, M. W., and Mitchell, M. E. (1993). Aggressive surgical and medical management of autosomal recessive polycystic kidney disease. Urology, 42, 309–12. Sweeney, W., Chen, Y., Nakanishi, K., Frost, P., and Avner, E. (2000). Treatment of polycystic kidney disease with a novel tyrosine kinase inhibitor. Kidney International, 57, 33–40. Warady, B., Alexander, S., Watkins, S., Kohaut, E., and Harmon, W. (1999). Optimal care of the pediatric end-stage renal disease patient on dialysis. American Journal of Kidney Diseases, 33, 567–83. Warady, B., Hebert, D., Sullivan, E., Alexander, S., and Tejani, A. (1997). Renal transplantation, chronic dialysis, and chronic renal insufficiency in children and adolescents. The 1995 Annual Report of the North American Pediatric Renal Transplant Cooperative Study. Pediatric Nephrology, 11, 49–64. Ward, C., Hogan, M., Rossetti, S., Walker, D., Sneddon, T., Wang, X., et al. (2002). The gene mutated in autosomal recessive polycystic kidney disease encodes a large, receptor-like protein. Nature Genetics, 30, 259–69. Zerres, K., Becker, J., Muecher, G., Rudnik-Schoneborn, S., Onuchic, L., Germino, G., et al. (1998). Haplotype-based prenatal diagnosis in autosomal recessive polycystic kidney disease (ARPKD). American Journal of Medical Genetics, 76, 137–44. Zerres, K., Hansmann, M., and Mallmann, R. (1988). Autosomal recessive polycystic kidney disease: problems of prenatal diagnosis. Prenatal Diagnosis, 8, 215–19. Zerres, K., Mucher, G., Bachner, L., Deschennes, G., Eggermann, T., Kaariainen, H., et al. (1994). Mapping of the gene for autosomal recessive polycystic kidney disease (ARPKD) to chromosome 6p21-cen. Nature Genetics, 7, 429–32. Zerres, K., Rudnik-Schoneborn, S., Deget, F., Holtkamp, U., Brodehl, J., Geisert, J., et al. (1996). Nephrologie AfP: autosomal recessive polycystic kidney disease in 115 children: clinical presentation, course and influence of gender. Acta Paediatrica, 85, 437–45. Zerres, K., Volpel, M. C., and Weiss, H. (1984). Cystic kidneys: genetics, pathologic anatomy, clinical picture, and prenatal diagnosis. Human Genetics, 68, 104–35. Zerres, K. (1992). Autosomal recessive polycystic kidney disease. Clinical Investigation, 70, 794–801.


11 Cystic renal diseases Anand K. Saggar-Malik

Introduction Renal cysts may result from a variety of causes. Table 11.1 shows a simple classification of renal cystic kidney disease. A diagnosis of multiple renal cysts is often confused with autosomal dominant polycystic kidney disease (ADPKD) which is a very common inherited cystic renal disease (Chapter 11). The clinical picture together with the location of the cyst may help in the differential diagnosis (see Table 10.1).

Table 11.1 Classification of renal cysts Polycystic kidney disease Autosomal dominant Autosomal recessive Syndromic renal cystic disease Tuberous sclerosis Von Hippel–Lindau disease Orofacialdigital syndrome Brachymesomelia–renal syndrome Glycogen storage diseases Beckwith–Weidman syndrome Meckels syndrome Trisomy 13 (And many more) Glomerular cystic disease Acquired renal cystic disease Simple Chronic dialysis Hypokalaemia induced (primary hyperaldosteronism) Renal medullary cysts Juvenile nephronophthisis (recessive) Medullary cystic disease (dominant) Senior-Loken syndrome Medullary sponge kidneys Renal cystic dysplasia Renal aplasia Multicystic dysplasia

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Acquired cystic kidney disease Acquired cysts are particularly common in elderly patients and those on chronic haemodialysis, the occurrence dependent upon the duration of dialysis, previous transplantation, and gender. There is no known genetic susceptibility. Cystic renal dysplasia Renal cystic dysplasia most probably represents a non-specific reaction to an early abnormality of development and so is seen in genetic and non-genetic syndromes. The genetic basis of isolated renal dysplasia remains unknown and the recurrence risk is low. However, in some families there have been described patterns of autosomal dominant dysplasia (termed adysplasia) (McPherson et al. 1987); autosomal recessive dysplasia (Cain et al. 1974) and also X-Linked inherited dysplasia (Pashayan et al. 1977). These conditions are characterized by highly variable degrees of kidney involvement ranging form virtually normal kidneys to aplasia. Glomerular cystic kidney disease This has historically been a general term used to describe a heterogeneous group of cystic kidney diseases, characterized by cystic dilatation of Bowman’s spaces. Such changes are often seen within other systemic renal disorders, for example, tuberous sclerosis, trisomy 13, orofacial-digital type 1 and brachymesomelia–renal to name but a few. Very often glomerular cystic kidney disease has been the first manifestation of ADPKD. The term ‘glomerular cystic kidney disease’ perhaps now should not really be used. Although usually sporadic, there is described a rare distinct dominant form with hypoplastic kidneys (Rizzoni et al. 1982). Occasionally the kidneys may be of normal size although histologically there are absent or have malformed renal papillae. All affected individuals have the same cystic phenotype. Renal function is usually impaired from infancy but stable. The gene is distinct from PKD1 and PKD2. More recently, hypoplastic forms have been mapped to the HGF gene in some families (Kolatsi-Joannou et al. 2001) but not in others (Gusmano et al. 2002). Clearly, much more work is yet to be done in this condition. Medullary cystic kidney disease There is a group of inherited cystic nephropathies characterized by juvenile onset recessive inheritance, previously termed familial juvenile nephronophthisis (FJN); or by adult onset dominant inheritance, termed medullary cystic disease, previously termed MCD. They share clinical and pathological features and were traditionally grouped together under the term FJN/MCD complex. The main clinical features are of renal cyst formation in the medulla or the corticomedullary junction, although this finding is inconstant as is salt wasting. Earlier reports had suggested that one single gene may be responsible for these diseases but increasingly it has become apparent that the recessive and dominant forms are not allelic and even within both the FJN and the MCD complexes, there is genetic heterogeneity. There is phenotypic variability in that juvenile and adult forms exist in families with either recessive FJN or dominant MCD. The age of onset is reportedly the major difference between FJN and MCD, which in MCD occurs much later, usually in the third or fourth decade of life. Histologically, thickening of the basement membrane is less or even absent in MCD. Haematuria, proteinuria, and hypertension occur

Cystic renal diseases

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more commonly and cysts are reportedly more numerous than in juvenile nephronophthisis (FJN). Extra renal abnormalities are more frequent but one notable exception is the association with gout and or hyperuricaemia. This however almost certainly represents co-inheritance of a separate entity called familial juvenile gouty nephropathy (Chapter 19). By contrast, FJN, although clinically a similar disease, leads to end-stage renal failure (ESRF) in adolescence, and has a recessive mode of inheritance with a neonatal juvenile or adolescent onset. The most reliable way to differentiate between FJN and autosomal dominant MCD is by family history and mode of inheritance. Familial juvenile nephronophthisis Autosomal recessive nephronophthisis is rare. It was described as the original disease in 10–32 per cent of children with uraemia and 7 per cent of children with ESRF in the European Dialysis and Transplantation Association (EDTA) registry (Broyer and Kleinknecht 1998). Equal sex distribution is reported in all races. Children initially develop polyuria and polydypsia because of reduced urine concentrating ability (hyposthenuria). These symptoms persist throughout the course of the disease and can be insidiously progressive and latent with 15 per cent of patients presenting with ESRF. Symptoms most commonly develop in the first decade of life if not in the first few months with ESRF occurring on average at the age of 12. However, the range is considerable, one study reporting a range from 4 to 23 years. Persistent hyposthenuria leads to growth retardation. Notably peripheral oedema, haematuria, proteinuria, and infection are absent. Similarly, hypertension is unusual before ESRF. Anaemia is usually severe. At a glomerular filtration rate (GFR) below 40 ml/min, metabolic acidosis is more commonly seen. The hyposthenuria also leads to persistently low serum sodium values which persist even at ESRF, and can mimic hypoaldosteronism. The fundamental defect appears to be abnormal tubular basement membrane. Histology usually confirms the diagnosis showing the typical features of tubular atrophy, cysts, interstitial fibrosis and marked thickening and wrinkling of the basement membrane. The family history is also an important aid to diagnosis. The absence of cysts on ultrasound does not exclude the diagnosis since they may develop later in life. FJN now more commonly called nephronophthisis (NPH) was recently mapped and localized to chromosome 2q13, (NPH1) (Antingnac et al. 1993). In the majority of familial cases and in sporadic NPH, large homozygous deletions spanning a region of 250 kb have been identified. Within NPH genetic heterogeneity has been identified (Antignac et al. 1993; Medhioub et al. 1994). Haider et al. (1998) identified linkage of the clinical phenotype to markers on 9q22–q31 (NPH2). Recently, from a large Venezuelan family, a third locus (NPH3) for the adolescent onset type has been described on chromosome 3p22 (Omran et al. 2000) and now there is reported a fourth locus (Schuermann et al. 2002) on 1p36. Two reports have also identified different loci for Senior-Loken syndrome, also known as ‘NPH with extra renal manifestations’, mapping to NPH3 (Omran et al. 2002) and NPH4 (Schuermann et al. 2002). Associated disorders described include tapetoretinal degeneration, which is almost certainly part of the Senior-Loken spectrum of nephronophthisis, Leber amaurosis, and tapetoretinal degeneration, where cystic kidneys are also commonly seen. Other ocular abnormalities described are coloboma, cataract, and retinitis pigmentosa (Broyer and Kleinknecht 1998). Mental retardation and cerebellar dysfunction has also been reported but these are also associated features of Leber amaurosis. Cone-shaped epiphyses have also been

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commonly described but not without the ocular, liver, or CNS abnormalities seen in lebers. Massive hepatosplenomegaly with congenital hepatic fibrosis has also been reported (Boichis et al. 1973) which appears distinct from that seen in autosomal recessive polycystic kidney disease.

Autosomal dominant medullary cystic kidney disease (ADMCKD) Medullary cystic kidney disease now often abbreviated to ADMCKD, is an adult onset inherited nephropathy, which leads to ESRF in adulthood. It was first recognized and described by Thorn et al. (1944) and then later by Smith and Graham (1945) and Fanconi (1951). Subsequent reports suggested a similarity between this condition and FJN, even though FJN followed a different mode of inheritance (Mongeau and Worthen 1967; Strauss and Sommers 1967; Burke et al. 1982). With retrospect, these individual papers can now be seen to be describing a mixture of patients with ADMCKD and NPH. ADMCKD is associated with polyuria, polydypsyia, and anaemia and on renal histology there is interstitial fibrosis, disintegration of the tubular basement membrane together with bilateral cortico-medullary cyst formation. All of these features are in common with NPH. However, it is now apparent that ADMCKD is a genetically distinct condition from NPH, it has an autosomal dominant mode of inheritance affecting mainly adults with a mean age of 30–47 years. Many patients are hypertensive at an early stage but some do later develop hypotension due to excessive salt wasting. Medullary cysts are present in over 75 per cent of patients but may not always be seen on ultrasound or CT scan due to their size (Gardner 1971; Swenson et al. 1974). ADMCKD does have its clinical variants and has been associated with such disparate renal manifestations as hyperuricaemia and gouty arthritis, peripheral dysostosis, mental retardation, post-axial polydactyly, cerebellar abnormalities, hypogonadism, obesity, renal tubular acidosis, parathyroid insufficiency, and congenital amaurosis. Earlier case reports have also shown an association of ADMCKD with gout and epilepsy (Burke et al. 1982) and spastic quadraparesis (Green et al. 1990). As yet it is still unknown whether this clinical variability is due to allelic or locus heterogeneity. Finding a specific gene locus was difficult in ADMCKD because of the absence of precise diagnostic criteria. Histological examination from renal biopsies does not allow differentiation between MCD and NPH. However Christodoulou et al. (1998), recently identified a locus on chromosome 1 for ADMCKD in two large Cypriot families. These families also had hyperuricaemia and gout and very late age of onset of 62 and 51 years. More recently there has been very clear demonstration of genetic heterogeneity within ADMCKD and none of these non-renal manifestations, particularly hyperuricaemia and gout appear to be locus specific. ADMCKD has now been shown to have three different loci, the first (MCKD1) localized on chromosome 1q21 and described above (Christodoulou et al. 1998) and a second locus (MCKD2) linked to chromosome 16p12 in an Italian pedigree (Scolari et al. 1999). This locus later confirmed by others (Hateboer et al. 2001). Scolari et al. (1999) noted that the uromodulin gene maps to this critical MCKD2 region and is expressed mainly in the kidney, localized to the epithelial cells of the thick ascending limb of the loop of Henle. Since uromodulin is functionally associated with water non-permeability in the loop of Henle, a function altered in ADMCKD, he postulated that uromodulin may be a candidate gene. A third ADMCKD locus has now also been suggested by Kroiss et al. (2000) where they excluded linkage to MCKD1 and MCKD2 in four European families and one Taiwanese kindred.


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Naturally, one of the obvious differential diagnoses for adult onset cystic kidney disease is ADPKD. However, important differences include a typical absence of flank pain, hypertension, or haematuria. Furthermore the kidneys are very often not enlarged. The average age of onset of symptoms is 23 years, with renal failure after the third decade. However, Christodoulou et al. (2001) described a very late age of onset in the fifth and sixth decade in his Cypriot families. Parvari et al. (2001) also described phenotypic variability within a Jewish family mapping to MCKD1 characterized by hypertension and progressive renal failure where some family members only had medullary cystic kidney disease with classical cortico-medullary cysts, tubular interstitial nephropathy, and anaemia. Interestingly, an average of 4.5 years elapsed between diagnosis and end stage renal disease. To complicate things a little further, Dahan et al. (2001), recently described a large Belgium family with familial juvenile hyperuricaemic nephropathy (FJHN), an autosomal dominant disorder characterized by hyperuricaemia during childhood and chronic interstitial nephritis leading to progressive renal failure during adulthood (see Chapter 19). This family showed tight linkage between the MCKD2 locus on 16p12 and FJHN, where previously FJHN had been mapped in a large Japanese family (Kamantani et al. 2000) and also in a Czech family (Stiburkowa et al. 2000). In this last report the locus was to a marker close by at 16p11. Dahan et al. (2001) suggest that FJHN and MCKD2 may be two facets of the same disease. Further refinement of the FJHN locus and examination of linkage to other loci within ADMCKD families with gout and/or hyperuricaemia is required. The demand for prenatal diagnosis may well be minimal in this condition but the usefulness of genetic testing would be most valuable for early presymptomatic diagnosis and treatment of abnormalities at an early stage. Control of hypertension and hyperuricaemia being perhaps the most important. Gene testing would also allow the possibility of identifying potential kidney transplant donors for those few unfortunate patients who do progress.

Glutaricaciduria type IIA (GA IIA) The glutaricacidurias are subdivided into type I and type II. The latter further subdivided on the basis of which electron transfer flavoprotein subunit (alpha IIA; beta IIB) or flavoprotein dehydrogenase (IIC) is defective. The phenotype of these three subtypes is largely indistinguishable. All can lead to mild or severe cases, dependent upon the intragenic mutation. The gene locus for GA IIA has been cloned and sequenced at chromosome 15q23–25 (Finochiaro et al. 1988). Glutaricaciduria type IIA is a rare, recessive condition, characterized by neonatal acidosis, hypoglycaemia, polycystic kidneys, and a strong ‘sweaty feet’ odour. Hepatomegaly, respiratory distress, muscle hypotonia are also typical clinical features (Niederwieser et al. 1983). The odour is due to high concentrations of glutaric acid in blood and urine. The fundamental defect in this disorder is due to abnormal metabolism of the acyl-CoA compounds. There is therefore often co-excretion of excess lactic, ethylmalonic, butyric, isobutyric, 2-metylbutyric, and isovaleric acids which differentiates it from glutaricaciduria type I. Inhibition of acyl-CoA pathways is also seen in cases of Jamaican vomiting caused by eating unripe ackee which contains the acyl-CoA dehydrogenase inhibitor hypoglycin. Those children that do not die of neonatal acidosis, symptoms later may include muscle weakness, nausea, vomiting, hypoglycaemia, and occasionally cataracts. Cerebral pachygyria, pulmonary hypoplasia, genital abnormalities, and dysmorphism also occur. Facial dysmorphic

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features include macrocephaly, large anterior fontanelle, high forehead, flat nasal bridge, and telecanthus (Wilson et al. 1989). However the dysmorphic features appear to be confined to the group of patients with the severe type IIC. Fatty infiltration of the liver, heart, and kidney invariably occurs (Mongini et al. 1992). The kidneys are enlarged with numerous cortical cysts. Selective tubular damage leads to glycosuria and aminoaciduria. Wilson et al. (1989) reported unique ultrasound findings suggestive of early renal cyst formation which the authors claim maybe useful for establishing a diagnosis when enzyme studies are not available. Improvement in muscle power using a low-fat diet has been observed (Mongini et al. 1992) as has successful treatment with riboflavin in a male child with progressive spastic ataxia and leukodystrophy but without acidosis (Uziel et al. 1995). References Antignac, C., Arduy, C. H., Beckmann, J. S., Benessy, F., Gros, F., Medhioub, M., et al. (1993). A gene for familial juvenile nephronophthisis (recessive medullary cystic kidney disease) maps to chromosome 2p. Nature Genetics, 3(4), 342–5. Boichis, H., Passwell, J., David, R., and Miller, H. (1973). Congenital hepatic fibrosis and nephronophthisis. A family study. Quarterly Journal of Medicine, 42(165), 221–33. Broyer, M. and Kleinknech, C. (1998). Structural tubulointerstitial disease: nephronopthisis. In Inherited disorders of the kidney (ed. S. H. Morgan and J. P. Grunfeld), pp. 341–8. Oxford University Press, Oxford. Burke, J. R., Inglis, J. A., Craswell, P. W., Mitchell, K. R., and Emmerson, B. T. (1982). Juvenile nephronophthisis and medullary cystic disease—the same disease (report of a large family with medullary cystic disease associated with gout and epilepsy). Clinical Nephrology, 18(1), 1–8. Cain, D. R., Griggs, D., Lackey, D. A., and Kagan, B. M. (1974). Familial renal agenesis and total dysplasia. American Journal of Diseases in Children, 128(3), 377–80. Christodoulou, K., Tsingis, M., Starrou, C., Eleftheriou, A., Papapavlou, P., Patsalis, P. C., et al. (1998). Chromosome 1 localization of a gene for autosomal dominant medullary cystic kidney disease. Human Molecular Genetics, 7(5), 905–11. Dahan, K., Fuchshuber, A., Adamis, S., Smaers, M., Koiss, S., et al. (2001). Familial juvenile hyperuricemic nephropathy and autosomal dominant medullary cystic kidney disease type 2: two facets of the same disease? Journal of the American Society of Nephrology, 12(11), 2348–57. Fanconi, G., Hanhart, E., Albertini, A., Uhlinger, E., Dolivo, G. and Prader, A. (1951). Familial Juvenile Nephronopthisis. Helvetica Paediatrics Acta, 6, 1–49. Finocchiaro, G., Ito, M., Ikeda, Y., and Tanaka, K. (1988). Molecular cloning and nucleotide sequence of cDNAs encoding the alpha-subunit of human electron transfer flavoprotein. Journal of Biological Chemistry, 263(30), 15773–80. Gardner, K. D., Jr. (1971). Evolution of clinical signs in adult-onset cystic disease of the renal medulla. Annals of Internal Medicine, 74(1), 47–54. Green, A., Kinirons, M., O’Meara, Y., Donohoe, J., Murphy, S., and Carmody, M. (1990). Familial adult medullary cystic disease with spastic quadriparesis: a new disease association. Clinical Nephrology, 33(5), 237–40. Gusmano, R., Caridi, G., Marini, M., Perfumo, F., Ghiggeri, G. M., Piaggio, G., et al. (2002). Glomerulocystic kidney disease in a family. Nephrology Dialysis Transplantation, 17(5), 813–8. Haider, N. B., Carmi, R., Shalev, H., Sheffield, V. C., and Landau, D. (1998). A Bedouin kindred with infantile nephronophthisis demonstrates linkage to chromosome 9 by homozygosity mapping. American Journal of Human Genetics, 63(5), 1404–10. Hateboer, N., Gumbs, C., Teare, M. D., Coles, G. A., Griffiths, D., Ravine, D., et al. (2001). Confirmation of a gene locus for medullary cystic kidney disease (MCKD2) on chromosome 16p12. Kidney International, 60(4), 1233–9.


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Hildebrandt, F., Strahm, B., Nothwang, H. G., Gretz, N., Schnieders, B., Singh-Sawhney, I., et al. (1997). Molecular genetic identification of families with juvenile nephronophthisis type 1: rate of progression to renal failure. APN Study Group. Arbeitsgemeinschaft fur Padiatrische Nephrologie. Kidney International, 51(1), 261–9. Kamatani, N., Moritani, M., Yamanaka, H., Takeuchi, F., Hosoya, T. and Hakura, M. (2000). Localization of a gene for familial juvenile hyperuricemic nephropathy causing underexcretion-type gout to 16p12 by genome-wide linkage analysis of a large family. Arthritis and Rheumatology, 43(4), 925–9. Kolatsi-Joannou, M., Bingham, C., Ellard, S., Bulman, M. P., Allen, L. I., Hattersley, A. T., et al. (2001). Hepatocyte nuclear factor-1beta: a new kindred with renal cysts and diabetes and gene expression in normal human development. Journal of American Society of Nephrology, 12(10), 2175–80. Konrad, M., Saunier, S., Calado, J., Gubler, M. C., Broyer, M., and Antignac, C. (1998). Familial juvenile nephronophthisis. Journal of Molecular Medicine, 76(5), 310–6. Kroiss, S., Huck, K., Berthold, S., Ruschendorf, F., Scolari, F., Caridi, G., et al. (2000). Evidence of further genetic heterogeneity in autosomal dominant medullary cystic kidney disease. Nephrology, Dialysis, Transplantation, 15(6), 818–21. McPherson, R., Carey, J., Kramer, A., Hall, J. G., Pauli, R. M., Schimke, R. N., et al. (1987). Dominantly inherited renal adysplasia. Journal of Medical Genetics, 26, 863–72. Medhioub, M., Cherif, D., Benessy, F., Silbermann, F., Gubler, M. C., Le Paslier, D., et al. (1994). Refined mapping of a gene (NPH1) causing familial juvenile nephronophthisis and evidence for genetic heterogeneity. Genomics, 22(2), 296–301. Mongeau, J. G. and Worthen, H. G. (1967). Nephronophthisis and medullary cystic disease. American Journal of Medicine, 43(3), 345–55. Mongini, T., Doriguzzi, C., Palmucci, L., De Francesco, A., Bet, L., Manfredi, L., et al. (1992). Lipid storage myopathy in multiple acyl-CoA dehydrogenase deficiency: an adult case. European Neurology, 32(3), 170–6. Niederwieser, A., Steinmann, B., Exner, U., Neuheiser, F., Redweik, U., Wang, M., et al. (1983). Multiple acyl-Co A dehydrogenation deficiency (MADD) in a boy with nonketotic hypoglycemia, hepatomegaly, muscle hypotonia and cardiomyopathy. Detection of N-isovalerylglutamic acid and its monoamide. Helvetica Paediatrics Acta, 38(1), 9–26. Omran, H., Sasmaz, G., Haffner, K., Volz, A., Olbrich, H., Melkaoui, R., et al. (2002). Identification of a gene locus for Senior-Loken syndrome in the region of the nephronophthisis type 3 gene. Journal of the American Society of Nephrology, 13(1), 75–9. Omran, H., Fernandez, C., Jung, M., Haffner, K., Fargier, B., Villaquiran, A., et al. (2000). Identification of a new gene locus for adolescent nephronophthisis, on chromosome 3q22 in a large Venezuelan pedigree. American Journal of Human Genetics, 66(1), 118–27. Parvari, R., Schnaider, A., Basok, A., Katchko, L., Borochovich, Z., Kanis, A., et al. (2001). Clinical and genetic characterization of an autosomal dominant nephropathy. American Journal of Medical Genetics, 99(3), 204–9. Pashayan, H. M., Dowd, T., and Nigro, A. V. (1977). Bilateral absence of the kidneys and ureters. Three cases reported in one family. Journal of Medical Genetics, 14(3), 205–9. Rizzoni, G., Loirat, C., Levy, M., Milanesi, C., Zachello, G., and Mathieu, H. (1982). Familial hypoplastic glomerulocystic kidney. A new entity? Clinical Nephrology, 18(5), 263–8. Schuermann, M. J., Otto, E., Becker, A., Saar, K., Ruschendorf, F., Polah, B. C., et al. (2002). Mapping of gene loci for nephronophthisis type 4 and Senior-Loken syndrome, to chromosome 1p36. American Journal of Human Genetics, 70(5), 1240–6. Scolari, F., Puzzer, D., Amoroso, A., Caridi, G., Ghiggeri, G. M., Maiorca, R., et al. (1999). Identification of a new locus for medullary cystic disease, on chromosome 16p12. American Journal of Human Genetics, 64(6), 1655–60. Smith, C. A. and Graham, J. B. (1945). Congenital medullary cysts of the kidneys with severe refactory anaemia. American Journal of Diseases in Children, 69, 369–77.

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Stiburkova, B., Majewski, J., Sebesta, I., Zhang, W., Ott, J., and Kmoch, S. (2000). Familial juvenile hyperuricemic nephropathy: localization of the gene on chromosome 16p11.2-and evidence for genetic heterogeneity. American Journal of Human Genetics, 66(6), 1989–94. Strauss, M. B. and Sommers, S. C. (1967). Medullary cystic disease and familial juvenile nephronophthisis. New England Journal of Medicine, 277(16), 863–4. Swenson, R. S., Kempson, R. L., and Friedland, G. W. (1974). Cystic disease of the renal medulla in the elderly. JAMA, 228(11), 1401–4. Thorn, G. W., Koepf, G. F., and Clinton, M. C. (1944). Renal failure stimulating adrenocortical insufficiency. New England Journal of Medicine, 231, 76–85. Uziel, G., Garavaglia, B., Ciceri, E., Moroni, I., and Rimoldi, M. (1995). Riboflavin-responsive glutaric aciduria type II presenting as a leukodystrophy. Pediatric Neurology, 13(4), 333–5. Wilson, G. N., De Chadarevian, J. P., Kaplan, P., Loehr, J. P., Frerman, F. E., and Goodman, S. I. (1989). Glutaric aciduria type II: review of the phenotype and report of an unusual glomerulopathy. American Journal of Medical Genetics, 32(3), 395–401.

12 Primary inherited metabolic diseases of the kidney Margaret Town and William van’t Hoff

Introduction Renal disease is most commonly associated with structural, immunological, or infective causes but this chapter deals with those in which there is a metabolic defect. There are several such metabolic disorders in which substrate accumulation or an enzyme deficiency can be demonstrated in the kidney but without any significant renal dysfunction (e.g. some of the lysosomal storage disorders). This review focusses on a few disorders in which renal dysfunction is a major symptom; several other such diseases are described in detail elsewhere in this book. Table 12.1 lists by category, the renal manifestations of metabolic disease (see also, van’t Hoff 1999). Cystinosis Clinical background Nephropathic cystinosis is a rare (incidence approximately 1 in 150,000) autosomal recessive disorder characterized biochemically by defective lysosomal cystine transport (Gahl et al. 1995). Affected children present towards the end of the first year of life with symptoms attributable to the renal Fanconi syndrome (generalized proximal tubular dysfunction), including poor feeding, recurrent vomiting, failure to thrive, or even weight loss, and delayed motor development due to rickets. The disorder affects all racial groups but Caucasian children often have a fair complexion and blond hair. Initial investigations demonstrate a hypokalaemic, hyperchloraemic metabolic acidosis with hypophosphataemia. Urinalysis may reveal glycosuria and proteinuria, further tests will show aminoaciduria, phosphaturia, and tubular proteinuria, a combination all suggestive of the renal Fanconi syndrome. There are several causes of generalized proximal tubular dysfunction (see Table 12.1) but the features are sufficiently different to usually allow prompt diagnosis. Aside from the complexion, the best clinical sign in cystinosis is the presence of corneal cystine crystals. These are best seen on slit-lamp examination but may not be very obvious in infants. Treatment of the Fanconi syndrome involves rehydration, free access to fluids, and correction of the electrolyte and mineral losses (Loirat 1999). A synthetic preparation of vitamin D (1--calcidol or calcitriol) is required to treat rickets. Correction of these deficits has to be supervised carefully since the biochemical abnormalities can be exacerbated in the early phase (e.g. correction of acidosis with bicarbonate and addition of phosphate supplements can further lower plasma calcium levels). Young children with a severe Fanconi syndrome

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Table 12.1 Renal manifestations of metabolic disorders (see van’t Hoff 1999 for review) Tubulopathy

Proteinuria/ haematuria

Nephrocalcinosis/ calculi

Cystic disease

Cystinosis Tyrosinaemia type 1

Fabry disease Imerslund–Grasbeck syndrome Lecithin:cholesterol acyltransferase deficiency Galactosialidosis

Cystinosis Tyrosinaemia type 1

Cystinosis Zellweger syndrome

Primary Hyperoxaluria types 1 and 2 Familial hyperuricaemic nephropathy Hypoxanthine-guanine phosphoribosyl transferase HGPRT deficiency Wilson disease

Alagille syndrome

Fructosaemia Galactosaemia Lowe syndrome

Glutamyl ribose-5phosphate glycoproteinosis

Wilson syndrome Mitochondrial cytopathies

Mucopolysaccharidosis type I (Hurler syndrome) Mitochondrial cytopathies

Glycogen storage disease type 1

Infantile sialic acid storage disease

Adenine phosphoribosyltransferase deficiency Glycogen storage disease type 1

Fanconi Bickel syndrome Carbonic anhydrase II deficiency Methylmalonic acidaemia Pyroglutamic aciduria

Cystic fibrosis

Xanthinuria

1-antitrypsin deficiency Cobalamin C deficiency Carbohydratedeficient glycoprotein syndrome type 1 Prolidase deficiency

Hereditary orotic aciduria Congenital lactase deficiency Hypophosphatasia (infantile form)

Carnitine palmitoyl transferase deficiency type 1 Adenosine deaminase deficiency Lysinuric protein intolerance

Gaucher disease

Carbohydratedeficient glycoprotein syndrome Glutaric aciduria type II (Neonatal onset) Smith–Lemli–Opitz syndrome Pearson syndrome Renal cystic disease and diabetes (RACAD)

Hereditary renal hypouricemia Blue diaper syndrome

Lysinuric protein intolerance Lipoprotein glomerulopathy

(such as cystinosis) also require significant nutritional support (e.g. nasogastric or gastrostomy feeds) as their appetite is often poor and they commonly have swallowing and feeding problems. Children with cystinosis have progressive glomerular damage leading to end-stage renal failure (ESRF) by the age of about 10 years. The combination of severe electrolyte imbalance, acidosis, poor feeding and the biochemical consequences of renal damage, and intracellular cystine accumulation all contribute to severe short stature.

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Cysteamine acts to reduce the intralysosomal cystine concentration and if given in sufficient doses early on in the disease, can promote growth and reduce the progression of glomerular damage (Markello et al. 1993). For those patients in ESRF, renal transplantation is successful and the disorder does not recur in the graft (although interstitial deposits of cystine are sometimes seen). However, renal transplantation does not correct the disorder and cystine continues to accumulate in non-renal tissues, causing multisystem dysfunction (delayed puberty, hypothyroidism, diabetes mellitus, myopathy, and central nervous system involvement; Gahl et al. 1995). It is therefore essential for patients to continue their cysteamine therapy even after transplantation. In the rarer late onset form of cystinosis, patients present after the age of 2 years with features of the Fanconi syndrome or simply with glomerular renal impairment (Goldman et al. 1971). An adult ‘benign’ form of cystinosis exists in which individuals have corneal cystine crystal deposition but no renal manifestations (Cogan et al. 1957). Lysosomal cystine accumulation occurs in cystinosis as a result of a defect in the transport of cystine across the lysosomal membrane (Gahl et al. 1982). Subsequently, lysosomal membrane transport systems have been described for other amino acids, monosaccharides, nucleosides, and various small molecules. Of these carriers only the sialic acid transporter has been isolated (Verheijen et al. 1999), and it is not known how these membrane proteins are synthesized, modified, targeted, or processed. Genetics Isolation of the cystinosis gene While extensive and elegant studies were carried out on the biochemical defect in cystinosis, it proved impossible to directly isolate the protein responsible for lysosomal cystine transport. A linkage study involving 23 families from Europe and North America, was therefore used to localize the cystinosis gene to the short arm of chromosome 17 with a Zmax of 9.87–10.89 for three markers (Cystinosis Collaborative Research Group 1995). No evidence for genetic heterogeneity was found, suggesting that all cases of nephropathic cystinosis are likely to result from mutations at this locus. One family, where the affected individuals were diagnosed as having late-onset cystinosis, also showed linkage to these markers. This supported earlier suggestions that the nephropathic and late-onset forms of cystinosis are allelic (Pellett et al. 1988). Recombination events in two cystinosis families defined the interval in which the gene should lie as a 4 cM region between markers AFMb307zg5 and AFMa061za9 (McDowell et al. 1996). Further, typing with a higher density of microsatellite markers provided new recombination events and using homozygosity mapping and linkage disequilibrium techniques, the region containing the cystinosis gene was narrowed to 1 cM (Jean et al. 1996). A positional cloning approach was used to isolate the cystinosis gene. An integrated genetic and physical (YAC) map of the cystinosis candidate region was established and the cystinosis interval was narrowed to an estimated distance of 200–500 kb. A microsatellite marker, which maps to the cystinosis region, was found to be deleted in 40 per cent of cystinosis patients, but not in unrelated controls. A series of overlapping cosmid clones (genomic DNA inserts of ~40 kb) which surround this microsatellite were identified and used to generate new markers, sequence-tagged sites (STSs), and to identify the position of potential candidate genes by exon trapping. By testing for the presence/absence of the STSs in DNA from patients the extent of the deletion was determined. Exons which fell within the deleted region were used

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to screen human cDNA libraries and a full-length cDNA was identified, which mapped to the deletion interval. Sequencing and characterization revealed a new gene, designated CTNS (Town et al. 1998). The CTNS and the predicted structure of cystinosin The CTNS gene is relatively small with 12 exons and an open reading frame of 1101 bp which starts in exon three. The gene is predicted to encode an integral membrane protein of 367 amino acids, cystinosin. A hypothetical structure for the protein, cystinosin is given in Fig. 12.1. This representation is based upon modelling with hydrophobicity algorithms, comparative analysis with the transmembrane proteins of yeast and C. elegans and observation of sequence motifs (Attard et al. 1999). The predicted model of cystinosin suggests a specific orientation across the lysosomal membrane based on the observation of a higher positive charge density (24 per cent) on the cytosolic side compared to the lysosomal side (8 per cent) of the membrane. Other lysosomal membrane proteins (Lamps and Limps) have a similar orientation and both cystinosin and the Lamps/Limps carry the lysosomal targeting motif at the cytosolic C-terminal (Hunziker and Geuze 1996). However, these proteins are even more heavily glycosylated towards the N-terminus than the seven or eight sites found in cystinosin, and they have only one, two, or four transmembrane domains compared to seven in cystinosin. Similarly, the plasma membrane cystine transporter -rBAT (mutated in cystinuria type 1) has at most four membrane-spanning domains (reviewed in Goodyer et al. 2000). This has now been shown to be a heterodimer which is active when complexed with a 40 kDa molecule which has 12 membrane spanning domains (reviewed in Goodyer et al. 2000). This is much more like the structure other membrane transport molecules cloned to date, for example, the recently cloned sialin, which is the sialic acid–glucuronic acid lysosomal membrane transporter defective in Salla disease and infantile sialic acid storage disoder (ISSD) (Verheijen et al. 1999). The G-protein coupled adrenergic receptors of endosomes,

Cytoplasm C–terminus Lysosomal membrane Lysosomal lumen

N–terminus

N–glycosylation sites

Fig. 12.1 Predicted structure of cystinosin (see text for details).


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have seven transmembrane alpha helices which have been suggested to form a ligand-binding ‘pocket’ (Lefkowitz and Caron 1988). They also have an extracellular N-terminus which is potentially N-glycosylated, and they have a cytoplasmic C-terminus. Similarly, CLN3, the integral membrane protein mutated in Batten disease, has six transmembrane domains with short cytosolic and intraorganelle domains (Janes et al. 1996). Although there is no amino acid identity between cystinosin and these receptors, the structural similarities support the hypothesis that the cystinosin protein functions as a membrane transporter or as part of a complex that binds cystine. Mutation analysis in cystinosis Initially, a variety of mutations in the CTNS gene were found in the cystinosis patients. These included a large deletion, encompassing exons 1–10 and another encompassing exons 1–3; small deletions of 1–9 bp; point mutations resulting in premature stop codons; and splice-site mutations. All of these mutations segregate with the disease in the families and are predicted to cause severe disruption of the gene, which provided definitive proof that CTNS is the cystinosis gene (Town et al. 1998). Subsequent work has confirmed that the most frequent mutation found in the CTNS gene of cystinotic patients is a large deletion which spans exons 1–10 of the cystinosis gene and actually encompasses approximately 57 kb of genomic DNA upstream (5) of the gene. Forty per cent of the cystinosis patients were found to be homozygous for this deletion. Many patients were also identified as heterozygous for the deletion (Anikster et al. 1999; Forestier et al. 1999). Characterization of the sequences around the breakpoints of this deletion showed that the 5 breakpoint of the large deletion mapped upstream of the CTNS gene, between two expressed sequence tags (ESTs) AA099495 and T85505. The 3 breakpoint occurs a few bases away from the end of CTNS exon 10. The breakpoints occur in regions containing several repetitive elements either from Alu, MIR, or LINE2 families (Smit 1996), but do not involve unequal recombination between two of them. The 57-kb deletion represents a clean breakage and end-joining mechanism with one nucleotide homology at the junction. This type of deletion junction, exhibiting limited homology (1–6 nucleotides) has been found in various hereditary diseases (Henthorn et al. 1990; Woods-Samuels et al. 1991), and indicates that a non-homologous recombination event underlies the mechanism of strand breakage and repair which gave rise to this deletion. Contrary to the recombination between homologous regions giving rise to recurrent rearrangements (Lakich et al. 1993), this mechanism favours the possibility of a founder effect for the mutation. This hypothesis is strengthened by the demonstration that the breakpoint sequence is strictly identical in all unrelated patients for whom the DNA has been sequenced. A founder effect probably occurred in a European patient, as all except two of the patients bearing the mutation, to date, are of European origin. To test the hypothesis of a founder effect, a common haplotype flanking the CTNS locus was sought. Strong linkage disequilibrium was not detected between the CTNS deletion and any haplotype across the region. However, a 215-bp allele of marker D17S1828 occurred more frequently (47 per cent) on the deleted chromosomes, as compared to the control chromosomes (26 per cent). This observation allowed an estimation of the age of the mutation using a simple derived formula (Risch et al. 1995), albeit with a broad 95 per cent confidence interval. The data inferred that the deletion event probably took place in the middle of the first millenium. This is in agreement with the estimation provided by Shotelersuk et al. (1998) who proposed that the deletion may

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have initially occurred in Germany prior to 700 AD, on the basis of the family history of their cystinotic patients of European descent. A high proportion (76 per cent) of the European patients carry the large deletion, and it is likely that this will be true of the general European population. By combining data from just two PCRs it is now possible to use a simple rapid molecular diagnosis test for cystinosis in Europe and for patients from European descent in other countries. One set of PCR primers (A) corresponds to an intragenic microsatellite marker located within the deletion and the other a set of primers (B), which were designed outside of the deletion boundary in nonrepeated areas, spans the deletion breakpoint junction. The latter set will give a 360-bp PCR product from DNA which contains the large deletion, but no product with normal DNA due to the large distance (58 kb) between the primers. By this method cystinotic individuals who are homozygous (B A) or heterozygous (B A) for the deletion can be detected and distinguished from non-deleted (B A) individuals (N). This methodology will be very useful for prenatal diagnosis and detection of carrier individuals in affected families, as well as for the diagnosis of new cases. Several groups have now carried out mutation analysis in cohorts of cystinotic patients and 55 different mutations have been described in approximately 300 patients (Attard et al. 1999; McGowan-Jordan et al. 1999; Thoene et al. 1999; Anikster et al. 2000). The first step is to identify those individuals who are homozygous for the large deletion by PCR. Patients who are heterozygous for the deletion or who do not have the major deletion are then screened using single strand conformation polymorphism (SSCP) analysis (Orita et al. 1989) covering the complete CTNS coding sequence and intron/exon boundaries (Town et al. 1998). In order to characterize each mutation direct sequencing is carried out for each exon displaying an abnormal SSCP band pattern. Overall, mutations have been detected in 90 per cent of cystinotic patients using this strategy. Patients recruited for the two largest studies (Shotelersuk et al. 1998; Attard et al. 1999) originate from various ethnic backgrounds but the majority are of European origin and the most common mutation (65 per cent) is the major deletion. A third study looked only at French Canadian patients where the incidence of this deletion is a lot lower (25 per cent). However, in this group an amino acid substitution, W138X, is present on 50 per cent of chromosomes and evidence for a common genetic background indicates that this mutation, like the major deletion, is due to founder effect (McGowan-Jordan et al. 1999). Two other mutations, a 4-bp deletion at nucleotides 357–360, and an insertion of C at nucleotides 1035–1036, occur in a repeat region and a CpG dinucleotide, respectively. No evidence for a common haplotype was found for these mutations indicating that these may represent mutational ‘hot spots’ within the CTNS gene (Attard et al. 1999). Correlation of genotype with phenotype In several studies (Shotelersuk et al. 1998; Town et al. 1998; Attard et al. 1999; McGowanJordan et al. 1999), it has been found that all cystinotic individuals who have truncating mutations on both alleles, likely to result in a complete loss of protein, have the most severe form of this disease, that is infantile nephropathic cystinosis. Although the function of cystinosin has yet to be defined, these mutations in the CTNS gene are consistent with the very severe phenotype of the disease and with the observations that patients have completely defective lysosomal cystine transport whereas obligate heterozygotes exhibit 50 per cent of normal transport activity (Gahl et al. 1995).


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However, among patients with this severe form of the disease, different missense mutations and in-frame deletions which are predicted to be non-truncating have also been found (Attard et al. 1999). Patients are either homozygous for the mutation or are heterozygous with a truncating mutation on the other allele. The missense mutations each result in substitution of an amino acid that is conserved between cystinosin and the transmembrane proteins of yeast and C. elegans (Town et al. 1998). These substitutions occur in putative transmembrane domains. Similarly, the in-frame deletions all occur at residues which are conserved between the three species and result in the loss of a highly conserved residues within or adjacent to transmembrane domains. Disruption of a transmembrane domain is likely to cause severe structural disruption to the protein. Since this protein is involved in cystine transport across the lysosomal membrane, then disruption to its orientation or localization would be predicted to severely reduce or ablate the function of cystinosin. In these cases the genotype is consistent with loss of protein function and, therefore, is consistent with the severe, infantile nephropathic phenotype of these patients. Mutations have also been described in the CTNS gene of patients whose phenotype is either typical of late-onset cystinosis or for whom the clinical course is generally milder than the classical nephropathic form of the disease (Attard et al. 1999; Thoene et al. 1999; Anikster et al. 2000). Five of these patients have at least one non-truncating mutation which is predicted to affect an amino acid within a non-conserved, functionally unimportant region of the protein molecule. These mutations include an in-frame deletion which results in the loss of seven amino acids within the first lysosomal luminal domain, a new splice site which results in the addition of three amino acids to the first cytosolic domain, and three missense mutations. Two patients with a milder phenotype have missense mutations which affect transmembrane domains, tm1 and tm7. In both cases a severely truncating mutation was found on the second allele, so it is presumably these missense mutations that still permit some functional protein to be made. A further two patients with a mild cystinotic phenotype and one patient with benign, ocular cystinosis have splice-site mutations predicted to result in the premature termination of the protein and the effective loss of the last two transmembrane domains and lysosomal targetting sequence (Thoene et al. 1999; Anikster et al. 2000). It is likely that in these cases an alternative splice site is used in some tissues, thus allowing the production of some full-length functional protein. Two patients with ocular cystinosis were found with the large deletion on one allele and a mutation that causes the substitution of arginine for glycine at amino acid 197. Again, this substitution is in a non-conserved region of the protein and likely to allow the production of sufficient cystinosin to prevent kidney damage but not enough to prevent crystal formation in the cornea. All of the late-onset and benign cystinosis patients have at least one mutation that is not predicted to result in the absence of mRNA or protein truncation. Although these mutations must affect the structure or function of the cystinosin protein, we speculate that for these patients there will be some protein product and hence some residual cystine transport activity. Functional studies will be required to confirm this but we believe that this would account for the heterogeneity in the severity of their symptoms and for their milder phenotypes. Prenatal diagnosis Accurate prenatal diagnosis for cystinosis is available. Two biochemical techniques can be used on chorion villus samples, either pulse-chase studies using 35S cystine or direct

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measurement of the intracellular cystine content, using the cystine binding protein assay (Gahl et al. 1995; Dumoulin et al. 1999). The high frequency of a mutation in the CTNS gene in European patients means that molecular prenatal diagnosis is also now feasible. Tyrosinaemia Clinical Tyrosinaemia type 1 is an autosomal recessive disorder due to a deficiency of fumaryl acetoacetase (FAH). Children present early in the first year of life with a combination of liver disease and renal tubulopathy. They have hepatosplenomegaly and develop hepatic cirrhosis with a risk of hepatocellular carcinoma. Renal involvement is with a severe Fanconi syndrome with rickets but in the long term, a substantial number of patients develop chronic renal failure. Affected children also suffer from recurrent episodes of polyneuropathy and from a cardiomyopathy. Fumaryl acetoacetase is a key enzyme in the degradation of tyrosine and its deficiency causes a build-up of metabolites, in particular succinylacteone and succinylacetoacetate (Lindblad et al. 1977). These substances can simulate a renal Fanconi syndrome when administered to animals (Wyss et al. 1992) and also inhibit porphobilinogen synthetase thereby causing the episodic neuropathy (akin to hepatic porphyria). The accumulation of succinylacetoacetone and subsequent metabolic derangements, can be prevented by treatment with 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) which inhibits 4-hydroxyphenylpyruvate dioxygenase (Lindstedt et al. 1992). Prior to the use of NTBC, patients were treated by liver transplantation but a number of survivors developed chronic renal failure (Freese et al. 1991; Laine et al. 1995). In a review of NTBC treatment, Holme and Lindstedt (1998) reported that more than 220 patients had received the drug with 90 per cent of those with acute tyrosinaemia showing a good response. If used early, NTBC also reduced the incidence of hepatocellular carcinoma. Genetics The FAH gene maps to chromosome 15q23–q25 (Berube et al. 1989). Tanguay et al. (1990) demonstrated molecular heterogeneity of mutations causing tyrosinaemia. Studies of liver tissue from tyrosinaemic patients has shown a mosaic pattern in immunoreactive FAH protein (Kvittingen et al. 1994). In some patients, areas of immunonegative liver tissue contained the FAH mutations demonstrated in fibroblasts of the patients, whereas in the immunopositive, regenerating liver tissue, the mutated alleles had reverted to the normal genotype. There is also evidence of a ‘pseudodeficiency’ FAH allele, which leads to production of FAH mRNA but a deficiency of immunoreactive protein (Rootwelt et al. 1994). The pseudodeficiency alleles had a C-to-T transition in nucleotide 1021, predicting an arg341-to-trp substitution which was shown to reduce FAH activity and produce reduced amounts of the full-length protein. There is a high prevalence of type 1 tyrosinaemia in a region of Quebec (1/1846 liveborn with a carrier rate of 1/20 inhabitants) due to a founder effect (De Braekeleer and Larochelle 1990). In other parts of the world, the incidence is estimated to be between 1 in 100,000 and 1 in 120,000. Using cDNA probes for the FAH gene, Demers et al. (1994) identified 10 haplotypes with 5 RFLPs in 118 normal chromosomes from the French-Canadian population.


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Among 29 children with hereditary tyrosinaemia, haplotype 6 was found to be strongly associated with disease, at a frequency of 90 per cent as compared with approximately 18 per cent in 35 control individuals. This frequency increased to 96 per cent in the 24 patients originating from the Saguenay-Lac St Jean region. Most patients were found to be homozygous for a specific haplotype in this population. Analysis of 24 tyrosinaemia patients from 9 countries gave a frequency of approximately 52 per cent for haplotype 6, suggesting a relatively high association worldwide. Genotype analysis has shown a number of mutations, spread throughout the gene, consisting of single-base substitutions resulting mainly in amino acid replacements (St-Louis and Tanguay 1997). The IVS12,G-A,5 mutation is the most frequent mutation in FrenchCanadian patients and is also the most common mutation in patients from Europe, Pakistan, Turkey, and the USA. The IVS6,G-T,1 transversion, encountered in 14 alleles, was common in Central and Western Europe (Rootweld et al. 1996). Although some of the mutations seemed to predispose for acute and others for more chronic forms of tyrosinaemia, there is no obvious correlation between genotype and phenotype. Detection of succinylacetone in the amniotic fluid (Gagne et al. 1982) or measurement of FAH in cultured amniotic cells (Kvittingen et al. 1983) allows prenatal diagnosis. For families with compound heterozygote genotypes for type I tyrosinaemia and pseudodeficiency, testing for the arg341-to-trp mutation may be helpful. Glycogen storage diseases affecting the kidney Clinical The glycogen storage diseases (GSD) are inherited disorders of the enzymes and transporters involved in the metabolism and regulation of glycogen (Chen and Burchell 1995). Of the many different forms, only two, GSD type 1 and the Fanconi–Bickel syndrome, have significant renal manifestations. In GSD type 1, there is a deficiency of glucose 6-phosphatase in the liver, kidney, and intestine, causing excessive glycogen storage. Children develop hepatomegaly, hypoglycaemia, lactic acidosis, hyperuricaemia, hyperlipidaemia, and have growth retardation. Variants include a form (type Ib) with neutropaenia leading to recurrent infections. Long-term complications include delayed puberty, hepatic adenomata, and renal disease. Renal manifestations are common but rarely serious in childhood. The kidneys are enlarged (principally due to glycogen accumulation) and patients have glomerular hyperfiltration, proteinuria, and histological changes of focal and segmental glomerular sclerosis. Chronic renal failure is a rare complication. Tubular dysfunction is also common, ranging from a mild proximal tubulopathy to hypercalciuria, distal renal tubular acidosis, and nephrolithiasis (Chen 1991; Reitsma-Bierens et al. 1992; Restiano et al. 1993; Lee et al. 1995). The key to treatment is to maintain a normal blood glucose level and this can be achieved by continuous administration of glucose or by frequent feeds with uncooked cornstarch. Several patients have had liver transplants but this has not prevented the long-term renal complications (Faivre et al. 1999). The Fanconi-Bickel syndrome (FBS) is a very rare autosomal recessive monosaccharide transport disorder. Children present in the first year of life with hepatomegaly (due to glycogen storage), hypoglycaemia, and a severe generalized proximal tubulopathy leading to rickets. There is marked glycosuria and galactosuria (Manz et al. 1987; Santer et al. 1998).

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Treatment is directed towards frequent feeds (and the use of uncooked cornstarch) together with management of the tubulopathy. Genetics The incidence of glycogen storage disorders is between 1 in 20,000 and 1in 25,000; GSD type I is the common form whereas FBS is extremely rare with less than 100 reported cases. The human D-glucose-6-phosphatase gene and the expressed protein were characterized by Lei et al. (1993). Several mutations in the G6PT gene that completely inactivate the enzyme, were identified in patients with type Ia glycogen storage disease. The gene consists of 5 exons, spans 12.5 kb and maps to chromosome 17q21. Using SSCP analysis and DNA sequencing to characterize the G6PT gene in 70 unrelated GSD Ia patients, Lei et al. (1995) detected mutations in 88 per cent alleles. Several of these mutations were shown to abolish or greatly reduce G6Pase activity. The most common mutations varied according to ethnic origin of the patients (R83C and Q347X in Caucasians, 130X and R83C in Hispanics; R83H in Chinese). Some mutations were specific to ethnic groups (Q347X in Caucasians, 130X mutation in Hispanics (Lei et al. 1995). In a Japanese group, a 91-nucleotide deletion in exon 5 of the G6PT cDNA, was found to be the disease-causing mutation in 91 per cent of patients and carriers of GSD Ia (Kajihara et al. 1995). In a French group of patients, 14 different mutations were found (100 per cent alleles) but five (Q347X, R83C, D38V, G188R, and 158Cdel) accounted for 75 per cent of the mutated alleles (Chevalier-Porst et al. 1996). Parvari et al. (1997) studied 12 Israeli GSD Ia patients of different families and found that all 9 Jewish patients, as well as a Muslim Arab patient, were found to have the R83C mutation. The ability to detect 100 per cent of mutations means that molecular genetic analysis can be considered an alternative to biochemical enzyme assay in liver biopsy material (Seydewitz et al. 2000). Santer et al. (1997) considered the GLUT2 facilitative glucose transporter, expressed in hepatocytes, pancreatic beta cells, and the basolateral membranes of intestinal and renal tubular epithelial cells, as a candidate for FBS. His group demonstrated mutations in the GLUT2 gene in three families with FBS. A functional defect in glucose and galactose transport explains the observed biochemical abnormalities in patients. Post-prandial hyperglycaemia (and hypergalactosaemia) occur due to first, decreased monosaccharide uptake by the liver and second, inappropriately low insulin secretion as the glucose-sensing mechanism in the pancreatic beta cells is abnormal. Pre-prandial hypoglycaemia occurs due to abnormal glucose transport out of the liver and due to excessive losses from the renal tubule. This could also increase the intracellular glucose level which inhibits glycogen breakdown, thereby causing glycogen storage and hepatomegaly (Santer et al. 1997).

Fabry disease Clinical Fabry disease is an X-linked lysosomal storage disorder due to a deficiency of the enzyme -galactosidase A (-gal A) which causes glycosphingolipids (predominantly globotriaosylceramide) to accumulate in plasma and tissues (Desnick et al. 1995). Affected males have distinctive but variable skin lesions (angiokeratoma), recurrent episodes of pain in the hands and


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feet (acroparaestheiae), hypohidrosis, and corneal and lenticular opacities. Older patients may develop proteinuria, renal failure, airway obstruction, and cardiac involvement (angina, left ventricular hypertrophy, valvular and conduction defects). Affected females are heterozygous and have a milder disorder (with renal but rarely skin involvement). The diagnosis may be confirmed by histological examination (e.g. in a renal biopsy) which shows birefringent glycosphingolipid deposits and on electron microscopy, the characteristic lysosomal inclusions or by demonstration of reduced -gal A activity in plasma, urine, leucocytes, or skin fibroblasts. The clinical presentation can be atypical. Some patients exhibit only the cardiac manifestations and in others the skin changes can be minimal and easily overlooked. This heterogeneity and the difficulty of heterozygote detection emphasize the importance of molecular investigations in this disorder. Genetics The incidence of Fabry disease has been estimated to be approximately 1 in 40,000 (Desnick et al. 1995). The GLA gene, mapped to Xq2 is 12 kb long and contains seven exons encoding a precursor protein of 429 amino acids. The -gal A exists as a homodimer comprised of two approximately 50-kDa subunits. A variety of gene re-arrangements have been identified in affected individuals. Mutations of all types (point mutations, deletions, insertions, etc.) and in all exons, have been identified in patients with typical Fabry disease (Germain et al. 1996; Topaloglu et al. 1999). Some, located at the central or 5-prime end of exon 6, have been associated with an atypical cardiac form of the disease. However, a clear genotype–phenotype link is not apparent. Mutations in the 3-prime end of exon 6 have been found in patients with typical Fabry disease. The same mutation (a G-to-A transition in exon 6 (codon 301) resulting in replacement of an arginine residue by glutamine) has been identified in a Japanese man who presented with late-onset cardiac involvement and in a 45-year-old man whose only manifestation was nephropathy (Sakuraba et al. 1990; Sawada et al. 1996). Miyamura et al. (1996) identified a mutation (tyr365ter) predicted to cause truncation of the C terminus by 65 amino acids. A heterozygote in this family had 30 per cent lymphocyte -gal A activity and a severe phenotype, despite evidence that the mutant and normal alleles were equally transcribed in cultured fibroblasts. In a series of transfection studies, these workers demonstrated that small deletions of the C-terminus enhanced enzyme activity whilst larger deletions (12 amino acids) led to a complete loss of enzyme activity. These experiments suggested that the C-terminal region of the GLA protein has an important regulatory role and help to explain some of the phenotypic heterogeneity. A GLA-knockout mouse has been created and shows a complete lack of -gal A activity and substrate accumulation (Oshima et al. 1997). Globotriaosylceramide (Gb3) accumulation and disease progression increased with age but bone marrow transplantation from wild-type mice led to an increase in GLA activity and a reduction in the accumulated Gb3, raising the possibility that bone marrow transplantation may be a useful therapy (Oshima et al. 1999). Schifferman et al. (2000) undertook a clinical trial to assess the efficacy of -gal A produced by transfection of human skin fibroblasts and administered by intravenous infusion. The enzyme replacement was well tolerated, led to detectable -gal A levels and was associated with a reduction in substrate in a variety of tissues.

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Lecithin-cholesterol acyl transferase (LCAT) deficiency Clinical LCAT is involved in the removal of excess cholesterol from peripheral tissues to the liver. It is synthesized in the liver and circulates in plasma as a complex with components of high density lipoprotein (HDL). Cholesterol from peripheral cells is transferred to HDL where LCAT causes the esterification of cholesterol with fatty acids derived from lecithin. The HDL complex can then be transported to the liver. Patients with LCAT deficiency are unable to esterify cholesterol in HDL, so that unesterified cholesterol and phosphatidylcholine accumulate in plasma and tissues (Glomset et al. 1995). The deficiency may be in alpha-LCAT, (specific for HDL) in fish-eye disease, or also involve beta-LCAT (specific for combined very low density lipoprotein (VLDL) and low density lipoprotein (LDL) in generalized LCAT deficiency (Norum disease). Patients with fish-eye disease only have ocular manifestations whereas those with Norum disease develop symptoms in late childhood including greyish corneal opacities, a target cell haemolytic anaemia, and proteinuria leading to renal failure in adult life (Imbasciata et al. 1986; Glomset et al. 1995). Manifestations typical of other disorders of lipoprotein metabolism (e.g. tendon xanthomata and atherosclerosis) can occur. Genetics The LCAT gene consists of six exons, maps to chromosome 16q22 (Azoulay et al. 1987), and is the site of the mutation in both Norum disease and fish-eye disease. Norum disease is inherited in an autosomal recessive manner. Mutations (mostly point) have been described throughout the gene. Some links between the genotype and phenotype have emerged. Two patients with severe renal manifestations were found to have separate mutations (the first a 3bp insertion in exon 4 causing the insertion of a glycine residue and the second, a replacement of asparagine-228 with positively charged lysine), both predicted to lead to complete loss of enzyme activity (Gotoda et al. 1991). In contrast, a patient who had corneal opacities and anaemia but no proteinuria was found to have a conservative amino acid substitution met293ile which would be predicted to cause only a partially defective enzyme. Bujo et al. (1991) contrast the complete loss of LCAT activity on HDL particle (alpha-LCAT activity) in fish-eye with a reduced activity in what is generalized LCAT deficiency (Norum disease).

Smith–Lemli–Opitz syndrome Clinical Smith–Lemli–Opitz syndrome is an autosomal recessive syndrome with multiple dysmorphic features resulting from a cholesterol synthesis defect (reviewed in Kelley and Hennekam 2000). Affected children have a characteristic facial appearance with a high forehead, ptosis, small mouth and mandible, cleft palate, abnormal ears, microcephaly, and cardiac and limb abnormalities. A variety of genito-urinary abnormalities have been described including hypospadias, ambiguous genitalia (male pseudohermaphroditism), renal hypoplasia or agenesis, pelvi-ureteric junction obstruction, duplex ureters, vesicoureteric reflux, and cystic dysplasia (Joseph et al. 1987; Cunniff et al. 1997). Patients have a low plasma cholesterol


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concentration with an elevation of plasma 7-dehydrocholesterol (the penultimate sterol in the cholesterol biosynthetic pathway) (Irons et al. 1993). This occurs due to a deficiency of 7-dehydrocholesterol-delta(7)-reductase (DHCR7). Cholesterol may be an essential nutrient in these patients and indeed treatment with cholesterol and bile salts can improve growth, speech, and well-being (Kelley and Hennekam 2000). Genetics and prenatal diagnosis Several families with SLO and translocations affecting chromosome 7 have been described (Berry et al. 1989; Wallace et al. 1994; Alley et al. 1995). The human DHCR7 gene is localized on 11q13 and mutations found in patients with SLO reduce protein expression by more than 90 per cent (Fitzky et al. 1998). Prenatal diagnosis of SLO syndrome can be undertaken by measurement of 7-dehydrocholesterol in amniotic fluid (Dallaire et al. 1995; Kratz and Kelley 1999) or in chorionic villus samples (Irons and Tint 1998). Methylmalonic acidaemia Clinical Methylmalonic acidaemia (MMA) is an autosomal recessive disorder of the metabolism of methylmalonyl coenzyme A due to a partial (mut-) or complete (mut-0) deficiency of methylmalonyl coenzyme A mutase or of the cofactor, adenosyl cobalamin (Fenton and Rosenberg 1995). Children usually present in the neonatal period or early infancy with lethargy, vomiting, poor feeding, failure to thrive, and recurrent metabolic acidosis. Patients who have a deficiency of the cofactor respond favourably to cobalamin supplements and have a good prognosis. In contrast, those with a complete deficiency of the methylmalonyl coenzyme A (mut 0) have a poor outcome, with progressive neurological and renal complications and approximately 50 per cent die within the first decade. In this group, there is evidence of both renal tubular and glomerular dysfunction. The tubulopathy is characterized by a urinary concentrating defect and/or acidification defect with huge salt and bicarbonate losses during episodes of metabolic decompensation (Ohura et al. 1990; D’Angio et al. 1991). In addition, patients with cobalamin-unresponsive MMA have progressive deterioration in glomerular filtration rate although this is not necessarily evident from routine monitoring of the plasma creatinine concentration (as the low protein intake and poor muscle mass reduces creatinine production) (Walter et al. 1989; van’t Hoff et al. 1998). Some patients develop hypertension, but typically there is no proteinuria or haematuria. Renal histopathology shows changes of tubulo-interstitial nephritis (Walter et al. 1989; Rutledge et al. 1993). The cause of renal dysfunction in MMA is not yet clear but may be related to toxicity of methylmalonate or an associated metabolite, recurrent episodes of acute tubular necrosis (due to dehydration during metabolic decompensation) or to hyperuricaemia. Patients are treated with a low protein and high (non-protein) diet together with broad spectrum antibiotics (to reduce gut flora production of propionate, a precursor of methylmalonate), allopurinol, and carnitine. The correct strategy for the management of their renal failure is not yet clear. Both isolated renal transplantation and combined liver–kidney transplants have been used (van’t Hoff et al. 1998, 1999). One other very rare disorder involving methylmalonic aciduria is associated with renal disease. Patients with a defect in

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the synthesis of the adenosylcobalamin and methylcobalamin have defective function of the two enzymes dependent on these cofactors (methylmalonyl CoA mutase and N5-methyltetrahydrofolate: homocysteine methyltransferase). These children therefore have homocystinuria in addition to methylmalonic aciduria and, in addition to multiple other problems, suffer from haemolytic uraemic syndrome (Fenton and Rosenberg 1995). Genetics The incidence of MMA has been estimated from neonatal screening programmes to be approximately 1 in 50,000 births (Fenton and Rosenberg 1995). Human methylmalonyl CoA mutase exists as a dimer (~145 to 150 kDa) of identical subunits (~72–77 kDa) (Kolhouse et al. 1980; Fenton et al. 1982). There are binding sites for methylmalonate and for the coenzyme (adenosylcobalamin). The MUT gene coding for the enzyme has been mapped to chromosome 6p12–p21.2 (Ledley et al. 1988) and mutations described in patients. Many patients are compound heterozygotes; Fenton et al. (1987) identified a mutation predicted to prevent transcription of the leader peptide necessary for processing of the enzyme precursor. Cell fusion studies using cells from a patient with mutase deficiency, with mut- and mut-0 cell lines led to complementation with 4 out of 5 mut-lines and 3 out of 9 mut-0 lines, suggesting interallelic complementation (Raff et al. 1991). The mutation in this line was a transition G354A, predicted to cause a substitution (R93H ) (Raff et al. 1991). Subsequent work has shown that most mutations that complement R93H, are located near to the C terminus of the protein, suggesting that these mutations complement a discrete function required for activity of R93H (Crane and Ledley 1994; Ledley and Rosenblatt 1997). Abramowicz et al. (1994) demonstrated paternal uniparental isodisomy in a newborn female with a mut-0 form of MMA and diabetes mellitus (due to complete absence of insulin-producing pancreatic islet beta cells). They concluded that the MMA was due to duplication of a mutated allele of the corresponding gene on the paternal chromosome 6.

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13 Genetics of stone forming diseases Pasquale Strazzullo and Pietro Vuotto

Introduction Nephrolithiasis is a common and heterogeneous disease manifesting itself most commonly during middle age (Preminger 1992). In particular, calcium nephrolithiasis is a health problem and a source of disability in all affluent countries with a reported prevalence between 4 and 10 per cent (Smith 1989). There is convincing evidence that nutritional and other environmental factors played an important role in the kidney stone disease (KSD) which developed in particular after the Second World War alongside improved living conditions and larger food availability. Nevertheless, it is clear that, given similar exposure to nutritional and other lifestyle risk factors, certain individuals are more susceptible than others to form renal calculi (with a high rate of recurrence). Most studies so far have focussed on the biochemical composition of the urine in an attempt to elucidate the reason for this greater predisposition to KSD and much is known today about the pathogenic role of several urinary constituents that exert either a stimulatory or an inhibitory effect on the formation of stones. Although some correlation can be appreciated between the urine biochemical composition and nutritional/environmental factors, the degree of association is insufficient to explain the large interindividual variation in this regard and thus the different individual predisposition to KSD. On the other hand, frequent familial occurrence of nephrolithiasis has emerged from numerous studies (Curhan et al. 1997) and, in fact, a positive family history is the most powerful predictor of risk after accounting for lifestyle and nutritional factors (Resnick et al. 1968). A group of rare Mendelian disorders, with an overall rate of 1 : 7000 to 1 : 200,000 individuals, set the stage for the role of genetic factors in the causation of KSD through alterations of the normal urine composition. The study of these disorders has provided fundamental information for the understanding of the molecular bases of nephrolithiasis: a short review of these disorders is thus one of the objectives of this chapter. Nevertheless, these disorders are very rare and do not account for the large majority of cases of KDS which are likely to have a polygenic mode of inheritance. Along this direction, recently a number of genes have been the object of intensive investigation, mainly through a classical candidate-gene approach and family linkage studies. Most studies have focussed on genes encoding for proteins involved in ion transport through the tubular and intestinal epithelia and/or in the regulation of urinary acid/base balance or in the modification of key urinary chemical constituents. A brief discussion of the main findings of these studies is the other objective of this chapter. Despite undeniable progress in the understanding of the biochemical basis of KSD in the last few decades, it is clear that much has still to be done for a thorough understanding of its heterogeneous genetic origins: to this purpose, both large scale population studies and carefully designed investigations of nuclear families are needed, with particular attention to gene–gene and gene–environment interactions.

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Pasquale Strazzullo and Pietro Vuotto

Hypercalciuric disorders Epidemiological and clinical features The term ‘idiopathic hypercalciuria’ was introduced to define the normocalcemic hypercalciuria commonly observed in calcium stone formers (Henneman et al. 1958). Given the common familial recurrence of idiopathic hypercalciuria, the term ‘familial hypercalciuria’ was also proposed (Coe et al. 1979). Later on, it was suggested that these definitions should be discarded on the basis of the evidence that distinguishes three main forms of hypercalciuria on pathophysiological grounds: absorptive, renal, and resorptive hypercalciuria (Pak 1998). We discuss all these conditions under the heading ‘primary hypercalciuric disorders’ to highlight both their etiopathogenetic heterogeneity and the exclusion from this group of hypercalciurias secondary to other concomitant disease states (Table 13.1). Hypercalciuria is detected in about 30–40 per cent of patients with calcium nephrolithiasis (Asplin et al. 1999). Diagnostic criteria are urinary calcium above 300 mg /24 h in men or 250 mg /24 h in women or 4 mg /kg of body weight /24 h with normal serum calcium concentration after exclusion of known causes of normocalcemic hypercalciuria. Three different pathogenetic mechanisms have been proposed for primary hypercalciuria: increased intestinal calcium absorption, decreased renal tubular calcium reabsorption, and increased bone turnover (Pak 1998). Patients with hypercalciuria have on average a rate of intestinal calcium absorption higher than that of normal subjects although with major overlap. On the other hand, they also excrete in the urine a greater percentage of absorbed calcium than normal subjects and manifest a trend to negative calcium balance and to lower bone mineral density (Bataille 1991; Borghi 1991; Zanchetta 1996). Pak and co-workers have described an ambulatory protocol to distinguish the different types of hypercalciuria (Levy et al. 1995). Absorptive hypercalciuria is the type most commonly diagnosed using this protocol; however, a number of patients show intermediate characteristics. Primary hypercalciuria is a multifactorial condition resulting from the interaction of genetic susceptibility with several metabolic and nutritional factors. Dietary sodium, potassium, and protein intake have all been shown to affect urinary calcium excretion (Leman 1979; Goldfarb 1988). Alterations of calcium metabolism including hypercalciuria have been described in patients with essential hypertension who are at increased risk of nephrolithiasis (Strazzullo 1991; Strazzullo and Cappuccio 1995; Borghi et al. 1999). Some hypercalciuric patients have increased production and serum concentration of vitamin D (1,25(OH)2-D3) (Kaplan et al. 1977; Pak 1979; Insogna et al. 1985), whereas hypercalciuric rats have been found to have high levels of vitamin D receptors with normal levels of vitamin D (Li et al. 1993). Increased activity of erythrocyte calcium–magnesium–ATPase and a significant direct correlation between pump activity and rate of calcium excretion was reported in patients with hypercalciuria (Bianchi et al. 1988), suggesting a role for the Ca-pump in the pathogenesis of hypercalciuria in as much as it is expressed both in the renal distal tubule and the intestine. Genetics The genetic bases of primary hypercalciuria have not been elucidated (Table 13.2). Polygenic inheritance appears to be very likely and the involvement of many plausible candidate genes has been proposed. One of these is the vitamin D receptor (VDR) gene. Spontaneously hypercalciuric rats have an increased number of vitamin D receptors in the intestine, bone, and

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Table 13.1 Primary hypercalciuric disorders: clinical features Urinary calcium 300 mg/24 h in men or 250 mg/24 h in women or 4 mg/kg of body weight/24 h with normal serum calcium concentration Calcium oxalate nephrolithiasis Increased intestinal calcium absorption Low bone mineral density Negative calcium balance Table 13.2 Genetic bases of stone forming disorders Candidate genes Primary hypercalciuria Vitamin D Receptor (VDR) gene Calcium Sensing Receptor (CaSR) gene Not known Not known Primary hyperuricosuria Not known Not known Primary hyperoxaluria Type I: AGXT Type II: GRHPR gene Type III: not known Primary hypomagnesaemia Paracellin-1 gene (PCLN-1) Cystinuria Type I: solute carrier family 3, member 1 gene (SLC3A1) Non type I (type II and type III): solute carrier family 7, member 9 gene (SLC7A9) and solute carrier family 7 member 10 gene (SLC7A10) Hartnup disease Not known Not known Dent disease Chloride channel gene (CLCN-5) Xanthinuria Type I: xanthine dehydrogenase gene (XDH) Type II: human molybdenum cofactor sulfurase gene (HMCS)

Chromosomal loci 12q12–14 3q13.3–q21 4q33–qter 1q23.3–q24

Mode of inheritance

Multifactorial

10q21–q22 20q13.1–13.3

Not known

2q36–q37 9 cen Not known

Autosomal recessive Autosomal recessive Not known

3q27

Autosomal recessive

2p16.3

Autosomal recessive

19q13.1

Incompletely autosomal recessive

11q13 5p15

Autosomal recessive Autosomal recessive

Xp11.22

X-linked recessive

2p22.3–p22.2

Autosomal recessive

Not known

Autosomal recessive

kidney and respond to very low doses of calcitriol by an upregulation of VDR gene expression (Yao et al. 1998; Bushinsky et al. 1999). Whereas Zerwekh and co-workers reported no evidence for VDR mutations or altered VDR expression in patients with absorptive hypercalciuria (Zerwekh et al. 1995, 1998), more recently the, VDR TaqI polymorphism has been associated with greater susceptibility and a higher rate of recurrence of calcium nephrolithiasis

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(Jackman et al. 1999). Moreover, evidence has been provided for linkage of calcium nephrolithiasis with microsatellite marker D12S339 near the VDR locus on chromosome 12q12–14, as well as with the flanking markers D12S1663 and D12S368 (Scott et al. 1999). The calcium sensing receptor (CaSR) gene is also a plausible candidate gene for primary hypercalciuria. CaSR is a 1078 amino acid protein that plays a key role in calcium homeostasis being expressed in parathyroid and C-cells, along almost the entire nephron and gastrointestinal tract and within numerous regions of the brain (Brown et al. 1998). It mediates the inhibitory effect of calcium on parathyroid hormone secretion and the stimulatory action of calcium on calcitonin secretion (De Luca and Baron 1998); it also directly controls the renal handling of divalent mineral ions in the cortical thick ascending limb and in the distal convoluted tubule. CaSR has a large extracellular amino-terminal domain, which binds calcium and polycationic agonists, a central core with seven membrane-spanning helices, identifying it is a G-protein-coupled receptor, with an approximately 200 amino acid carboxyl-terminal tail. The mechanism of signal transduction following calcium binding to extracellular sites is mediated by activation of phosphatidylinositol turnover and a rise in intracellular calcium levels. The CaSR gene is located on the long arm of chromosome 3 (3q13.3–q21) (Janicic et al. 1995). It spans about 20 kb and consist of 6 exons (Pollak et al. 1993). CaSR loss-offunction mutations cause familial hypocalciuric hypercalcaemia (Pollak et al. 1996). CaSR gene gain-of-function mutations have been reported that are associated with a leftward shift in the receptor dose-response curve causing hypocalcaemia with normal serum parathormone (PTH) levels, hypercalciuria and risk of nephrocalcinosis (Pearce et al. 1996; Scheinman et al. 1999). On the other hand, another recent study on a French Canadian population, using nonparametric linkage analysis could not confirm an association between the CaSR gene and idiopathic hypercalciuria and KSD (Petrucci et al. 2000). Thus, the pathogenic role of CaSR gene in hypercalciuria and KSD remains elusive. An unbalanced translocation with deletion of the 4q33–qter chromosomal segment has been associated with multiple malformations and absorptive hypercalciuria (Imamura et al. 1998). Reed et al. (1999) have also described in three kindreds a linkage of absorptive hypercalciuria with 1q23.3–q24 chromosomal locus in association with D1S318 and D1S196 markers in the region between D1S2681 and D1S2815. Management The treatment of familial hypercalciuria is essentially based on the administration of thiazide diuretics which have been proved in long-term trials to reduce calcium excretion and prevent the formation of stones. Some authors also advocate a low sodium diet (Saggar-Malik et al. 1996). Potassium citrate (15–30 mEq/day) is useful to prevent hypokalaemia and to increase urinary citrate excretion. Administration of cellulose phosphate has been used in absorptive forms of hypercalciuria (Pak 1998).

Primary hyperuricosuria Epidemiological and clinical features Isolated hyperuricosuria is found in approximately 10 per cent of patients with kidney stones. Most of these patients have calcium oxalate and/or calcium phosphate stones, provided that


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the urinary pH is greater than 5.5, which is the dissociation constant for the first proton of uric acid: under these circumstances urinary supersaturation with monosodium urate occurs and initiates the formation of calcium stones (Finlayson and Smith 1974). In other patients, who produce urine with unusually and persistently low pH (less than 5.5), both radiolucent uric acid calculi and calcium stones may be formed in different occasions. The reason for this very acid urine production has not been clarified. Hyperuricosuria is defined as a uric acid excretion greater than 600 mg (3.6 mmol)/day in repeated 24 h collections, with normal serum calcium, urinary calcium and oxalate excretion and normal fasting and calcium load response. Although the most common cause of hyperuricosuria is probably excess consumption of purine-rich foods, in a significant proportion of patients uric acid overproduction may be operating (Pak 1998). Uric acid stones are often observed in patients with gouty arthritis.

Genetics Apart from secondary causes of hyperuricosuria, such as all the disease states characterized by increased cellular turnover, well-studied enzymatic disorders of uric acid metabolism are hypoxanthine–guanine phosphoribosyl transferase deficiency (Lesch–Nyhan syndrome), phosphoribosyl pyrophosphate synthetase overactivity, and glucose-6-phosphatase deficiency (type 1 glycogen storage disease), in which the affected individuals have very high rates of uric acid production. In addition to these inborn errors of uric acid metabolism, a role for genetics in hyperuricosuric kidney stone disease is likely. A recent study in children with either hypercalciuria or hyperuricosuria and their first- and second-degree relatives showed that family history of nephrolithiasis was three-fold more common in both categories compared with control children (Polito et al. 2000). Another study, carried out by genome-wide search in a deep-rooted pedigree from a small isolated founder population in Sardinia with a high prevalence of uric acid stones, identified two chromosomal regions that may harbour loci with susceptibility genes for uric acid stones. The strongest evidence was observed on 10q21–q22 with a LOD score of 3.07 for D10S1652 under an affected-only dominant model. Suggestive evidence was also obtained in an approximately 20-cM region on 20q13.1–13.3 region (Ombra et al. 2001). An autosomal dominant disorder referred to as familial urate nephropathy (sometimes called familial hyperuricaemic juvenile nephropathy) was described which is characterized by hyperuricaemia, gouty arthritis, and nonspecific features of chronic interstitial nephropathy leading to early onset renal failure (Duncan and Dixon 1960; see also Chapter 19, this volume). The metabolic abnormality in these patients consists in a marked reduction in the fractional uric acid clearance in the absence of alterations in the uric acid production pathway (Moro et al. 1991). The underlying molecular mechanism is nevertheless still unknown. Management The management of hyperuricosuria is mainly based on the prescription of allopurinol (300 mg/day), particularly in patients with increased uric acid production but also in those with high consumption of purine, as dietary purine restriction is impractical and rarely successful. Potassium citrate is an effective alternative as it increases uric acid solubility, thus preventing the formation of both uric acid and calcium stones (Pak 1998).

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Hyperoxaluria Epidemiological and clinical features Hyperoxaluria is defined as a urinary oxalate output greater than 44 mg/day. It is clearly associated with increased risk of calcium nephrolithiasis because of enhanced urinary saturation of calcium oxalate. This in turn is determined by several concomitant factors such as increased oxalate excretion, hypocitraturia, hypomagnesuria, low urinary pH, and low urinary volume: all of these factors increase the likelihood of calcium oxalate stone formation (Table 13.3). Whereas no primary derangement in renal oxalate handling has been recognized, hyperoxaluria is due to increased serum concentration and increased renal filtered load of oxalate resulting from: (i) increased intestinal absorption associated with ileal disease (enteric oxaluria), the most frequent cause of hyperoxaluria; (ii) enzymatic disturbances in oxalate biosynthesis leading to overproduction (primary hyperoxaluria); (iii) more rarely, overindulgence in oxalate-rich foods or excess vitamin C supplementation. The primary hyperoxalurias are autosomal recessive disorders characterized by elevated urinary oxalate excretion due to inborn overproduction. Affected individuals present with recurrent calcium oxalate stones, nephrocalcinosis, and progressive renal failure (Milliner et al. 2001). Systemic oxalosis occurs at later stages. Death from renal failure may occur in childhood or in early adult life. Heart block may be caused by deposition of oxalate in the cardiac conduction system. Raynaud phenomenon, livedo reticularis, acrocyanosis, spasm of large arteries, intermittent claudication, and gangrene are expression of peripheral arterial deposition of oxalate with consequent spasm or occlusion (Shih et al. 2000). Soft tissue calcifications limiting motion of involved joints and frequent pathologic fractures, occurring in localized areas of massive calcium oxalate deposition and histiocytic destruction of bone, have been described (Chesney et al. 1983), as also have crystalline retinopathy and optic neuropathy (Small et al. 1990). In humans, oxalate is synthesized mainly via glyoxylate oxidation (Hagler and Herman 1973a,b). Overproduction may be caused by alterations at one of several steps in glyoxylate metabolism. Type I primary hyperoxaluria is characterized by glycolicaciduria (Danpure and Jennings 1988) and is caused by deficient activity of the peroxisomal alanine : glyoxylate aminotransferase (AGT), a pyridoxine-dependent enzyme which is expressed only in the liver (Danpure 1989). The AGT converts glyoxylate to glycine: a reduction of its activity leads to increased oxidation of glyoxylate to oxalate catalysed by cytosolic L-lactate dehydrogenase. The Table 13.3 Primary hyperoxalurias: clinical features Urinary oxalate output 44 mg/day Recurrent calcium oxalate stones, nephrocalcinosis and progressive renal failure Heart block Raynaud phenomenon, livedo reticularis, acrocyanosis, spasm of large arteries, intermittent claudication, and gangrene Soft tissue calcifications Pathologic fractures Crystalline retinopathy and optic neuropathy


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mechanisms of AGT deficiency are heterogeneous: in fact, 60 per cent of patients completely lack hepatic AGT activity, most of them showing no peroxisomal AGT immunoreactivity on liver biopsy whereas a few have some peroxisomal AGT immunoreactivity with no detectable enzymatic activity (Cooper et al. 1988). The remaining 40 per cent show detectable levels of AGT activity, but the enzyme immunoreactivity is localized in the mitochondria: this error in intracellular compartmentalization causes a marked reduction of enzyme activity because of the negative influence of mitochondrial micro-environment on AGT function (Danpure et al. 1989). Type II primary hyperoxaluria is characterized by L-glycericaciduria and is due to deficient D-glyceric dehydrogenase activity (Williams and Smith 1968). This enzyme is responsible for the conversion of hydroxypyruvate to D-glycerate but also has glyoxylate reductase activity (Seargeant et al. 1991). It is thus commonly referred to as glyoxylate reductase/ hydroxypyruvate reductase (GRHPR). The decreased D-glyceric dehydrogenase activity leads to an accumulation of hydroxypyruvate which is then converted to L-glyceric acid by L-lactate dehydrogenase: this reaction generates NAD. On the other hand, the decrease in glyoxylate reductase activity leads to accumulation of glyoxylate. Simultaneous availability of large amounts of glyoxylate and NAD promotes the conversion of glyoxylate to oxalate catalysed by L-lactate dehydrogenase. The D-glyceric dehydrogenase with glyoxylate reductase activity is mainly confined to the liver whereas other enzymes with only D-glyceric dehydrogenase activity are present in other tissues (Giafi and Rumsby 1998). Another type of primary hyperoxaluria characterized by hyperglycoluria with normal L-glyceric acid and AGT activity has been described (van Acker et al. 1996). The metabolic defect of this type of hyperoxaluria has not been elucidated. It seems to be inherited as an autosomal dominant trait. Enzymatic assays on percutaneous hepatic needle biopsy are still the most effective diagnostic tool for the diagnosis of hyperoxaluria pending progress in genetic analysis (Danpure et al. 1987; Rumsby 2000). Techniques for prenatal diagnosis are based on metabolite analysis of amniotic fluid in the second trimester, AGT assay, immunoassay, and immunoelectron microscopy of fetal liver biopsy in the second trimester, and linkage and mutational analysis of DNA from chorionic villi in the first trimester (Danpure and Rumsby 1996). Genetics The molecular bases of hyperoxaluria type I (Table 13.2) have been clarified to a large extent. The human liver-specific peroxisomal AGT gene (AGXT), is localized to the 2q36–q37 region (Purdue et al. 1991). The AGXT coding region is constituted of 11 exons spanning about 10 kb. Several mutations of AGXT have been described (Purdue et al. 1990; Nishiyama et al. 1991; Purdue et al. 1992; Danpure et al. 1993; van Schnakenburg et al. 1997; Pirulli et al. 1999; Basmaison et al. 2000; Nogueira et al. 2000; Amoroso et al. 2001; Coulter-Mackie et al. 2001). Some of these are nonsense mutations or missense mutations resulting in amino acidic substitutions that directly impair AGT catalytic activity. Moreover, other mutations interfere with the intracellular trafficking of AGT. Pro11-Leu substitution generates an amphiphilic alpha-helix with characteristics similar to mitochondrial targeting sequences. Gly170Arg and Phe152Ile substitutions, located in the same highly conserved internal region of 58 amino acids, disrupt peroxisomal targeting sequence. If these two types of mutation are present in the same allele, the result is the peroxisome to mitochondrion mistargeting of

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expressed AGT. Gly41Arg substitution, either in combination with the Pro11Leu polymorphism or by itself, is responsible for the intraperoxisomal aggregation of the AGT protein (Purdeu et al. 1990; Purdue et al. 1992; Danpure et al. 1993). Important information has been obtained recently on the genetics of hyperoxaluria type II. The human GRHPR gene has been localized on chromosome 9: it is structured in nine exons and eight introns and encodes a predicted 328 amino acid protein. Four patients from two independent families were found to be homozygous for a single nucleotide deletion at codon 35 in exon 2, resulting in a premature stop codon at codon 45 (Cramer et al. 1999). Later on, other six mutations of this gene have been described in affected patients: a nonsense mutation (C295T) resulting in a premature stop codon at codon 99, a 4-bp deletion in the 5 consensus splice site of intron D resulting in a predicted spicing error, three missense mutations including a T965G transversion in exon 9 (Met322Arg), a G494A transition in the putative cofactor binding site in exon 6 (Gly165Asp) and an A → G substitution in the 3 splice site of intron G (Webster et al. 2000), and finally the deletion of the last two nucleotides of exon 8 resulting in a frameshift and the introduction of a stop codon at codon 310 (Lam et al. 2001). Management Treatment is essentially based on pyridoxine supplementation in patients with type I primary hyperoxaluria and residual AGT activity in liver biopsy samples (Yendt et al. 1985). Longterm neutral orthophosphate and pyridoxine supplementation is useful in preventing end stage renal disease (Milliner et al. 1994). Oral potassium and magnesium citrate administration and maintenance of a high fluid intake reduce the rate of stone formation (Leumann et al. 1993; Pak 1998). Successful treatment with combined liver and renal transplantation has been reported (Watts et al. 1987; McDonald et al. 1989). Primary hypocitraturia Epidemiological and clinical features Hypocitraturia is a powerful risk factor for kidney stone disease. It is defined as a urinary citrate excretion of less than 320 mg (1.7 mmol)/day. Citrate reduces the urinary saturation of calcium oxalate and calcium phosphate and is an effective inhibitor of crystal growth (Meyer et al. 1975; Pak et al. 1982). Alkalosis, PTH, and vitamin D enhance urinary citrate excretion whereas acidosis, hypokalaemia, and urinary tract infection tend to decrease it. Acidosis lowers urinary citrate by both enhancing tubular reabsorption and impairing peritubular uptake and synthesis of citrate, a mechanism that accounts for the hypocitraturia occurring in distal renal tubular acidosis (Morrissey et al. 1963) and in chronic diarrhoeal disorders with intestinal alkali loss (Rudman et al. 1980) and hypokalaemia (Nicar et al. 1984). Distal renal tubular acidosis (type I) is the only acidification defect associated with KSD: the formation of renal calculi is the result of both elevated urinary pH and calcium and low urinary citrate concentration. The typical calculus is made of hydroxyapatite and, to a lower extent, of calcium oxalate. This subject is not discussed here as it is extensively treated in Chapter 14. In many patients presenting with KSD and hypocitraturia, the cause of the low urinary citrate excretion is not known (idiopathic hypocitraturia). Often in these patients low urinary citrate excretion is associated with hypercalciuria or hyperuricosuria. Typically, in these forms the renal calculi are made of calcium oxalate (Pak and Fuller 1986).


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Genetics The genetic background of idiopathic hypocitraturia remains elusive at present time. For the molecular alterations in patients with renal distal tubular acidosis. Patients with autosomal dominant polycystic kidney disease have a greater risk of nephrolithiasis: recently, it has been seen that reduced urinary concentration of inhibitors of of stone formation such as citrate and magnesium are common features in these patients (Grampsas et al. 2000). Hypocitraturia has also been reported as a risk factor for the more common occurrence of nephrocalcinosis and nephrolithiasis in metabolically compensated older patients with type 1 glycogen storage disease (Weinstein et al. 2001). Management Potassium citrate, given orally in three to four divided doses, is an effective treatment for all forms of hypocitraturia and is able to restore normal urinary citrate excretion and pH (Pak et al. 1994). Primary hypomagnesaemia Epidemiological and clinical features Primary hypomagnesaemia, also termed familial hypomagnesaemia with hypercalciuria and nephrocalcinosis (Praga et al. 1995), is an autosomal recessive disorder characterized by renal magnesium wasting and hypercalciuria (Table 13.4) (Friedman et al. 1967; Shalev et al. 1998). Calcium nephrolithiasis and bilateral nephrocalcinosis in these patients are frequently complicated by recurrent urinary tract infections, nephrogenic diabetes insipidus, and distal tubular acidosis (Manz et al. 1978; Bianchetti et al. 1993). Progression to end-stage renal disease has been described (Bianchetti et al. 1993; Praga et al. 1995). Hypocalcaemia and hypomagnesaemia may lead to spasms, tetany, seizures, paresthesias, muscle weakness, and

Table 13.4 Primary hypomagnesaemia: biochemical and clinical features Renal magnesium wasting with hypomagnesaemia Hypercalciuria and hypocalcaemia High serum PTH levels Hypocitraturia Calcium nephrolithiasis, nephrocalcinosis, recurrent urinary tract infections, nephrogenic diabetes insipidus, distal tubular acidosis, progression to end-stage renal disease Spasms, tetany, seizures, paresthesias, muscle weakness and permanent neurologic impairment beginning in the first 3 months of life Tissue deposition of calcium: calcification of the basal ganglia, corneal calcification and chondrocalcinosis with crystal arthropaty Chorioretinitis, keratoconus, macular colobomata, myopia, and nystagmus Male infertility due to severe oligospermia Sensorineural deafness

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permanent neurologic impairment beginning in the first 3 months of life (Friedman et al. 1967; Shalev et al. 1998). Calcification of the basal ganglia, corneal calcification, and chondrocalcinosis with crystal arthropathy are expression of the abnormal tendency to tissue deposition of calcium. Chorioretinitis, keratoconus, macular colobomata, myopia, and nystagmus are frequently observed (Praga et al. 1995; Torralbo et al. 1995). Male infertility due to severe oligospermia and sensorineural deafness have also been reported (Evans et al. 1991). Low citrate excretion (Manz et al. 1978) and high serum PTH levels (Praga et al. 1995) complete the typical biochemical picture. Genetics Primary hypomagnesaemia is caused by mutations in paracellin-1 gene (PCLN-1) (Table 13.2). This gene is localized to the long arm of chromosome 3 (3q27) and consists of five exons. The 3.5 kb mRNA encodes the 305-aminoacid protein paracellin-1 (claudin-16), which has four transmembrane domains and intracellular N- and C-termini. Expression studies have shown that paracellin-1 mRNA is expressed in the kidney only in the thick ascending limb of Henle’s loop and in the distal convoluted tubule. Paracellin-1 shares sequence and structural homologies with members of the claudins family, a class of transmembrane proteins which localize to tight junctions. It also co-localizes with occludin, an integral membrane protein localizing at tight junctions. These data indicate that paracellin-1 is a specific structural component of tight junctions of some renal tubule tracts. Magnesium reabsorption is predominantly due to paracellular conductance in the thick ascending limb of Henle’s loop, a site of high expression of PCLN-1. Tight junctions regulate paracellular conductance and paracellin-1 is involved in the selective regulation of magnesium absorption. Ten different mutations, including nonsense, missense, and splice-site mutations of PCLN-1 were first described in ten kindreds with primary hypomagnesaemia (Simon et al. 1999). More recently, comprehensive clinical data have been reported for 33 affected individuals from 25 families first seen at a median age of 3.5 years (Weber et al. 2001). Genotype analysis revealed PLCN-1 mutations in all except three mutant alleles, 48 per cent of them involving a Leu151Phe substitution. Eight novel mutations were observed. Noteworthy, in 13 of 23 families, hypercalciuria and/or nephrolithiasis were observed in otherwise unaffected family members suggesting a possible role of heterozygous PCLN-1 mutations in predisposing to KSD. Management Treatment is essentially based on oral or intravenous replacement (Oster and Epstein 1988). In refractory patients potassium sparing diuretics may be useful, in reducing renal magnesium wasting (Bundy et al. 1995). Aminoacidurias Cystinuria Epidemiological and clinical features Cystinuria is a hereditary form of aminoaciduria characterized by recurrent urinary stones and frequently complicated by urinary tract obstruction, infection, and renal failure. It is an


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autosomal recessive disease caused by defective transport of cystine and the dibasic aminoacids ornithine, lysine, and arginine across the luminal membrane of renal proximal tubule and small intestine (Dent and Rose 1951; Milne et al. 1961; Bonnardeaux and Bichet 1999). Cystinuria is one of the most frequent mendelian disorders (1 in 7000). Cystine is sparingly soluble in urine; it is increased at higher pH but rarely exceeds 400 mg/L. At greater concentrations cystine stones may form. Three different types of cystinuria have been described, with regard to amounts of cystine in the urine of the patient and his/her parents. Type I cystinuria is a completely recessive disease in which the homozygous proband excretes large amounts of cystine (more than 250 mg/g creatinine, on average 1000 mg) and parents, who are heterozygous obligate carriers, or other heterozygous relatives excrete normal amounts of the aminoacid. Type II and type III cystinuria are incompletely recessive diseases in which not only the homozygous proband but also his/her parents, who are obligatory heterozygotes, and other heterozygous relatives excrete greater than normal amounts of cystine (usually 240–420 mg/g creatinine in type II and 24–144 mg/g creatinine in type III). Accordingly, contrary to type I heterozygotes, types II and III heterozygotes share with the proband an increased risk of developing urinary stones. Moreover, differences in intestinal amino acid transport have been observed. In type I cystinuria, there is a severe defect of the intestinal transport of all three dibasic amino acids; in types II and III, some cystine is taken up by the jejunal mucosa but at a reduced rate. In type III patients, an oral cystine load causes a blunted-to-nearly-normal elevation of serum cystine at variance with type I and type II patients (Goodyer et al. 1998). Phenotypic variation may be the expression of genetic heterogeneity in this condition (Goodyer et al. 1998; Goodyer et al. 2000; Langen et al. 2000). Genetics Mutations in the genes encoding for members of the heteromeric amino acid transporter (HAT) family are the molecular bases of cystinuria (Table 13.2). The heteromeric amino acid transporters are composed of two polypeptide units: a heavy subunit (HSHAT) and a light subunit (LSHAT) linked by a disulphide bridge. HSHATs are N-glycosylated type II membrane glycoproteins, whereas LSHATs are nonglycosylated polytopic membrane proteins. The HSHATs have been known since 1992 whereas the LSHATs have been described only in the last few years. Functional alterations in either a heavy or a light subunit may determine a defect in amino acid transport. Two different genetic loci involved in cystinuria have been characterized so far. The type I cystinuria gene has been identified as the solute carrier family 3, member 1 gene referred to as SLC3A1. This gene spans about 45 kb and is located to the short arm of chromosome 2 (2p16.3) (Calonge et al. 1995; Pras et al. 1996). The coding region is composed of 10 exons and encodes a 633 amino acid protein sharing 86 per cent homology with the rabbit homologue rBAT. The human amino acid transporter protein, also referred to as human rBAT, is expressed strongly in the kidney and intestine. It elicits sodium-independent, high affinity obligatory exchange of cystine, dibasic amino acids, and some neutral amino acids (Chillaron et al. 1996). It has been proposed that human rBAT protein forms part of heteromultimeric amino acid transporters (Palacin et al. 2001). The non-type I cystinuria gene has been localized to the long arm of chromosome 19 (19q13.1) (Bisceglia et al. 1997; Stoller et al. 1999; Font et al. 2001). It is referred to as solute carrier family 7, member 9 gene (SLC7A9), is composed of 13 exons and encodes

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a 487 amino acid protein, designated b(o)AT for b(o) amino acid transporter, belonging to the HAT family light subunits and expressed in kidney, liver, small intestine, and placenta. This protein has been supposed to form a heterodymeric complex with rBAT mediating the uptake of cystine and the dibasic amino acids at the luminal surface: in contrast to this view, however, recent immunofluorescence studies showed that while rBAT is most abundant in the proximal straight tubule, b(o)AT is most abundant in the proximal convoluted tubule of the nephron. Thus, it is possible that other components of both the heavy and the light subunit families of the HAT system are involved in the determination of cystinuria and related abnormalities of dibasic amino acid transport (Palacin et al. 2001). Over 60 missense mutations, deletions, insertions, donor splice-site mutations, nonsense mutations, and complex mutations of SLC3A1 have been described in patients with type I cystinuria (Goodyer et al. 2000; Langen et al. 2000; Bisceglia et al. 2001; Harnevick et al. 2001), the two most common mutations being M467T and T216M. Thirty five missense and two frameshift mutations of SLC7A9 have been identified in nontype I cystinuric patients, mutations G105R, V170M, A182T, and R333W being the most frequent (Font et al. 2001). It has recently been observed that a different gene (SLC7A10), similar to SLC7A9, also maps to the 19q13.1 chromosomal region and is highly expressed in the kidney. The homologies between SLC7A9 and SLC7A10 may be the result of gene duplication. The SLC7A10 contains 11 exons and encodes a protein with a function similar to that of the SLC7A9 gene product. One missense mutation and one intronic change in this gene have been associated with cystinuria in a few patients. On this basis it has been suggested that the SLC7A10 gene might be a second candidate gene for non-type I cystinuria (Leclerc et al. 2001). Management Treatment of cystinuria is based on dietary restriction of methionine and sodium intake, increased fluid intake up to 4 l a day, urine alkalinization with potassium citrate, sodium bicarbonate, and possibly carbonic anhydrase inhibitors and thiols, for example, D-penicillamine and tiopronin (Bonnardeaux and Bichet 1999). Recently, data have been reported about 27 adult patients followed for 1.3–32 years (mean 11.6, overall 312 patient-years). The study showed that compliance with a medical programme based on high diuresis and alkalization with second line addition of thiols may arrest or markedly decrease cystine stone formation and preclude the need for urological procedures in more than half of the patients. By contrast, patients poorly compliant with hyperdiuresis remain at high risk for recurrence (Barbey et al. 2000). Hartnup disease Epidemiological and clinical features Hartnup disease is an autosomal recessive disorder characterized by photosensitive erythematous pellagra-like skin rash, emotional instability, intermittent cerebellar ataxia, and increased urinary excretion of monoamino, monocarboxylic neutral alpha-amino acids (Table 13.5). Its incidence in metabolic screening programmes of newborns is 1 in 26,000 (Levy 1995). Most patients with Hartnup disease are symptom-free and newborns identified in metabolic screening programs remain asymptomatic during long periods of follow-up (Wilcken et al. 1977). Hartnup disease is a benign condition: its clinical expression seems to


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Table 13.5 Hartnup disease: clinical features Photosensitive erythematous pellagra-like skin rash Emotional instability Intermittent cerebellar ataxia Increased urinary excretion of monoamino, monocarboxylic neutral alpha-amino acids

be modulated by associated environmental and genetic modifiers (Scriver et al. 1987). The pathogenic mechanism of Hartnup disease involves an impairment of intestinal and renal transport of neutral amino acids alanine, asparagine, glutamine, histidine, isoleucine, leucine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, and valine. Some patients, while presenting full spectrum aminoaciduria, have no evidence of intestinal transport defects or have only partial intestinal transport defects under loading conditions. These findings suggest genetic heterogeneity of the disease (Levy 1995). Thus, there appear to be two distinct major forms of Hartnup disease: a classic form characterized by evidence of amino acid transport defects both in kidney and intestine, and a variant form with only a renal defect (Scriver 1985). Clinical expression of disease is related to abnormalities in the intestinal absorption of neutral amino acids, particularly tryptophan, rather then to increased urinary output. The clinical features are caused by nicotinamide deficiency, which is a consequence of tryptophan deficiency. Full expression of the clinical manifestations is triggered by inadequate dietary intake or by increased metabolic needs, related to physiological states, such as lactation, or increased physical activity (Oakley and Wallace 1994). Genetics Hartnup disease is transmitted as an autosomal recessive character (Table 13.2) but the heterogeneity of clinical expression suggests a multifactorial mechanism, with underlying polygenic control of amino acid metabolism and triggering environmental factors, that influence the phenotypic expression of the ‘Hartnup gene’ (Scriver et al. 1987). An animal model of Hartnup disease, the HPH2 mouse, has been described: the pattern of aminoaciduria and niacin deficiency symptoms are similar to those of patients with Hartnup disease. Interestingly HPH2 mouse exhibits a niacin-reversible syndrome that is modified by diet and by genetic background. The increased urinary excretion and the normal plasma concentration of neutral amino acids and the reduction of glutamine uptake by kidney cortex brush border membrane vesicles suggest an amino acid transport defect. The genetic locus involved in this recessive disorder, the HPH2 locus, maps to mouse chromosome 7, close to D7Nds4, a marker in the fibroblast growth factor 3 (FGF3) gene. In the human genome fibroblast growth factor 3 gene is located to the long arm of chromosome 11 (11q13). On these bases, it was proposed that the candidate gene of Hartnup disease may map to 11q13 (Symula et al. 1997). More recently, linkage analysis was performed in two Japanese first cousin-marriage families with several affected members. Genome-wide screening by homozygosity mapping located a locus associated with Hartnup disease on 5p15 (Nozaki et al. 2001). Management Treatment is essentially based on nicotinamide supplementation both in symptomatic subjects and, as a preventive measure, in asymptomatic ones (Bonnardeax and Bichet 1999).

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Dent disease (X-linked hypercalciuric nephrolithiasis) Epidemiological and clinical features Dent disease is an uncommon form of Fanconi syndrome characterized by low molecular weight proteinuria and hypercalciuria with calcium nephrolithiasis (Dent and Friedman 1964). It is caused by inactivating mutations of a renal chloride channel gene (CLCN-5) (Tables 13.6 and 13.7). Three other clinical pictures caused by the same mutations have been described besides Dent disease: X-linked recessive nephrolithiasis with renal failure (Frymoyer et al. 1991), X-linked recessive hypophosphataemic rickets (Bolino et al. 1993), and low molecular weight proteinuria with nephrocalcinosis (Igarashi et al. 1995). These syndromes differ in the extent and severity of bone deformities and in the degree of renal impairment (Thakker 2000). These different clinical expressions of similar inactivating mutations of chloride channels are probably due to genetic and/or environmental modifiers (Bonnardeaux et al. 1997). This group of renal tubular disorders is further characterized by other variable signs of impaired proximal tubular solute reabsorption, namely aminoaciduria, glycosuria, and phosphate wasting. Hypophosphataemic rickets with severe limb deformities, shortness of stature, and hypokalaemia occurs in some affected subjects. Abnormalities of urinary acidification are rarely observed. Genetics An X-linked recessive inheritance for Dent disease was suggested by the marked predominance and the greater severity of clinical features of the disease in males (Table 13.1), and by the absence of male-to-male transmission (Wrong et al. 1994). Family linkage studies detected a chromosomal microdeletion of approximately 515 kb in size involving the hypervariable locus DXS255 in association with Dent disease, leading to localization of the candidate gene to the short arm of the X chromosome (Xp11.22) (Pook et al. 1993; Scheinman et al. 1993). This gene encodes a protein of 746 amino acids, with significant Table 13.6 Clinical syndromes related to chloride channel gene (CLCN-5) mutations Dent disease X-linked recessive nephrolithiasis with renal failure X-linked recessive hypophosphataemic rickets Low molecular weight proteinuria with nephrocalcinosis

Table 13.7 Dent disease and related renal tubular disorders: clinical features Low molecular weight proteinuria Aminoaciduria, glycosuria and phosphate wasting Hypercalciuria Hypophosphataemic rickets with severe limb deformities, shortness of stature Hypokalaemia Abnormalities of urinary acidification Calcium nephrolithiasis


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homologies with all known members of the voltage-gated chloride channel gene family (Fisher et al. 1995). The novel chloride channel was referred to as CLC-5 and the gene as CLCN5. The human CLCN5 consists of 12 exons, spanning 25–30 kb of genomic DNA (Fisher et al. 1995). The coding region of 2238 bp is organized in 11 exons (2–12) and 10 introns. The 9.5 kb mRNA transcript is expressed predominantly in the kidney and to a lesser extent in the placenta and in the skeletal muscle. The CLC channels, consisting of about 12 transmembrane domains, function as multimeric complexes (Middleton et al. 1996). Heterologous expression of wild-type CLCN5 in Xenopus oocytes showed that CLC-5 conducts outwardly rectifying chloride currents, which are either completely suppressed or markedly reduced if mutant CLCN5 is expressed (Lloyd et al. 1996). At present, 49 inactivating mutations of CLCN5 have been described in 60 families (Lloyd et al. 1996; Hoopes et al. 1998; Igarashi et al. 1998; Scheinman 1998; Cox et al. 1999; Yamamoto et al. 2000). Lloyd and co-workers showed that all disease-related missense mutations affect the predicted transmembrane domain; donor splice site mutations result in in-frame deletion of predicted transmembrane domain D2; whereas nonsense mutations delete the highly conserved cytoplasmic domain D13. CLCN5 is expressed in the epithelial cells lining the proximal tubule and the thick ascending limb of Henle’s loop, and in acid-secreting alpha-intercalated cells of the collecting duct. It co-localizes with proton ATPase and with internalized proteins early after uptake in the subapical endosomes of epithelial cells of proximal tubule. In alpha-intercalated cells of the collecting duct it also co-localizes with the proton pump. CLC-5 channels have a distribution closely related to that of Rab4, a marker of recycling early endosomes. It may be essential for proximal tubular endocytosis dissipating the positive charges generated during the acidification of vesicles by the proton ATPase (Gunther et al. 1998; Devuyst et al. 1999). Loss of function of CLCN5 could impair endosome acidification and thus membrane protein recycling: in turn, this could results in defective reabsorption of proteins and other solutes. The mechanism of hypercalciuria in X-linked hypercalciuric nephrolithiasis is not yet clarified. It may be a consequence of hypophosphataemia and of increased serum levels of 1,25(OH)2D3 due to renal phosphate leak. On the other hand, as CLC-5 is expressed in the thick ascending limb of Henle’s loop, a site of calcium reabsorption, impairment of the endosomal pathway could result in abnormal recycling of transporters and/or channels involved in tubular calcium handling or in decreased reabsorption of a regulatory protein (Gunther et al. 1998). Noteworthy, a recent study was unable to detect mutations of CLCN5 in a series of 32 patients with idiopathic hypercalciuria (Scheinman et al. 2000). Management Treatment of X-linked hypercalciuric nephrolithiasis is based on supportive measures. Hypercalciuria and renal stones are better treated with increased fluid intake. It has been suggested to avoid calcium restricted diet and thiazide diuretics because of increased risk of metabolic bone disease and salt loosing nephropathy (Wrong et al. 1994; Scheinman 1998). Small doses of vitamin D may be required to treat rickets, if present, but appropriate dosage requires frequent monitoring of urinary calcium output because of the enhanced risk of nephrolithiasis and nephrocalcinosis (Wrong et al. 1994).

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Xanthinuria Epidemiological and clinical features Xanthinuria is an autosomal recessive disorder of purine metabolism characterized by urinary excretion of large amounts of xanthine and recurrent xanthine lithiasis (Dent and Philpot 1954). End-stage pyelonephritis, non-functioning hydronephrotic kidney and a myopathy with crystalline deposits may occur (Simmonds et al. 1995). Xanthine calculi account for less than 0.04 per cent of analysed urinary calculi (Gleeson et al. 1992). Xanthinuria is due to a deficit of xanthine oxidase, an enzyme involved in the purine degradation pathway, which catalyses the oxidation of hypoxanthine to xanthine and of xanthine to uric acid (Dent and Philpot 1954). Therefore, serum and urine levels of uric acid are diminished whereas serum and urine levels of xanthine and hypoxanthine are increased (Gleeson et al. 1992): only 20 per cent of the purine load is excreted as hypoxanthine whereas about 50–80 per cent is excreted as xanthine. Experimental data support the hypothesis that in hereditary xanthinuria there is enhanced hypoxanthine salvage via hypoxanthine guanine phosphoribosyltransferase. Degradation of guanine nucleotides to xanthine bypasses the hypoxanthine salvage pathway and may explain the predominance of this urinary purine compound in xanthinuria (Mateos et al. 1987). Two clinically similar but distinct types of xanthinuria have been described. Type I is an isolated defect of xanthine dehydrogenase whereas type II is a combined defects of xanthine dehydrogenase and aldehyde oxidase: these two forms differ because type I patients metabolize allopurinol to oxypurinol whereas type II patients do not (Simmonds et al. 1995). Genetics The human xanthine dehydrogenase gene, referred to as XDH gene (Table 13.2) spans approximately 60 kb and is composed by 36 exons and 35 introns (Xu et al. 1996). XDH gene is localized to the short arm of chromosome 2 (2p22.3–p22.2) (Rytkonen et al. 1995). It encodes a protein of 1333 amino acids, referred to as xanthine dehydrogenase (XDH), which is 90 per cent identical to rat XDH. The XDH is a molybdenum-containing hydroxylase, that functions as a homodimer. In humans and other mammalian species XDH can be converted to xanthine oxidase, referred to as XO, by reversible sulfydryl oxidation or by irreversible proteolytic modification (Xu et al. 1996). The regions involved in reversible and irreversible conversion from dehydrogenase to oxidase region are conserved between rat and human enzyme. At present four mutations of XDH gene have been discovered in patients with xanthinuria: a C-to-T transition at nucleotide 682 that causes a CGA (R) to TGA (stop) nonsense change of codon 228, a C-to-T transition at nucleotide 445 determining an arginine to cysteine substitution at codon 149, a 1658insC mutation predicting a truncated inactive XDH protein, and a deletion of C at nucleotide 2567 that generates a stop codon from nucleotide 2783 (Ichida et al. 1997; Levartovski et al. 2000; Sakamoto et al. 2001). The molecular bases of the combined defects in type II xanthinuria have not been fully elucidated. Drosophila ma-l gene was suggested to encode an enzyme for sulfuration of the desulfo molybdenum cofactor for XDH and aldehyde oxidase (AO). The human molybdenum cofactor sulfurase (HMCS) gene, the human ma-l homologue, is therefore a candidate gene responsible for classical xanthinuria type II, which involves both XDH and AO deficiencies. Recently, the HMCS gene has been cloned from a cDNA library prepared from liver, and in


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two independent patients with classical xanthinuria type II a C-to-T substitution at nucleotide 1255 in the HMCS gene has been identified that should cause a CGA (Arg) to TGA (Ter) nonsense substitution at codon 419 (Ichida et al. 2001). These results seem to indicate that a functional defect of the HMCS gene is responsible for classical xanthinuria type II, and that HMCS protein functions to provide a sulfur atom for the molybdenum cofactor of XDH and AO. Xanthinuria also occurs in molybdenum cofactor deficiency, a complex inborn metabolic disorder, resulting in severe neurologic damage and early death, characterized by pleiotropic loss of all activities of molybdenum-containing enzymes: sulfite oxidase, xanthine dehydrogenase, and adehyde oxidase. This syndrome shows a severe clinical picture characterized by mental retardation, seizures, spastic tetra-paresis, brain atrophy, abnormal muscle tone, myoclonic spasms, dislocation of lenses, feeding difficulties, urinary xanthine calculi, increased urinary excretion of sulfite, thiosulfate, S-sulfocysteine, taurine, hypo-xanthine, and xanthine, decreased urinary excretion of sulfate and urate, and absence of urinary urothione (Aukett et al. 1988). The molybdenum cofactor is a unique pterin, termed molybdopterin, which binds catalytically active metal molybdenum and is responsible for the correct anchoring and positioning of the molybdenum center within the holoenzyme. Two genes encoding the enzymes of the molybdopterin biosynthetic pathway, molybdenum cofactor synthesis-1 gene (MOCS1) and molybdopterin synthase gene (MOCS2), have been localized respectively to the short arm of chromosome 6 (6p21.3) and to the long arm of chromosome 5 (5q11). The genomic organization of MOCS1 and MOCS2 has been clarified (Reiss et al. 1998a, 1999). Mutations of MOCS1 and MOCS2 genes have been identified in patients with molybdenum cofactor deficiency (Reiss et al. 1998b). Management Treatment is based on maintenance of urine output of 3–4 l a day and on dietary restriction of purine. Allopurinol therapy may also be indicated (Gleeson et al. 1992). References Amoroso, A., Pirulli, D., Florian, F., Puzzer, D., Boniotto, M., Crovella, S., et al. (2001). Journal of the American Society of Nephrology, 12, 2072–79. Asplin, J. R., Favus, M. J., and Coe, F. L. (1999). Nephrolithiasis. In The kidney (ed. B. M. Brenner), pp. 1786–94. Saunders, Philadelphia, PA. Aukett, A., Bennett, M. J., and Hosking, G. P. (1988). Molybdenum cofactor deficiency: an easily missed inborn error of metabolism. Developmental Medicine and Child Neurology, 30, 531–5. Bai, M., Pearce, S. H. S., Kifor, O., Trivedi, S., Stauffer, U. G., Thakker, R. V., et al. (1997). In vivo and in vitro characterization of neonatal hyperparathyroidism resulting from a de novo, heterozygous mutation in the Ca(2)-sensing receptor gene: normal maternal calcium homeostasis as a cause of secondary hyperparathyroidism in familial benign hypocalciuric hypercalcemia. Journal of Clinical Investigation, 99, 88–96. Barbey, F., Joly, D., Rieu, P., Mejean, A., Daudon, M., and Jungers, P. (2000). Medical treatment of cystinuria: critical reappraisal of long-term results. Journal of Urology, 163, 1419–23. Basmaison, O., Rolland, M. O., Cochat, P., and Bozon, D. (2000). Identification of five novel mutations in the AGXT gene. Human Mutation, 15, 577.

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14 Disorders of tubular transport David S. Geller, Mark A. J. Devonald, and Fiona E. Karet

Introduction The advent of molecular genetic analysis has had a major impact in advancing our understanding of renal homeostatic mechanisms, because techniques permitting gene discovery and identification of the genetic basis of Mendelian monogenic disorders have provided a novel set of physiologic tools. In this chapter, we discuss three major categories of inherited renal tubular transport disorders: those primarily affecting sodium and potassium (whether retention or wasting), water balance defects, and the primary renal acidopathies. While the disorders described here are in general rare, the insights gained in understanding their molecular bases have added significantly to our understanding of normal human physiology. These findings may prove relevant to the understanding and treatment of more common disorders, and we have attempted to highlight these advances where possible. In general, though not always, the disorders that give rise to over-activity of a channel or transporter show a dominant mode of inheritance, while loss-of-function genetic defects are more likely to be recessive. While all the recessive diseases described in this chapter are rare, they are encountered more commonly in areas of the world where parental consanguinity is prevalent. The investigation of kindreds where affected children are the offspring of consanguineous union of unaffected parents has therefore allowed us to investigate more easily the genetic causes of such disorders, by employing homozygosity mapping (Lander and Botstein 1987). This technique uses the likelihood that an affected individual who is the offspring of consanguineous union will inherit identical copies of a mutated gene from a single carrier ancestor by descent down both paternal and maternal lines. Genotyping studies seeking regions of homozygosity by descent therefore offer an attractive and powerful tool. The kidney does not of course carry out any of its myriad homeostatic functions in a vacuum, and interactions with other systems form an important part of the overall picture. This is perhaps most marked in the context of salt homeostasis, where the endocrine system, particularly the mineralocorticoid axis and aldosterone, are closely dovetailed. We have therefore included reference to some inherited endocrine disorders that have a major impact on the kidney, and have indicated where else in this volume a fuller discussion may be found. In addition, although several of the diseases described here affect divalent cation balance, primary disorders of calcium and magnesium handling are covered in Chapters 12 and 13. Disorders of salt homeostasis Hypertension remains a leading contributor to cardiovascular disease, yet the molecular mechanisms underlying this trait remain elusive in the majority of patients. Studies to identify

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the molecular bases of rare Mendelian forms of hypertension and hypotension were initiated in an attempt to clarify the physiologic basis of blood pressure regulation. The molecular bases of almost all known Mendelian disorders with a primary effect on blood pressure have now been elucidated, and remarkably, they converge upon a final common pathway: the regulation of sodium reabsorption in the kidney. Mutations that increase renal sodium reabsorption increase blood pressure, whereas those that decrease renal sodium reabsorption serve to decrease it. Insights gained from the study of these disorders have reinforced the primary role of the kidney in blood pressure regulation via its effect on sodium homeostasis, and have provided new insights into the physiologic mechanisms underlying sodium transport in the kidney. Here, we review monogenic disorders of hypertension and hypotension (Tables 14.1 and 14.2), and where possible, point towards our attendant novel understanding of renal physiology. Hypertensive disorders Liddle syndrome Clinical features. Liddle syndrome is characterized by the autosomal dominant transmission of early-onset hypertension and hypokalaemia (Table 14.1). Despite the clinical picture of aldosteronism, renin, and aldosterone levels are suppressed, and the syndrome is thus referred to as pseudoaldosteronism (Liddle et al. 1963). Liddle syndrome is caused by gain of function mutations in subunits of the distal nephron epithelial sodium channel (see below). Table 14.1 Monogenic hypertensive disorders Transmission Serum aldosterone

Serum potassium

GRA

Dominant

Elevated

Normal or slightly low

Liddle Syndrome Hypertension exacerbated by pregnancy PHA2

Dominant

Suppressed

Dominant

Suppressed

Dominant

Variable

FH2

Dominant

Aldosterone/ Normal PRA ratio 20 Suppressed Low

Syndrome of Autosomal apparent recessive mineralocorticoid excess 11- hydroxylase Autosomal deficiency recessive 17- hydroxylase Autosomal deficiency recessive

Gene

Treatment

CYP11B2/ Dexamethasone CYP11B1 Spironolactone hybrid Amiloride Normal or mildly or ENaC Amiloride low Normal or mildly Mineralocorticoid ? Amiloride low Receptor High

WNK1, WNK4 Chr. 1 locus Chr. 7 locus

Thiazide diuretics Spironolactone

11 -HSD2

Spironolactone, Dexamethasone

Suppressed

Low

CYP11B1

Dexamethasone

Suppressed

Low

CYP17

Dexamethasone

GRA glucocorticoid remediable aldosteroism; PHA2 pseudohypoaldosteronism type 2; and FH2: familial hyperaldosteronism type 2.

Table 14.2 Clinical and biochemical features of salt wasting disorders Gitelman

Type I Bartter

Type II Bartter

Type III Bartter

Dominant PHA1

Recessive PHA1

Presentation

Older/adult

Neonatal/ infancy

Neonatal/ infancy

Childhood/older

Neonatal/infancy

Pregnancy

Normal None? Weakness Tetany

Polyhydramnios Prematurity Vomiting Dehydration Polyuria

Usually normal

Symptoms/ signs

Polyhydramnios Prematurity Vomiting Dehydration Polyuria

Neonatal/infancy (may be self-limiting) Normal

Chondrocalcinosis Salt craving Alkalosis Low Cl

Polydipsia Failure to thrive Alkalosis Low Cl

Polydipsia

Low K

Low K

Mg level Urine Ca Nephrocalcinosis

Low Low No

Gene

SLC12A3

Biochemistry

Normal

Variable

Vomiting Dehydration May be asymptomatic

Vomiting Dehydration

Alkalosis Low Cl

Alkalosis Low Cl

Mild acidosis High Cl

Low K

Normal/high K

Normal High Yes

Low K (after salt replacement; initially high) Normal High Yes

Acidosis High Cl Low Na High K

Normal or low Normal No

Normal

Normal

No

No

SLC12A1

KCNJ1

CLCNKB

NR3C2

SCNN1, NR3C2

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The identification of the molecular basis of Liddle syndrome has allowed correlation of phenotype with genotype, permitting a precise description of disease hallmarks. While earlyonset hypertension is generally present and is often severe, there can be wide variation in blood pressure among genotypically affected individuals, even within a kindred, suggesting the importance of other genetic or environmental factors. Hypokalaemia, although frequently present, is not required for diagnosis. In one study, the mean serum potassium of 18 affected individuals was 3.6 mEq/l vs a mean of 4.2 mEq/l in unaffected family members (BoteroVelez et al. 1994), but in another, only four of seven affected individuals were hypokalaemic (Findling et al. 1997). That Liddle syndrome can present with mild hypertension and/or normokalaemia raises the possibility that the syndrome is underdiagnosed in the general population, but it is nonetheless a rare disorder. The best discriminants for the presence of disease in at-risk individuals (aside from genetic analysis) are low 12 h urinary aldosterone excretion (369 vs 1903 ng) and a low urine aldosterone:potassium ratio (22 vs 171, Botero-Velez et al. 1994). Genetics. The finding that the blood pressure and electrolyte abnormalities are corrected by inhibitors of the collecting duct epithelial sodium channel (ENaC), but not by mineralocorticoid receptor antagonists, suggested that the disorder is caused by a specific transport defect in the renal distal nephron rather than by an unknown mineralocorticoid. This was confirmed by the demonstration that activating mutations in the - or -subunit of ENaC cause Liddle syndrome (Shimkets et al. 1994; Hansson et al. 1995a). The disease is transmitted in autosomal dominant fashion, but de novo cases have been described (Hansson et al. 1995b; Yamashita et al. 2001). The mutations causing Liddle syndrome are restricted to the C-terminus of these subunits of ENaC; no mutations in the subunit causing Liddle syndrome have been identified. A gain-of-function mutation in the mineralocorticoid receptor has been described, which causes a syndrome similar to that seen in Liddle syndrome (see section on ‘Hypertension exacerbated by pregnancy’). Cell biology. The characterization of the molecular basis of Liddle syndrome has yielded important insights into the physiology of blood pressure regulation. All mutations thus far described causing Liddle syndrome disrupt or delete a highly conserved proline-rich sequence in the C-terminus of the - or -subunit of ENaC termed the ‘PY’ motif. The significance of this was clarified with the finding that the PY motif mediates removal of ENaC from the cell surface via interaction with a ubiquitin protein ligase called Nedd4. ENaC lacking the PY motif are inefficiently removed from the cell surface, leading to increased channel concentration on the cell surface and increased sodium reabsorption (Staub et al. 2000). The physiologic pathways thus identified are currently under investigation for their role in blood pressure regulation and as possible targets for new antihypertensive agents. While a complete review of this work is beyond the scope of this text, it does serve to highlight the role genetics can play in identifying novel physiologic pathways important in cellular homeostasis. Management. Therapy of Liddle syndrome requires efficient blockade of ENaC, which is generally attempted pharmacologically via the use of amiloride or triamterene. As these drugs compete with sodium for access to ENaC, they should be used in conjunction with a sodiumrestricted diet. When used early in the course of disease, these agents alone may be adequate for blood pressure control; patients untreated for some time prior to diagnosis may develop

Disorders of tubular transport

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resistant hypertension due to secondary end-organ damage, requiring the use of multiple antihypertensive medicines. In those rare cases where renal insufficiency develops, renal transplantation may be curative of the hypertension (Botero-Velez et al. 1994). Hypertension exacerbated by pregnancy Clinical features. Not all cases of pseudoaldosteronism are due to mutations in ENaC (Table 14.1). A single kindred with a variant form of Liddle syndrome has been described (Geller et al. 2000). Clinically, these patients present with a history identical to that seen in ‘classical’ Liddle syndrome: the autosomal dominant transmission of early onset and frequently severe hypertension with mild hypokalaemia, hyporeninaemia, and hypoaldosteronaemia. A particularly striking characteristic of the disorder is the effect of pregnancy on affected women. While at baseline, blood pressure in affected men and women is similar, pregnancy induces remarkable blood pressure elevations in affected women with accompanying hypokalaemia and hyperkaliuria. Genetics. In this family, early-onset hypertension co-segregates with an activating mutation in the mineralocorticoid receptor (MR). The mutation identified, a substitution of leucine for serine at codon 810 in the hormone-binding domain, alters the hormone binding specificity of MR, allowing progesterone and its derivatives to agonize rather than antagonize the receptor. The pregnancy-induced increase in renal potassium wasting provides dramatic clinical evidence in support of progesterone-mediated activation of the mutant receptor. Spironolactone and other MR antagonists agonize the L810 mutant receptor (MRL810), and thus, the use of these medications is contraindicated in this disorder. Cell biology. Investigation into the novel activities of MRL810 have provided useful insights into understanding MR physiology. Progesterone-mediated activation of MRL810 requires a novel intramolecular van der Waals interaction. This interaction is conserved among many other nuclear receptors, including all steroid hormone receptors, suggesting a general mechanism important in steroid hormone receptor activity (Geller et al. 2000). However, this rare syndrome should not be considered as a form of pre-eclampsia. Syndrome of apparent mineralocorticoid excess Clinical features. Apparent mineralocorticoid excess (AME) is transmitted in autosomal recessive fashion, the vast majority of reported cases arising in children born of consanguineous union. Birth weights of affected children are low and growth retardation is common. Mild forms of the disorder involving only mild hypertension have been described (Wilson et al. 1998), but more severe hypertension with end-organ damage in the eye, heart, kidney, and central nervous system is common (Dave-Sharma et al. 1998). As with other forms of pseudoaldosteronism, patients with the syndrome of AME present with early-onset hypertension and hypokalaemia despite very low renin and aldosterone levels. AME differs, however, in that hypertension in these patients is improved by the mineralocorticoid antagonist spironolactone, suggesting the presence of a circulating mineralocorticoid. The missing mineralocorticoid proved to be cortisol, a potent in vitro MR agonist that normally has little in vivo mineralocorticoid activity. The mineralocorticoid receptor is protected in the kidney in vivo from the normal high circulating cortisol levels by the type 2 isoform of the enzyme 11- hydroxysteroid dehydrogenase (11-HSD2), which converts cortisol to its inactive metabolite cortisone (Stewart et al. 1987; Funder et al. 1988). Patients with AME lack this enzyme, and cortisol therefore gains access to MR in the kidney. Affected individuals thus

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excrete reduced amounts of cortisone metabolites such as tetrahydrocortisone (THE) in the urine compared with the cortisol metabolites tetrahydrocortisol (THF) and 5-alpha tetrahydrocortisol (THF). Elevations in the (THF THF)/THE ratio are characteristic of AME (Dave-Sharma et al. 1998), although glycerrhetinic acid (found in black liquorice) and carbenoxolone, which inhibit 11-HSD2, can cause this picture as well. AME must also be distinguished from the salt-retaining forms of congenital adrenal hyperplasia caused by the absence of either steroid 11- hydroxylase or steroid 17- hydroxylase enzymes (see below). Genetics. AME is caused by homozygous loss-of-function mutations in the gene encoding the 11- hydroxysteroid dehydrogenase type 2 enzyme (Mune et al. 1995). Most mutations identified are missense mutations, but truncating mutations have been seen (Dave-Sharma et al. 1998). Patients with identical disease-causing mutations display varying severity of clinical and biochemical features, suggesting the importance of other genetic and/or environmental factors in blood pressure determination. While it has proven difficult to correlate genotype with clinical phenotype, urinary (THF THF)/THE ratio strongly correlated with in vitro enzymatic activity of the corresponding mutant (r 0.839, P 0.001 with cortisol as the substrate, Mune and White 1996; Nunez et al. 1999). Management. Early aggressive treatment may limit the most dangerous side effects of the disease (Dave-Sharma et al. 1998). Therapy generally relies on spironolactone to block activation of the MR and/or dexamethasone to suppress cortisol secretion. Kidney transplantation may be curative of hypertension in patients with end-stage renal disease (Palermo et al. 1998). Pseudohypoaldosteronism type 2 Clinical aspects. Pseudohypoaldosteronism type 2 (PHA2), also referred to as Gordon syndrome, is a rare familial form of volume-dependent hypertension characterized by hyperkalaemia and hyperchloraemic metabolic acidosis. Serum potassium and chloride levels are well above normal, with a concomitant fall in bicarbonate. Renal function is otherwise normal. A hallmark of the disorder is the sensitivity of both the hypertension and hyperkalaemia to thiazide diuretics. Short stature and dental abnormalities have been described (Gordon et al. 1970; Weinstein et al. 1974; Iitaka et al. 1980). These patients thus in many ways represent the clinical inverse of Gitelman syndrome, in which patients have salt-wasting, hypotension, hypocalciuria, and hypokalaemia due to loss of function mutations in the thiazide-sensitive cotransporter gene NCCT (see below). Genetics. Given the opposite phenotypes of PHA2 and Gitelman syndrome, activating mutations in NCCT represented the obvious initial candidate for PHA2. However, linkage analysis ruled out this possibility in most families (Mansfield et al. 1997). PHA2 is in fact a genetically heterogeneous disorder that has been linked to three distinct chromosomal loci. Mansfield et al. (1997) demonstrated linkage to chromosomes 1q31–42 and 17p11–q21, and a locus on chromosome 12p13.3 was later identified (Disse-Nicodème et al. 2000). The molecular bases of the chromosome 12 and 17 forms of PHA2 have been recently described. Wilson et al. (2001) demonstrated that two PHA2 kindreds which link to the chromosome 12 locus have independent overlapping large deletions in the first intron of a gene coding for a kinase known as WNK1 (Xu et al. 2000). This kinase, whose physiological role is not yet known, is a ubiquitously expressed salt-sensitive serine kinase distinguished from other known serine kinases by the absence of a conserved lysine in a sub-domain crucial for binding to ATP (hence WNK—with no lysine [K]). In the kidney, WNK1 is found


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in the cytoplasm of distal convoluted tubule and principal cells. Affected individuals with this form of PHA2 demonstrated a 5-fold increase in WNK1 cellular mRNA expression, suggesting a functional effect of the intron 1 deletion on gene expression. A WNK1 paralogue, WNK4, which lies at the centre of the chromosome 17 locus has also been identified. WNK4 proved to be a renal-specific gene localized mainly to tight junctions in the distal nephron, and four kindreds were identified with novel mutations in WNK4 co-segregating with disease (Wilson et al. 2001). In three of these kindreds, the mutations alter a 10 amino acid sequence that is perfectly conserved throughout the entire WNK family. Cell biology. The links between mutations in WNK1 and WNK4, blood pressure regulation and potassium homeostasis remain unknown at present, and are certain to be an area of intense future study. It is noteworthy that the actions of a number of other proteins involved in sodium homeostasis in the kidney, including ENaC, NKCC2 and the thiazide-sensitive cotransporter, are regulated by phosphorylation. Whether the WNK kinases are involved in these regulatory pathways is not currently known. The studies on WNK4’s contribution to blood pressure regulation will be particularly intriguing, as the chromosome 17 locus on which it lies has been linked to hypertension in humans (Julier et al. 1997; Levy et al. 2000) and rats (Jacob et al. 1991). Future studies will no doubt focus on the possible relationship of this gene with essential hypertension in the general population. Other Mendelian disorders of hypertension A number of other Mendelian disorders cause hypertension (Table 14.1). These are not true renal tubular disorders, but they must be distinguished from those described above. In some of these disorders, the link to hypertension and the kidney is not understood. For example, an autosomal dominant disorder characterized by severe hypertension and type E brachydactyly has been described and linked to chromosome 12, but the molecular basis of this disorder and the involvement, if any, of the kidney have not been determined (Schuster et al. 1996). Other disorders may mimic the renal tubular disorders described above, and these are summarized below. Glucocorticoid-remediable aldosteronism Clinical features. Glucocorticoid remediable aldosteronism (GRA) is a rare autosomal dominant condition characterized by early onset hypertension, hyporeninaemia, and intermittently elevated aldosterone levels (Sutherland et al. 1966; New and Peterson 1967). GRA is distinguished from other forms of aldosteronism by the rapid normalization of blood pressure and aldosterone levels after the administration of physiologic doses of glucocorticoids. Patients have mild to very severe hypertension and are generally normokalaemic or only slightly hypokalaemic. Interestingly, these patients are at significant risk of cerebral vascular accidents as a result of cerebral haemorrhage, and pre-symptomatic screening for cerebral aneurysms is recommended (Litchfield et al. 1998). Diagnosis of GRA is made by demonstration of a fall in plasma aldosterone levels to 4 ng/dl during a 2–4 day dexamethasone-suppression test (Litchfield et al. 1997). The stringent criteria are necessary to avoid a false positive test in patients with aldosterone-producing adenomas, in whom aldosterone levels frequently decline in response to glucocorticoids as well. Demonstration of the chimaeric gene product characteristic of GRA is also diagnostic.

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Genetics. GRA is caused by a genetic recombination event between the contiguous and highly homologous aldosterone synthase and steroid 11- hydroxylase genes. The crossover places coding sequences of the aldosterone synthase gene under the control of the ACTHresponsive promoter of the steroid 11- hydroxylase gene (Lifton et al. 1992). The resultant ectopic expression of the aldosterone synthase’s 18-hydroxylase activity in the adrenal zona fasciculata triggers the conversion of cortisol to 18-oxocortisol and 18-hydroxycortisol; these hybrid steroids are characteristic of GRA and are otherwise seen only in a subset of patients with aldosterone-producing adrenal adenomas (Stowasser et al. 1996). Familial hyperaldosteronism type 2 A second form of familial aldosteronism has been described, which has been termed familial hyperaldosteronism type 2, or FH-2 (Gordon et al. 1991). Affected individuals have hypertension and non-dexamethasone suppressible hyperaldosteronism, as defined by an elevated aldosterone/renin ratio 25. The onset of clinical aldosteronism is variable, and may occur at any point in life. Using these criteria, the diagnosis of aldosteronism is not particularly rare, and thus, some of the noted familial incidence of aldosteronism may be due to chance alone. In one such kindred, however, nine affected individuals share the diagnosis in a pattern consistent with autosomal dominant transmission of the trait (Torpy et al. 1998). Affected individuals demonstrated an elevated aldosterone/renin ratio with non-suppression of aldosterone levels during a fludrocortisone suppression test. Genotypic studies identified a locus on chromosome 7p22, which co-segregates with disease. The nature of the mutation leading to this phenotype is presently unknown (Lafferty et al. 2000). Congenital adrenal hyperplasia 11--hydroxylase and 17- hydroxylase deficiencies. Congenital adrenal hyperplasia refers to a diverse group of disorders caused by an inability of the adrenal gland to synthesize cortisol due to an inherited deficiency of a necessary biosynthetic enzyme. Many of these disorders, such as 21-hydroxylase deficiency or 3--hydroxysteroid dehydrogenase deficiency, cause salt-wasting and hypotension. However, two forms of congenital adrenal hyperplasia, result in excess mineralocorticoid production, leading to salt retention and hypertension. Each is transmitted in autosomal recessive fashion. In steroid 11--hydroxylase deficiency syndrome, the absence of the steroid 11- hydroxylase activity leads to an accumulation of 11-deoxycortisol and 11-deoxycorticosterone (DOC), both of which possess substantial mineralocorticoid activity, leading to activation of the MR and hypertension. Zona glomerulosa function is suppressed, leading to reduced serum aldosterone levels. Steroid precursors are shunted to the intact 17-hydroxylation pathway, and resulting excess sex steroids cause virilization. In steroid 17-hydroxylase deficiency overproduction of DOC and corticosterone leads to mineralocorticoid hypertension, but the inability to produce sex steroids leads to primary amenorrhoea and sexual infantilism. This diagnosis is confirmed by the demonstration of low levels of 17-ketosteroids and 17-hydroxycorticosteroids with large amounts of tetrahydro-corticosterone and tetra-hydro-deoxycorticosterone in the urine (Bondy 1981). Salt-wasting disorders The search for the genetic causes of salt-wasting syndromes became focused by the observation of a wide variation in the blood pressures of individuals who were known to harbour mutations causing various forms of inherited hypertension (see section on ‘Hypertensive disorders’).


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In other words, if carriers of such abnormal genes possess variants in other genes that serve to lower blood pressure, they may be protected from the development of hypertension. Searching for causes of single gene disorders responsible for hypotensive syndromes therefore represents the opposite end of the spectrum of renal salt homeostatic disorders by virtue of the profound renal salt wasting and hypotension that are characteristic. As with the inherited hypertensive syndromes, these have been thought to be relatively rare, and attempts to unravel their molecular pathology over the past 60 years have been hampered by the degree of phenotypic overlap between them. Until molecular genetic studies became available, it was not fully appreciated how much genetic heterogeneity was present, nor what the real prevalence might be. These genetic studies have served to clarify much of the confusion in the relevant literature, which has often (in retrospect) failed adequately to differentiate between several different disorders. Pseudohypoaldosteronism type 1 Clinical aspects. Pseudohypoaldosteronism type 1 (PHA1) represents the clinical inverse of Liddle syndrome. It is a rare inherited disorder characterized by renal salt wasting and hyperkalaemic metabolic acidosis despite markedly elevated renin and aldosterone levels (Cheek and Perry 1958). The clinical picture is thus one of renal resistance to the action of mineralocorticoids. Two clinically distinct forms of the disease have been described, an autosomal recessive form and an autosomal dominant form. Both generally present in the first weeks of life, with dehydration, sodium wasting, hyponatraemia, and hyperkalaemic metabolic acidosis despite elevated renin and aldosterone levels. In the autosomal recessive form of the disease, patients have severe sodium wasting from the kidney, colon, sweat, and salivary glands. These children have recurrent life-threatening episodes of salt wasting and hyperkalaemia and require lifelong sodium supplementation and potassium binding resins. In the autosomal dominant form, sodium wasting occurs primarily from the kidney. While these patients may be quite ill at birth, they generally respond well to sodium supplementation and are usually able to discontinue supplements within the first few years of life. Recessive PHA1 The diagnosis of recessive PHA1 is made by the demonstration of renal salt wasting with hyperkalaemia and metabolic acidosis despite elevated renin (10 ng/ml/h) and aldosterone (100 ng/dl) levels in the setting of normal renal function and otherwise normal adrenal function. These patients are frequently the result of consanguineous parental union. Recessive PHA1 is further distinguished from the dominant form of the disease by the demonstration of elevated sweat and salivary sodium and chloride levels. Both parents will have normal aldosterone levels and there is no known phenotype associated with the heterozygous condition. In autosomal dominant PHA1 (discussed below) one parent will frequently have elevated aldosterone levels. Recessive PHA1 must also be distinguished from Type 2 Bartter syndrome in which patients may present initially with the hyperkalaemia and salt-wasting characteristic of PHA1, but after volume resuscitation, revert to the hypokalaemia more typical of Bartter syndrome. Genetics. The autosomal recessive form of the disease is caused by homozygous loss of function mutations in either the -, -, or -subunit of the ENaC (Chang et al. 1996; Strautnieks et al. 1996). The mutations identified include frameshift, splice-site, non-conservative missense mutations, and premature termination codons. Grunder et al. (1997) described a

316


-ENaC missense mutation in which serine was substituted for a conserved glycine at codon 37; the resulting subunit had reduced but not absent sodium transport activity in vitro, thus explaining the mild PHA1 phenotype in the patient carrying this mutation. To date, all patients with autosomal recessive PHA1 have had disease causing mutations identified in one of the subunits of the ENaC, suggesting that this disorder is caused chiefly if not exclusively by mutations in ENaC. It is notable that mice lacking the sodium–hydrogen exchanger NHE3 present with a clinical picture of salt-wasting and hyperkalaemic metabolic acidosis reminiscent of recessive PHA1, but this mutation has not been observed in humans (Schultheis et al. 1998). Physiology. Affected individuals carry homozygous mutations in ENaC that cause truncation of the channel proteins and presumably a null allele, causing a marked reduction of sodium reabsorption in the cortical collecting duct. As potassium and hydrogen ion secretion in this segment are linked to sodium reabsorption, these processes are blocked as well. The ensuing hyperkalaemic volume depleted state stimulates the renin–angiotensin system, leading to high serum aldosterone levels and maximal activation of MR. Mineralocorticoid receptor, however, is unable to stimulate sodium reabsorption in the collecting duct due to the absence of ENaC, thus ensuring persistent sodium wasting and hyperkalaemic acidosis. The severity of the clinical course of recessive PHA1 patients highlights the crucial role of ENaC in sodium homeostasis, even in individuals ingesting a high-salt diet. The determination of the molecular basis of recessive PHA1 has also clarified an extrarenal role of ENaC. Patients with recessive PHA1 have a novel pulmonary phenotype characterized by increases in airway liquid volume and mucociliary clearance with frequently positive methacholine challenge tests and mild air trapping on pulmonary function tests. The clinical picture in some ways resembles that seen in cystic fibrosis (CF; Hanukoglu et al. 1994), but the classic infections of CF are not seen (Kerem et al. 1999). These findings are significant because they have clarified issues of sodium and volume transport in the lung, helping to explain the pathophysiology of disease and even suggesting opportunities for therapeutic intervention in patients with CF (Kerem et al. 1999). It should be noted that these findings came primarily from individuals with mutations in -ENaC. Mice deficient in the -subunit of ENaC have enormous lung secretions that lead to their death soon after birth from asphyxiation, but mice lacking either the -or -subunits of ENaC have no pulmonary phenotype (McDonald et al. 1999; Barker et al. 1998). It thus remains unclear whether PHA1 patients with -and -ENaC mutations have the identical pulmonary phenotype that Kerem et al. (1999) observed in their patients. Management. There is some variability in the prognosis of recessive PHA1. For patients with homozygous null mutations, the prognosis is often poor. Even minor illness can bring on rapid deterioration with hypotension and hyperkalaemia; nausea and vomiting often herald and then accelerate the clinical decline. Neonatal death from volume depletion and hyperkalaemia is not uncommon, and thus, massive saline supplementation and careful attention to electrolyte abnormalities are continually necessary. Even with vigilant observation, children may still suffer untoward effects, but some have survived into the adult years (U. Kuhnle, pers. comm.). Some patients have a milder course, and in at least one case, a partial loss-of-function mutation in -ENaC has been demonstrated (Grunder et al. 1997, see below).


317

Dominant PHA1 In contrast to the severe salt-wasting characteristic of recessive PHA1, patients with autosomal dominant PHA1 typically have a much milder course. Patients may be asymptomatic or quite ill from salt wasting in the neonatal years, but symptoms typically resolve soon thereafter, and patients remain asymptomatic through their adult years. Genetics. Heterozygous loss-of-function mutations in the human MR cause this form of the disease (Geller et al. 1998). The mutations identified all cause premature termination of translation of the gene. In at least one PHA1 patient, the mutant RNA is degraded, demonstrating that haploinsufficiency at the MR locus is sufficient to cause PHA1 (D. Geller, unpubl. obser.). More recently, missense loss-of-function mutations in the hormone-binding domain of the receptor have been reported (Tajima et al. 2000). While genetic heterogeneity has been proposed (Viemann et al. 2001), no linkage data ruling out the MR locus has been demonstrated in a dominant PHA1 kindred. The ability to assign phenotype unambiguously on the basis of genotype has enabled us to assess genotype–phenotype correlation in an extended kindred. Some children had severe electrolyte derangements requiring high levels of sodium supplementation and potassium binders, while other children were entirely asymptomatic and received no treatment for the disease. Of note, a number of at-risk neonates died, suggesting that at-risk infants for PHA1 should be treated empirically with salt until a definitive diagnosis can be made. Although pregnancy is a time of intrinsic progesterone-mediated aldosterone resistance, we are not aware of any pregnancy-related problems in women with PHA1. All affected adults were asymptomatic, and were clinically indistinguishable from their unaffected relatives, exhibiting similar blood pressure and serum chemistries. An elevated aldosterone level was the only biochemical marker of disease, a random seated aldosterone level above 30 ng/dl correlating well with the presence of disease within these kindreds. Inherited metabolic alkaloses: Bartter and Gitelman syndromes Bartter and Gitelman syndromes were originally described as variations of a single disease process (Bartter et al. 1962; Gitelman et al. 1966) resulting in hypokalaemic metabolic alkalosis. More recent biochemical and latterly genetic studies have permitted their separation into distinct disorders, with separable phenotypic characteristics (Bettinelli et al. 1992; Gitelman 1992). In both diseases, inheritance is autosomal recessive. All affected subjects have variably severe metabolic alkalosis with low serum potassium, renal salt wasting, low blood pressure and an activated renin–angiotensin system. Features that differentiate Bartter and Gitelman syndromes are concerned with renal calcium handling and deposition, serum magnesium, and clinical presentation (Table 14.2). To date, three genes have been implicated in the pathogenesis of Bartter syndrome in different kindreds, whereas all cases of Gitelman syndrome studied, which now number several hundreds, are accounted for by mutations in one gene. All four have some phenotypic differences that may be useful diagnostically. Bartter syndrome Clinical features. In Bartter syndrome, affected individuals may present in infancy or early childhood with severe volume depletion, failure to thrive and hyperchloraemic hypokalaemic alkalosis. They may have been delivered prematurely, and maternal polyhydramnios is

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common. Their metabolic dysfunction is usually accompanied by hypercalciuria with normal serum calcium and magnesium levels. Nephrocalcinosis is very common, and may be present even in neonates. Hyperprostaglandinuria and a therapeutic response to indomethacin may also be seen. In the majority of families where the genetic basis has been revealed, there is parental consanguinity, and affected offspring of related unaffected parents are homozygous for the causative mutation. Calculations of allele frequency are difficult to obtain, due to the ascertainment bias of recent research and inaccessibility of some populations where parental consanguinity is particularly prevalent. A figure of 1 : 1000 has been cited. Since genetic heterogeneity has become evident in Bartter syndrome, with three genes thus far identified and at least a fourth likely to exist, some genotype–phenotype correlations have emerged that permit division into three separate types. Some phenotypic differences between Bartter syndrome types 1–3 are listed in Table 14.2, which may be helpful in identifying the likely genetic culprit (see below). For example, infants presenting with nephrocalcinosis are more likely to have hypercalciuria and type 1 or 2 Bartter. By contrast, those with type 3 Bartter may well be normocalciuric and devoid of renal calcium deposition. Often, patients with type 2 actually are hyperkalaemic in the neonatal period, developing the classical biochemical picture only after they have been fluid and salt repleted, possibly because of immaturity of the collecting duct in early life. Genetics. The biochemical picture in untreated Bartter syndrome is reminiscent of that occasionally seen in otherwise normal people on long-term loop diuretic therapy. The target for loop diuretics is the electroneutral sodium-potassium-chloride cotransporter (NKCC2), which is expressed apically in the thick ascending limb of Henle’s loop. This gene was therefore the first candidate examined. It and the disease locus were co-localized to a short region of chromosome 15 (Simon et al. 1996a). In five kindreds, frameshift or non-conservative missense mutations were found which would ablate or severely reduce channel function. This is now referred to as type I Bartter. Homozygosity mapping in additional families showed that the syndrome must be a heterogeneous disorder, prompting a search for mutations in related or interacting genes. Subsequently, two further defective genes causing loss of function have been identified in different kindreds. The first was SLC12A3, which encodes the inward-rectifier potassium channel ROMK2 that regulates NKCC2’s activity by recycling K ions back in to the tubular fluid (type 2 Bartter) (Simon et al. 1996b). ROMK2 mutations, like those found in NKCC2, are scattered throughout the coding sequence, and represent premature stop codons, frameshift, and missense alterations. The third gene implicated in Bartter syndrome encodes the basolateral chloride channel CLC-KB in these same loop of Henle cells (type 3) (Simon et al. 1997). Here, mutations reported also include whole gene deletions, and an unusual chimaeric gene produced by unequal crossing over between ClCN-KB and the highly homologous and tightly-linked adjacent ClCN-KA gene. (Notably, a mouse knock-out of this latter gene results in nephrogenic diabetes insipidus, see section on ‘Hypertension exacerbated by pregnancy’.) Management. The treatment of Bartter syndrome can be difficult, as the degree of salt wasting may be severe. However, aggressive replacement of salt, and K in particular is essential. Some patients respond well to the administration of indomethacin, especially in type 2 Bartter.


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Further heterogeneity. A few families remain where none of the known causative genes have been implicated, suggesting the existence of a fourth locus (Lifton, pers. comm.) In addition, Landau et al. (1995) reported an extended consanguineous Bedouin family where in five children from several branches, a combination of the infantile variant of Bartter syndrome and sensorineural deafness occurred. Using a DNA-pooling strategy, Brennan et al. (1998) showed linkage of the syndrome to 1p31. They excluded the kidney-specific chloride channel genes ClCN-KA and ClCN-KB and also the amiloride-sensitive Na/H exchanger gene SLC9A1, which all map to 1p36–35. Most recently. Birkenhager and colleagues have identified the causative gene (BSND), which is novel and is expressed in cochlear epithelium as well as the loop of Henle. It is an essential subunit for the activity of ClC-KA and ClC-KB (Estevez et al. 2001). Gitelman syndrome Clinical features. By far the majority of patients suspected of having Bartter syndrome in fact have Gitelman syndrome, which is also autosomal recessive. This was first clinically described in Gitelman’s paper of 1966 (although in retrospect, it is far from clear whether any of Bartter’s original patients might in fact really have had Gitelman syndrome). The phenotype in Gitelman syndrome is often much milder, usually being identified in late childhood or even in adulthood. Some affected individuals are asymptomatic, the disorder coming to light in the search for causes of hypokalaemia. However, some patients may be more severely affected, with growth problems and not uncommonly with joint problems and/or neuromuscular abnormalities (Gitelman et al. 1966). Tetany is a commonly cited problem. A recent survey of presenting symptoms (Cruz et al. 2001) highlights the differences in perception between patients and their physicians; the latter often consider Gitelman syndrome to be asymptomatic, whereas most patients would disagree! Anecdotally, it is reported that affected individuals note a longstanding preference for salty over sweet foods and snacks. Biochemically, Gitelman syndrome is characterized by low urinary calcium excretion and low serum magnesium levels with renal magnesium wasting. These features often allow an initial biochemical distinction between Gitelman and Bartter syndromes, though there may be some degree of overlap (Bettinelli et al. 1992). Occasionally, frank hypocalcaemia is seen, usually in association with severe hypomagnesaemia, which impairs parathyroid function. Patients with Gitelman syndrome display many of the biochemical changes seen in individuals on thiazide diuretics; indeed the surreptitious abuse of diuretics (or laxatives) remains the commonest differential diagnosis. The fact that thiazides have been used therapeutically in osteoporosis predicts that Gitelman patients may have an increase in bone density. This has recently been demonstrated to be the case in a cohort of Gitelman families where affected homozygotes had the highest densities when compared with their unaffected wildtype homozygote relatives (Cruz 2001), with heterozygote carriers in an intermediate range. These carrier relatives are also likely to have a lower serum K and blood pressure than those with two copies of the normal gene. Genetics. The similarity between the Gitelman syndrome phenotype and thiazide treatment led to the search for the human gene encoding the renal thiazide-sensitive Na–Cl cotransporter NCCT. This gene mapped to a region of chromosome 16 and found to co-localize with the linked locus for Gitelman syndrome (Simon et al. 1996c). The electroneutral ion transporter NCCT is present on the apical epithelial surface of distal renal convoluted tubule cells

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(Fig. 14.1) and mediates reabsorption of sodium and chloride (Gamba et al. 1993). Patients with Gitelman syndrome have been shown to harbour a diverse array of non-conservative missense mutations, premature termination codons, and splice site mutations in SLC12A3 that co-segregate with the disease and will result in loss of cotransporter function (Simon et al. 1996c; Mastroianni et al. 1996; Lemmink et al. 1998). In general, those with unrelated parents are compound heterozygotes, whereas those from consanguineous kindreds (much less common, compared with Bartter’s) are likely homozygous. Thus far, NCCT mutations account for all reported cases of Gitelman syndrome. The carrier frequency for SLC12A3 mutations may be as high as 1 : 50. Despite a recessive inheritance pattern, it is not clear why the occurrence of affected individuals in a sibship is often observed to exceed the expected 1 : 4. Management. Loss-of-function mutations in SLC12A3, whose product serves to reabsorb some 7 per cent of filtered sodium, demonstrate that the primary defect in these patients is renal salt wasting, and that the other biochemical manifestations of the disease must therefore derive from this primary abnormality. However, the interactions between NCCT and the mediators both of calcium transport into the urine and of magnesium reabsorption in the distal convoluted tubule remain to be fully elucidated, and thus the basis for the observed

Distal tubule

NCCT Gene SLC12A1 KCNJ1 CLCNKB BSND SLC12A3 SCNN1A,B,G NR3C2

Protein NKCC2 ROMK2 CLC-KB Barttin NCCT ENaC MR

Cl– Na+

Lumen Disorder Blood Bartter type 1 Bartter type 2 Bartter type 3 Bartter type 4 Gitelman Liddle (β,γ), PHA1 (α,β,γ) PHA1, HT exacerbated by pregnancy Thick limb

Collecting duct principal

ENaC

Na+

< MR

K+ ROMK2

of Henle

NKCC2

Na+ K+ 2Cl–

K+

CLC-KB Cl– Barttin

ROMK2 Fig. 14.1 Gene products mutated in salt-handling disorders. Circles indicate transporters, parallel lines are channels, and the MR is shown as . In each cell, the urinary (luminal) surface is at left. Gain of function in ENaC causes Liddle syndrome whereas loss of function results in recessive PHA1. Loss of function of the MR results in dominant PHA1 while gain of function is associated with hypertension exacerbated in pregnancy. Barttin is an essential accessory subunit of the loop basolateral chloride channel. The sodium-potassium-chloride cotransporter NKCC2 and the sodium chloride cotransporter NCCT are the targets for loop and thiazide diuretics respectively, while amiloride and triamterene antagonize ENaC. WNK1 and WNK4 mutations cause PHA2 but are not depicted as their functions are yet to be elucidated (see text).


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hypocalciuria and hypomagnesaemia are not fully explained as yet. This has made directed therapy to correct the biochemical abnormalities more difficult, and treatment is often limited by what patients will tolerate, both in terms of palatability and severity of hypotension. As with Bartter syndrome, aggressive salt replacement would be expected to be useful, but the delivery of additional Na to the collecting duct may in fact exacerbate renal K wasting. In addition therefore, some patients respond well to amiloride, spironolactone, or indomethacin. Mg2 supplementation is also a useful adjunct in elevating K levels, and assists in restoring suppressed parathyroid function (Bettinelli et al. 1999). However oral K, and Mg2 in particular, may be difficult for patients to tolerate due to unwelcome gastrointestinal disturbances, and compliance is often a problem. Disorders of water balance In addition to its essential role in regulating electrolyte transport, the kidney plays a crucial role in maintaining water balance. Water conservation in humans is regulated by the arginine vasopressin system. The hypothalamus, responding to osmolality sensors, releases vasopressin, a nine amino acid polypeptide. Vasopressin is recognized in the principal cell by the V2 receptor (V2R), a 371 amino acid protein expressed only in the collecting duct of the kidney (Lolait et al. 1992). The receptor has the classic structure of a G-protein coupled receptor with seven transmembrane domains. In the presence of vasopressin, the receptor activates, by a stimulating G-protein, adenylate cyclase, which increases cellular cyclic adenosine monophosphate (cAMP) levels. cAMP triggers protein kinase A to induce the insertion of aquaporin-2 (AQP2) water channel containing vesicles into the water-tight apical plasma membrane, resulting in an increase in water permeability. AQP2 is absolutely required for vasopressin-dependent urine concentration (Deen et al. 1994). Nephrogenic diabetes insipidus Clinical. Diabetes insipidus (DI), a failure of the kidney to reclaim water properly, is caused by a failure of any part of the renal water-handling system. Central (or neurogenic) DI is a heterogeneous condition characterized by polyuria and polydipsia due to an inability of the hypothalamus to secrete sufficient vasopressin, and can be treated with the administration of exogenous vasopressin, generally given in the form of a synthetic analogue 1-Desamino-8D arginine vasopressin (DDAVP). Nephrogenic diabetes insipidus (NDI) occurs when the kidney is unable to respond to the effects of endogenous or exogenous vasopressin (Fig. 14.2), and can be primary or acquired (Morello and Bichet 2001). Here, we review primary nephrogenic DI, of which three different forms have been described: X-linked recessive, autosomal recessive, and autosomal dominant. The different forms of the disease do not differ with respect to the time of onset or clinical symptoms, but they can be distinguished by response to desmopressin (van Lieburg et al. 1995, 1996). Patients generally present within the first 2.5 years of life. While polyuria and secondary polydipsia are the primary clinical sequelae of NDI, anorexia, vomiting, failure to thrive, fever, and constipation are the most common initially reported symptoms. Growth retardation is common, likely due to inadequate food intake, dehydration, and hypernatraemia, with an average of 1 on height standard deviation scores. Serious urological

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David S. Geller, Mark A. J. Devonald, and Fiona E. Karet Collecting duct principal

AQP2 AVPR2

Recessive NDI Dominant NDI X–linked NDI

AQP2

H2O

< AVPR2

Fig. 14.2 Gene products mutated in nephrogenic diabetes insipidus. (Symbols are as in Fig. 14.1.)

complications and acute urinary retention are not uncommon, and mental retardation has been frequently described (van Lieburg et al. 1999). It has been observed, however, that all complications of congenital NDI can be prevented by an adequate water intake (Morello and Bichet 2001). For this reason, an attempt to make an early genetic diagnosis has been recommended in all at-risk individuals. In addition, patients should be provided with unrestricted amounts of water from birth to ensure normal development. In addition to a low-sodium diet, the use of diuretics (thiazides) or indomethacin may reduce urinary output, although the benefits of these therapies must be weighed against the side effects of these drugs (thiazides: electrolyte disturbances; indomethacin: reduction of glomerular filtration rate and gastrointestinal symptoms). As described below, some rare patients may have a partial response to administered DDAVP. The differential diagnosis includes primary polydipsia, central DI and acquired NDI. The last results from failure of urinary concentrating ability associated with severe hypokalaemia or hypercalcaemia, obstructive uropathy, or drugs such as lithium (Morello and Bichet 2001). X-linked NDI Genetics. Approximately 90 per cent of cases of congenital NDI are X-linked. This disease is caused by loss-of-function mutations in the V2R gene (AVPR2) located on the long arm of the X chromosome (Rosenthal et al. 1992; van den Ouweland et al. 1992; Holtzman et al. 1993). To date 155 mutations in this gene have been identified in 239 families. Mutations occur in all regions of the gene. Missense mutations account for 50 per cent of the mutations identified, whereas frameshift mutations (27 per cent), non-sense mutations (11 per cent), in frame deletions or insertions (4 per cent), splice site mutations (2 per cent) and one complex mutation account for the remainder. Patients with a particular mutation (G185C) may have a milder form of the disease, with delayed time of disease presentation and a partial response to exogenous DDAVP (van Lieburg et al. 1999). X-linked NDI is a rare disorder; the incidence in the general population has been estimated in the Canadian province of Quebec to be approximately 8.8 per million male live births, although it may be higher in certain geographic regions (Arthus et al. 2000). Consistent with X-linked recessive transmission, females do not, in general, present with clinical symptoms. However, some female carriers have been identified with clinical features resembling those of affected males. The presence of asymptomatic female carriers within these same kindreds suggests that this finding is due to skewed X-inactivation rather than the specific mutation carried (van Lieburg et al. 1995). Therefore, in female NDI patients, the possibility of heterozygosity for an AVPR2 gene mutation must be considered.


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Cell biology. Studies on the mechanism of disease with mutations derived from congenital NDI patients have greatly added to our understanding of vasopressin receptor cell biology. V2 receptor mutations have been divided into three distinct classes, based on a classification system devised for the low-density lipoprotein receptor (Hobbs et al. 1990; Ala et al. 1998). Type 1 mutant receptors reach the cell surface but display impaired ligand binding and are consequently unable to induce normal cAMP production. Type 2 mutant receptors have defective intracellular transport. It is likely that these mutant type 2 receptors accumulate in a pre-Golgi compartment because they are initially glycosylated but fail to undergo glycosyltrimming maturation. Type 3 mutant receptors are ineffectively transcribed. This subgroup seems to be rare because Northern blot analysis of transfected cells reveals that most V2R mutations do not affect the quantity or molecular size of the receptor mRNA (Morello and Bichet 2001). The vast majority of naturally occurring mutations in the V2R that cause NDI are type 2 mutations. Missense mutations and short in-frame deletions or insertions that impair the propensity of the affected polypeptide to fold into its functional conformation have been termed conformational diseases (Carrell and Lomas 1997). Morello et al. (2000) reasoned that V2R ligands might alter the conformation of certain misfolded V2R mutants and allow them to be folded properly, thus releasing them from the endoplasmic reticulum quality control apparatus and allowing them to migrate to the cell surface. They therefore assessed the in vitro effect of cell-permeant selective V2R antagonists such as SR121463 on folding of mutant proteins which are responsible for NDI and which are retained in the endoplasmic reticulum. They found that these antagonists were able to convert precursor forms of mutant V2R into fully glycosylated mature receptor proteins, and furthermore, that the receptors were targeted to the cell surface. Once at their correct cellular location, these receptors were able to bind to AVP and produced an intracellular cAMP response that was 15 times higher than that produced in cells not exposed to these antagonists. This effect could neither be mediated nor overcome by V2R antagonists that are membrane impermeant, indicating that SR121463A was mediating its effects intracellularly. These findings raise the intriguing possibility that patients with congenital NDI caused by certain conformational mutants could someday be treated effectively with cell-permeant receptor ligands. Autosomal recessive NDI Genetics. In 10 per cent of patients with inherited NDI, autosomal recessive transmission is observed. This form of the disease is caused by homozygous loss of function mutations in AQP2 (Deen et al. 1995). Most mutations identified have been missense alterations, but frameshift mutations and nonsense mutations have also been seen. As with AVPR2 mutations causing X-linked NDI, the major cause underlying NDI in these patients is misrouting of AQP2 mutant proteins. Mutations in other genes in the water reclamation pathway can also affect water transport. Humans lacking aquaporin-1 have recently been described (King et al. 2001). While they do not have overt NDI, they exhibit an inability to maximally concentrate their urine in response to water deprivation. Mice bearing homozygous mutations in a variety of other genes also present with NDI. For example, knockout mice lacking aquaporin-3 have severe polyuria while an aquaporin-4 knockout mouse has mild polyuria (Chou et al. 1998; Ma et al. 2000; Verkman et al. 2000). Similarly, mice lacking the ClC-K1 gene (Matsumura et al. 1999) have overt NDI, which is thought to be due to the requirement for loop of Henle NaCl transport for

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urinary concentration, in a passive model of the counter-current multiplication system. While it remains possible that patients with autosomal recessive NDI carry mutations in one of these genes instead of in AQP2, to date none has been identified. Autosomal dominant NDI Genetics. Autosomal dominant transmission of NDI has been observed and is due to dominant negative mutations in the AQP2 gene. One such mutation, a substitution of glutamic acid for lysine at codon 258, causes a conformational shift in the peptide, leading to retention of aquaporin-2 in the Golgi apparatus (Knoers and Monnens 1999). Wild-type aquaporin is retained in the Golgi along with the mutant channel in a heterotetramer, leading to loss of distal nephron water reabsorption. Three C-terminal frameshift mutations causing autosomal dominant NDI have been described (Kuwahara et al. 2001). Each produces a novel 61 amino acid C-terminus that apparently disrupts intracellular trafficking. Treatment. In contrast to patients with X-linked or recessive NDI, who were unable to increase urine osmolality above 150 mmol/kg in response to DDAVP infusion, patients with dominant disease transiently increased urine osmolality to approximately 350 mmol/kg (Mulders et al. 1998), suggesting a possible therapeutic benefit of DDAVP in this population. Renal tubular acidosis (RTA) The kidney plays a key role in the regulation of acid-base balance homeostasis, being able to vary bicarbonate reclamation and net acid excretion over a wide range. This takes place at two main sites: the proximal convoluted tubule and the collecting duct. Renal acid-base balance may become deranged in a number of ways, some of which are the consequence of inherited disorders. These are relatively rare in Western populations, but occur more commonly in areas of the world where rates of parental consanguinity are high. Two main defects are either proximal bicarbonate loss (causing type 2 or proximal RTA), or inability to secrete acid in the distal nephron (giving rise to type 1 or distal RTA). Distal RTA most commonly arises as a secondary phenomenon, for example in the context of drugs or hypo-aldosteronism, when it is referred to as type 4 RTA. In this context it is usually accompanied by hyperkalaemia. However, there are many reports of primary (inherited) type 1 distal RTA (dRTA). At the time of early descriptions, dRTA was thought to be due to ‘back-leak’ of normally secreted protons across a leaky tubular epithelium. Over the following five decades it became evident that acid secretion itself is abnormal, due to failure of hydrogen ion secretion in the cortical collecting duct. Whether this was a primary or secondary event at the molecular level has, however, remained an enigma, and until recently, the molecular pathophysiology remained elusive. It is now clear that defective function in at least two components of the -intercalated cell’s polarized membrane transporters can cause dRTA (Fig. 14.3). On the apical surface, the multi-subunit proton pump transfers H into the urine. At least two of its subunits have been found to have different tissue-specific isoforms (the B and a subunits). This proton pump function is functionally coupled to the basolateral reclamation of bicarbonate ions (in exchange for chloride) via the anion exchanger AE1.


NBC1 HCO3–

Collecting duct α-intercalated

Proximal tubule B1 a4

Cl–

CLC5

Na+

H+

H+ + HCO3– CAII H2O + CO2

Blood

325

ATP ADP

Cl–

H++HCO3–

AE1 HCO3–

CAII H2O+CO2

Lumen SLC4A4 CLCN5 CA2 ATP6V1B1 ATP6V0A4 SLC4A1

NBC1 CLC5 CAII H+ATPase B1 subunit H+ATPase a4 subunit AE1

Lumen Prox RTA Dent Osteopetrosis dRTAw/deafness dRTA dRTA

Blood

Fig. 14.3 Gene products mutated in primary acidopathies. Symbols are as in Fig. 14.1, with pumps shown as grey circles. Carbonic anhydrase 2 catalyses the conversion of CO2 and H2O to H and HCO 3. Dent disease is described in Chapter 13. ATP6V1B1 and ATP6V0A4 are kidney-specific isoforms of the apical proton pump’s B and a subunits respectively.

Distal RTA (Type 1 or dRTA) Clinical features Primary dRTA arises when the collecting duct fails to remove excess acid into the urine. It is therefore characterized biochemically by failure of the kidney to produce appropriately acid urine in the presence of systemic metabolic acidosis or following acid loading (e.g. with ammonium chloride), and was first recognized some 70 years ago (Lightwood 1935; Butler et al. 1936). This failure to excrete appropriately the normal acid products of the diet results in hyperchloraemic metabolic acidosis of varying severity. Primary dRTA is almost always accompanied by nephrocalcinosis and/or nephrolithiasis. Urinary citrate is low in dRTA, because citrate reabsorption is up-regulated in the proximal tubule to provide new bicarbonate (1 citrate 2 bicarbonate). Abnormal calcium deposition in dRTA is attributed in large part to this hypocitraturia and to urine alkalinity, but the exact mechanisms for, and precise sites of, this deposition are unclear. Other biochemical findings in primary dRTA include hypokalaemia and osteomalacia or rickets, with normal serum calcium and phosphate levels. Both autosomal dominant and autosomal recessive inheritance patterns have been reported in primary dRTA. The spectrum of clinical severity is very wide, ranging from compensated mild acidosis, absence of symptoms and the incidental finding of renal tract calcification or stones on the one hand, to major effects in infancy, with severe acidosis, impaired growth and early nephrocalcinosis causing eventual renal insufficiency (Table 14.3). In general, though not invariably, patients with dominant (type 1a) dRTA display a milder phenotype than do those with recessively inherited disease. In addition, among patients with recessive but not dominant dRTA, a substantial fraction has progressive and irreversible bilateral sensorineural hearing loss (SNHL; type 1b) (Zakzouk et al. 1995). One report (Berretini et al. 2001)

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Table 14.3 Clinical and biochemical features of primary acidopathies Type 1a (dominant dRTA)

Presentation Symptoms/ signs

Older/adult None? Nephrolithiasis Nephrocalcinosis Sometimes rickets Sometimes osteomalacia Biochemistry Mild or compensated hyperchloraemic acidosis Low/normal K Min. urine pH 5.5 Treatment Citrate/bicarbonate

Type 1b (recessive dRTA w/deafness

Type 1c (recessive dRTA w/ preserved hearing)

Type 2 (proximal RTA)

Infancy/early childhood Early nephrocalcinosis Vomiting/dehydration Poor growth

Infancy/early childhood Early nephrocalcinosis Vomiting/dehydration Poor growth

Childhood Skeletal deformity Blindness Conductive hearing loss

Rickets Bilateral SNHL Severe hyperchloraemic acidosis Low K Min. urine pH 5.5 Citrate/bicarbonate

Rickets Severe hyperchloraemic Severe hyperchloraemic acidosis acidosis Low K Min. urine pH 5.5 Citrate/bicarbonate

Normal/low K Min. urine pH 5.5 Much bicarbonate K

describes the radiological observation of vestibular aqueduct widening in association with recessive dRTA, but this abnormality is not pathognomonic, also being seen in Branchio-OtoRenal syndrome (Chapter 5), Pendred syndrome and in isolation. Autosomal dominant dRTA (type 1a) Genetics. Many investigators have hypothesized that the underlying mechanism in dRTA was most likely defective apical proton pump function, either of the H- or H/K-ATPase. However, two earlier reports of patients with coexistent dRTA and ovalocytosis or elliptocytosis prompted the initial consideration of SLC4A1 as a candidate gene for dRTA (24), although these kindreds did not include the demonstration of co-segregation of these traits, nor mutation analysis. Three groups have identified ten dominant dRTA kindreds, in which affected individuals are heterozygous for mutations in AE1 (Bruce et al. 1997; Karet et al. 1997; Jarolim et al. 1998). This represented the first molecular evidence that anion rather than cation transport function is at fault in dominant dRTA. There is no current evidence for genetic heterogeneity in dominant dRTA. Cell biology. Strikingly, a single base change alters the identical AE1 residue, Arg589, in 8 of the 10 reported kindreds, supporting the importance of this residue in the normal acidification process. Arg589 lies at the intracellular border of the sixth transmembrane domain of the protein, adjacent to Lys590; this latter residue is the target for the specific AE1 inhibitor phenyl isothiocyanate. These basic residues are conserved in all the known vertebrate anion exchanger isoforms and are thought to form part of the site of intracellular anion binding. It is notable that expression of these mutations in Xenopus oocytes did not generally result in significant loss of function, suggesting that simple haploinsufficiency cannot explain the dRTA phenotype. Furthermore, mutations in SLC4A1 can cause the dominant red cell morphologic diseases hereditary spherocytosis and ovalocytosis, including mutations that result in very early termination (codon 81) or frameshift (codon 170) (Jarolim et al. 1996). These would probably result in severe disruption or absence of the encoded protein, but are not


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usually associated with classical dRTA. It is possible, but not yet established, that the apparent dominant negative effects of AE1 mutations in dominant dRTA are due to mis-targeting of the mutant protein away from its usual basolateral location, or some alteration of the interaction of AE1 with other intracellular components (Tanphaichitr et al. 1998). Autosomal recessive dRTA Genetics. In approaching the genetic causes of recessive dRTA, the potential involvement of AE1 (both by linkage analysis and direct screening) has been assessed. In a cohort of kindreds of largely Middle Eastern origin, linkage was conclusively excluded. However, a few recessive kindreds from Thailand with coexistent haemolytic anaemia and RTA have now been described, with loss-of-function changes in AE1 that manifest in Xenopus in vitro but not red cell anion transport (Tanphaichitr et al. 1998; Vasuvattakui et al. 1999). To date, however, this phenotypic combination seems to be confined to south-east Asia. A genome-wide search to localize a gene or genes for recessive dRTA revealed evidence for linkage to two loci, on 2p and 7q, in a cohort of mainly consanguineous kindreds. The responsible genes have both been identified, accounting for types 1b (deaf) and 1c (hearing) disease, respectively. DRTA with deafness (type 1b) Genetics. ATP6V1B1, the gene encoding the B1-subunit of H-ATPase resides on chromosome 2. This gene was of particular interest because it encodes the kidney-associated B1 isoform of the secretory proton pump in the kidney. Radiation hybrid mapping definitively placed ATP6V1B1 within the linked dRTA interval. Screening for mutations in this gene revealed fifteen different mutations in kindreds where almost all the affected individuals had documented bilateral SNHL, and in all but one kindred had homozygous mutations (Karet et al. 1999). The majority of these mutations are likely to disrupt the structure, or abrogate the production, of the normal B1-subunit protein. DRTA with preserved hearing (type 1c) Genetics. By a similar linkage approach in a cohort of dRTA kindreds where hearing was essentially normal, the defective gene in this subset of families was found, and encodes the newly identified kidney-specific a4 isoform of the proton pump’s 116 kDa accessory a-subunit (ATP6V0A4). Although this genetic finding shows that the a4 subunit must be essential for proper proton pump function in the kidney, its role within the multi-subunit pump structure is at present unclear (Smith et al. 2000). Apart from the presence or absence of hearing loss, there do not appear to be major phenotypic differences between recessive patients with and without hearing loss. Treatment. Simple alkali replacement (by administration of citrate or bicarbonate orally) is sufficient to reverse most of the biochemical abnormalities and associated bone disease in both dominant and recessive dRTA, leading to the resumption of normal growth. Potassium salts are preferable to sodium as the latter can exacerbate hypokalaemia. However, while alkali administration prevents further calcium deposition, it does not appear either to ameliorate or to prevent the progression of the hearing impairment. Further heterogeneity. Some families do not appear to link to either ATP6V1B1 or ATP6V0A4. There are numerous other candidate genes for recessive dRTA, including (a) genes for all the subunits of the proton transporters; (b) genes whose products are required for

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trafficking of proton pumps to the apical membrane of the -intercalated cell (without altering any of the elements of the pump itself); and (c) genes encoding molecules necessary for the generation of protons, absorption of bicarbonate, recycling of chloride or maintenance of the electrochemical gradient across both apical and basolateral membranes. Unfortunately, many of these potential candidates remain unidentified, uncharacterized or as yet unmapped in humans. In addition, known genes may have as yet unrevealed novel isoforms in the kidney. Proximal RTA (type 2) Clinical. Proximal RTA is characterized by presentation in childhood, bicarbonate wasting in to the urine (unless the serum bicarbonate is so low that the filtered load can be reabsorbed), with preserved ability to acidify the urine. Nephrocalcinosis and stones are rare, but rickets and osteomalacia may be seen. Hypokalaemia is usually mild, but may become marked if large sodium bicarbonate loads have been administered. Genetics. In general, proximal RTA arises as a secondary consequence of related proximal tubular dysfunction, as in the Fanconi syndrome (see Chapter 12). However, a recent report has studied two kindreds in which the affected offspring of normal parents have proximal RTA associated with glaucoma, cataracts and band keratopathy (Igarashi et al. 1999). Homozygous loss-of-function mutations were found in SLCA4, the gene encoding a sodiumbicarbonate co-transporter present in kidney. Interestingly, the corneal endothelium normally transports Na and HCO3 from corneal stroma to aqueous humour, probably via this same sodium bicarbonate co-transporter SLC4A4, which is therefore thought to play a role in preservation of corneal clarity. Proximal RTA is more difficult to treat than distal, and may require very large quantities of bicarbonate supplementation, which is desirable to maintain growth. Vitamin D and phosphate may also be useful. Mixed RTA (type 3) The entity of primary mixed proximal and distal RTA, which enjoyed some popularity a few decades ago, is not now thought to represent a distinct disease process, but rather a developmental hiatus in that distal nephron function continues to mature after birth. However, RTA with the characteristics of both proximal and distal tubular dysfunction usually accompanies one form of autosomal recessive osteopetrosis. (Guibaud–Vainsel syndrome or marble brain disease.) Here, both defective urinary acidification and bicarbonate wasting are observed. This condition is characterized by fractures, short stature, mental retardation, dental malocclusion, and visual impairment from optic nerve compression. Basal ganglion calcification may develop. Sly et al. (1983) identified loss of carbonic anhydrase 2 (CA2) as the biochemical defect, and loss-of-function mutations have subsequently been described (Hu et al. 1997). The commonest of these involves loss of the splice donor site in intron 2, nicknamed the Arabic mutation. It introduces a novel Sau3A1 restriction site that is useful in PCR-based screening, carrier detection, and antenatal diagnosis. The presence of mental retardation and relative infrequency of skeletal fractures distinguishes the clinical course of patients with the Arabic mutation from that of Caucasian patients with other CA2 defects.


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15 Tuberous sclerosis complex Astrid P. Weber and Robert F. Mueller

Introduction Tuberous sclerosis complex (TSC) is a neurocutaneous syndrome characterized by the development of hamartomatous lesions in numerous organ systems. It was first described by Bourneville in 1880 who described the association between mental retardation and fits with post-mortem findings of tuber-like growths and areas of sclerosis (Bourneville 1880). Vogt (1908) later described the classical triad of fits, mental retardation, and facial angiofibromata. It has since been recognized that TSC is a multisystem disorder and can involve the brain, skin, eyes, lungs, heart, kidneys, and endocrine organs. Diagnostic criteria first proposed by Gomez (1988) have recently been updated and revised (Roach et al. 1998). Systematic ascertainment studies have reported population prevalence figures as high as 1 in 5000 under the age of 5 years and 1 in 25,000 for individuals of all ages (Hunt 1983; Hunt and Lindenbaum 1984; Sampson et al. 1989; Shepherd et al. 1991a; Ahlsen et al. 1994). Genetics of tuberous sclerosis TSC is an autosomal dominant disorder which arises as a new mutation in 60–70 per cent of cases (Fleury et al. 1980). It exhibits variable expressivity, with considerable variation in the clinical features in affected individuals, both within and between families (Sampson et al. 1989). Linkage Two loci have been identified as causing TSC, TSC1 and TSC2. Linkage of TSC to the ABO blood group locus on the long arm of chromosome 9 at 9q34 was first described in 1987 (Fryer et al. 1987). In a significant proportion of multigeneration families with TSC linkage to that locus was excluded. The occurrence of polycystic kidney disease (PKD) in some individuals with TSC suggested that the short arm of chromosome 16 containing the gene for PKD (PKD1) was a potential candidate region for a second gene for TSC. Linkage to the TSC2 locus at 16p13.3 was reported by Kandt et al. in 1992. Multigeneration families with TSC demonstrate linkage to TSC1 and TSC2 in approximately equal proportions (Povey et al. 1994) (Table 15.1). The possibility of other previously suggested loci being responsible for TSC has been excluded (Janssen et al. 1994). Gene cloning The TSC2 and PKD1 genes were identified through a family with a chromosomal translocation involving 16p in which affected individuals had either TSC or PKD. The TSC2 gene was

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Astrid P. Weber and Robert F. Mueller Table 15.1 Proportion of familial and sporadic cases of tuberous sclerosis linked to or due to a mutation in either TSC1 or TSC2 TSC gene

Proportion of familial cases*† (%)

Proportion of sporadic cases† (%)

TSC1 TSC2

50 50

10 68

*Povey †Jones

et al. 1994. et al. 1999.

isolated in 1993 (The European Chromosome 16 Tuberous Sclerosis Consortium 1993) and consists of 41 exons spanning 43 kb of genomic DNA. The protein, tuberin, contains a relatively hydrophobic N-terminal domain and a conserved 163-amino acid region close to the C-terminus. It has two predicted coiled-coil domains (van Slegtenhorst et al. 1998). The TSC1 gene was identified in 1997 by positional cloning and sequencing (van Slegtenhorst et al. 1997) and found to contain 23 exons spanning 45 kb of genomic DNA. The protein encoded, hamartin, is relatively hydrophilic. The predicted protein structure includes a coiled-coil domain at the C-terminus and a single potential membrane spanning domain. No homologous vertebrate protein has been identified (van Slegtenhorst et al. 1997). Biological function of TSC1 and TSC2 Tuberin and hamartin share no structural homology. However, they appear to associate closely in vivo via their coiled-coil structures to exist and function as a complex (van Slegtenhorst et al. 1998). This is thought to explain the similar phenotypic features seen in persons with mutations in either gene. The C-terminal part of tuberin has homology to rap1 GTPase activating protein (GAP) and has recently been shown to interact with rabaptin-5 (Maheshwar et al. 1997). The latter is a cytosolic protein which acts as an effector for the endosomal small GTPase, rab5 (Stenmark et al. 1995). Rab5 is involved in endocytosis. Tuberin and hamartin, possibly interacting with other endosomal proteins, have been postulated to reduce the rate of fluid-phase endocytosis (Xiao et al. 1997). Investigations are underway to determine whether dysregulation of endocytosis is important in the aetiology of TSC (van Slegtenhorst et al. 1998). Mutational basis of disease Of patients with tuberous sclerosis 60–80 per cent have an identifiable mutation in either the TSC1 or TSC2 gene (Jones et al. 1999) (Table 15.1). Although it was initially suggested that the variable phenotypic features seen in persons with TSC could be due to it occurring as the result of a mutation in one or other of the two genes (Jones et al. 1997), this has not been a consistent finding. Sporadic cases of TSC have been found to be most commonly due to mutations in the TSC2 gene (Jones et al. 1999). The nature of mutations found in the TSC1 and TSC2 genes differs. Mutations reported in the TSC1 gene include deletions causing frame-shifts which lead to premature termination

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codons and splice-site mutations resulting in a truncated protein. A large proportion of the mutations reported occur in exons 15 and 17. Exon 15 accounts for 16 per cent of the coding structure. To date no missense mutations have been shown to alter the function of hamartin (van Slegtenhorst et al. 1999). There are no significant clinical differences between patients with deletions which maintain the reading frame and truncating mutations of the TSC1 gene (van Slegtenhorst et al. 1999). A germline deletion of the TSC2 gene has been reported in approximately 5 per cent of TSC patients (The European Chromosome 16 Tuberous Sclerosis Consortium 1993). A diverse mutational spectrum has been reported in persons with TSC due to mutations in the TSC2 gene, including large deletions, insertions and non sense mutations leading to a truncated tuberin protein, along with missense mutations. Deletions of the whole of the TSC1 gene, and adjacent PKD1 gene, have been reported in individuals with features of TSC and polycystic kidney disease (Sampson et al. 1997). Tumour suppressor phenotype There is substantial evidence that both TSC1 and TSC2 function as tumour suppressor genes, according to Knudson’s two-hit hypothesis (Knudson 1971). The majority of TSC1 and TSC2 mutations are likely to inactivate the protein (van Slegtenhorst et al. 1998). Loss of heterozygosity (LOH) has been found in approximately 50 per cent of hamartomata associated with germline mutations of TSC2. Although less than 10 per cent of TSC1associated hamartomata demonstrate LOH, many of these tumours have an additional inactivating mutation in the wild-type allele. A second mutation may be the main mechanism of tumorigenesis in TSC1 instead of LOH (van Slegtenhorst et al. 1998). The tumour suppressor hypothesis predicts that similar tumours to those seen in people with TSC should be found in the general population. Sporadic tumours would be postulated to arise from two separate TSC gene mutations. Many of the tumours found in people with TSC do indeed occur sporadically, including renal angiomyolipoma. The clonal origin of TSC lesions is further evidence for the tumour suppressor hypothesis. Despite the mixture of cell types in TSC lesions, such as renal angiomyolipomata, there is evidence that they arise from a common progenitor cell (Green et al. 1994; Kattar et al. 1999). The prevalence of angiomyolipomata correlates positively with age, which is also consistent with the two-hit hypothesis (Stillwell et al. 1987). The Eker rat hereditary renal carcinoma model demonstrates autosomal dominant inheritance of a strong predisposition towards renal and other tumours (Eker and Mossige 1961). The mutated gene is the rat homologue of human TSC2 (Tsc2) (Hino et al. 1994). LOH has been demonstrated in the neoplastic and early preneoplastic renal lesions of affected rats (Yeung et al. 1994). Renal disease in tuberous sclerosis Types of renal disease in tuberous sclerosis Renal involvement in TSC is usually asymptomatic, although the prevalence has been estimated at 40–80 per cent. Three main lesions occur, either singly or together; renal cystic disease, angiomyolipoma, and renal cell carcinoma. Less common complications include adenocarcinoma, oncocytoma, interstitial fibrosis, and focal segmental glomerulosclerosis (Torres et al. 1994).

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Renal disease is the second most common cause of death in persons with TSC, after neurological complications, and the commonest cause of death in those over 30 years of age (Shepherd et al. 1991b). There is no apparent correlation between the severity of renal disease and that of neurological or cutaneous manifestations (Webb et al. 1994). End-stage renal failure (ESRF) is a rare complication of TSC, occurring in an estimated 1 per 100 patients (Schillinger and Montagnac 1996; Clarke et al. 1999). Angiomyolipoma Angiomyolipomata are benign vascular tumours composed histologically of smooth muscle, often with cellular atypia, mature adipose tissue, and thick-walled blood vessels (Fig. 15.1) in varying proportions (Morgan et al. 1951). Angiomyolipomata occur in 40–80 per cent of patients with TSC, but TSC accounts for only 20 per cent of renal angiomyolipomata (Webb et al. 1994). Sporadic angiomyolipomata are usually single lesions occurring in women in their third or fourth decade but can be multiple. The female preponderance probably reflects a hormonal influence (Webb et al. 1994). Female patients with TSC are also more prone to angiomyolipomata than males. Females tend to have a greater number of angiomyolipomata, present at an earlier age with them, and have more complications than males with TSC (Webb et al. 1994). In persons with TSC renal angiomyolipomata tend to be bilateral and multiple, and increase in prevalence with age (Stillwell et al. 1987). Patients with angiomyolipomata due to TSC tend to present at a younger age, are more frequently symptomatic, have larger tumours, and more frequently require surgery (Steiner et al. 1993). Several studies have shown that angiomyolipomata with a diameter greater than 4 cm are more likely to enlarge and result in complications requiring medical or surgical intervention than lesions under 4 cm (Oesterling et al. 1986; Steiner et al. 1993; Webb et al. 1994; Lemaitre et al. 1995). The development of angiomyolipomata has been found to start in children with TSC as young as two years of age (Ewalt et al. 1998). However, such lesions usually remain small until puberty and virtually never cause serious complications in childhood (Ewalt et al. 1998).

Fig. 15.1 A histological section of kidney showing angiomyolipomata from an individual with TSC (the clear cystic areas are fat which has been lost in the processing of the section).


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Despite their benign nature, angiomyolipomata may extend into the renal vein and have rarely been found in lymph nodes (Busch et al. 1976). They can also extend beyond the renal capsule and metachronous lesions have been found in other organs such as the spleen and liver. This seems to result from a multicentric site of origin rather than metastasis (Sant et al. 1986). Complications of angiomyolipoma. The most common symptom of renal angiomyolipoma(ta) is flank or abdominal pain, due to spontaneous perinephric haemorrhage. Angiomyolipomata are prone to bleeding due to their high vascularity, tortuous blood vessels, and vascular malformations such as aneurysms and arteriovenous malformations (Van Baal et al. 1994). Haemorrhage may be life-threatening, patients presenting with hypovolaemic shock (Van Baal et al. 1994). Approximately a third of symptomatic patients have microscopic or macroscopic haematuria (Fazeli-Matin and Novick 1998). Some present with hypertension, a palpable mass, or anaemia. Patients may also complain of weight loss, fever, or nausea. There is an increased risk of rupture of angiomyolipomata in pregnancy when haemodynamic compromise can result in fetal distress (Forsnes et al. 1996). End-stage renal failure secondary to angiomyolipomata in persons with TSC is uncommon. When it occurs it is usually secondary to replacement of the renal parenchyma by angiomyolipomata, or due to bilateral nephrectomy for intractable haemorrhage (Balligand et al. 1990; Neumann et al. 1995). Diagnosis of angiomyolipoma. Urinary dipstick testing does not usefully distinguish larger lesions or those at greater risk of bleeding (Webb et al. 1994). Renal ultrasound scanning demonstrates an echodense intra-renal mass (Fig. 15.2). The echogenicity may not be uniform (Hartman et al. 1981). Computerized tomography shows lipomatous elements which are highly specific for angiomyolipoma. MRI does not distinguish fatty tissue well (Clarke et al. 1999). Management of angiomyolipoma. A balance must be struck between treating or preventing symptoms and complications, and conserving renal function. Acute severe haemorrhage from an angiomyolipoma can be treated by total nephrectomy or angioembolization (Fig. 15.3). Angiographic demonstration of one or more accessible

Fig. 15.2 Ultrasound scan of the left kidney of an individual with TSC showing multiple areas of nonuniform echogenicity consistent with multiple angiomyolipomata.

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Fig. 15.3 Kidney removed from an individual with TSC due to an uncontrollable haemorrhage.

feeder arteries may allow selective embolization. This procedure is attractive because it is minimally invasive, successful and has a low complication rate. Selective angioembolization can be used to shrink large lesions to make them amenable to renal-sparing surgery, or as a definitive procedure (Fazeli-Matin and Novick 1998). It has some disadvantages, however, which includes additional procedures, such as repeat embolization, percutaneous drainage of liquefactive necrosis, or nephrectomy, either partial or total. Renal function may not be conserved by the procedure, particularly if the artery embolized also supplies extensive normal parenchyma. Surgery has been successfully employed to remove even large lesions, allowing renal function to be preserved (Blute et al. 1988; Fazeli-Matin and Novick 1998). Partial nephrectomy is suitable for a cluster of lesions in one pole of the kidney or for lesions not amenable to embolization (Oesterling et al. 1986). Enucleation can be performed in single peripheral lesions. Cystic disease Renal cystic disease occurs in approximately 15–20 per cent of patients with TSC (Stapleton et al. 1980). Radiologically and macroscopically this resembles autosomal dominant PKD (Torres et al. 1994). However, the histological appearance is considered to be unique with the cysts being lined with hyperplastic epithelium consisting of large eosinophilic cells with large hyperchromatic nuclei. There is often increased mitotic activity and the epithelium can appear ‘piled up’ with nodular extensions of epithelium into the cyst lumen (Stapleton et al. 1980). The cysts can arise from all portions of the nephron. They are usually bilateral, multiple, and of various sizes (Stapleton et al. 1980). There is no correlation with age. Isolated cystic involvement occurs in only a minority of patients with TSC (Sampson 1996). The severity of cystic disease in TSC varies widely between families and also within families. The type of mutation and the presence of mosaicism partly explains this. Somatic mosaicism has been reported in 10–30 per cent of families with a combined TSC/PKD phenotype (Sampson et al. 1997). On rare occasions enlarged cystic kidneys are the first presenting feature of TSC in infancy, leading to an initial misdiagnosis of early onset autosomal dominant polycystic kidney disease


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(ADPKD), or autosomal recessive PKD (Webb et al. 1993). Many such cases have been found to have deletions encompassing both the TSC2 and PKD1 genes (Brook-Carter et al. 1994). Deletions of TSC2 and PKD1 may involve only the 3 untranslated region (UTR) of PKD1, in which case renal cystic disease tends to be less severe. TSC2 gene inversion is associated with moderately severe renal cystic disease. This may result from the disruption of regulatory sequences upstream of the PKD1 gene (Sampson 1996). Mild cystic disease may be associated with mutations of TSC1 or TSC2 without involvement of PKD1 (Brook-Carter et al. 1994). The increased severity and different histology of cystic disease in cases with a combined deletion of TSC2 and PKD1 compared to cases of ADPKD may reflect the loss of the TSC2 gene in addition to PKD1. The difference may also be attributable to the loss of the entire PKD1 gene (Brook-Carter et al. 1994). In ADPKD mutations of the PKD1 gene are most commonly missense mutations (Peral et al. 1996). Complications of cystic disease. Renal impairment may occur, complicated by hypertension necessitating medical therapy (Sampson et al. 1997). Severe cystic disease can progress to ESRF due to destruction of the renal parenchyma. ESRF may also result from glomerulosclerosis as a secondary phenomenon accompanying renal cystic disease (Schillinger and Montagnac 1996). Diagnosis of cystic disease. Ultrasound is very sensitive in identifying and measuring renal cysts (Hartman et al. 1981). Campos et al. (1993) have suggested renal biopsy in cases of cystic kidney disease in infancy where the diagnosis remains in doubt. Management of cystic disease. Monitoring of renal function in individuals with TSC with severe renal cystic disease allows anticipation of ESRF to be able to provide support by dialysis and/or renal transplantation. Patients with TSC are good candidates for dialysis and renal transplantation. In those patients undergoing renal transplant, many advocate removal of the native kidneys to avoid later risks of bleeding from angiomyolipomata or the development of renal malignancy (Balligand et al. 1990). Others argue that the native kidneys should be left in situ in case of transplant failure, in which case any remaining native kidney function would be of benefit to the patient (Schillinger and Montagnac 1996). Concurrent renal angiomyolipomata should be monitored by ultrasound and treated to preserve renal function. Decompression of large cysts to protect the function of normal renal parenchyma has been reported (Stillwell et al. 1987). Renal malignancy The true prevalence of malignant renal disease in people with TSC is unknown but has been said to occur in about 2–4 per cent of patients with TSC (Cook et al. 1996). Several factors point towards a predisposition to renal malignancy in TSC. There is a higher number of reported cases of renal malignancy than would be expected by chance alone (Bjornsson et al. 1996). LOH has been found at either the TSC1 or the TSC2 locus in some sporadic renal cell carcinomata (Bjornsson et al. 1996). The Eker rat shows mutations at the rat equivalent of TSC2 (Hino et al. 1994). Evidence that renal malignancies in TSC arise from dysplastic cysts and angiomyolipomata also strongly suggests a predisposition to malignancy in persons with TSC (Al-Saleem et al. 1998). Renal cell carcinoma has been described as occurring in patients with TSC as young as 5 years of age (Robertson et al. 1996). Overall, renal malignancies in persons with TSC occur on average 20 years earlier than the sporadic form (Bjornsson et al. 1996). It is bilateral in

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approximately 40 per cent of patients with TSC, compared to 2–4 per cent of sporadic cases (Weinblatt et al. 1987; Washecka and Hanna 1991). Washecka and Hanna (1991) have reported a female preponderance. Sporadic renal cell carcinoma stains positive with anti-cytokeratin antibody and negative with anti-HMB-45 antibody (Pea et al. 1991). HMB-45 is a marker of neural crest or melanocyte derivation. Angiomyolipomata stain positive for HMB-45. Renal cell carcinoma in patients with TSC can show either pattern of immunoreactivity. It is postulated that those with typical immunostaining are derived from dysplastic renal tubules, while lesions that histologically resemble renal cell carcinoma but demonstrate the staining pattern of angiomyolipomata, are derived from the latter (Al-Saleem et al. 1998). Complications of renal malignancy. Weinblatt (1987) have reported a more benign course of renal neoplasms in persons with TSC compared with sporadic renal cell carcinoma. Metastases are infrequent at presentation, possibly because renal neoplasia are picked up at an early stage in persons with TSC because of coincident benign renal disease (Washecka and Hanna 1991; Sampson et al. 1995). Diagnosis of renal malignancy. Renal cell carcinoma is echolucent on ultrasound scan and may contain intratumoral cysts (Yamashita et al. 1993). The presence of an anechoic rim supports the diagnosis (Yamashita et al. 1993). Although renal cell carcinoma can contain fat, visible at gross pathological examination, this does not produce low attenuation values on CT. In the majority of cases CT can differentiate renal cell carcinoma from angiomyolipoma (Stillwell et al. 1987). If doubt remains, fine needle aspiration biopsy (FNAB) can be undertaken to exclude malignancy (Murphy et al. 1985). However, the histology at the edge of an angiomyolipoma can look like renal cell carcinoma (Nguyen 1984). It has been suggested that immunohistochemistry with anti-HMB-45 can be used to distinguish them (Pea et al. 1991). Management of renal malignancy. mandatory.

Early diagnosis and removal of malignant lesions is

Screening for renal disease in tuberous sclerosis All patients with TSC should have regular clinical review. Ewalt (1998) suggested that all children with tuberous sclerosis should have two to threeyearly renal ultrasound scans before puberty, followed by annual renal ultrasound scanning post-pubertally. The long-term benefits of pre-pubertal screening have yet to be established (Ewalt et al. 1998). The guidelines for screening post-pubertal patients have been more extensively formulated. Angiomyolipomata less than 3.5–4 cm tend to be asymptomatic and rarely require intervention (Steiner et al. 1993). Annual evaluation by ultrasound scan is advised. Medium-sized lesions (4–8 cm) are less predictable. Approximately half will require intervention for haemorrhagic complications (Dickinson et al. 1998). Lesions that are steadily increasing in size should be treated, even if asymptomatic. Six-monthly CT or ultrasound scanning can be employed, unless significant changes in lesion size or patient symptoms are noted, in which case more frequent scanning is necessary (Van Baal et al. 1994). Large lesions (8 cm) are associated with significant morbidity. Even if asymptomatic, they are likely to become symptomatic. Elective treatment can prevent symptoms and complications (Dickinson et al. 1998).


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All lesions should be monitored closely during pregnancy when large increases in angiomyolipoma size have been observed (Forsnes et al. 1996). Genetic counselling in tuberous sclerosis Mutations in TSC1 and TSC2 are very diverse and many are unique to an individual family. The large size of both genes and the high rate of somatic mosaicism in TSC have hampered the development of a clinical molecular diagnostic test, although molecular testing is becoming available on a service rather than a research basis. If a mutation has been identified in an individual, prenatal and testing of family members at risk can be offered. The recurrence risks for familial (50 per cent) and sporadic (1–2 per cent) TSC are very different. The degree to which the relatives of a proband should be investigated is debated (Al-Gazali et al. 1989). Cassidy et al. (1983) advocate clinical examination, especially of skin, and investigation, including renal ultrasound scan, dilated ophthalmoscopy, and cranial CT scan. Other studies have not found a significant diagnostic yield from such investigations in the presence of a normal detailed clinical examination, including skin examination with a Wood’s lamp and routine ophthalmoscopy (Fryer et al. 1990). There is a consensus that a detailed physical examination is the most sensitive means of diagnosis (Cassidy et al. 1983). References Ahlsen, G., Gillberg, I. C., Lindblom, R., and Gillberg, C. (1994). Tuberous sclerosis in Western Sweden. A population study of cases with early childhood onset. Archives of Neurology, 51, 76–81. Al-Gazali, L. I., Arthur, R. J., Lamb, J. T., Hammer, H. M., Coker, T. P., Hirschmann, P. N., et al. (1989). Diagnostic and counselling difficulties using a fully comprehensive screening protocol for families at risk for tuberous sclerosis. Journal of Medical Genetics, 26, 694–703. Al-Saleem, T., Wessner, L. L., Scheithauer, B. W., Patterson, K., Roach, E. S., Dreyer, S. J., et al. (1998). Malignant tumors of the kidney, brain, and soft tissues in children and young adults with the tuberous sclerosis complex. Cancer, 83, 2208–16. Balligand, J.-L., Pirson, Y., Squifflet, J.-P., Cosyns, J.-P., Alexandre, G. P. G., and van Ypersele de Strihou, C. (1990). Outcome of patients with tuberous sclerosis after renal transplantation. Transplantation, 49, 515–8. Bjornsson, J., Short, M. P., Kwiatkowski, D. J., and Henske, E. P. (1996). Tuberous sclerosis-associated renal cell carcinoma. Clinical, pathological, and genetic features. American Journal of Pathology, 149, 1201–8. Blute, M. L., Malek, R. S., and Segura, J. W. (1988). Angiomyolipoma: clinical metamorphosis and concepts for management. The Journal of Urology, 139, 20–4. Bourneville, D. M. (1880). Sclérose tubéreuse des circonvolutions cérébrales: idiotie et épilepsie hémiplégique. Archives of Neurology, 1, 81–91. Brook-Carter, P. T., Peral, B., Ward, C. J., Thompson, P., Hughes, J., Maheshwar, M. M., et al. (1994). Deletion of the TSC2 and PKD1 genes associated with severe infantile polycystic kidney disease—a contiguous gene syndrome. Nature Genetics, 8, 328–32. Busch, F. M., Bark, C. J., and Clydine, H. R. (1976). Benign renal angiomyolipoma with regional lymph node involvement. The Journal of Urology, 116, 715–7. Campos, A., Figueroa, E. T., Gunasekaran, S., and Garin, E. H. (1993). Early presentation of tuberous sclerosis as bilateral renal cysts. The Journal of Urology, 149, 1077–9. Cassidy, S. B., Pagon, R. A., Pepin, M., and Blumhagen, J. D. (1983). Family studies in tuberous sclerosis. Evaluation of apparently unaffected parents. Journal of the American Medical Association, 249, 1302–4.

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Clarke, A., Hancock, E., Kingswood, C., and Osborne, J. P. (1999). End-stage renal failure in adults with the tuberous sclerosis complex. Nephrology Dialysis Transplantation, 14, 988–91. Cook, J. A., Oliver, K., Mueller, R. F., and Sampson, J. (1996). A cross-sectional study of renal involvement in tuberous sclerosis. Journal of Medical Genetics, 33, 480–4. Dickinson, M., Ruckle, H., Beaghler, M., and Hadley, H. R. (1998). Renal angiomyolipoma: optimal treatment based on size and symptoms. Clinical Nephrology, 49, 281–6. Eker, R. and Mossige, J. (1961). A dominant gene for renal adenomas in the rat. Nature, 189, 858–9. Ewalt, D. H., Sheffield, E., Sparagana, S. P., Delgado, M. R., and Roach, E. S. (1998). Renal lesion growth in children with tuberous sclerosis complex. The Journal of Urology, 160, 141–5. Fazeli-Matin, S. and Novick, A. C. (1998). Nephron-sparing surgery for renal angiomyolipoma. Urology, 52, 577–83. Fleury, P., de Groot, W. P., Delleman, J. W., Verbeeten, B. Jr., and Frankenmolen-Witkiezwicz, I. M. (1980). Tuberous sclerosis: the incidence of sporadic versus familial cases. Brain Development, 2, 107–17. Forsnes, E. V., Eggleston, M. K., and Burtman, M. (1996). Placental abruption and spontaneous rupture of renal angiomyolipoma in a pregnant woman with tuberous sclerosis. Obstetrics and Gynaecology, 88, 725. Fryer, A. E., Chalmers, A., Connor, J. M., Fraser, I., Povey, S., Yates, A. D., et al. (1987). Evidence that the gene for tuberous sclerosis is on chromosome 9. Lancet, 1(8534), 659–61. Fryer, A. E., Chalmers, A. H., and Osborne, J. P. (1990). The value of investigation for genetic counselling in tuberous sclerosis. Journal of Medical Genetics, 27, 217–23. Gomez, M. R. (1988). Tuberous Sclerosis (2nd edn). Raven Press, New York, NY. Green, A. J., Smith, M., and Yates, J. R. W. (1994). Loss of heterozygosity on chromosome 16p13.3 in hamartomas from tuberous sclerosis patients. Nature Genetics, 6, 193–6. Hartman, D. S., Goldman, S. M., Friedman, A. C., Davis, C. J. Jr., Madewell, J. E., and Sherman, J. L. (1981). Angiomyolipoma: ultrasonic-pathologic correlation. Radiology, 139, 451–8. Hino, O., Kobayashi, T., Tsuchiya, H., Kikuchi, Y., Kobayashi, E., Mitani, H., et al. (1994). The predisposing gene of the Eker rat inherited cancer syndrome is tightly linked to the tuberous sclerosis (TSC2) gene. Biochemical and Biophysical Research Communication, 203, 1302–8. Hunt, A. (1983). Tuberous sclerosis: a survey of 97 cases. I: Seizures, pertussis immunisation and handicap. Developmental Medical Child Neurology, 25, 346–9. Hunt, A. and Lindenbaum, R. H. (1984). Tuberous sclerosis: a new estimate of prevalence within the Oxford region. Journal of Medical Genetics, 21, 272–7. Janssen, B., Sampson, J., van der Est, M., Deelen, W., Verhoef, S., Daniels, I., et al. (1994). Refined localization of TSC1 by combined analysis of 9q34 and 16p13 data in 14 tuberous sclerosis families. Human Genetics, 94, 437–40. Jones, A. C., Daniells, C. E., Snell, R. G., Tachataki, M., Idziaszczyk, S. A., Krawczak, M., et al. (1997). Molecular genetic and phenotypic analysis reveals differences between TSC1 and TSC2 associated familial and sporadic tuberous sclerosis. Human Molecular Genetics, 6, 2155–61. Jones, A. C., Shyamsundar, M. M., Thomas, M. W., Maynard, J., Idziaszczyk, S., Tomkins, S., et al. (1999). Comprehensive mutation analysis of TSC1 and TSC2—and phenotypic correlations in 150 families with tuberous sclerosis. American Journal of Human Genetics, 64, 1305–15. Kandt, R. S., Haines, J. L., Smith, M., Northrup, H., Gardner, R. J., Short, M. P., et al. (1992). Linkage of an important gene locus for tuberous sclerosis to a chromosome 16 marker for polycystic kidney disease. Nature Genetics, 2, 37–41. Kattar, M. M., Grignon, D. J., Eble, J. N., Hurley, P. M., Lewis, P. E., Sakr, W. E., et al. (1999). Chromosomal analysis of renal angiomyolipoma by comparative genomic hybridization: evidence for clonal origin. Human Pathology, 30, 295–9. Knudson, A. G. Jr. (1971). Mutation and cancer: statistical study of retinoblastoma. Proceedings of the National Academy of Sciences, USA, 68, 820–3.


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Lemaitre, L., Robert, Y., Dubrulle, F., Claudon, M., Duhamel, A., Danjou, P., et al. (1995). Renal angiomyolipoma: growth followed up with CT and/or US. Radiology, 197, 598–602. Maheshwar, M. M., Cheadle, J. P., Jones, A. C., Myring, J., Fryer, A. E., Harris, P. C., et al. (1997). The GAP-related domain of tuberin, the product of the TSC2 gene, is a target for missense mutations in tuberous sclerosis. Human Molecular Genetics, 6, 1991–6. Morgan, G. S., Straumfjord, J. V., and Hall, E. J. (1951). Angiomyolipoma of the kidney. Journal of Urology, 65, 525. Murphy, W. M., Zambroni, B. R., Emerson, L. D., Moinuddin, S., and Lee, L. H. (1985). Aspiration biopsy of the kidney. Simultaneous collection of cytologic and histologic specimens. Cancer, 56, 200–5. Neumann, H. P. H., Brüggen, V., Berger, D. P., Herbst, E., Blum, U., Morgenroth, A., et al. (1995). Tuberous sclerosis complex with end-stage renal failure. Nephrology Dialysis Transplantation, 10, 349–53. Nguyen, G. K. (1984). Aspiration biopsy cytology of renal angiomyolipoma. Acta Cytologica, 28, 261–4. Oesterling, J. E., Fishman, E. K., Goldman, S. M., and Marshall, F. F. (1986). The management of renal angiomyolipoma. The Journal of Urology, 135, 1121–4. Pea, M., Bonetti, F., Zamboni, G., Martignoni, G., Riva, M., Colombari, R., et al. (1991). Melanocytemarker-HMB-45 is regularly expressed in angiomyolipoma of the kidney. Pathology, 23, 185–8. Peral, B., San Millan, J. L., Ong, A. C., Gamble, V., Ward, C. J., Strong, C., et al. (1996). Screening the 3 region of the polycystic kidney disease 1 (PKD1) gene reveals six novel mutations. American Journal of Human Genetics, 58, 86–96. Povey, S., Burley, M. W., Attwood, J., Benham, F., Hunt, D., Jeremiah, S. J., et al. (1994). Two loci for tuberous sclerosis: one on 9q34 and one on 16p13. Annals of Human Genetics, 58, 107–27. Roach, E. S., Gomez, M. R., and Northrup, H. (1998). Tuberous sclerosis complex consensus conference: revised clinical diagnostic criteria. Journal of Child Neurology, 13, 624–8. Robertson, F. M., Cendron, M., Klauber, G. T., and Harris, B. H. (1996). Renal cell carcinoma in association with tuberous sclerosis in children. Journal of Pediatric Surgery, 31, 729–30. Sampson, J. R. (1996). The kidney in tuberous sclerosis: manifestations and molecular genetic mechanisms. Nephrology Dialysis Transplantation, 11(Suppl. 6), 34–7. Sampson, J. R., Maheshwar, M. M., Aspinwall, R., Thompson, P., Cheadle, J. P., Ravine, D., et al. (1997). Renal cystic disease in tuberous sclerosis: role of the polycystic kidney disease 1 gene. American Journal of Human Genetics, 61, 843–51. Sampson, J. R., Patel, A., and Mee, A. D. (1995). Multifocal renal cell carcinoma in sibs from a chromosome 9 linked (TSC1) tuberous sclerosis family. Journal of Medical Genetics, 32, 848–50. Sampson, J. R., Scahill, S. J., Stephenson, J. B., Mann, L., and Connor, J. M. (1989). Genetic aspects of tuberous sclerosis in the west of Scotland. Journal of Medical Genetics, 26, 28–31. Sant, G. R., Ucci, A. A. Jr., and Meares, E. M. Jr. (1986). Multicentric angiomyolipoma: renal and lymph node involvement. Urology, 28, 111–3. Schillinger, F. and Montagnac, R. (1996). Chronic renal failure and its treatment in tuberous sclerosis. Nephrology Dialysis Transplantation, 11, 481–5. Shepherd, C. W., Beard, C. M., Gomez, M. R., Kurland, L. T., and Whisnant, J. P. (1991a). Tuberous sclerosis complex in Olmsted County, Minnesota, 1950–1989. Archives of Neurology, 48, 400–1. Shepherd, C. W., Gomez, M. R., Lie, J. T., and Crowson, C. S. (1991b). Causes of death in patients with tuberous sclerosis. Mayo Clinical Proceedings, 66, 792–6. Stapleton, F. B., Johnson, D., Kaplan, G. W., and Griswold, W. (1980). The cystic renal lesion in tuberous sclerosis. Journal of Pediatrics, 97, 574–9. Steiner, M. S., Goldman, S. M., Fishman, E. K., and Marshall, F. F. (1993). The natural history of renal angiomyolipoma. The Journal of Urology, 150, 1782–6.

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Stenmark, H., Vitale, G., Ullrich, O., and Zerial, M. (1995). Rabaptin-5 is a direct effector of the small GTPase Rab5 in endocytic membrane fusion. Cell, 83, 423–32. Stillwell, T. J., Gomez, M. R., and Kelalis, P. P. (1987). Renal lesions in tuberous sclerosis. The Journal of Urology, 138, 477–81. The European Chromosome 16 Tuberous Sclerosis Consortium. (1993). Identification and characterization of the tuberous sclerosis gene on chromosome 16. Cell, 75, 1305–15. Torres, V. E., King, B. F., Holley, K. E., Blute, M. L., and Gomez, M. R. (1994). The kidney in the tuberous sclerosis complex. Advances in Nephrology from the Necker Hospital, 23, 43–70. Van Baal, J. G., Smits, N. J., Keeman, J. N., Lindhout, D., and Verhoef, S. (1994). The evolution of renal angiomyolipomas in patients with tuberous sclerosis. The Journal of Urology, 152, 35–8. van Slegtenhorst, M., de Hoogt, R., Hermans, C., Nellist, M., Janssen, B., Verhoef, S., et al. (1997). Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34. Science, 277, 805–8. van Slegtenhorst, M., Nellist, M., Nagelkerken, B., Cheadle, J., Snell, R., van den Ouweland, A., et al. (1998). Interaction between hamartin and tuberin, the TSC1 and TSC2 gene products. Human Molecular Genetics, 7, 1053–7. van Slegtenhorst, M., Verhoef, S., Tempelaars, A., Bakker, L., Wang, Q., Wessels, M., et al. (1999). Mutational spectrum of the TSC1 gene in a cohort of 225 tuberous sclerosis complex patients: no evidence for genotype-phenotype correlation. Journal of Medical Genetics, 36, 285–9. Vogt, H. (1908). Zur Diagnostik der Turberösen Sklerose. Zeitschrift für Erforsch Jugendl Schwachsinns, 2, 1–12. Washecka, R. and Hanna, M. (1991). Malignant renal tumors in tuberous sclerosis. Urology, XXXVII, 340–3. Webb, D. W., Kabala, J., and Osborne, J. P. (1994). A population study of renal disease in patients with tuberous sclerosis. British Journal of Urology, 74, 151–4. Webb, D. W., Super, M., Normand, I. C., and Osborne, J. P. (1993). Tuberous sclerosis and polycystic kidney disease. British Medical Journal, 306, 1258–9. Weinblatt, M. E., Kahn, E., and Kochen, J. (1987). Renal cell carcinoma in patients with tuberous sclerosis. Pediatrics, 80, 898–903. Xiao, G.-H., Shoarinejad, F., Jin, F., Golemis, E. A., and Yeung, R. S. (1997). The tuberous sclerosis 2 gene product, tuberin, functions as a Rab5 GTPase activating protein (GAP) in modulating endocytosis. Journal of Biological Chemistry, 272, 6097–100. Yamashita, Y., Ueno, S., Makita, O., Ogata, I., Hatanaka, Y., Watanabe, O., et al. (1993). Hyperechoic renal tumours: anechoic rim and intratumoral cysts in US differentiation of renal cell carcinoma from angiomyolipoma. Radiology, 188, 179–82. Yeung, R. S., Xiao, G.-H., Jin, F., Lee, W.-C., Testa, J. R., and Knudson, A. G. (1994). Predisposition to renal carcinoma in the Eker rat is determined by germ-line mutation of the tuberous sclerosis 2 (TSC2) gene. Proceedings of the National Academy of Sciences, USA, 91, 11413–6.

16 Neurofibromatosis Susan M. Huson and Natalie Canham

Introduction The neurofibromatoses are a group of dominantly inherited genetic disorders defined by the presence/absence of specific types of skin pigmentation, tumours of the nervous system and ophthalmalogical features. Although historically eponymous or descriptive terminology was used, the NIH Consensus Conference (1988) recommended a numerical classification which is now internationally recognized. The same conference defined diagnostic criteria for the two main types of neurofibromatosis and these have been more recently updated (Gutmann et al. 1997). The main form of neurofibromatosis is type 1 (NF1, formerly known as von Recklinghausen disease or multiple neurofibromatosis). It has a birth incidence of around 1 in 3000 (Huson 1994). Type 2 neurofibromatosis (NF2, formerly called central or bilateral acoustic neurofibromatosis) has a birth incidence of around 1 in 30,000 and a symptomatic prevalence of about 1 in 210,000 (Evans et al. 1992). Other definite forms of neurofibromatosis are very rare, the other phenotypes seen most frequently in clinical practice are the mosaic/ segmental forms of NF1 and NF2 (reviewed by Ruggieri and Huson 2001). In these forms the disease manifestations are limited to specific segments of the body and result from somatic mutation. Primary involvement of the renal tract only occurs in NF1. Although patients with NF2 may have disturbance of bladder function secondary to spinal cord or nerve root compression. NF2 is therefore not discussed further. Readers requiring more clinical or molecular information about the neurofibromatoses in general should consult the most recent textbooks on the disease (Huson and Hughes 1994; Upadhyaya and Cooper 1998; Friedman et al. 1999). Neurofibromatosis type 1 Diagnostic criteria The diagnosis of NF1 can be made in a patient who has two or more of the following (NIH Consensus Development Conference statement 1988; Gutmann et al. 1997):

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Six or more café au lait macules greater than 5 mm in greatest diameter in pre-pubertal individuals or greater than 15 mm in greatest diameter after puberty. Two or more neurofibromas of any type or one or more plexiform neurofibromas.

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Freckling in the axilla or inguinal region.

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A tumour of the optic pathway.

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Two or more Lisch nodules (iris hamartomas). A distinctive osseus lesion such as sphenoid wing dysplasia or thinning of the cortex of the long bones (with or without pseudarthrosis). A first degree relative (parents, sib, or offspring) with NF1 by the above criteria.

Natural history The clinical features of NF1 and major disease complications are summarized in Table 16.1. The major features develop at different ages. Café au lait spots are the first to appear and begin to develop during the first 2 years of life, increasing in size and number throughout childhood. Skin-fold freckling appears from around the age of 2–3 years. Iris Lisch nodules are never symptomatic, but provide a useful diagnostic aid, they develop from around 3–4 years of age. Cutaneous neurofibromas are rarely obvious in childhood. They begin to develop from the late teens onwards. Approximately half the patients with NF1 only ever have major features and no disease complications. It is the occurrence of one or more of the disease complications which causes the significant morbidity and mortality associated with NF1. The individual frequency of many of the disease complications in an NF1 population is very low, often only 1 or 2 per cent. However, the relative risk of their occurrence compared with the general population is greatly increased. For example, Narod et al. (1991) found the relative risk for phaeochromocytoma in NF1 to be 1000 and malignant peripheral nerve sheath tumours 9000!—even though the frequency for each of complications in a population-based study of NF1 was only 1 and 2 per cent respectively

Table 16.1 Summary of the clinical and genetic features of NF1 Inheritance Gene location Gene Major features Renal and urinary tract complications

Other features

Dominant—variable even within families, 50% of cases have no family history 17q 11.2 Large gene with 60 exons; acts as a tumour suppressor; codes for the protein neurofibromin, a member of the GAP family Café au lait spots (birth onwards) and skin fold freckling (from around 3 years) Dermal neurofibromas (usually from mid teens) Lisch nodules (from around 3 years) Hypertension secondary to Renovascular disease 2% ( 20 years) Phaeochromocytoma 1% (9 years onwards) Tumours Wilms: No increased frequency Rhabdomyosarcomas 1% (birth to 7 years) Plexiform neurofibromas 1% (present from infancy) but become symptomatic in late childhood or adulthood Learning disability 30–65% (depending on definition) Macrocephaly 50% Slight shortening of stature 33% Other plexiform neurofibromas 26% Scoliosis 6% Other disease related cancers and brain tumours 4%

The percentages refer to frequency of given complications and the ages of usual presentation are also given when relevant.

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(Huson et al. 1988). Disease complications occur in all body systems. Their occurrence cannot be predicted even within families. The recommended management of patients with NF1 is based on the normal findings in previous studies which used screening (Riccardi 1981, 1992), clinical experience and expert consensus (Huson and Upadhyaya 1994; Gutmann et al. 1997; Wolkenstein et al. 1999). All people with NF1 should have an annual assessment with an interval history and physical examination including blood pressure measurement. They should also be aware to seek medical advice for any unusual symptoms between checks. Riccardi (1992) reports on the use of screening investigations in NF1. Relevant to the kidney, his clinic initially performed IVPs on all patients, but ceased to do so after 160 IVPs detected no unsuspected NF1 related renal problems. A subset of 50 patients underwent digital subtraction arteriography and no abnormalities were found in those without systemic hypertension. Genetics NF1 is an autosomal dominant disorder with 50 per cent of cases representing new gene mutations. The NF1 gene is in band 11.2 of the long arm of chromosome 17. It spans approximately 335 kilobases (kb) of genomic DNA, arranged in 60 exons (reviewed in Upadhyaya and Cooper 1998; Viskochil 1999). The processed transcript is approximately 12 kb long and encodes a peptide, neurofibromin, of 2818 amino acids (Marchuk et al. 1991; Bernards et al. 1992). All the functions of neurofibromin are still being determined. The studies to date have mainly focussed on the portion of the coding sequence which shows homology to the GTPase—activating protein (GAP) family. Neurofibromin acts as a negative regulator of the Ras/Raf/MAPK signalling pathway. NF1 acts as a tumour suppressor gene, homozygote in activating second mutations or loss of heterozygozity have been shown in a number of disease related tumours. Inactivating mutations of the NF1 gene result both in increased cell proliferation and cell survival. Although the tumour suppressor action of NF1 is likely to account for the different tumours, one copy of the abnormal gene must have some form of systemic effect to give rise to problems such as learning difficulties and short stature. NF1 mutation analysis was initially hampered by the large size of gene and lack of mutation hot spots. No genotype/phenotype correlation has emerged except in the group of patients with whole gene deletion (first reported by Kayes et al. 1994; Wu et al. 1995). Subsequent analysis has shown that these patients have a microdeletion involving the NF1 gene of approximately 1.5 Mb, and deletion break points clustering in flanking duplicated regions. Analysis of the genes from within the flanking region is now being undertaken as these may represent modifiers for the NF1 phenotype (Lopez-Correa et al. 1999; Dorschner et al. 2000; Jenne et al. 2001). Improved methods of mutation detection (Messiaen et al. 2000) mean that service provision of NF1 mutation analysis is becoming available. The authors use it for a patient they suspect having the NF1 microdeletion syndrome, for patients considering pre-natal diagnosis and to assist in diagnosis in unusual cases. NF1 complications affecting the kidney and urinary system These are listed in Table 16.1. They each occur in only 1–2 per cent of NF1 patients.

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Hypertension Patients with NF1 have an increased frequency of hypertension due to two causes which are rare in the general population, renal artery stenosis and phaeochromocytoma. As in the general population, the former is the most common and usually presents in patients under the age of 20 years. Phaeochromocytoma can occur in childhood but is much more common in adults. There is no evidence of an increase in essential hypertension, studies of NF1 (Friedman 1999) showing similar frequencies to the 20 per cent frequency in the adult population (Brown and Haydock 2000). Several older studies suggested that pregnancy-related hypertension may be more frequent in NF1 than in the general population but this has not been confirmed. Renovascular disease Hypertension due to renovascular disease occurred in 2 per cent of a population-based study of NF1 in South Wales, UK (Huson et al. 1988) and 1 per cent of an NF1 clinic population in Australia (the majority of whom were children, North 1993). Fossali et al. (2000) report a frequency of 11 per cent but in a selected clinic population who were intensively investigated. Conversely, in a series of 54 cases of renovascular hypertension investigated in London, eight (15 per cent) had NF1 (Deal et al. 1992). As in the general population, the majority of cases occur in childhood. Halpern and Curirano (1965) reported 10 cases, 9 of which had developed hypertension prior to the age of 20 years. When the NF1-related hypertension had presented clinically the symptoms were similar to those caused by hypertension in the general population. Recent series include more cases picked up through routine disease surveillance which is recommended to include blood pressure measurement at all ages (Huson and Upadhyaya 1994; Gutmann et al. 1997). In the general population, childhood renovascular disease has more extensive arterial involvement than that seen in adults. There is frequent bilateral renal artery involvement and small intrarenal artery involvement. There is also an association with more widespread arterial disease. The histology is of fibromuscular dysplasia. In NF1, the distribution is equally diverse but the histology has distinctive features. It can be categorized by vessel diameter (Reubi 1945; Green et al. 1974; Finlay and Dabbs 1988; Campos et al. 1994; Huffman et al. 1996). In larger vessel (the aorta and proximal renal arteries) neurofibromatous proliferation in the adventitia is the cause. Campos et al. (1994) present a case where they demonstrate this is principally schwann cell proliferation. Secondary thinning of the media and fragmentation of elastic tissue occurs and either vessel stenosis or aneurysm formation results. Small vessel disease in NF1 is due to smooth muscle and not neural proliferation (i.e. it represents a mesodermal dysplasia). It can present as either a stenotic or an occlusive lesion, rarely an aneurysm develops. Histological appearances are similar to but distinctive from fibro-muscular dysplasia. Firstly, the vessel distribution is different. In NF1 the disease tends to involve smaller (intraparenchymal) vessels whereas fibrous dysplasia involves the main renal artery and its proximal branches. Secondly on microscopy, the lesions characteristic for NF1 are nodular aggregates of smooth muscle cells. NF1 is primarily a disorder of neuroectodermal tissue. Schwann cell proliferation in large vessels fits with this. However, smooth muscle cells are of mesodermal origin. Hamilton and Friedman (2001) have proposed a hypothesis for the mesodermal vasculopathy seen in NF1. They point out that neurofibromin expression has been identified in the heart and endothelial and smooth muscle cells of blood vessels. Therefore aberrant neurofibromin expression could

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be causative in these lesions. As all cells in NF1 patients are haplo-insufficient and the lesions localized, it is unlikely that this alone accounts for the vasculopathy. Mechanisms proposed are a second hit in the NF1 or a related gene or abnormal response of the vascular wall in NF1 to endothelial injury from aberrant blood flow. The investigation and management of renovascular disease is the same in NF1 as in the general population and a detailed review is beyond the remit of this section. The abstract in Deal et al.’s (1992) review of 54 childhood cases with renovascular disease (8 with NF1) concludes: ‘renal vascular disease in children is often widespread, maybe associated with intracerebral disease, frequently affects both kidneys, including both intrarenal and extrarenal vessels and is therefore not always amenable to surgical intervention and cure’. Our literature review supports these observations in NF1 patients, who may have in increased risk of cerebro-vascular and intra-renal disease compared with other children with this disorder. The other important point is that these lesions can be progressive (Deal et al. 1992; Kurien et al. 1997). Kurien et al. describe a 4-year old girl with NF1 referred for investigation of hypertension. At presentation she had narrowing of the left renal artery near its origin from the aorta. Repeated angiography three and a half years later showed a three cm long stenosis of the abdominal aorta below the superior mesenteric artery at the level of the renal arteries. By then she had bilateral renal artery stenosis. Such cases stress the importance of long-term follow up of cases of renovascular disease. Phaeochromocytoma Phaeochromocytomas are an uncommon cause of hypertension in the general population (occurring in about 1 in a 1000 hypertensives, Ferner 1994). In a series of 18 hypertensive NF1 patients, Kalff et al. (1982) found phaeochromocytomas in 10. The frequency of NF1 in one series of patients with phaeochromocytoma was 10 per cent (Modlin et al. 1979). Conversely, in series of patients with NF1 the frequency is much lower, around 1 per cent at most (Samuelson and Axelsson 1981; Sorenson et al. 1986; Huson et al. 1988). It is our practice to screen for phaeochromocytomas using 24 h urine cathetecholamine estimations in all patients with NF1 in whom we detect hypertension. In patients referred already on hypertensive medication, we suggest investigation if they are under 60 years of age or the blood pressure is difficult to control. Previously undiagnosed phaeochromocytomas presenting with intra-operative hypertensive crises are well recognized (Hull 1986; Platts et al. 1995). It is imperative to check the blood pressure and enquire regarding relevant symptoms in all NF1 patients prior to general anaesthetics. There are no specific features about the presenting symptoms, anatomical location or treatment of phaeochromocytomas in NF1 compared with those occurring in the general population. The only difference is that in the context of NF1 there is an association with duodenal carcinoid tumours (Griffiths et al. 1987). It is therefore recommended that patients with NF1 and phaeochromocytomas are investigated for duodenal carcinoid tumours and vice versa. Pregnancy-related hypertension The medical literature has numerous case reports of series of NF1 patients with severe hypertension during pregnancy. Many of these were found to have underlying phaeochromocytomas

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or renovascular disease. Swapp and Maine (1973) reported pregnancy-related hypertension in 10 of 11 patients, only one of whom was found to have an underlying cause. This was much higher than expected compared to the general population and they postulated that the vascular changes of NF1 can be induced by pregnancy or become more marked. However, this association has not been confirmed in later, much larger studies (Dugoff and Sujansky 1996). However, pregnancy is a time when maternal health is particularly important and close monitoring of blood pressure during pregnancy in NF1 patients is advisable. Tumours of the kidney and urinary tract Wilms We do not believe there is an increased frequency of Wilms tumour in NF1. The literature contains several case reports (Miller et al. 1964; Walden et al. 1977; Szwagiel 1982) and one series (Stay and Vawter 1977) which suggests an association. Stay and Vawter reviewed 342 nephroblastoma cases and found three cases with co-existent NF1. This was 33.5 per cent more than accounted for by chance. Hope and Mulvihill (1981) reviewing the literature suggested that there probably was a weak association. They also commented on clinical similarities (hemihypertrophy and hamartomas) and that both had a dominant inheritance pattern. Further support for an association came from the work of Cardesa et al. (1989) on exposing pregnant rats to ethylnitrosurea at different gestations. Five of 180 rats exposed for 15 days and 1 of 172 exposed for 21 days developed Wilms tumour. The association with NF1 arose because 64 of the first group developed peripheral nervous systems tumours, 30 per cent of which had a plexiform neurofibroma pattern. Of the second group 130 developed peripheral nervous system tumours, 21 per cent of which were plexiform. The other tumours seen in significant numbers were gliomas and the authors argued that this was a model system for investigating some of the tumours occurring in NF1. Despite the above, Mulvihill on re-reviewing the literature in 1994 felt that there was no association. He cited that none of the large cross-sectional studies of NF1 populations had identified further cases with both conditions. Furthermore the study of Narod et al. (1991) looking at the heritable fraction of childhood cancer did not find an association. Occasional cases of both conditions will of course be seen by chance. When this occurs, there are several case reports which suggest there may be an increased risk of secondary tumours after chemotherapy and irradiation in NF1 children compared with those with isolated Wilms tumours (Perilongo et al. 1993; Maris et al. 1997). Rhabdomyosarcoma Rhabdomyosarcoma arising from the pelvic organs is a rare but significant disease complication occurring in less than 1 per cent of patients. McKeen et al. (1978) first raised the possibility of an association reporting 5 patients with NF1 in a series of 84 patients with rhabdomyosarcoma. Subsequent series of rhabdomyosarcoma (Hartley et al. 1987; Matsui et al. 1993; Yang et al. 1995) have confirmed the association. Narod et al. (1991) in their study of the heritable fraction of childhood cancer, estimated the relative risk of rhabdomyosarcoma in NF1 to be 23.4, which was highly significant. None of the large cross-sectional studies of NF1 have identified patients with rhabdomyosarcoma, this may reflect the poor prognosis and early age of presentation. In the Welsh population study, none of the

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living individuals but 2 of 25 deceased affected had died from rhabdomyosarcoma. None of their affected parents (who were part of the study cohort), were aware of the association with the tumour with NF1 (Huson et al. 1988; Huson 1989). A total of 16 cases of rhabdomyosarcoma and NF1 were reported by Hartley et al. (1988), Huson (1989), Matsui et al. (1993), and Yang et al. (1995). The average age of diagnosis was 1.5 years (range 0.05–6.06 years), 12 patients were male and 4 female. Fourteen of the 16 tumours were in the pelvic area (bladder nine, vagina one, uterus one, prostate two, and perineum one), one retro-peritoneal and one is described as being ‘in the extremities’. The young age of presentation means these tumours may present prior to the development of any, or at a time when there are less than six café au lait spots. Therefore it is important to consider the diagnosis of NF1 in any young child with rhabdomyosarcoma and enquire about the family history.

Neurofibromas and plexiform neurofibromas Symptomatic neurofibromas affecting the genito-urinary system are rare (much less than 1 per cent of patients) (Huson et al. 1988; Huson 1989). However, when present, plexiform neurofibromas in particular can cause major problems and it is important for nephrologists and neurologists to be aware of their presentation. Isolated, discreet intraneural neurofibromas can occur on any major peripheral nerve in the body (Woodruff 1999). They usually present with localized pain or neurological deficit. If they occur near the urinary tract they may cause symptoms secondary to compression, for example, of the urinary flow or produce hypertension due to external renal artery compression. As these lesions are relatively well-defined anatomically, once the diagnosis is made their removal is relatively straightforward. More problematic are plexiform neurofibromas. There are numerous case reports and small series of plexiform neurofibromas arising in the pelvis, involving the bladder and causing urinary outflow obstructions (Barone et al. 1995; Brown et al. 1997; Kaefer et al. 1997; Cheng 1999; Psycha et al. 2001). The term plexiform neurofibroma is used histologically (Woodruff 1999) to describe a multinodular growth that forms along a plexus of nerves, such as the sacral plexus, or along the fascicles of a single large peripheral nerve. Clinically, we refer to these as nodular plexiform neurofibromas. When present there are no external cutaneous clues to their presence and they are usually diagnosed through symptoms. Woodruff uses the term ‘massive soft tissue neurofibromas’ to describe lesions that we refer to as diffuse plexiform neurofibromas. In these lesions, in addition to plexiform change in the nerves, there is massive diffuse infiltration of soft tissue and the overlying epidermis is often pigmented, hypertrophied and may have excessive hair growth (Huson et al. 1988; Korf 1999; Woodruff 1999). One of the authors (SMH) has now reviewed over 1000 patients with NF1 and only seen two cases of urinary obstruction secondary to diffuse pelvic plexiform neurofibromas. Both were brought to her attention by colleagues in other centres. Both cases were only diagnosed when they presented with urinary obstruction. However, in both children there was a huge area of café au lait pigmentation (in a bathing trunk distribution) which had been present since infancy. It is now our practice, when we identify such large areas of pigmentation over the pelvis to monitor the internal organs with serial scans. Kaefer et al. (1997) identifed five patients with

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complex genito-urinary lesions in a specialist children’s hospital population of 260 NF1 patients. They describe specific management principles. Pelvic plexiform neurofibromas may present as genital hypertrophy. Kousseff and Hoover (1999) describe a neurofibroma of the penile shaft. In females perineal hypertrophy in infancy has lead to initial misdiagnosis of an intersex state (Kaneti et al. 1988; Nogita 1990).

References Barone, J. G., Massad, C. A., Parrott, T. S., Broecker, B. H., and Woodard, J. R. (1995). Symptomatic tumours affecting the urinary tract in children with neurofibromatosis. Journal of Urology, 154, 1516–17. Bernards, A., Haase, V., Murthy, A. Menon, A., Hannigan, G. E., and Gusella, J. F. (1992). Complete human NF1 cDNA sequence: two alternatively spliced mRNAs and absence of expression in a neuroblastoma line. DNA and Cellular Biology 11, 727–34. Brown, J. A., Levy, J. B., and Kramer, S. A. (1997). Genitourinary neurofibromatosis mimicking posterior urethral valves. Urology, 49, 960–2. Brown, M. J. and Haydock, S. (2000). Pathoaetiology, epidemiology and diagnosis of hypertension. Drugs, 59(Suppl 2), 1–12. Campos, A., Panzarino, V., Debich, D. E., and Gilbert B. E. (1994). Pathological case of the month. Case 2. Archives of Pediatric Adolescent Medicine, 148, 725–6. Cardesa, A., Ribalta, T., von Schilling, B., Palacin, A., and Mohr, U. (1985). Experimental model of tumours associated with Neurofibromatosis. Cancer, 63, 1737–49. Cheng, L., Scheithauer, B. W., Leibovich, B. C., Ramnani, D. M., Cheville, J. C., and Bostwick, D. G. (1999). Neurofibroma of the urinary bladder. Cancer, 86, 505–13. Deal, J. E., Snell, M. F., Barratt, T. M., and Dillon, M. J. (1992). Renovascular disease in childhood. Journal of Pediatrics, 121, 378–84. Dorschner, M. O., Sybert, V. P., Weaver, M., Pletcher, B. A., and Stephens, K. (2000). NF1 microdeletion breakpoints are clustered at flanking repetitive sequences. Human Molecular Genetics, 9, 35–46. Dugoff, L. and Sujansky, E. (1996). Neurofibromatosis type 1 and pregnancy. American Journal of Medical Genetics, 66, 7–10. Evans, D. G. R., Huson, S. M., Neary, W., Newton, V., Blair, V., Donnai, D., et al. (1992). A clinical study of type 2 neurofibromatosis. Quarterly Journal of Medicine, 304, 603–18. Ferner, R. E. (1994). Medical complications of neurofibromatosis 1. In The neurofibromatoses: a pathogenetic and clinical overview (ed. S. M. Huson and R. A. C. Hughes). Chapman and Hall, London. Finley, J. L. and Dabbs, D. J. (1988). Renal vascular smooth muscle proliferation in neurofibromatosis. Human Pathology, 19, 107–10. Fossali, E., Signorini, E., Intermite, R. C., Casalini, E., Lovaria, A., Maninetti, M. M., et al. (2000). Renovascular disease and hypertension in children with neurofibromatosis. Pediatric Nephrology, 14, 806–10. Friedman, J. M. (1999). Vascular and endocrine abnormalities in neurofibromatosis. In Neurofibromatosis: phenotype, natural history and pathogenesis (ed. J. M. Friedman, D. H. Gutmann, M. MacCollin, and V. M. Riccardi), 3rd edn. Johns Hopkins University Press, Baltimore, MD. Friedman, J. M., Gutmann, D. H., MacCollin, M., and Riccardi, V. M. (1999). Neurofibromatosis: phenotype, natural history and pathogenesis (3rd edn). Johns Hopkins University Press, Baltimore, MD. Greene, J. F., Fitzwater, J. E., and Burgess, J. (1974). Arterial lesions associated with neurofibromatosis. American Journal of Clinical Pathology, 62, 481–7.

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Griffiths, D. F. R., Williams, G. T., and Williams, E. D. (1987). Duodenal carcinoid, phaeochromocytoma and neurofibromatosis: islet cell tumour, phaeochromocytoma and the von Hippel Lindau complex: two distinctive neuroendocrine syndromes. Quarterly Journal of Medicine, 64, 769–82. Gutmann, D. H., Aylsworth, A., Carey, J. C., Korf, B., Marks, J., Pyeritz, R. E., et al. (1997). The diagnostic evaluation and multidisciplinary management of neurofibromatosis 1 and neurofibromatosis 2. Journal of the American Medical Association, 278, 51–7. Halpern, M. and Currarino, G. (1965). Vascular lesions causing hypertension in neurofibromatosis. New England Journal of Medicine, 273, 248–52. Hamilton, S. J. and Friedman, J. M. (2000). Insights into the pathogenesis of neurofibromatosis 1 vasculopathy. Clinical Genetics, 58, 341–4. Hartley, A. L., Birch, J. M., Marsden, H. B., Harris, M., and Blair, V. (1988). Neurofibromatosis in children with soft tissue sarcoma. Pediatric Hematology and Oncology, 5, 7–16. Hope, D. G. and Mulvihill, J. J. (1981). Malignancy in neurofibromatosis. Advances in Neurology, 29, 33–55. Huffman, J. L., Gahtan, V., Bowers, V. D., and Mills, J. L. (1996). Neurofibromatosis and arterial aneurysms. American Surgeon, 62, 311–14. Hull, C. J. (1986). Phaeochromocytoma: diagnosis, preoperative preparation and anaesthetic management. British Journal of Anaesthesia, 58, 1453–68. Huson, S. M. and Upadhyaya, M. (1994). Neurofibromatosis 1: clinical management and genetic counselling. In The neurofibromatoses: a pathogenetic and clinical overview (ed. S. M. Huson and R. A. C. Hughes). Chapman and Hall, London. Huson, S. M., Harper, P. S., and Compston, D. A. S. (1988). Von Recklinghausen neurofibromatosis: A clinical and population study in South East Wales. Brain, 111, 1355–81. Huson, S. M. (1989). Clinical and genetic studies of von Recklinghausen neurofibromatosis. MD Thesis, University of Edinburgh, Edinburgh. Huson, S. M. and Hughes, R. A. C. eds, (1994). The neurofibromatoses: a pathogenetic and clinical overview. Chapman and Hall, London. Huson, S. M. (1994). Neurofibromatosis 1: a clinical and genetic overview. In The neurofibromatoses: a pathogenetic and clinical overview (ed. S. M. Huson and R. A. C. Hughes), pp. 160–203. Chapman and Hall, London. Jenne, D. E., Tinschert, S., Reimann, H., Lasinger, W., Thiel, G., Hameister, H., et al. (2001). Molecular characterization and gene content of breakpoint boundaries in patients with neurofibromatosis type 1 with 17q 11.2 microdeletions. American Journal of Human Genetics, 69, 516–27. Kaefer, M., Adams, M. C., Rink, R. C., and Keating, M. A. (1997). Principles in management of complex pediatric genitourinary plexiform neurofibroma. Urology, 49(6), 936–40. Kalff, V., Shapiro, B., Lloyd, R., Sisson, J. C., Holland, K., Nakajo, M., et al. (1982). The spectrum of phaeochromcytoma in hypertensive patients with neurofibromatosis. Archives of Internal Medicine, 142, 2092–8. Kaneti, J., Lieberman, E., Moshe, P., and Carmi, R. (1988). A case of ambiguous genitalia owing to neurofibromatosis—review of the literature. Journal of Urology, 140, 584–5. Kayes, L. M., Burke, W., Riccardi, V. M., Bennett, R., Ehrlich, P., Rubenstein, A., et al. (1994). Deletions spanning the Neurofibromatosis 1 gene: Identification and phenotype of five patients. American Journal of Human Genetics, 54, 424–36. Korf, B. R. (1999). Plexiform neurofibromas. American Journal of Medical Genetics (Seminars in Medical Genetics), 89, 31–7. Kousseff, B. G. and Hoover, D. L. (1999). Penile neurofibromas. American Journal of Medical Genetics, 89, 23–30. Kurien, A., John, P. R., and Milford, M. V. (1997). Hypertension secondary to progressive vascular neurofibromatosis. Archives of Disease in Childhood, 76, 454–5.

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Lopez-Correa, C., Brems, H., Lazaro, C., Eshvill, X., Clementi, M., Mason, S., et al. (1999). Molecular studies in 20 submicroscopic neurofibromatosis type 1 gene deletions. Human Mutation, 14, 387–3. Marchuk, D., Saulino, A., Tavakkol, R., Swaroop, M., Wallace, M. R., Andersen, L. B., et al. (1991). cDNA cloning of the type 1 neurofibromatosis gene: complete sequence of the NF1 gene product. Genomics, 11, 931–40. Maris, J. M., Wiersma, S. R., Mahgoub, N., Thompson, P., Geyer, R. J., Hurwitz, C. G. H., et al. (1997). Monosomy 7 myelodysplastic syndrome and other second malignant neoplasms in children with neurofibromatotis type 1. Cancer, 79, 1438–46. Matsui, I., Tanimura, M., Kobayashi, N., Sawada, T., Nagahara, N., and Akatsuka, J. (1993). Neurofibromatosis type 1 and childhood cancer. Cancer, 72, 2746–54. McKeen, E. A., Bodurtha, J., Meadows, A. T., Douglass, E. C., and Mulvihill, J. J. (1978). Rhabdomysarcoma complicating multiple neurofibromatosis. Journal of Pediatrics, 93, 992–3. Messiaen, L. M., Callens, T., Mortier, G., Beysen, D., Vandenbroucke, I., Van Royn, et al. (2000). Exhaustive mutation analysis of the NF1 gene allows identification of the 95% mutations and reveals and high frequency of unusual splicing defects. Human Mutation, 15, 541–55. Miller, R. W., Fraumeni, J. F., and Manning, M. D. (1964). Association of Wilms’s tumour with aniridia, hemipertrophy and other congential malformations. New England Journal of Medicine, 270, 922–7. Modlin, I. M., Farndon, J. R., Shepherd, A., Johnston, I. D. A., Kennedy, T. L., Montgomery, D. A. D., et al. (1979). Phaeochromcytoma in 72 patients: clinical and diagnostic features, treatment and long term results. British Journal of Surgery, 66, 456–65. Mulvihill, J. J. (1994). Malignancy: epidemiologically associated cancers. In The neurofibromatoses: a pathogenetic and clinical overview (ed. S. M. Huson and R. A. C. Hughes), pp. 305–15. Chapman and Hall, London. Narod, S. A., Stiller, C., and Lenoir, G. M. (1991). An estimate of the heritable fraction of childhood cancer. British Journal of Cancer, 63, 993–9. NIH Consensus Development Conference: Neurofibromatosis. Conference Statement (1988). Archives of Neurology, 45, 575–8. Nogita, T., Kawabata, Y., Tsuchida, T., Otsuka, F., Ishibashi, Y., Minowada, S., et al. (1990). Clitoral and labial involvement of neurofibromatosis. Journal of the American Academy of Dermatology, 23, 937–8. North, K. (1993). Neurofibromatosis type 1: review of the first 200 patients in an Australian clinic. Journal of Child Neurology, 8, 395–402. Perilongo, G., Felix, C. A., Meadows, A. T., Nowell, P., Biegel, J., and Lange, B. J. (1993). Sequential development of Wilms’ tumour, T-cell acute lymphoblastic leukaemia, medulloblastoma and myeloid leukaemia in a child with type 1 neurofibromatosis: a clinical and cytogenetic case report. Leukemia, 7, 912–5. Platts, J. K., Drew, P. J. T., and Harvey, J. N. (1995). Death from phaeochromocytoma: lessons from a post-mortem survey. Journal of the Royal College of Physicians of London, 29, 299–306. Pycha, A., Klinger, C. H., Reiter, W. J., Schroth, B., Haitel, A., and Latal, D. (2001). Von Recklinghausen neurofibromatosis with urinary bladder involvement. Urology, 58(1), 106. Reubi, F. (1945). Neurofibromatosis et lesions vasculaires. Schweizerische Medizinische Wochenschrift, 75, 463. Riccardi, V. M. (1981). von Recklinghausen neurofibromatosis. New England Journal of Medicine, 305, 1617–27. Riccardi, V. M. (1992). Neurofibromatosis: phenotype, natural history and pathogenesis (2nd edn). The Johns Hopkins University Press, Baltimore, MD. Ruggieri, M. and Huson, S. M. (2001). The clinical and diagnostic implications of mocaisism in the neurofibromatoses. Neurology, 56, 1433–43. Samuelsson, B. and Axelsson, R. (1981). Neurofibromatosis. A clinical and genetic study of 96 cases in Gothenburg, Sweden. Acta Dermatovenereologica (Stockholm), 95(Suppl), 67–71.

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Sorenson, S. A., Mulvihill, J. J., and Nielsen, A. (1986). Long-term follow-up of von Recklinhausen neurofibromatosis: survival and malignant neoplasms. New England Journal of Medicine, 314, 1010–15. Stay, E. J. and Vawter, G. (1977). The relationship between nephroblastoma and neurofibromatosis (von Recklinghausen’s disease). Cancer, 39, 2550–5. Swapp, G. H. and Maine, R. A. (1973). Neurofibromatosis in pregnancy. British Journal of Dermatology, 80, 320. Szwagiel, J. P. (1982). Wilms’ tumor and neurofibromatosis. Journal of Clinical Cancer, 32, 320. Upadhyaya, M. and Cooper, D. N. (1998). Neurofibromotosis type 1 from genotype to phenotype. BIOS Scientific Publishers, Oxford. Viskochil, D. H. (1999). The structure and function of the NF1 gene: molecular pathoophysiology. In Neurofibromatosis: phenotype, natural history and pathogenesis (ed. J. M. Friedman, D. H. Gutmann, M. MacCollin, and V. M. Riccardi), 3rd edn. John Hopkins University Press, Baltimore, MD. Walden, P. A., Johnson, A. G., and Bagshawe, K. D. (1977). Wilms’ tumour and neurofibromatosis. British Medical Journal, 1, 813. Wolkenstein, P., Freche, B., Zeller, J., and Revuz, J. (1996). Usefulness of screening investigations in neurofibromatosis type 1: a study of 152 patients. Archives of Dermatology, 132, 1333–6. Woodruff, J. M. (1999). Pathology of tumors of the peripheral nerve sheath in type 1 neurofibromatosis. American Journal of Medical Genetics (Seminars in Medical Genetics), 89, 23–30. Wu, B.-L., Austin, M. A., Schneider, G. H., Boles, R. G., and Korf, B. R. (1995). Deletion of the entire NF1 gene detected by FISH: four deletion patients associated with severe manifestations. American Journal of Medical Genetics, 59, 528–35. Yang, P., Grufferman, S., Khoury, M. J., Schwartz, A. G., Kowalski, J., Ruymann, F. B., et al. (1995). Association of childhood rhabdomyosarcoma with neurofibromatosis type 1 and birth defects. Genetic Epidemiology, 12, 467–74.


17 The Bardet–Biedl and Alström syndromes Philip L. Beales, Patrick S. Parfrey, and Nicholas Katsanis

Introduction Six conditions have as their primary features, retinal dystrophy and obesity and the association of retinal dystrophy and renal dysplasia occurs in four disorders (Table 17.1). The Bardet–Biedl and Alström syndromes are associated with all three components; retinal dystrophy (RD), obesity, and renal dysplasia. There are many other similarities between the two entities and both are inherited in an autosomal recessive manner. The clinical manifestations and genetics of both syndromes will be discussed in this chapter. Bardet–Biedl syndrome Nomenclature The eponymous label ascribed to this condition has given rise to much confusion and it has been called at one time or other, the Laurence–Moon–Biedl syndrome, Laurence–Moon– Bardet–Biedl syndrome, or Bardet–Biedl syndrome (BBS). Laurence and Moon (1866) described the first cases in London in 1866. However, no further cases were published until the early 1920s when quite independently, George Bardet (1920) described two Parisian girls with the triad of obesity, polydactyly, and retinitis pigmentosa, and likewise the renowned Austrian endocrinologist, Arthur Biedl (1922) published a short case-report of two siblings with retinitis pigmentosa, polydactyly, obesity, hypogenitalism, and intellectual impairment. Soon after, the condition was ascribed to Bardet and Arthur Biedl, but in 1925, Solis-Cohen Table 17.1 Syndromes with coexisting retinal dystrophy, renal dysplasia, and obesity (A) Retinal dystrophy and obesity Bardet–Biedl syndrome Alström syndrome Cohen syndrome Pigmentary retinopathy with hypogonadism Choroideremia with obesity and deafness Laurence–Moon syndrome (B) Retinal dystrophy and renal dysplasia Bardet–Biedl syndrome Alström syndrome Allagille syndrome Mainzer–Saldino syndrome

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and Weiss (1925) pointed out that Laurence and Moon deserved recognition and coined the term, the Laurence–Moon–Bardet–Biedl syndrome. In subsequent literature, Bardet is often omitted and the eponym Laurence–Moon–Biedl syndrome employed. In 1970, Ammann (1970) and more recently Schachat and Maumenee (1982) following a review of the literature, highlighted essential differences between the Laurence–Moon syndrome and BBS. The medical and scientific communities have now adopted this split nomenclature. On the basis that the family described by Laurence and Moon subsequently developed a progressive spastic paraparesis and that there was no mention of polydactyly, the Laurence–Moon syndrome is considered to comprise RD, obesity, hypogenitalism, and spastic paraparesis without polydactyly. The cardinal manifestations of BBS include RD, obesity, renal dysplasia, polydactyly, male hypogenitalism, and learning difficulties (Green et al. 1989; Beales et al. 1999). Demography and prevalence Bardet–Biedl syndrome is found throughout the world. Prevalence rates in the USA, UK, Netherlands, and Switzerland range from 1 in 100,000 to 1 in 160,000 (Klein and Ammann 1969; Stigglebout 1972; Croft 1995; Beales et al. 1997). Nevertheless, there are communities in which BBS appears to be common. Green et al. (1989) identified 32 patients in Newfoundland and estimated the prevalence at 1 per 17,500 (See Newfoundland Paradox). Another region of relatively high prevalence of BBS is the Middle East, in particular Kuwait. Prior to the Gulf War, Farag and Teebi (1988) estimated the prevalence in the mixed Arab population as 1 in 36,000. However, among the Bedouin peoples of Kuwait, where consanguinity is frequent, the rate is estimated at 1 in 13,500 (Farag and Teebi 1989). Klein and Ammann (1969) provided more conservative estimates of prevalence in their report of 57 cases of BBS in 26 families from 5 isolated regions of Switzerland. Twenty-two of the cases were from the central mountainous region of Switzerland. They concluded, given that the Swiss population in 1965 was 6 million; the prevalence rate must be 1 in 160,000. Croft and Swift (1990) have suggested that the heterozygote frequency in North America may be as high as 1 per cent thus inferring a prevalence of approximately 1 in 40,000 although it is more likely that the true rate approaches that of Northern Europe. Genetics of BBS BBS has traditionally been considered an autosomal recessive disorder. Early expectations supposed BBS mutations might map to a single locus. On the contrary, subsequent linkage studies revealed substantial genetic heterogeneity with seven BBS loci mapped to date (Table 17.2). The first BBS locus (BBS2) was mapped in 1993 following a genome-wide scan using a large consanguineous Bedouin pedigree to 16q21 (Kwitek-Black et al. 1993). A second locus (BBS1) was reported using 31 North American nuclear families, which demonstrated linkage between BBS1 and PYGM on 11q13 (Leppert et al. 1994). BBS3 was identified on 3p12–13 (Sheffield et al. 1994) and the fourth (BBS4) on 15q23 (Carmi et al. 1995). Both these studies used a pooled sample homozygosity approach in two large consanguineous Bedouin kindreds. The fifth locus (BBS5) was mapped to 2q31 using homozygosity mapping in a single, large Newfoundland kindred (Young et al. 1999a). BBS6 was mapped to 20p12 and was the first BBS gene to be cloned (Katsanis et al. 2000; Slavotinek et al. 2000), using a cohort of Newfoundland BBS families that had been excluded from known BBS loci (Woods et al. 1999). Intriguingly, there are no reliable genotype–phenotype correlations that can be

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Table 17.2 Summary of the genetic position of all BBS loci Locus

Chromosome

Method

% Contribution

Status

BBS1

11q13

20–30

Novel transcript

BBS2 BBS3* BBS4

16q21 3p13 15q23

Linkage/sequence homology to BBS2 IBD IBD IBD

8–16 2–4 1–3

BBS5* BBS6 BBS7

2q31 20p12 4q27

IBD IBD Sequence homology to BBS2

3 4–5 19–42

Novel transcript CI ~ 2 cM Novel transcript— TPR repeats. OGT similarities CI ~ 6 cM Chaperonin? Novel transcript

Adapted from Katsanis et al. 2001 CI critical interval, cM centimorgan, IBD identity-by-descent. *Not yet cloned.

made and furthermore the genetic exclusion of several pedigrees from all known BBS loci suggests the presence of further loci. The ‘Newfoundland paradox’ In Newfoundland, BBS is encountered more frequently than possibly anywhere else in the world (Green 1989; Farag and Teebi 1989). A few British and Irish families who established small isolated communities around the coast colonized the island of Newfoundland in the 1700s. The limited gene pool and migration are thought to account for the increased incidence of recessive disorders by virtue of a founder effect (Bear et al. 1988). Contrary to the expectation that the high frequency of BBS in Newfoundland was also due to a founder mutation, genetic locus heterogeneity has been demonstrated both genetically and through mutational analysis. Two disease-associated haplotypes have been found in Newfoundland for BBS1 (Young et al. 1999b). Two families have been assigned genetically to BBS2 (Woods et al. 1999); another family has been linked to BBS3 (Young et al. 1998), whereas yet another family defined BBS5 (Young et al. 1999a). Three distinct BBS6 mutations have also been found in Newfoundland pedigrees (Katsanis et al. 2000; Slavotinek et al. 2000). Finally, digenic or multiallelic inheritance was suspected in one Newfoundland pedigree (Beales et al. 2001). A similar situation has been reported on the island of La Reunion with respect to inheritance of limb-girdle muscular dystrophy type 3 where multiple disease-associated haplotypes and mutations in Calpain 3 were found, contrary to expectation and dubbed ‘La Reunion Paradox’ (Richard et al. 1995). The ‘Newfoundland Paradox’ is even more pronounced in that not only are there multiple mutations in the same gene found in Newfoundland (exemplified by the three different mutations in BBS6), but also multiple loci. Identification of BBS genes Since 2000, positional cloning efforts have led to the identification of five BBS genes. The characterization of the gene for McKusick–Kaufman syndrome (MKKS) on 20p12 (Stone et al. 1999) facilitated the identification of the first BBS gene (BBS6). MKKS has

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some similarities with BBS such as polydactyly and hydrometrocolpos and several cases of MKKS have been re-classified as BBS owing to the late development of retinal dystrophy and obesity (Schaap et al. 1998; David et al. 1999). As MKKS fell within the BBS6 critical interval, two independent groups reported mutations in MKKS were responsible for a minority of BBS (Katsanis et al. 2000; Slavotinek et al. 2000). Both BBS2 and BBS4 were identified using classical positional cloning methods, whereby ancestral recombinants were used to refine the critical interval to a 2 and 1 cM region on 16q21 and 15q22.3–q23, respectively. Availability of genomic sequence permitted the identification of several candidate genes in each region, one of which was ultimately shown to harbour pathogenic mutations in each case (Mykytyn et al. 2001, 2002; Nishimura et al. 2001). By contrast, the cloning of BBS1 was achieved by a positional candidate approach in combination with in silico sequence homology to BBS2. Utilizing an entirely in silico sequence homology paradigm, Badano et al. (2003), identified a candidate (with modest similarity to BBS2) for which subsequent sequencing of several BBS families revealed pathogenic mutations and identification of the seventh BBS locus—BBS7. The identification of BBS1 was predicted to yield 40–56 per cent of mutations among Caucasian patients with BBS (Beales et al. 1997; Bruford et al. 1997; Katsanis et al. 1999). In the final analysis, mutations appear to account for between 18 per cent and 32 per cent of all BBS patients (Beales et al. 1999; Mykytyn et al. 2003). Intriguingly, the missense mutation giving rise to M390R, appears to be common (78–80 per cent of families with BBS1 mutations or 23–30 per cent of all BBS) and is almost exclusive to Caucasian populations on a common haplotype background suggesting this is an ancient mutation (Beales et al. 2003; Mykytyn et al. 2003). The BBS gene products Nucleotide and protein database comparisons reveal that BBS6/MKKS is similar to archeobacterial chaperonins and the eukaryotic T-complex-related proteins (TCPs). Threedimensional modelling also indicated that the protein whose fold best resembles MKKS is the archeal thermosome from Thermoplasma acidophilum (Stone et al. 1999). Archeal thermosomes and members of the TCP family belong to the type-II class of chaperonins. In contrast to type I chaperonins, which are often associated with conditions of cellular stress (such as heat-shock proteins), the type-II class is implicated in the facilitation of nascent protein folding (for reviews see Gutsche et al. 1999; Wickner et al. 1999; Agashe and Hartl 2000). Unlike BBS6, the amino-acid sequence of BBS2 offers no functional insights. Other than a strong conservation across phyla, indicating limited tolerance for variation, the primary sequence of BBS2 bears no homology to any known protein, nor does it contain any recognizable motifs (Nishimura et al. 2001). Further experiments will thus be required to establish the physiological role of BBS2. Domain and homology searches using the BBS4 amino acid sequence identified significant similarity to O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) from several species (Mykytyn et al. 2001). O-GlcNAc modifies a large number of nucleocytoplasmic proteins and is thought to play an important role in signalling by determining how a cell responds to extracellular stimuli (Wells et al. 2001). In addition, BBS4 contains a several tetratricopeptide repeat (TPR) (Mykytyn et al. 2001). Such structures are thought to mediate protein–protein interactions in a variety of cellular processes (Blatch and Lassle 1999). It is therefore possible that BBS4 docks with other proteins to glycosylate specific residues in order to either propagate or block an extracellular signal.


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Likewise, the most recently identified genes, BBS1–BBS7, do not contain functionally recognizable domains and have no close homology with the other BBS genes. Multiallelic inheritance in BBS The majority of mutations reported in the initial BBS6 studies suggested that a total loss of function results in BBS (Katsanis et al. 2000; Slavotinek et al. 2000). By contrast, the mutations found in MKKS patients were either missense alterations or a 2-bp deletion towards the N-terminus of the protein (Stone et al. 1999), suggesting that MKKS is a hypomorphic variant of BBS (Katsanis et al. 2000). However, it is likely that this explanation is too simplistic. First, all but one of the mutations reported in a later study using a large outbred patient cohort were missense alterations (Beales et al. 2001). Second, two mutant alleles associated with MKKS, Y37C and A242S were found in BBS patients (Katsanis et al. 2000; Beales et al. 2001), suggesting that it may be the combination of both alleles that determines the severity of the phenotype. Genetic and mutational data suggest that some cases of BBS may be caused by mutations at more than one locus, as several BBS6 mutations were detected in pedigrees excluded genetically from BBS6 on the basis of haplotype analyses. During analysis of 163 BBS patients for mutations in BBS6, Beales et al. (2001) observed a high frequency of pedigrees (7/8) in which only one BBS6 mutant allele was identified. Furthermore, they described one consanguineous pedigree in which both the affected and unaffected sibs carried a heterozygous A242S mutation, but the affected sib was homozygous for markers across the BBS2 locus. This observation led to the speculation that either multiallelic inheritance was a possibility for Bardet–Biedl syndrome, or the A242S allele was a rare polymorphism (Beales et al. 2001). The identification of BBS2 enabled the same investigators to test their hypothesis by screening the same cohort of BBS patients (Katsanis et al. 2001) for coding sequence alterations in BBS2, irrespective of prior linkage or mutational data. Alterations were detected in 19 unrelated patients, not present in 192 control-matched chromosomes. Of these, six pedigrees segregated two independent mutations in the affected individuals but not their unaffected sibs. However, in eight pedigrees a second mutation was elusive but the ‘singleton’ mutant alleles were predicted to be severe. For example, a single base deletion in one pedigree gave rise to a frameshift alteration (V158fsX200) and other pedigrees harbour nonsense alterations (R275X). Upon haplotype analysis of these pedigrees, surprisingly, only one was consistent with linkage to BBS2. The remaining five pedigrees could be excluded genetically from BBS2 either because the affected individuals shared only one parental chromosome at the locus or the affected and unaffected sibs had identical haplotypes. Intriguingly, haplotype construction across all known BBS intervals indicated that three of five pedigrees had haplotypes consistent with linkage to BBS1; one pedigree with BBS3, and a further pedigree could be assigned to BBS6. These data raised the possibility that mutations at more than one locus may be present in some BBS patients. Close scrutiny of the 19 pedigrees with one or two BBS2 mutations, led Katsanis et al. (2001) to identify four pedigrees harbouring a third mutation (Katsanis et al. 2001) (Table 17.3). For example, AR259 segregates two nonsense mutations in BBS2 present in both affected and unaffected sibs, however, only the affected sib has inherited a third BBS6 nonsense mutation not present in the unaffected child (Fig. 17.1). In addition, there was genetic evidence for involvement of another locus in a further five families (47.3% in total). Conversely, of the eight outbred pedigrees where mutations have been identified in BBS6,

Table 17.3 Mutational and genetic analysis of pedigrees with BBS2 mutations Pedigree

Allele 1

Allele 2

Allele 3

AR-171a PB-005a PB-020a PB-026a PB-057a K-059a AR-029a AR-724b PB-045b AR-050b AR-124b AR-238b AR-596b AR-237b,c AR-579c NF-B14c AR-241b,d AR-259c,d PB-043d

D104A (BBS2)* R275X (BBS2)** Y24X (BBS2)*** D170fsX171 (BBS2) C210fsX246 (BBS2) R315W (BBS2) Y24X (BBS2)*** Q59X (BBS2)**** IVS1–1G C R275X (BBS2)** V158fsX200 (BBS2) D104A (BBS2)* IVS4 1G C N70S (BBS2) L168fsX170 (BBS2) Y24X (BBS2)*** IVS1 1G C/R315Q Y24X (BBS2)*** T560I (BBS2)

R634P (BBS2) R275X (BBS2)** Y24X (BBS2)*** D170fsX171 (BBS2) C210fsX246 (BBS2) R315W (BBS2) BBS2-linkage unknown unknown BBS1 BBS1 BBS1 BBS3 Y37C (BBS6) R216X (BBS2) Y24X (BBS2)*** R315Q (BBS2) Q59X (BBS2)**** T560I (BBS2)

(IBD for BBS1) (IBD for BBS4)

BBS1 BBS1 BBS1 BBS3 Y37C (BBS6) C499S (BBS6) A242S (BBS6) Unmapped Q147X (BBS6) (IBD for BBS4)

Pedigrees have been divided into 4 groups BBS2 mutations segregating with disease. bOne BBS2 mutation detected but genetically excluded from the BBS2 locus. cThree BBS mutations. dTwo BBS mutations found in unaffected persons. *, **, ***, and **** indicate recurrent mutations. Adapted from Katsanis et al. (2001). aTwo

AR259 – BBS2 01 wt wt Q59X wt

03 wt Y24X Q59X wt

02 wt Y24X wt wt

04 wt wt wt wt

05 wt Y24X Q59X wt

AR259 – BBS6 01

02

Q147X wt

03 Q147X wt

wt wt

04 wt wt

05 wt wt

Fig. 17.1 AR-259, a North American pedigree in which both siblings 03 and 05 harbour BBS2 compound, nonsense mutations (Y24X, Q59X) although 05 is unaffected. However, 03 carries an additional heterozygous, nonsense mutation (Q147X) on BBS6, whereas 05 is wildtype. This suggests that the affected sibling requires the presence of three mutant alleles to manifest the BBS phenotype (triallelic inheritance) (reprinted with permission from Katsanis et al. (2001).


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three harboured BBS2 mutations (37.5%). The combination of the genetic and mutational data for the only two genes known at that time to be mutated in BBS suggest that multiple alleles may be necessary for the full phenotype to emerge. A model of ‘triallelic’ inheritance was proposed, in which three mutations segregate with the disorder. Since the first report describing cases of multiallelic inheritance with BBS, further examples have been published implicating BBS4 (Katsanis et al. 2002) in combination with BBS2 (tetra-allelic inheritance?) and BBS1. However, when BBS1 was identified in 2002, Mykytyn et al. (2002) did not observe any cases requiring three mutations to manifest disease. In a larger study, Mykytyn et al. (2003) systematically screened 129 patients for BBS1 mutations and once again did not find patients segregating three mutations with disease or asymptomatic individuals with two mutations, concluding that BBS1 does not participate in triallelic inheritance. Beales et al. (2003) offered evidence to the contrary, following the mutation analysis of 259 BBS families in which they found eight families with non-Mendelian trait inheritance. Six of these harboured sequence variations at a second BBS gene in addition to two BBS1 mutations. Furthermore, they identified two asymptomatic individuals with two M390R mutations. The same study also reported a family harbouring two BBS7 mutations and a third BBS1 heterozygous mutation thus confirming the participation of all cloned BBS genes in non-Mendelian multiallelic inheritance. Clinical presentation The clinical manifestations of BBS are heterogeneous with as much intra- as inter-familial variation observed. Neither does there appear to be any inter-racial variation or clues as to the underlying genotype (Beales et al. 1997; Bruford et al. 1997) (see section on ‘phenotype–genotype correlations’). Diagnosis Bardet (1920) initially characterized four cardinal features: retinitis pigmentosa, polydactyly, obesity, and genital hypoplasia. In 1922, Biedl then added mental retardation. In a comprehensive review of the literature, Schachat and Maumenee (1982) proposed that specific diagnostic criteria should include at least four of the following: mental retardation, obesity, hypogenitalism, polydactyly, and pigmentary retinopathy. It was not until 1973, that an association with renal disease was first noted by Bauman and Hogan (1973) and then described later in detail by Hurley et al. (1975). Churchill et al. (1981) and Green et al. (1989) suggested that renal disease was sufficiently common to be regarded as a 6th cardinal manifestation. Many minor features have also been described: diabetes mellitus (Klein and Ammann 1969; Green 1989), nephrogenic diabetes insipidus (Hurley et al. 1975; Harnett et al. 1988), hepatic fibrosis, dental anomalies (Magnusson 1960; Kobrin et al. 1990; Lofterod et al. 1990; Borgstrom et al. 1996), psychosis (Klein and Ammann 1969), and deafness (Burn 1950). In an attempt to incorporate the renal manifestations as well as the many minor features, Beales et al. (1999) have suggested the presence of four primary features or three primary plus two secondary features as diagnostic of BBS (Table 17.4). Genetic counselling Among European and North American populations, the prevalence of BBS is in the order of 1 in 100,000. By inference (Hardy–Weinberg principle) the gene carrier frequency will be

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Table 17.4 Modified diagnostic criteria for Bardet–Biedl syndrome Primary features Four features are required to be present of: Rod-cone dystrophy Polydactyly Obesity Learning disabilities Hypogonadism in males Renal anomalies or Three primary plus two secondary features are required of: Secondary features Speech disorder/delay Strabismus/cataracts/astigmatism Brachydactyly/syndactyly Developmental delay Polyuria/polydipsia (nephrogenic diabetes insipidus) Ataxia/poor coordination/imbalance Mild spasticity (especially lower limbs) Diabetes mellitus Dental crowding/hypodontia/small roots/high arched palate Left ventricular hypertrophy/congenital heart disease Hepatic fibrosis

approximately 1 in 160. As it is thought to be an autosomal recessive condition the risk of two carriers having an affected child is clearly 1 in 4. Two out of three offspring will themselves be carriers. However, given the phenomenon of triallelism, it is no longer possible to be certain that the recurrence risk in an affected family will be as high as 25 per cent as it may be a requirement in up to 10 per cent of pedigrees for three mutant alleles to co-segregate. In such a case the odds will be reduced to a 1 in 8 chance of affected offspring. In practice, given the ever increasing number of BBS genes and the impracticability of testing each of these, it seems reasonable to counsel on an autosomal recessive basis. Prenatal diagnosis in an at-risk family is now feasible providing the exact mutation(s) have been identified. Again the same caveats apply in that there cannot be any certainty whether or not a given fetus requires two or three mutations and therefore there is always a risk of terminating a normal biallelic fetus. These issues may be resolved as the mechanisms underlying inheritance in BBS become clearer. Major features Retinal degeneration The fundus abnormality in BBS has been described as an atypical pigmentary RD with early macular involvement (Ammann 1970; Bergsma and Brown 1975; Campo and Aaberg 1982). Macular hypopigmentation (Ammann 1970; Bergsma and Brown 1975; Campo and Aaberg 1982) and macular wrinkling (Ackernan et al. 1980) have also been described. The retinal disease in BBS is primarily a degeneration of the photoreceptors (Runge et al. 1986). It has been suggested that the pattern of photoreceptor degeneration is predominantly one of cone


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dysfunction (Berson et al. 1968; Schachat and Maumenee 1982), rod dysfunction (Campo and Aaberg 1982), or both (Fulton and Hansen 1983). Full-field rod and cone electroretinograms are the investigation of choice and may be abnormal as early as 14 months of age (Runge et al. 1986). Both central (acuities) and more peripheral (thresholds) functions are affected. The appearance of the fundus does not predict visual function and in Fulton et al.’s (1983) study, maculopathy was occasionally present in the first decade but was present in all patients in the second decade. This is accompanied by acuities of 20/200 or worse. Disc pallor is not a feature in infants but develops with age, as do attenuated retinal vessels. In the peripheral fundus there may be bone spicule-like pigmentary changes of the retinal pigment epithelium (Fig. 17.2) (Jacobson et al. 1990). In the macula, there may only be minimal pigmentary disturbances, a wrinkled appearance or depigmentation in a ring around an intact central area (bull’s eye) (Campo and Aaberg 1982; Jacobson et al. 1990). The visual prognosis for children with BBS is poor. Jacobson et al. (1990) determined that the visual fields were usually abnormal by age 10 years and that as early as 17 years, there was rarely more than a central island of vision remaining. Beales et al. (1999) reported that parents first noted night blindness in their child at a mean age of 8.5 years. From the same study, the mean age at which patients were registered legally blind in the UK was 15.5 years. They concluded that the mean time for progression from diagnosis to blindness was 7 years. O’Dea et al. (1996) in a study of 36 Newfoundland patients reported that 25 per cent of BBS patients were registered blind by 13 years, 50 per cent by 18 years and 100 per cent by 30 years. Retinal degeneration is reported to be almost universally present (Bell 1958; Ammann 1970; Green 1989; Beales et al. 1999) but these data may be a result of bias as the source of patients is often institutions for the visually impaired. Furthermore, without retinal signs

Fig. 17.2 Rod-cone dystrophy associated with Bardet–Biedl syndrome taken from a 32-years-old patient. Pigmentary changes are not always apparent, especially in young patients. (See Plate 9 of the Colour Plate Section at the centre of this book.)

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many physicians would be reluctant to make a definite diagnosis. Extra-fundal abnormalities have been frequently described, including strabismus, myopia, nystagmus, and cataract. Obesity Obesity is a consistent feature of BBS; its frequency varies from 72 to 96 per cent (Klein and Ammann 1969; Green 1989; Beales et al. 1999) depending on measurement criteria (Fig. 17.3). Obesity usually begins in childhood and the severity increases with age. Bauman and Hogan (1973) reviewed 73 cases in the literature (and added two of their own) in which birth weight and subsequent weight gain were recorded. Seventy-one percent had a birthweight at or above the 50th centile, whereas 38 per cent were born with a weight above the 90th centile. Among those with birth weights below the 50th centile (21 cases), approximately one third were obese by 1 year. Thus, in the majority of cases, obesity was apparent within the first year of life. Distribution of adipose tissue is widespread in childhood but becomes most prominent in the trunk and proximal limbs in adulthood. The cause of obesity in BBS is

Fig. 17.3 Typical stature associated with Bardet–Biedl syndrome (note truncal and rhizomelic distribution of obesity).


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unknown and both abnormalities of the pituitary and hypothalamus have been implicated (Burn 1950; Bell 1958). The basal metabolic rate (BMR) and body composition was shown to be normal in 20 adult BBS volunteers (11 male and 9 female) but nutrient intake may be higher in patients than controls (Grace et al. 1998). Limb abnormalities Post-axial polydactyly is commonly reported in BBS but is not always present. Both Beales et al. (1999) and Ammann (1970) noted its presence in 69 per cent of British and Swiss patients and Green et al. (1989) noted polydactyly in 58 per cent of Newfoundland patients. In a survey of 109 patients, Beales et al. (1999) reported an extra digit was present over twice as often in the feet alone (21 per cent) than in the hands (8 per cent), findings similar to those of Klein and Ammann (1970) and Green et al. (1989). Polydactyly is usually post-axial, manifesting itself as a 6th digit on the lateral border of the hand or foot (Fig. 17.4). Occasionally, a bifid thumb has been reported (Klein and Ammann 1969). The accessory digit is often hypoplastic, but ranges from a fleshy tag of skin to a fully formed and functional finger or toe. Moreover, brachydactyly of both hands and feet may be a more common finding. Green et al. (1989), using precise measurements, reported 100 per cent occurrence in their patient cohort. Other common findings are partial syndactyly (most usually between the second and third toes) (Fig. 17.5), fifth finger clinodactyly (incurved finger) and a prominent sandal-gap between the first and second toes. Cognitive impairment Mental retardation has been widely described as a major feature of BBS, but often visual acuity has not been taken into account in its characterization. In Bell’s (1958) series, 86 per cent were said to have a mental defect but she concedes that this is a difficult component to assess using retrospective studies. Klein and Ammann also reported a high proportion (78 per cent)

Fig. 17.4 Post-axial polydactyly of feet.

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Fig. 17.5 Brachydactyly and second/third toe partial syndactyly. (See Plate 10 of the Colour Plate Section at the centre of this book.)

with mental retardation and tried to categorize the severity of impairment into mild, moderate, and severe (Bell 1958). They concluded that most cases (~55 per cent) showed only ‘mild feeble-mindedness’. Green et al. (1989) administered objective IQ tests and determined that only a minority was mentally retarded. Furthermore, they showed patients scored better on performance subtests than verbal subtests. Beales et al. (1999) reported that 62 per cent of patients had significant learning disabilities and half of these had received special education. Altered behaviour in some BBS patients has been reported. Such traits include emotional immaturity, frequent volatile outbursts, inappropriate behaviour, shallow affect, and a preference for fixed routines (Green 1989; Beales et al. 1999). Hypogenitalism and genital abnormalities Hypogenitalism is more frequent in BBS males than females (Bell 1958; Klein and Ammann 1969). Klein and Ammann (1969) suggested this difference was not explained by an easier diagnosis in males but that other physiological and endocrinological factors must be responsible. Several affected women have given birth to children but there have been only two reports of affected males fathering children (Bell 1958; Klein and Ammann 1969; Green 1989; O’Dea et al. 1996; Beales et al. 1999). Seven of eight men in Green’s study had small volume testes and a very small penis size (Green 1989). Two had low serum testosterone levels, three had high basal follicle stimulating hormone (FSH) levels, and one had a high basal luteinizing hormone (LH) level. All men responded to pituitary stimulation (gonadotrophinreleasing hormone). The lack of evidence for hypopituitarism in this study suggests that the hypogonadism is primary in origin, an hypothesis supported by our own observations and Toledo et al. (1977) (L. Albon et al. unpubl. data). In the latter study, the hypothalamic– pituitary–gonadal axis was evaluated in three BBS siblings born to consanguineous parents. Serum LH levels were raised in the two elder brothers and FSH levels were within normal


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limits for all three. Each responded to pituitary stimulation. Plasma testosterone was normal in all three, but two failed to show an expected rise in testosterone level after repeated human chorionic gonadotrophin (HCG) stimulation. Testicular biopsies were performed in the three siblings and showed degeneration of the seminiferous tubules with marked reduction of spermatogenesis in the two elder brothers. Frequent spermatids but few spermatozoa were seen and the basement membrane was thickened. The testis of the youngest sibling showed an almost normal prepubertal tubular appearance, though hyalinization of the basement membranes was apparent in some areas. These findings appeared to be progressive with age and the authors suggested that testicular failure might progress throughout adult life. All post-pubertal males developed normal or female-like secondary sexual characteristics. This contrasts with the report by Perez-Palacios et al. (1977) in which they performed similar endocrinological studies on two pre-pubertal brothers and one unrelated 19-year-old male. The adult patient presented with a lack of secondary sexual characteristics, azoospermia, small genitalia. The pituitary gonadotrophins were not elevated and the gonadal response to HCG was normal thus ruling out a primary testicular dysfunction and suggesting a hypothalamic– pituitary gonadotrophin disorder. However, pituitary gonadotrophin release was normal in response to exogenous LH–RH stimulation whereas, clomiphene administration did not result in raised pituitary and gonadal hormones, confirming failure of the hypothalamic–pituitary axis. Soliman et al. (1996) concurred that there was a co-existence of a hypothalamic disorder of gonadotrophin-releasing hormone secretion with primary testicular failure; however, in their study, all five children also had empty sellae on CT scanning. Earlier reports of testicular biopsies in BBS males suggested changes similar to that seen in Klinefelter syndrome (Roth 1947; Francke 1950). Hypogonadism and reproductive abnormalities in females Clearly hypogonadism cannot be readily determined in BBS females on clinical examination alone. In one study, 12 women had normal secondary sexual characteristics but irregular menses (Green 1989). Of 11 women of reproductive age, three (27 per cent) had low serum oestrogen levels, one had low peak FSH and LH responses to gonadotrophin-releasing hormone (Gn-RH), findings compatible with hypogonadotropism. A further patient had supranormal peak FSH and LH levels after receiving Gn-RH, findings compatible with ovarian dysfunction. Genital abnormalities have been described in 13 BBS females and the range includes hypoplastic fallopian tubes, uterus and ovaries, partial and complete vaginal atresia, septate vagina, duplex uterus, haematocolpos, persistent urogenital sinus, vesico-vaginal fistula, absent vaginal orifice, and absent urethral orifice (McLoughlin et al. 1967; Klein and Ammann 1969; Nadjmi et al. 1969; Campo and Aaberg 1982; Srinivas et al. 1983; Cramer et al. 1988; Green 1989; Stoler et al. 1995; Mehrotra et al. 1997). Nadjmi et al. (1969) point out the value of detailed urological and gynaecological investigations. Many cases of BBS have been misdiagnosed as McKusick–Kauffman Syndrome on this basis (David et al. 1999). Renal dysfunction Prior to the 1980s, the renal component of BBS had been infrequently reported (Brattgard 1949; Landau et al. 1949; Bimbi-Kovac and Hardyment 1956; Ross 1956) although a high frequency of structural abnormalities was observed at post-mortem (MeLoughlin and Shanklin 1967; Nadjmi et al. 1969). In 1969, Nadjmi et al. (1969) reviewed 330 cases of BBS reported in the literature and found that in 14 an autopsy was performed. Ten of these had

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urinary tract malformations (71 per cent), which varied from renal hypoplasia and hydronephrosis to chronic pyelonephritis and glomerulonephritis. In Klein and Ammann’s (1969) study, three cases (out of 57) had cystic kidneys, one had proteinuria and hypertension, two had glomerulonephritis and one had nephrosclerosis with unilateral hydronephrosis. Imaging Renal dysplasia can be present without clinical evidence of renal disease thus careful imaging of the urinary tract is warranted (Magro and Peres 1970; Labrune et al. 1974). Beales et al. (1999) reported that out of 57 patients imaged, 26 (46 per cent) had renal structural abnormalities. However, only 5 per cent had renal impairment at the time of assessment. Alton and McDonald (1973) and Bluett et al. (1977) described seven patients with renal anomalies consisting of cystic spaces communicating with the collecting system (Fig. 17.6). These spaces were frequently joined to the upper and lower calyceal groups. Other urographic features of BBS include persistence of fetal lobulation, small kidneys with parenchymal scarring, prolonged nephrogram, and blunting and clubbing of the calyces (Hurley et al. 1975; Bluett et al. 1977; Harnett et al. 1988; Linne et al. 1986). Persistent fetal lobulation has been described in isolation (MeLoughlin and Shanklin 1967; Okuyama et al. 1983) and Harnett et al. (1988) have suggested that this lobulation may reflect a defect in maturation. Ultrasound (USS) is not as successful in identifying the communicating cysts and diverticula as intravenous pyelography (IVP) but USS is comparable to IVP in documenting cortical changes (Cramer et al. 1988). Large echogenic kidneys can be seen on prenatal ultrasound scanning but may mimick recessive polycystic kidney disease. Ritchie et al. (1988) reported a single case in which the kidneys were enlarged and echogenic by the end of the second trimester but did not continue to enlarge thereafter. In one of the siblings described by Gershoni–Baruch et al. (1992) the prenatally enlarged kidneys were later found to be of normal size (at 28 months age).

Fig. 17.6 Abdominal CT scan taken from an 11-year-old boy with normal renal function. Note the irregular shaped kidneys containing multiple cysts.


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The combination of calyceal clubbing, cystic diverticula, and persistent fetal lobulation is characteristic and may be pathognomonic of BBS. Therefore early and serial renal imaging is important, especially in the young patient (Cramer et al. 1988). Histopathology Much variation has been described in the histopathology of the renal lesion (Hurley et al. 1975; Falkner et al. 1977; Price et al. 1981; Tieder et al. 1982; Roussel et al. 1985; Sata et al. 1988; Barakat et al. 1990). Hurley et al. (1975) described one patient with mesangial proliferation and sclerosis, who had only mild renal impairment. Other patients with more severe renal insufficiency had mesangial proliferation and sclerosis with cystic dilatation of the tubules, cortical and medullary cysts, periglomerular, and interstitial fibrosis. One patient had focal areas of dysplasia with the presence of primitive ducts and metaplastic cartilage. Linne et al. (1985) reported that reduced glomerular filtration rates and reduced maximal concentrating capacity (nephrogenic diabetes insipidus) were characteristic findings and that 50 per cent of the patients they studied had hyperaminoaciduria. An association with cystinuria has been described (De Marchi et al. 1992). Harnett et al. (1988) also showed that 14 of 17 patients could not concentrate urine above 750 mOsm per kg body weight after vasopressin administration, furthermore they found variable degrees of renal acidosis in almost a third of patients. All patients they studied had some abnormality of renal structure, function or both, but only 15 per cent had severe, progressive renal disease.

Cumulative % affected

Progression of renal impairment Alton and McDonald (1973) reported that over 30 per cent of patients with BBS die of uraemia. O’Dea et al. (1996) followed 36 BBS patients and reported that by 48 years, 25 per cent had developed chronic renal failure (O’Dea et al. 1996) (Fig. 17.7). Other reports have described CRF in up to 100 per cent of cases (Hurley et al. 1975; Harnett et al. 1988; Linne et al. 1986; Garber and de Bruyn 1991). Reports of the benefits of renal replacement therapy are few. For end-stage renal disease, hospital-based haemodialysis is reported to be the treatment of choice (Collins et al. 1994). Chronic ambulatory peritoneal dialysis may be difficult because of visual impairment, 100 90 80 70 60 50 40 30 20 10 0

Blindness

Diabetes

Renal impairment

0

5

10 15 20 25 30 35 40 45 50 55 60 65 Age (years)

Fig. 17.7 Age at diagnosis of blindness, diabetes mellitus (DM), and renal impairment in BBS. (Reprinted with permission from O’Dea et al. (1996).)

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learning difficulties, obesity and reduced manual dexterity. Renal transplantation has been successful in several patients but no study of long-term follow-up has been reported (Anonymous 1988; Williams et al. 1988; Norden et al. 1991; Collins et al. 1994). We know of five patients who have all undergone successful transplantation and remain well. Collins et al. (1994) also reported that immunosuppressive steroid treatment exacerbated obesity and suggested that perhaps an adjuvant appetite suppressant be given prior to transplantation. Urinary tract infections and hypertension are major concomitant problems, which also need to be addressed. Renal abnormalities in unaffected relatives Beales et al. (2000) reported that five of 123 (4.1 per cent) unaffected siblings of 109 BBS patients had one or more congenital renal anomalies. Significantly, two had unilateral renal agenesis (representative of a 20-fold excess) and three, vesicoureteric reflux. Moreover, one parent (obligate carrier) had unilateral renal agenesis, and a second parent had a duplicated renal pelvis and ureter. In addition, three parents (two males and one female) developed renal cell adenocarcinoma (RCC) of the same clear cell histopathological type at a relatively young age. The authors concluded the age-adjusted relative cumulative risk to BBS parents is 17 times that of the general population. They went onto suggest that these significant renal abnormalities may be a manifestation of the heterozygous state and predispose carriers to increased risks for RCC. Minor features The presence of distinct minor features may be helpful in arriving at a BBS diagnosis. Some of the better-defined areas are outlined below. Developmental delay Beales et al. (1999) reported that half of the BBS patients studied were late in reaching major developmental milestones and that 42 per cent had gross motor delay of up to one year. They also noted slow development of speech and language in 47 per cent (up to two years before first speech). Speech and language deficit A large proportion of patients (54 per cent) required some form of speech and language therapy as a child (Beales et al. 1999). In several patients some form of speech deficit persists into adulthood but the majority responded well to therapy. Facial features Subtle facial dysmorphism is apparent in some but not all patients (Fig. 17.8). There may be deep-set eyes, hypertelorism with downward-sloping palpebral fissures, a flat nasal bridge with anteverted nares, prominent nasolabial folds, a long philtrum and a thin upper lip. Many patients have a prominent forehead and males display early frontal balding (Beales et al. 1999).


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Fig. 17.8 Facial features in BBS are often subtle but may include enophthalmos, hypertelorism, downsloping palpebral fissures, flattened nasal-bridge, prominent nasolabial folds and frontal balding (adult males) (reprinted with permission from Beales et al. (1999)).

Neurological abnormalities Despite the classification into Laurence–Moon and BBS on the basis of the presence of spastic paraplegia in the former, there are several reports of disturbances of the central nervous system. Ataxia, difficult or scanning speech, dysdiadochokinesis, excessive blinking, tongue tremor, and Rombergism have all been reported in the 1940s (Lurie and Levy 1942; Garein et al. 1945; Roth 1947). Klein and Ammann (1969) found 10 per cent had slight spasticity of the extremities, 12 per cent had minor extrapyramidal signs and one case had a spastic diplegia. Bell (1958) in her large review found cases of ataxia, paraplegia, hesitant gait, spasticity, absent tendon-jerks, choreiform movements, and facial tics. She noted that in families with two or more affected siblings, both (or more) had a neurological defect. Both Kowal and Sikora (1989) and Rizzo et al. (1986) noted ataxia and found evidence of cerebellar atrophy on brain imaging. Hearing loss Deafness is not usually associated with BBS and the differential diagnoses to consider would be Usher syndrome or Alström syndrome if there was evidence of a sensorineural loss. Nevertheless, Burn (1950) reviewed the BBS case literature up to 1950 and found 10 out of 611 affected cases (1.6 per cent) reported sensorineural deafness. Klein and Ammann (1969) found 9 per cent BBS patients with progressive nerve deafness. A further study reported 24 per cent with deafness; 21 per cent conductive (glue-ear) and 3 per cent unexplained sensory-neural loss (Beales et al. 1999).

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Brain structure Few post-mortems have been reported in which abnormalities of the brain have been detected (Fraccaro and Gastaldi 1953; MeLoughlin and Shanklin 1967; Klein and Ammann 1969; Soliman et al. 1996). A number of pituitary and hypothalamic abnormalities have been reported including pituitary stalk necrosis, reduced pituitary tissue, increased proportion of basophils, pituitary eosinophilic adenoma, cyst in sella, and a hypothalamic hamartoma (Brattgard 1949; MeLoughlin and Shanklin 1967; Leroith et al. 1980; Sundar et al. 1981; Lee et al. 1986; Whitaker et al. 1987; Radetti et al. 1988; Mulaisho and Taha 1989; Diaz et al. 1991). Metabolic disturbances Abnormal glucose tolerance was reported in the 1940s (Attiah et al. 1940; Lurie and Levy 1942). Klein and Ammann (1969) reported that five cases (14 per cent) in their study had diabetes mellitus (DM); but did not define the type. O’Dea et al. (1996) revisited the families reported earlier by Green et al. and found that 12 of 38 (32 per cent) patients investigated were diabetic. Two were insulin dependent, four were maintained on oral hypoglycaemic agents and six were managed with diet alone. They concluded that 25 per cent of BBS patients were diabetic by the age of 35 years and 50 per cent by the age of 55. The cause of diabetes mellitus is not known and pancreatic abnormalities have not been observed in post-mortem reports (MeLoughlin and Shanklin 1967). Diabetes in BBS is probably of type II secondary to insulin resistance (Green 1989; Hauser et al. 1990). Diabetes insipidus has been described frequently and has now been determined to be mainly of nephrogenic origin (Viduta 1965; Radice et al. 1971; Harnett et al. 1988; De Marchi et al. 1992) but one report suggests a hypothalamic-pituitary cause (Koepp 1975). Cardiovascular anomalies Hypertension in BBS is common and was reported in 50 and 66 per cent of the patients studied by Harnett et al. (1988) and O’Dea et al. (1996), respectively (Price et al. 1981; Tieder et al. 1982; Fralick et al. 1990; Riise 1996). McLoughlin et al. (1964, 1967) described tetralogy of Fallot and transposition of the great vessels in two cases of BBS. On the basis of echocardiographic studies of 22 BBS patients, Elbedour et al. (1994) found cardiac abnormalities in 50 per cent. In addition to congenital heart disease, they also noted interventricular hypertrophy and dilated cardiomyopathy. Dental anomalies Poor dentition is common amongst BBS patients. Dental crowding, malocclusion, enamel hypoplasia, small teeth, and short roots have all been described (Bell 1958; Magnusson 1960; Kobrin et al. 1990; Lofterod et al. 1990; Borgstrom et al. 1996; Riise et al. 1997). In addition, a high arched palate is present in the majority of patients (Beales et al. 1999). Hepatic fibrosis Perilobular fibrosis, periportal fibrosis with small bile ducts, bile duct proliferation with cystic dilatation, biliary cirrhosis, portal hypertension, congenital cystic dilations of both the intrahepatic and extrahepatic biliary tract in patients with BBS, have all been described


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(Meeker and Nighbert 1971; Tsuchiya et al. 1977; Pagon et al. 1982; Roussel et al. 1985; Croft and Swift 1990; Nakamura et al. 1990). Atresia ani and Hirschsprung disease Atresia ani was described in the original report of Biedl (1922) and later by Raab (1924) and Radner (1940) (Biedl 1922). An association with Hirschsprung disease has been reported several times (Radetti et al. 1988; Islek et al. 1996; Lorda-Sanchez et al. 2000). Alström syndrome (ALS) The syndrome was first formally described in 1959, but in fact Alström himself had encountered the first case in 1946 (Alström et al. 1959). This was a 14-year-old boy from an area near Stockholm with a clinical picture, which had been interpreted as an atypical case of BBS. The boy had retinal degeneration, obesity, normal intelligence, and slight hearing impairment. Further investigation revealed impaired glucose tolerance. His parents were related. He also had two elder cousins with a similar clinical picture but whose parents were unrelated. The proband weighed 6 kg at birth and developed nystagmus and photophobia in the first year. By 7 years he was completely blind. Ophthalmological examination revealed peripheral degenerative changes with some pigmentation. The ERG was extinguished bilaterally. Both cousins had a similar clinical picture but in addition they developed chronic renal failure to which they succumbed in their early thirties. Demography and prevalence Less than a hundred cases of ALS have been reported. No formal estimates of the prevalence of Alström syndrome have been given but by comparison with BBS it is rare. Alström syndrome occurs with approximately 1/5th the prevalence of BBS in the UK (i.e. ~1/600,000— Personal observation). Alström Syndrome has been described in Africa, Japan, Middle-East, Russia, India, Europe, and the US (Garg et al. 1991; Cohen and Kisch 1994; Farah et al. 1996; Awazu et al. 1997; Marshall et al. 1997; Macari et al. 1998; Russell-Eggitt et al. 1998). Marshall et al. (1997) described a large Acadian pedigree, which included eight affected members and supported a high degree of consanguinity (average kinship coefficient 0.01). The ancestry was traced back over 13 generations to a small group of seventeenth century Acadian settlers who emigrated from Northern France. Within the kindred, only one ancestral pair born in the 1660s were found to be common to both the maternal and paternal lineages of all affected children, providing good evidence for a founder effect. Many of the affected kindreds described in the study originated from France, Germany, England, and Ireland. Genetics Alström et al. (1959) clearly showed that the syndrome is inherited in an autosomal recessive fashion. Using the pedigree described by Marshall et al. (1997), a genome-wide search for a region of homozygosity-by-descent was performed which identified the gene locus on 2p13–14

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between D2S286 and D2S393, a distance of 14.9 cM (Collin et al. 1997). A maximum lodscore of 3.84 ( 0.00) was generated at D2S292 (two-point analysis). The region was independently confirmed and refined using a consanguineous Algerian family with three affected siblings (Macari et al. 1998) and located in a 6.1 cM interval from D2S291 to D2S2114. A maximal two-point lodscore of 3.0 ( 0.00) was obtained at D2S1209. These combined data suggest that the ALS gene is located between D2S2114 and D2S2113. To confirm these findings, a large linkage study was performed in twelve further Alström syndrome families (Collin et al. 1999). A maximum two-point lod score of 7.13 ( 0.00) for marker D2S2110 and a maximum cumulative multipoint lod score of 9.16 for marker D2S2110 were observed, further supporting linkage to chromosome 2p13. No evidence of genetic heterogeneity was observed in these families. Cloning of ALMS1 In 2002, two groups coincidentally cloned ALMS1. The first mapped the breakpoints in an individual with Alström syndrome who carried a balanced reciprocal translocation (46, XY,t(2;11)(p13;q21)mat) (Hearn et al. 2002). They postulated that the affected individual would carry a maternally derived physical disruption (breakpoint) of one allele and that to accompany this there must also be a second constitutive paternal mutation in ALMS1. This was indeed proved with the breakpoint falling between exons 4 and 5 in the maternally inherited chromosome and a 2 bp deletion in exon 8 of the paternal ALMS1 allele (2141delCT) was detected. Sequencing of an RT-PCR product from this patient confirmed expression of only the paternal transcript as predicted. Five further ALS families harboured either compound heterozygous or homozygous mutations, each leading to premature transcription termination. Notably one family did not have mutations in ALMS1. Collin et al. (2002), using a positional candidate approach, reported mutations in six families including one insertion and three deletions; five were homozygous and one compound heterozygous. Expression of ALMS1 in adult human tissues appears to be ubiquitous and consistent with the diverse pattern of organ involvement seen in AS. ALMS1 is a large gene comprising 12.9 kb in length containing a 12.5 kb open reading frame and 23 exons. The ALMS1 protein consists of 4,169 amino acids (461 kD) and is predicted to contain a leucine zipper motif and a potential signal peptide. Further analysis of the ALMS1 sequence reveals a striking large tandem-repeat domain comprising 34 imperfect repetitions of 47 amino acids devoid of cysteine residues. This constitutes ~40 per cent of the protein and is entirely encoded by exon 8. In addition, ALMS1 contains a polyglutamine tract followed by a polyalanine run. Size variation in the glutamic acid tract was detected in the normal population but subsequent analysis in obligate carriers and an affected individual without ALMS1 mutations did not detect abnormal GAG allele sizes (Hearn et al. 2002). Although the function of ALMS1 is not known at present, there are some structural organization similarities of ALMS1 to the mucin genes, in particular the presence of low cysteine tandem repeats. Clinical presentation Like BBS, ALS is also pleiotropic with differing ages of emergence of key clinical features.


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Ophthalmology Nystagmus and photophobia occur early in infancy and are usually the first signs of disease, often noted by 4 months of age (Marshall et al. 1997; Russell-Eggitt et al. 1998). Cone degeneration has been reported as early as 6 months and is followed closely by rod degeneration (Tremblay et al. 1993). Russell-Eggitt et al. (1998) suggest that the earliest fundal change is narrowing of the retinal vessels followed by patchy atrophy of the retinal pigment epithelium (RPE) with increased visibility of choroidal vessels with diffuse increased pigmentation in the mid-periphery and macula (Fig. 17.9). The optic disc is often pale and there was no bone-corpuscular pigmentation seen. Most of the patients in that study had a visual acuity of less than 3/60 and all patients older than 11 years were completely blind. Posterior subcapsular cataracts were present in the majority of patients reported (63 per cent—(Marshall et al. 1997); 100 per cent—(Sebag et al. 1984)) and may be a consequence of DM (Sebag et al. 1984). A number of other ophthalmic histopathological findings have been described (Table 17.5). Hearing loss Bilateral sensory hearing loss is slowly progressive and often arises during the first 5 years of life (Marshall et al. 1997). By the end of the first to second decade, hearing loss is often in the moderately severe range (40–70 dB) (van den Abeek et al. 2001). Obesity The birth weights of most cases reported were normal although, Alström’s original proband was over 6 kg at birth. However, weight gain is rapid in the first year and has been associated

Fig. 17.9 Fundus photograph from a 5-year-old child with ALS (acuity 2/36). One of the earliest changes observed is narrowing of the retinal vessels. (Photo courtesy of Isabelle Russell-Eggitt, Great Ormond Street Hospital, London.)

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Philip L. Beales, Patrick S. Parfrey, and Nicholas Katsanis Table 17.5 Range of ophthalmic abnormalities reported in ALS Cone and rod dystrophy/atrophy Nystagmus Photophobia Posterior subcapsular cataracts (usually bilateral) Small globes Lacy vacuolation of iris Ciliary process hyalinization Asteroid hyalosis Narrowing of retinal vessels Melanin pigmentation RPE atrophy Preretinal fibrosis Bilateral giant optic disc drusen Optic nerve atrophy

with hyperphagia although Marshall et al. (1997) found no excess caloric intake. ALS patients develop truncal obesity and find it difficult to maintain static body weight. The BMI ranges from 23.8 to 36.5 kg/m2. The lipid profile was abnormal in all children studied by Marshall et al. (1997) and was observed as early as 4 years. Whilst total cholesterol and HDL levels were normal, each affected child had raised triglycerides and low HDL-cholesterol concentrations. Stature All young children in the study had an advanced bone age and were tall for their age (Marshall et al. 1997) (Figs 17.10 and 17.11). However, older patients were found to have a below average height-for-age suggesting that they reached their final adult height too early. They also noted scoliosis in all of their study subjects who were over 14 years of age suggesting some kind of gene effect on bone growth. Acanthosis nigricans The presence of acanthosis nigricans is a common finding in ALS and is probably a consequence of hyperinsulinaemia. Endocrine abnormalities Although only two of nine subjects within the same family had raised fasting serum glucose, all were significantly hyperinsulinaemic (Marshall et al. 1997). The youngest patient with raised insulin levels was 4-years-old and hyperinsulinaemia appears to persist into adulthood with the majority going on to develop overt NIDDM. In another study, only 5 of 22 patients had diabetes but most of these were under 15 years and may subsequently develop NIDDM (Russell-Eggitt et al. 1998). Other endocrinological disturbances cited include hypothyroidism, and low sex steroid hormone levels. Gonadotrophin levels are usually normal or elevated, pointing to a primary gonadal failure.


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Fig. 17.10 Short stature in a 31-year-old Italian male. (Photo courtesy of Pietro Maffei, University of Padua, Italy.)

Hepatic function There have been 24 cases cited of ALS and associated liver disease (Ikeda et al. 1974; Horiuchi et al. 1976; Puech et al. 1982; Sebag et al. 1984; Connolly et al. 1991; Farah et al. 1996; Awazu et al. 1997; Marshall et al. 1997; Russell-Eggitt et al. 1998). On liver biopsy, disease findings include bridging portal fibrosis, hepatocellular steatosis, and active hepatitis (Connolly et al. 1991; Marshall et al. 1997). Indication of underlying liver disease is usually reflected in raised hepatic transaminases (ALT, AST). Renal disease Renal disease infrequently presents before the second decade and is slowly progressive. The severity and type of nephropathy is variable. Goldstein and Fialkow (1973) described three patients all of whom had normal urinalyses, urea and electrolyte levels in the first two decades. End-stage renal failure was present in one subject at 31 years but only mild impaired function was seen in her two affected sisters at ages 28 and 26 years. Amongst the tubular abnormalities present, they observed aminoaciduria and abnormalities of renal concentration, characterized by partial resistance to exogenous vasopressin. They concluded that there were intrinsic abnormalities of proximal and distal tubular function.

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Fig. 17.11 Facial appearance in a 31-year-old male with ALS. (Photo courtesy of Pietro Maffei, University of Padua, Italy.)


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Fig. 17.12 Glomerular hyalinization and fibrosis in ALS. (Courtesy of Jan Marshall, Jackson Labs, Bar Harbor, US.) (See Plate 11 of the Colour Plate Section at the centre of this book.)

Renal biopsies from the two youngest sisters revealed a chronic nephropathy characterized by thickening of glomerular and tubular membranes. Furthermore, there was hyalinization of the glomeruli, diffuse atrophy of tubules with moderate interstitial fibrosis, but only minimal inflammation was noted (Fig. 17.12). They concluded that as the renal small vessels were unremarkable, the histopathology seen was not a consequence of diabetic nephropathy. EM studies did not reveal inclusion bodies or immune complexes. There appears to be a pattern of generalized thickening of cell membranes together with dense hyalinization of connective tissue observed in extra-renal tissues such as skin (dermis layer—(Goldstein and Fialkow 1973)), and testis (Alström et al. 1959; Weinstein et al. 1969). Cardiomyopathy Warren et al. (1987) reported follow-up information on the two brothers (33 and 19 years), previously described by Weinstein et al. (1969). The original description revealed no cardiac abnormality but later both brothers (the older brother at 36 years and the younger at 37 years) developed dilated cardiomyopathy. The 36-year-old brother died soon after emergency hospital admission and histopathology indicated patchy interstitial fibrosis, hypertrophic changes of myocytes, and focal endocardial fibrosis of the left ventricular wall and interventricular septum. The second brother presented with a more insidious onset of heart failure. A right ventricular biopsy revealed myocardial hypertrophy and focal endocardial, subendocardial, and interstitial fibrosis. EM studies were again normal. Michaud et al. (1996) reported 5 children (of 8, onset between 3 weeks and 4 months) and Marshall et al. (1997) reported 5 children (of 12; infantile onset in three, and age 14 and 23

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in the fourth and fifth, respectively) that developed dilated cardiomyopathy. Again myocardial biopsies revealed focal myocyte hypertrophy, interstitial fibrosis and vacuolar degeneration. No inflammatory infiltrate was seen and EM studies showed morphologically normal mitochondria. Russell-Eggitt et al. (1998) reported 18 out of 22 (82 per cent) presented with cardiac failure with onset between 2 weeks and 7 months of age. Other reports of dilated cardiomyopathy have been made (Puech et al. 1982; Aynaci et al. 1995). After initial resuscitation and treatment, the clinical course of the cardiomyopathy was characterized by gradual improvement with few long-term sequelae in all the cases described by Michaud et al. (1996) Marshall et al. (1997), and Russell-Eggitt et al. (1998) (see section on ‘management’); however, up to 10 per cent may have a recurrence of cardiomyopathy at a later age (Cathy Carey, pers. commn.). Learning difficulties Although generally considered to be a discriminating feature between ALS and BBS, developmental delay and learning difficulties have been cited for ALS (Ikeda et al. 1974; Pfeiffer and Pusch, 1978; Sebag et al. 1984; Marshall et al. 1997). Interestingly, Marshall et al. (1997) also noted four of their subjects had disordered language and delay, and one had difficulty in initiation and problem solving.

Diagnosis Alström et al. described the key components of the syndrome as: retinal degeneration, severe nerve deafness, obesity, and DM. Ten years later further distinguishing features were described including acanthosis nigricans, hyperuricaemia, hypertriglyceridaemia, and male hypogonadism (Klein 1968; Weinstein et al. 1969). Goldstein and Fialkow (1973) also reported chronic nephropathy, insulin resistance, vasopressin, and gonadotrophin resistance and baldness.

Investigation of BBS and ALS A summary of the investigations required for a patient suspected of having BBS or ALS is given in Table 17.6. If either condition is suspected in a child, then a prompt ophthalmological assessment should be requested—however if the child is under 4 years of age performing an electroretinogram will be difficult and may need to be postponed until a later age. A renal ultrasound and intravenous pyelogram should be performed to look for fetal lobulation (BBS), general kidney morphology and calyceal dilatation. Height and weight should be measured at frequent intervals. A full developmental assessment should be performed as early as possible and be diligently repeated at proscribed ages. In particular, an experienced therapist should perform a formal speech and language assessment, as early intervention may influence the child’s subsequent educational needs. A formal IQ should also be requested at this point as later visual deterioration may bias the results. Follow-up investigations of patients should include eye examination (although the exact interval cannot be generalized), height, weight, BMI measurement, and evaluation of body


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Table 17.6 Investigation of Bardet–Biedl and Alström syndromes Clinical feature

Investigation

Ophthalmology

Visual acuity Strabismus test Fundoscopy (direct/indirect) Tonometry (intra-ocular pressure) Visual fields Colour vision Dark adaptation Electroretinogram (ERG) with visually evoked responses (VER) Body mass index calculation Body composition (using bioimpedance, DEXA or CT) IQ measurement (visual impairment to be considered) Formal developmental assessment Formal educational assessment Blood pressure (6 monthly) Blood urea Serum creatinine Creatinine clearance Serum bicarbonate Urine osmolality (after water deprivation) Intravenous pyelogram Renal ultrasound scan Glucose tolerance test Growth hormone (approaching puberty) Testosterone (in males before and after puberty) Bone age (advanced before puberty in AS) Serum luteinizing and follicle stimulating hormones Serum oestrogen (females) Serum prolactin (females) Pelvic ultrasound in females (urogenital anomalies?) Liver function test biopsy

Obesity

Cognitive function and development

Renal impairment

Endocrinology and assessment of the hypothalamic–pituitary gonadal axis

Liver function

composition (by bioimpedance if available). Blood pressure should be monitored at least half yearly, blood urea, and serum creatinine level estimations made to assess glomerular filtration rate, serum sodium to determine the presence of nephrogenic diabetes insipidus, serum bicarbonate to monitor any renal tubular acidosis, and serum glucose for DM. The development of polyuria, in the absence of diabetes mellitus, is an indication to test the ability of the kidneys to concentrate urine (i.e. water deprivation test). The failure of urinary osmolality to rise to normal following an overnight fast, and the failure to rise above 750 mOsm/Kg of body weight after ADH administration, confirms the presence of nephrogenic diabetes insipidus. The integrity of the hypothalamic–pituitary–gonadal axis should be assessed before and after puberty. Growth hormone and testosterone levels (in boys) should be measured at around 10–11 years and if abnormal the child should be promptly referred to a childhood endocrinologist for assessment of the need for supplementation. An assessment of bone-age should be sought (especially in ALS). Following puberty, measurement of serum

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follicle-stimulating hormone, luteinizing hormone, and testosterone will determine the presence of primary/secondary gonadal failure in males. Semen analysis can help determine infertility. In post-pubertal females, serum oestrogen, FSH, LH and prolactin should be measured and a pelvic ultrasound performed to determine the structure of gonads, presence of follicles, and the presence of urogenital structural anomalies (BBS). Management As the underlying biochemical defect causing both BBS and ALS is unknown, management strategies must concentrate on individual problems or systems. The principles of management remain the same for both diseases (summarized in Table 17.7). Educational needs These must be assessed early and where possible, well before visual impairment ensues. There is no clear recommendation as to whether an individual requires special education as many have coped well in mainstream education. However, many patients say they have benefited greatly from extra input and attendance at a specialist blind school or college. In a young child suspected of having BBS, the diagnosis should be discussed with the school authorities and provision made for visual deterioration and classroom support (such as seating position, lighting, visual aids). Ophthalmology Atypical retinitis pigmentosa is the hallmark of BBS and cone-rod dystrophy, ALS, for which no proven treatments exist. Early ophthalmological assessment is important to document the rate of deterioration and to plan for a life with little or no vision. It is particularly hard for a young BBS child who has largely normal vision until his/her second decade, to have to adapt to night blindness and then patchy field deficit. Nystagmus and photophobia occurring in infancy suggests a diagnosis of ALS. It is important to seek advice of low-vision specialists to decide what is the best communications medium (i.e. Braille, keyboard, electronic books) for that person and when to implement acquisition of skills to use them. Other ophthalmological conditions such as glaucoma, myopia cataracts and diabetic retinopathy are to some extent treatable and so should not be overlooked by the ensuing RD. In some countries the registration of a person as legally blind can help in the provision of visual aids. Table 17.7 Management summary of BBS and ALS Educational support Visual impairment aids and skills training (i.e. Braille or keyboard) Regular ophthalmological follow-up Obesity reduction programme (dietetics, behaviour therapy, physical exercise, drugs?) Diabetes control (regular half-yearly clinic follow-up) Nephrological assessment (where chronic renal failure is imminent, early discussion of end-stage disease treatment options is imperative) Discussion of contraception with adult females Genetic counselling


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Obesity Obesity left untreated will eventually lead to multiple morbidity and even mortality. As with multifactorial obesity there is no single recommended therapy. In our experience, those with BBS can successfully lose weight if referred to a dietician early. However, the best management strategy is probably a multidisciplinary approach involving the whole family where possible. Such a strategy will include a careful dietary assessment, diet control, behavioural therapy, and exercise. The role of therapeutic agents (appetite suppressants or lipid inhibitors) is still being evaluated. Renal disease The presence of renal disease in both BBS and ALS is variable and should be closely assessed before pronouncing a patient clear of disease. In general, renal disease associated with BBS can emerge at any age but tends to develop in the second and third decades in ALS. Hypertension is a common sequel and may be controlled by weight loss measures alone. Blood pressure should be monitored at least half yearly. Serum urea, creatinine, and electrolytes should be measured at least annually. Chronic renal disease may necessitate renal replacement therapy. The type offered will depend on the extent of concomitant disabilities. Often this will begin with some form of dialysis but the issue of renal transplantation should also be discussed as early as possible. Many patients have successfully undergone transplantation but many difficulties may arise out of the immunosuppressant therapy (e.g. unmasking of diabetes with glucocorticoids). Fertility and contraception Females with BBS should not be assumed infertile or sexually inactive. Contraception should therefore not be overlooked, but the type offered will depend on a number of social and medical factors. Likewise, whilst most men with BBS will be infertile, some males have fathered children. If there is doubt, endocrine and semen analyses should be sought. To our knowledge no affected patient with ALS has produced live offspring but until specific studies are complete, a similar management strategy as described for BBS should be employed. Cardiomyopathy (ALS) The onset of dilated cardiomyopathy often appears in infancy and is indistinguishable from sporadic and familial forms of DC. In fact Michaud et al. (1996) suggest that the cardiac defect is a consequence of gene expression at a specific developmental stage. They felt that the residual minimal dilatation seen in four of their patients was a sequel to the original episode rather than the result of an ongoing process. The majority of patients with cardiomyopathy reported by Marshall et al. (1997) and Russell-Eggitt et al. (1998) suffered few cardiac sequelae provided initial resuscitation and prompt treatment of cardiac failure was implemented. Echocardiography is required in all suspected cases of ALS (and is advised in BBS) and any subclinical impairment rigorously followed-up. Hearing loss Hearing assessments (including brainstem-evoked auditory responses and audiometry) should be performed as early as possible in both conditions. Russell-Eggitt et al. (1998)

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Table 17.8 Comparison of clinical features of Bardet–Biedl syndrome and Alström syndrome Feature

BBS

ALS

Ocular anomalies

Rod-cone dystrophy; onset (night blindness) typically 6–8 years with rapid progress (7–10 years) to complete visual loss Onset 1–2 years generalized/ truncal Glue ear common Occasionally aortic stenosis, LVH Gross and fine motor skills, speech and language delay; learning difficulties in ~70%

Cone-rod dystrophy; photophobia/ nystagmus onset typically birth—15 months. Progressive visual loss

Obesity Hearing loss Cardiac abnormalities Developmental delay/ cognitive impairment

Stature Renal anomalies

Genital anomalies

Type 2 diabetes Hepatic disease

Tall and short Persistent fetal lobulation, cystic tubules, polyuria/polydipsia, aminoaciduria, RTA, ESRF in 5–10% Vaginal atresia, bicornuate uterus, hydrometrocolpos, hypospadias, urethral valves, micropenis (hypogonadotrophic hypogonadism) + Uncommon—abnormal liver function tests—usually self-limiting, hepatic fibrosis

Onset 1–2 years generalized/ truncal Sensorineural loss in 70% Dilated cardiomyopathy in 60% Gross and fine motor skills, speech and language delay; learning difficulties in ~30% Mainly short Late onset—polyuria/polydipsia, interstitial fibrosis, glomerular hyalinosis, tubular atrophy, ESRF minority Micropenis (hypogonadotrophic hypogonadism)

++ insulin resistance/acanthosis nigricans Common—liver function deranged in early childhood. Can progress to hepatic failure in second decade; hepatic fibrosis, cirrhosis, CAH, fatty liver

detected hearing loss as early as 9 months of age. They also stated that many ALS patients required grommet insertion for middle-ear disease during the first decade. Beales et al. (1999) reported that 20 per cent of BBS children developed middle-ear disease in their first decade. Nevertheless, the majority of middle-ear disease resolves with time. However, isolated sensorineural hearing loss may not be easily recognized and may only involve certain frequency ranges but may be amenable to correction with aids. Endocrine A comprehensive assessment of endocrine function should be sought in all patients. These should include thyroid function, LH and FSH, testosterone, oestradiol, fasting glucose and insulin, growth hormone. Dynamic tests should include glucose tolerance and GnRH stimulation. In children, a bone assessment should be made especially in those with ALS. Growth hormone supplementation may be warranted in some BBS patients and timing is of


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essence. Although most patients have normal secondary sexual characteristics, libido is often said to be lacking. A trial of testosterone supplementation has been initiated in many of our male BBS patients with mixed results. Hyperinsulinaemic states usually respond to oral hypoglycaemic therapy (sulfonylureas and metformin) although occasional control of blood glucose can only be achieved with insulin. Conclusions Although ALS and BBS have some similar manifestations, nevertheless there are fundamental differences, which should be sought before deciding on the ultimate diagnosis. Table 17.8 lists a comparison of clinical features common to both diseases. Perhaps the most discriminating features are the ocular findings, in particular infantile onset of nystagmus and photophobia in ALS (compared with BBS in which early normal vision is usual) and the development of cardiomyopathy (infantile onset) in ALS. The combination of features specific to ALS, potentially implicate a mitochondrial respiratory-chain abnormality in disease pathogenesis. The heterogeneity encountered in BBS (and to a lesser extent ALS) suggests the presence of novel parallel pathways involved at key developmental steps. Ultimately the cloning and functional elucidation of all the cognate genes will shed light on the aetiology of these conditions and may provide potential targets for the design of therapeutic interventions. References Ackerman, J., Brody, P., Kanarek, I., and Gottlieb, F. (1980). Macular wrinkling and atypical retinitis pigmentosa in Laurence–Moon–Biedl–Bardet syndrome. Annals of Ophthalmology, 12, 632. Agashe, V. R. and Hartl, F. U. (2001). Roles of molecular chaperones in cytoplasmic protein folding. Seminars in Cell and Developmental Biology, 11, 15–25. Alström, C., Hallgren, B., Nilsson, L., and Asander, H. (1959). Retinal degeneration combined with obesity, diabetes mellitus, and neurogenous deafness. A specific syndrome distinct from Laurence–Moon–Biedl syndrome. A clinical endocrinological and genetic examination based on a large pedigree. Acta Psychiatrica et Neurologica Scandinavica, 34, 1–35. Alton, D. J. and McDonald, P. (1973). Urographic findings in the Bardet–Biedl syndrome, formerly the Laurence–Moon–Biedl syndrome. Radiology, 109, 659–63. Ammann, F. (1970). Investigations clinique et genetique sur le syndrome de Bardet–Biedl en Suisse. J Genet Hum, 18(Suppl), 1–310. Anonymous. (1988). Laurence–Moon and Bardet–Biedl syndromes (editorial). Lancet, 2, 1178. Attiah, M., Attiah, H., and El Gamal, Y. (1940). The Laurence–Moon–Biedl syndrome. Bulletin of the Ophthalmological Society of Egypt, 33, 155. Awazu, M., Tanaka, T., Sato, S., Anzo, M., Higuchi, M., Yamazaki, K., et al. (1997). Hepatic dysfunction in two sibs with Alström syndrome: case report and review of the literature. American Journal of Medical Genetics, 69, 13–16. Aynaci, F. M., Okten, A., Mocan, H., Gedik, Y., and Sarpkaya, A. O. (1995). A case of Alström syndrome associated with diabetes insipidus. Clinical Genetics 48, 164–6. Badano, J. L., Ansley, S. J., Leitch, C. C., Lewis, R. A., Lupski, J. R., and Katsanis, N. (2003). Identification of a novel Bardet–Biedl syndrome protein, BBS7, that shares structural features with BBS1 and BBS2. American Journal of Human Genetics, 72(3), 650–8. Barakat, A. J., Arianas, P., Glick, A. D., and Butler, M. G. (1990). Focal sclerosing glomerulonephritis in a child with Laurence–Moon–Biedl syndrome. Child Nephrology and Urology, 10, 109–11.

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Radice, C., D’amico, E., and Torti, G. (1971). Secondary nephrogenic diabetes insipidus in the Laurence– Moon–Biedl syndrome (exploration of renal hydric homeostasis). Minerva Pediatrica, 23, 1968–73. Radner, S. (1940). On Laurence–Moon–Biedl’s syndrome. Acta Medica Scandinavica, 55, 141–52. Richard, I., Broux, O., Allamand, V., Fougerousse, F., Chiannikulchai, N., Bourg, N., et al. (1995). Mutations in the proteolytic enzyme calpain 3 cause limb-girdle muscular dystrophy type 2A. Cell, 81, 27–40. Riise, R. (1996). The cause of death in Laurence–Moon–Bardet–Biedl syndrome. Acta Ophthalmologica Scandinavica Supplement, 74(219), 45–7. Riise, R., Andreasson, S., Borgastrom, M. K., Wright, A. F., Tommerup, N., Rosenberg, T., et al. (1997). Intrafamilial variation of the phenotype in Bardet–Biedl syndrome. British Journal of Ophthalmology, 81, 378–85. Ritchie, G., Jequier, S., and Lussier-Lazaroff, J. (1988). Prenatal renal ultrasound of Laurence–Moon–Biedl syndrome. Pediatric Radiology, 19, 65–6. Rizzo, J. F. d., Berson, E. L., and Lessell, S. (1986). Retinal and neurologic findings in the Laurence–Moon–Bardet–Biedl phenotype. Ophthalmology, 93, 1452–6. Ross, C., Crome, L., and Mackenzie, B. (1956). The Laurence–Moon–Biedl syndrome. Journal of Pathology and Bacteriology, 72, 161–72. Roth, A. (1947). Familial eunuchoidism. The Laurence–Moon–Biedl syndrome. Journal of Urology, 57, 427–45. Roussel, B., Leroux, B., Gaillard, D., and Fandre, M. (1985). Laurence–Moon–Bardet–Biedl syndrome, chronic diffuse tubulo-interstitial nephritis and liver involvement. Helvetica Paediatrica Acta, 40, 405–13. Runge, P., Calver, D., Marshall, J., and Taylor, D. (1986). Histopathology of mitochondrial cytopathy and the Laurence–Moon–Biedl syndrome. British Journal of Ophthalmology, 70, 782–96. Russell-Eggitt, I. M., Clayton, P. T., Coffey, R., Kriss, A., Taylor, D. S., and Taylor, J. F. (1998). Alström syndrome: Report of 22 cases and literature review. Ophthalmology, 105, 1274–80. Sato, H., Saito, T., Yamakage, K., Kyogoku, Y., Furuyama, T., and Yoshinaga, K. (1988). Renal histopathology of Laurence–Moon–Biedl syndrome: tubulointerstitial nephritis without specific glomerular changes. Nephron, 49, 337–8. Schaap, C., Tusscher, M. P., Schrander, J. J., Kuijten, R. H., and Schrander-Stumpel, C. T. (1998). Phenotypic overlap between McKusick–Kaufman and Bardet–Biedl syndromes: are they related? (letter; comment). European Journal of Pediatrics, 157, 170–1. Schachat, A. P. and Maumenee, I. H. (1982). Bardet–Biedl syndrome and related disorders. Archives of Ophthalmology, 100, 285–8. Sebag, J., Albert, D. M., and Craft, J. L. (1984). The Alström syndrome: Ophthalmic histopathology and retinal ultrastructure. British Journal of Ophthalmology, 68, 494–501. Sheffield, V. C., Carmi, R., Kwitek-Black, A., Rokhlina, T., Nishimura, D., Duyk, G. M., et al. (1994). Identification of a Bardet–Biedl syndrome locus on chromosome 3 and evaluation of an efficient approach to homozygosity mapping. Human Molecular Genetics, 3, 1331–5. Slavotinek, A. M., Stone, E. M., Mykytyn, K., Heckenlively, J. R., Green, J. S., Heon, E., et al. (2000). Mutations in MKKS cause Bardet–Biedl syndrome. Nature Genetics, 26, 15–16. Soliman, A. T., Rajab, A., AlSalmi, I., and Asfour, M. G. (1996). Empty sellae, impaired testosterone secretion, and defective hypothalamic-pituitary growth and gonadal axes in children with Bardet–Biedl syndrome. Metabolism: Clinical and Experimental, 45, 1230–4. Solis-Cohen, S. and Weiss, E. (1925). Dystrophia adiposogenitalis, with atypical retinits pigmentosa and mental deficiency. The Laurence–Biedl syndrome: a report of four cases in one family. American Journal of Medical Science, 169, 489–505. Srinivas, V., Winsor, G. M., and Dow, D. (1983). Urologic manifestations of Laurence–Moon–Biedl syndrome. Urology, 21, 581–3. Stigglebout, W. (1972). The Bardet–Biedl syndrome; including Hutchinson–Laurence–Moon syndrome. in Neuroretinal degeneration. Handbook of clinical neurology (ed. P. J. Vinken and G. W. Bruyn), Vol. 13, pp. 1277–91. North Holland, Amsterdam.

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18 Genetic syndromes with a renal component Richard Sandford and Robin G. Woolfson

Sickle cell disease Introduction Inherited defects in haemoglobin synthesis result in sickle cell disease and thalassaemia, and these conditions can occur alone or in combination. The heterozygote states have few clinical implications but homozygotes have profound anaemia, which is more severe in thlassaemia where dyserythropoiesis is greater. Improvements in the management of patients with haemoglobinopathies, particularly sickle cell disease have resulted in a much greater prevalence of renal complications. In broad terms, the renal complications of sickle cell disease result from intrarenal vaso-occlusive disease whereas those in thalassaemia are the sequelae of repeated blood transfusion, leading to iron overload and viral infection. Epidemiology The sickle-cell gene occurs widely throughout Africa and in countries with African immigrant populations, some Mediterranean countries, the Middle East, and parts of India. The sicklecell mutation results in a single amino acid substitution at position 6 of the -globin chain on chromosome 11. The homozygotes produce mainly haemoglobin S with variable amounts of fetal haemoglobin (HbF). The heterozygotes have one normal ( A) and one affected -chain ( S) gene and produce about 60 per cent haemoglobin A and 40 per cent haemoglobin S with minimal clinical sequelae. The prevalence of other haemoglobinopathies in affected communities leads to compound heterozygous states. Compound heterozygotes for haemoglobins S and C produce almost equal amounts of each variant but heterozygotes for haemoglobin S and -thalassaemia make predominantly haemoglobin S. Patients with these compound haemoglobinopathies tend to suffer fewer acute crises and be less anaemic, although HbSC disease predisposes to thrombotic events, particularly in the puerperium. Hypoxia and acidosis, due to cellular stress, inflammation or infection, promote the polymerization of deoxy HbS which deforms the red cell envelope to form a rigid, sickle-shaped structure (see Fig. 18.1). Sickled cells aggregate in the microcirculation, interact abnormally with vascular endothelium, haemolyse, and cause acute and chronic tissue ischaemia. Acute crises cause bone pain and less commonly acute lung or hepatic sequestration or stroke; recurrent sickling results in avascular necrosis of the femoral heads, retinal ischaemia leading to proliferative retinopathy and organ failure, particularly affecting the kidneys, heart, and cerebral vasculature.

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Fig. 18.1 Micrographs showing the appearance of normal and sickled erythrocytes. (See Plate 12 of the Colour Plate Section at the centre of this book.)

Clinical outcome The prognosis in homozygous sickle cell disease has improved dramatically over the last 40 years, rising from a life expectancy in the second decade in the 1960s to survival into the sixth or seventh decade presently. This reflects improved management of acute crises based on early attention to oxygen therapy, intravenous fluids, antibiotics, appropriate analgesia with avoidance of non-steroidal anti-inflammatory drugs and opiates whenever possible.

Factors which predict outcome: Data regarding life expectancy and risk factors for early death come from the multicentre Cooperative Study of Sickle Cell Disease (CSSCD) in which 3764 patients were enrolled between 1978 and 1988 (Platt et al. 1994). There were 209 deaths in the HbSS group, occurring at a mean age of 42 years for men and 48 years for women; survival for HbSC patients was longer at 60 and 68 years for men and women, respectively. Within the entire study group, 964 adults were followed for a minimum of 2 years of whom 85 died and from this cohort risk factors for early death were defined. The most important of these was the presence of renal failure, defined as a 20 per cent increase in baseline creatinine or a creatinine clearance 100 ml/min (see Table 18.1).

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Table 18.1 Risk factors for early death in HbSS patients aged 20 years or older (Platt et al. 1994)

Renal failure Seizures Acute chest syndrome Sepsis 30% fall in haemoglobin Painful crises

Risk of early death

p value

1.6 0.04 1.32 0.36 0.97 0.26 0.70 0.36 0.66 0.27 0.59 0.22

0.001 0.002 0.001 0.07 0.02 0.01

Chronic renal failure The hypoxic, hypertonic, and haemoconcentrated micro-environment of the renal medulla promotes red cell sickling. Nephropathy is more likely in patients with the HbSS homozygote, compared with either HbSC or HbSThal, but depends on the degree of anaemia, the amount of HbF present and other undefined host factors. Although early findings suggested an increased risk of nephropathy with particular -globin gene haplotypes (e.g. Central African Republic), surprisingly these haplotypes do not predict the development of macroalbuminaemia (300 g/g Cr) (Powars et al. 1991; Guasch et al. 1999) which usually presages renal failure. This apparent anomaly may be explained by co-inheritance of microdeletions in the -globin gene locus in HbSS which confer ‘renoprotection’. A prospective study of 934 patients from South California over a 25 year period provides important observational data about sickle nephropathy (Powars et al. 1991). Chronic renal failure (CRF) was defined as an irreversible increase in creatinine of 88.4 mol/l for children and 132.6 mol/l for adults. In 725 patients with HbSS, the incidence of CRF was 4.2 per cent with a median age of 23.1 years at diagnosis; in contrast, CRF only affected 2.4 per cent of 209 patients with HbSC and was diagnosed at a median age of 49.9 years. The median survival of HbSS patients with CRF was only 3.3 years compared to 8 years for those without CRF which represents a 1.42 increased relative risk of death ( p 0.02). Using multivariate analysis, predictors for the subsequent development of CRF were identified as: worsening anaemia (Hb 5 g/dl present in 74 per cent of CRF patients); proteinuria between 1.3 and 31 g/day (present in 68%); nephrotic syndrome (present in 40%); macroscopic haematuria (present in 36%); and, hypertension (present in 31%). The presence of CRF is associated with a significant increase in hospitalizations, acute chest syndrome, painful crises, multi-organ failure, stroke, leg ulcers, and death. Interestingly, the presence of CRF did not increase the frequency of episodes of acute renal failure or papillary necrosis. CRF should be managed actively with early dietetic referral to ensure adequate nutrition, treatment of hypocalcaemia, and phosphate retention to prevent the development of renal osteodystrophy, reversal of metabolic acidosis with oral bicarbonate, avoidance of nephrotoxic drugs and dose reduction of drugs which are excreted by the kidneys. Increased anaemia both predicts and complicates deterioration in renal function; and although most patients will respond to treatment with recombinant erythropoietin, some will require increasingly frequent blood transfusion.

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Acute renal failure Although not more common in patients with established sickle nephropathy, episodes of acute renal failure (ARF) may result in further loss of renal function. ARF is usually non-oliguric and is caused by volume depletion and sepsis in the context of an acute crisis, often exacerbated by drug nephrotoxicity and rhabdomyolysis secondary to intramuscular sickling. Upper renal tract obstruction by necrotic papillae or blood clots must always be excluded by ultrasound. Management involves re-salination, antibiotic therapy and possibly exchange blood transfusion if the percentage of circulating sickle cells remains high. Hypertension Blood pressure tends to be low in sickle cell disease and probably reflects chronic hypovolaemia secondary to tubular salt wasting, decreased peripheral pressure response to angiotensin II, and systolic cardiac dysfunction. Prospective data from 3317 patients with HbSS or HbSC aged 2 years or older enrolled in the CSSCD has confirmed lower blood pressures compared to age, sex, and race matched controls (Pegelow et al. 1997). The risk of occlusive stroke increased progressively with systolic blood pressure and the overall risk of death increased with both systolic and diastolic blood pressure, more markedly in males than females. Since blood pressure above the 90th centile in sickle cell disease patients overlapped with values considered normal, the finding of a high-normal blood pressure in a patient with sickle cell disease should provide cause for concern. However, there are as yet no data to support clinical outcome benefit from blood pressure lowering. Proteinuria Early glomerular functional changes in HbSS resemble those observed in diabetic nephropathy. Increased glomerular filtration rate, due to both haemodynamic changes and glomerular hypertrophy, and a greater increase in effective renal plasma flow leads to a reduced filtration fraction (Schmitt et al. 1998). Basement membrane permeability increases with increased fractional excretion of albumin and IgG (Guasch et al. 1997). These functional changes are mirrored by the development of massive glomerular hypertrophy, capillary loop dilatation, and progressive segmental sclerosis, probably in response to increased capillary hydrostatic pressure and protein traffic through the mesangium (see Fig. 18.2). Increased intrarenal synthesis of pGI2 and pGE2 may underlie the increased renal blood flow and glomerular filtration rate, particularly since these changes can be reversed by NSAID administration which also reduces natriuresis and improves urinary concentrating ability. There is, however, no evidence of therapeutic benefit from prolonged NSAID administration which is more likely to contribute to chronic tubulo-interstitial ischaemia when used frequently in the treatment of painful crises. There is experimental evidence from transgenic mice, but no clinical evidence, that increased renal nitric oxide synthesis contributes to renal vasodilatation. Microalbuminuria is present in 25 per cent of children aged 2–18 years with HbSS; its prevalence increases with age with some data to suggest that severity may correlate with blood pressure. Microalbuminuria is less prevalent in HbSC and HbSThal disease and affects less than 10 per cent of individual with HbAS who never develop CRF. The successful management of diabetic nephropathy with angiotensin converting enzyme (ACE) inhibition has prompted similar but smaller studies in patients with sickle nephropathy. These have shown consistent reduction in levels of proteinuria, possibly independent of blood pressure lowering,


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Occlusion of vasa recta Loss of concentrating ability

Natriuresis

Increased vasodilatory prostaglandins Increased blood flow Increased GFR Hyperfiltration-induced glomerulosclerosis Loss of GFR Progressive ischaemia and fibrosis End-stage renal failure Fig. 18.2 Proposed pathogenesis of progressive nephropathy in sickle cell disease.

sustained for periods of up to 6 months, which reversed when the drug was withdrawn (Falk et al. 1992; Aoki and Saad 1995). However, there are no data to show either improved renal survival or improved patient survival with blood pressure lowering. Nephrotic syndrome The appearance of perihilar focal and segmental glomerulosclerosis consistent with hyperfiltration injury is not the only lesion observed in sickle nephropathy. Several studies have reported acute nephrotic syndrome with biopsy evidence of mesangial proliferation, proliferative segmental glomerulonephritis, and mesangiocapillary glomerulonephritis (Tejani et al. 1985; Wierenga et al. 1995). The pathogenesis is unknown but may include undefined environment factors, including viral infection (such as Parvovirus B19 and Hepatitis C) and iron complexes, or familial factors. There is no established role for immunosuppression and management commonly involves ACE inhibition and blood pressure lowering. Tubulo-interstitial disease Red cell sickling in the vasa recta causes local acidosis and hypoxia, increased blood viscosity, and decreased medullary blood flow. Occlusion and obliteration of the microcirculation by microthrombi leads to defective counter current exchange and loss of the medullary concentration gradient. The clinical consequence is a loss of urinary concentrating ability, an obligate natriuresis and an increased risk of hyponatraemia (see Fig. 18.3). Concentrating defect The maximum urine concentration that patients can generate is around 414 10 mOsmol/kg which leads to an obligate urine output of 2–4 l/m2/day with nocturia, enuresis, and a reduced response to diuretics if indicated. Until an average age of 15 years, hyposthenuria may be reversed by blood transfusion. Although unable to produce concentrated urine, there is no defect in urinary dilution and patients can excrete a free water load without difficulty.

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Richard Sandford and Robin G. Woolfson Medullary hyperosmosis Red cell sickling ↑ Blood viscosity

Local acidosis Local hypoxia

Decreased medullary blood flow Microthrombi Vasa recta occlusion Papillary necrosis Haematuria

Defective countercurrent exchange Reduced concentrating ability Hyponatraenia

Fig. 18.3 Pathogenesis of tubular dysfunction and papillary necrosis in sickle cell disease.

Metabolic acidosis Proximal tubular generation of bicarbonate is normal but there is an incomplete distal renal tubular acidosis (reduced acid excretion) which means that urinary pH will not fall below 5.3 on acid loading. Hyperchloraemic metabolic acidosis is not a clinical problem except under conditions of severe stress, for example, septicaemia, when it should be actively corrected with intravenous sodium bicarbonate. Hyperkalaemia Patients are frequently hyperkalaemic which is occasionally due to hyporeninaemic hypoaldosteronism but usually reflects an unexplained redistribution of potassium from the intracellular compartments. Hyperkalaemia must be considered if treatment with ACE inhibitors or diuretics is indicated. Other tubular defects Proximal tubular function is supranormal with increased reabsorption of phosphate and 2-microglobulin. Increased creatinine secretion (together with hyperfiltration) makes creatinine clearance a greater than usual overestimate of glomerular filtration rate. Serum urate levels are normal as increased uric acid secretion compensates for increased urate production due to expanded erythropoiesis and increased erythrocyte turnover. There may be consequences for drug pharmacokinetics including renal handling of cimetidine and penicillin. Haematuria, papillary necrosis, and cancer The obliterative medullary vasculopathy can lead to papillary necrosis (see Fig. 18.4). This is best diagnosed by intravenous urography and affects 25 per cent of patients with HbSS. Ectatic collateral vessels are the source of both asymptomatic microscopic and episodic macroscopic haematuria which originate in the left kidney in 80 per cent of cases, due to its higher venous pressure. The management of persistent or severe macroscopic haematuria involves reduction of urinary tonicity and urinary alkalinization. In severe cases,


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Fig. 18.4 Radiograph demonstrating bilateral papillary necrosis in a patient with sickle cell disease.

ε-aminocaproic acid has been used to inhibit thrombolysis by endogenous urokinase, and rarely, embolization or nephrectomy may be required. Necrotic papillary debris can cause renal colic or acute upper renal tract obstruction. Management includes fluids, antibiotics, and ultrasound examination to exclude obstruction which should be decompressed if present. NSAIDs and radiocontrast contribute to medullary vasoconstriction and should be avoided if possible. Although papillary bleeding can occur in all types of sickle cell disease, other pathologies, including renal tuberculosis, nephrolithiasis, malignancies, and other unrelated pathologies, must be excluded. In a small number of cases, haematuria is due to an aggressive and rapidly fatal renal medullary carcinoma, originating in the medulla and metastasizing early, which has been reported mostly in patients with HbAS (Coogan et al. 1998). Infection Underlying urinary tract infection should be excluded in the patient presenting with acute sickle cell crisis. The incidence of asymptomatic bacteruria in women with HbSS and HbAS disease is twice normal and pregnant patients should be screened regularly and treated actively to prevent acute pyelonephritis. Renal replacement therapy As survival improves, an increasing proportion of HbSS patients will require renal replacement therapy. Most patients select haemodialysis but given their relative youth, outcome is

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disappointing with under 60 per cent survival after 36 months. This survival is comparable to that for older patients with end stage renal failure secondary to type II diabetes mellitus (Nissenson and Port 1990). Both cadaveric and live-related renal transplantation have been successfully undertaken in sickle patients with allograft survival similar to non-sickle Afro-Americans at 12 months but reduced by 36 months. However, patient survival was reduced at both 12 and 36 months compared to controls (Ojo et al. 1999), which reflects an increased frequency of acute crises as the haematocrit improves. In the longer term sickle nephropathy may recur. Conclusion The last 40 years have seen dramatic progress in the management of acute sickle cell crises but as a result organ failure, in particular renal failure, is becoming more prevalent. Although the risk factors for the development of renal failure are well recognized, the optimum management of the sickle patient with microalbuminuria, proteinuria, or renal impairment is not established. Haemophilia Haemophilia is due to deficiency of Factor VIII (type A) or Factor IX (type B, Christmas disease). The phenotypes are identical identical although haemophilia A is about six times more common with an incidence of 20–130 per million, occurring in all races and parts of the world. Although both types are sex-linked recessives with the gene located on the X chromosome, almost half of cases are sporadic. Haemophilia affects the generation of thrombin via the intrinsic pathway and this leads to the delayed formation of friable clots which fail to achieve haemostasis. The frequency and severity of bleeding depends on the residual amount of Factor VIII or IX generated. Bleeding can be severe and spontaneous when factor concentration is less than 1 per cent of normal but occurs only after severe trauma or major surgery when factor levels are 5–20 per cent of normal. Common sites for spontaneous bleeds include joints, deep muscles, bowel, renal tracts, and brain. Treatment is by intravenous infusion of adequate doses of the missing factor derived from plasma concentrates or recombinant techniques, anti-fibrinolytic therapy, bed rest, and analgesia for severe pain. Optimum management depends on residual factor levels and includes prophylactic factor replacement as well as rapid treatment when bleeding occurs. The aim is to avoid the crippling consequences of musculoskeletal bleeding which include muscle necrosis, contractures, and progressive destructive arthropathy. Although plasma products are now heat-treated and high risk donors excluded, their earlier use was accompanied by an increased incidence of serious viral infection. These include HIV and hepatitis C with renal complications associated with both the infection and its treatment. Renal and urological complications Haematuria is a common urological complication of both haemophilia A and B. Studies have shown that about one third of haemophiliacs demonstrate abnormalities on intravenous urography, with correlation between severity of haematuria and underlying pathology (Dholakia et al. 1979). Observed abnormalities include blood clots within the collecting systems, renal papillary necrosis, hydronephrosis, and non-functioning kidneys. Neither renal papillary necrosis


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which may progress over time nor haematuria are associated with progressive renal insufficiency (Small et al. 1982; Roberts et al. 1983). Upper tract obstruction may be due to intra-luminal obstruction by clot or sloughed papilla or due to extrinsic upper tract compression following retroperitoneal haemorrhage and subsequent peri-ureteric fibrosis (Roberts et al. 1983). Other nephro-urological disorders associated with haemophilia include analgesic nephropathy, chronic pyelonephritis, and spontaneous bleeding into the bladder wall. Of course, haematuria may be due to unrelated urinary tract disorders, such as nephrolithiasis, tumours, and nephritis, which must be excluded. Although extra-corporeal shock wave lithotripsy has been used to treat nephrolithiasis in haemophilia, the high incidence of intra- and peri-renal bleeding in normal individuals relatively contra-indicate its use in haemophiliacs. Finally, there is an increased prevalence of hypertension in haemophilia which presumably reflects underlying parenchymal disease described above and may contribute to the increased incidence of stroke. Acute intermittent porphyria Enzymatic defects in the synthesis of haem, subsequent to the first step by which aminolaevulinic acid (ALA) is synthesized from glycine and succinyl CoA, give rise to the porphyrias. Failure of negative feedback results in elevated levels of ALA and subsequent precursors until the level of the defective enzyme, and their accumulation is directly responsible for tissue injury. The pathogenesis remains unclear but may include oxidative stress and direct damage to neuronal bodies, perhaps resulting from reduced haem availability for essential intracellular cytochrome-containing enzymes. The enzyme defects responsible for porphyria are inherited and the diagnosis depends on the specific pattern of precursor accumulation. The porphyrias can be categorized into acute or non-acute on the basis of their clinical presentation. Of the acute forms, acute intermittent porphyria (AIP) is both the commonest and also the most severe. Reduced activity of hydroxymethylbilane synthase results in the accumulation of porphobilinogen and to a lesser extent, ALA, and during an acute attack urinary levels of these are raised in the absence of fecal porphyrins. These substances characteristically turn the urine dark red or brown on exposure to light and laboratory samples must therefore be shielded in transit to the laboratory. The mutation on chromosome 11 responsible for AIP is inherited as an autosomal dominant and over 100 different family-specific mutations have been identified; mutations also occur sporadically. AIP is of low penetration and is not expressed clinically in the vast majority of affected individuals but if symptoms do present, this usually occurs in the fourth or fifth decade, and is much commoner in women. First attacks before puberty or in the sixth decade are very rare. Attacks of AIP may be precipitated by many commonly prescribed drugs (including antibiotics, diuretics, antihypertensives, anti-epileptics, psychotropics, and hypoglycaemics), menstruation, pregnancy, infection, reduced calorie intake, stress, and alcohol, although it is common for no precipitant to be identified. Clinical presentation Gastrointestinal symptoms with pain, vomiting, constipation, or diarrhoea, are prominent affecting 95 per cent of individuals and may precipitate unnecessary surgery. Neuromuscular symptoms include cramps and muscular weakness, respiratory failure, sensori-motor neuropathy, cranial nerve palsies, and upper motor neurone lesions. Seizures, depression, anxiety, and psychosis

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may occur in the context of the acute attack. Hyponatraemia, secondary to inappropriate ADH secretion (SIADH), can occur during acute episodes and may provoke seizures. Sinus tachycardia and hypertension, sometimes severe enough to cause left ventricular failure and papilloedema, might occur as can symptomatic hypotension. Renal sequelae Fluid depletion during an acute attack can lead to acute renal failure and recovery may be incomplete with persistently elevated plasma creatinine and proteinuria. A significant proportion of AIP patients develop chronic renal failure, probably due to a combination of hypertension which is present in up to 70 per cent of patients (Andersson and Lithner 1994), analgesic nephropathy and direct nephrotoxicity of accumulated porphyrin precursors (Laiwah et al. 1983). In one series, uraemia was the cause of death in 9.1 per cent of patients (Andersson and Lithner 1994). Management Acute episodes require hospital admission to identify and remove the precipitant factor and provide supportive care until resolution occurs. Underlying infection must be diagnosed and treated appropriately. Porphyrogenic drugs must be avoided but opiates can be given for analgesia, certain phenothiazines for acute psychiatric disturbance, benzodiazepines and sodium valproate for seizures, and propanolol for the treatment of tachycardia and hypertension. If respiratory muscles are affected, then vital capacity must be monitored and mechanical ventilation may be required. Fluid balance and electrolytes must be carefully monitored; hyponatraemia due to SIADH should be managed by water restriction. Both glucose and haem arginate are used to inhibit ALA synthase which reduces flux through the synthetic pathway. In mild attacks, oral or intravenous glucose (10% solution) to maintain a daily intake of 1500–2000 kcal may be adequate but in more severe episodes, slow infusion of intravenous haemarginate over 15–30 min at a dose of 3 mg/kg/day for 4 days is commonly given, and this has been used in pregnancy. Outcome Data from the USA indicates that there is a three-fold increase in mortality in patients with symptomatic AIP compared to the general population, although the availability of haemarginate therapy since 1971 has improved survival. Most deaths occur during acute episodes and 1 per cent of these may be fatal; sadly, suicide is another important cause of premature death (Jeans et al. 1996). Biochemical and genetic screening of relatives of patients with AIP provides an opportunity to identify those with latent disease as well as unaffected members. Given the potentially serious neuro-psychiatric complications of acute attacks and the significant restrictions required to prevent them, family screening is of clear benefit to all. Nail-patella syndrome (hereditary osteo-onychodysplasia) Introduction Nail-patella syndrome (NPS) or hereditary osteo-onychodysplasia is a rare skeletal malformation syndrome condition affecting 1 : 50,000 of the population, which is characterized


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by abnormalities of the knees, elbows, pelvis, and nails. Other more variable features include a nephropathy that presents with proteinuria and may occasionally progress to end-stage renal failure (ESRF). This represents the most severe complication of the disease. More recently glaucoma and other eye signs have been suggested as part of the clinical spectrum. It is an autosomal dominant disorder that is caused by mutations in the LIM-homeodomain transcription factor gene, LMX1B (Chen et al. 1998; Dreyer et al. 1998). Mice homozygous for a targeted LMX1B mutation also demonstrate an NPS-like phenotype. This has shed considerable light on the pathogenesis of the disorder, which seems to result from abnormal developmental dorso-ventral patterning and a direct effect on the regulation of expression of type IV collagen (Morello et al. 2001). Clinical features NPS is characterized by four distinct features (Table 18.2). Dysplasia of the nails and absent or hypoplastic patellae are the cardinal features with radial head dislocation and iliac horns being the other most consistent features of this condition. All these features are usually bilateral and symmetrical and may be apparent from birth (Meyrier et al. 1990; Guidera et al. 1991). Nails are hypoplastic or absent from birth in almost all individuals with NPS. The ulnar border of the nails of the thumb and index fingers are typically affected (Fig. 18.5). Abnormalities in the knees are seen in over 90 per cent of cases. This mainly comprises patella aplasia or hypoplasia but may also include dysplasia of the femoral condyles and tibial epiphysis. The most frequent problems associated with these abnormalities are joint pains and recurrent dislocation of the patella with resulting joint instability. Premature degenerative changes may also occur. Elbow dysplasia is also common affecting 80–94 per cent of cases. A small radial head may predispose to dislocation and other associated minor abnormalities can cause loss of movement at the elbow with associated disability. Iliac horns, present in about 80 per cent of cases, are pathognomonic of the disease and can be detected by pelvic X-ray (Fig. 18.6). Other skeletal features in NPS families have been described including foot deformities and scoliosis. Despite the typical features of the condition often being present at birth they may be unrecognized for many years only being observed when the diagnosis is made in another family member. The renal manifestations of NPS are also very variable even within the same family. Approximately 25 per cent of individuals with NPS have the typical nephropathy. Proteinuria is the commonest manifestation occasionally being severe enough to cause nephrotic syndrome. Up to a third of individuals with renal involvement may progress to renal failure but it is difficult to predict who will develop this complication and at what rate. Using a single large NPS family with a history of nephropathy and 34 other published families with nephropathy, Looji et al. (1988) calculated the risks of having a child with nephropathy for any affected family member as being about 1:4 and the risk of having a child who subsequently develops renal failure as being 1:10. Further data is required to confirm these figures. Recent reports have identified open-angle glaucoma as a feature of NPS in up to 30 per cent of cases (Lichter et al. 1997). This is supported by the Table 18.2 Characteristic features of NPS Absent or dysplastic nails Absent or dysplastic patellae Elbow dysplasia Iliac horns

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Fig. 18.5 Hypoplastic thumb nails in a patient with NPS showing the typical distribution over the ulnar border of the nail. In this case the index finger seen is normal. (Picture courtesy of Dr J. Rankin.)

Fig. 18.6 A plain pelvic radiograph demonstrating the bilateral osseous horns arising from the posterior iliac wings that are pathognomonic of NPS. (Picture courtesy of Professor A. Green.)


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finding of anterior segment ocular abnormalities in mice with targeted disruption of the LMX1B gene (McIntosh et al. 1998). The role of screening for this condition remains to be evaluated. Diagnosis and management A diagnosis of NPS is made by identifying the typical clinical features of the disease. This is simply done by clinical examination and radiology. Iliac horns are pathognomonic for the disease. Orthopaedic review may be necessary for some individuals. Renal disease should be sought by regular urinalysis and if present suggests the need for continued follow-up. The role of renal biopsy is uncertain. The presence of glomerular sclerosis and other non-specific changes by light microscopy predicts the development of renal failure but the typical glomerular EM changes of collagen fibres within the GBM may be present in individuals with no obvious renal disease. No reliable clinical screening test for the prenatal diagnosis of NPS exists. If a pathogenic mutation in the LMX1B gene has been discovered or linkage studies in a family confirm linkage to the NPS locus, prenatal testing can be offered but the often mild clinical disease and lack of correlation between the LMX1B mutation and the presence of kidney disease, or overall NPS severity makes genetic counselling difficult. Molecular genetics Linkage of NPS to the ABO blood group locus was described in 1955. Mapping of these and other genes to the long arm of chromosome 9 lead to the identification of several candidate genes for NPS. The NPS gene was finally identified because of similarities of a mouse mutation in the LIM-homeodomain transcription factor gene, LMX1B, to the human disease. Lmx1b plays a central role in dorsal/ventral patterning of the vertebrate limb. Targeted disruption of Lmx1b results in skeletal defects, including hypoplastic nails, absent patellae, and a unique form of renal dysplasia (Chen et al. 1998). Dreyer et al. (1998) showed that the LMX1B gene maps to 9q in the same region as the NPS locus by fluorescence in situ hybridization (Dreyer et al. 1998). Furthermore, they demonstrated that three unrelated NPS patients carried de novo heterozygous mutations in this gene. Functional studies showed that one of these mutations disrupted sequence-specific DNA binding, while the other two mutations resulted in premature termination of translation. However no clear genotype–phenotype correlations have been identified (McIntosh et al. 1998). Mice homozygous for a targeted LMX1B mutation demonstrate an NPS-like phenotype and suggests that the disease results from abnormal developmental dorsoventral patterning. In addition, LMX1B has a direct effect on the regulation of collagen expression (Morello et al. 2001). It directly regulates the co-ordinated expression of alpha3(IV) and alpha4(IV) collagens that are required for normal GBM development providing a direct link between pathogenic mutations and abnormal collagen expression in NPS kidneys. Alpha-1-antitrypsin deficiency Introduction Whilst great progress has been made in the understanding of the pathogenesis of the characteristic features of this disorder, emphysematous lung disease and cirrhosis, the aetiology of the accompanying renal disease remains largely unknown. Alpha-1-antitrypsin is a 52 kD acute phase protein that is a member of the serine protease inhibitor (serpin) family. Its

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function is to protect tissues against the actions of neutrophil elastase. Alpha-1-antitrypsin deficiency, an autosomal recessive disease resulting from point mutations in the PI gene located on the distal long arm of chromosome 14, is associated with proteolytic damage to the lungs due to the lack of enzyme activity. It is a conformational abnormality in the mutant protein that leads to cirrhosis due to the accumulation of protein polymers in the endoplasmic reticulum of hepatocytes (Lomas et al. 1992). Alpha-1-antitrypsin deficiency and diseases due to mutations in other members of the serpin protein family are part of a growing list of disorders that result from abnormal protein folding. These ‘conformational diseases’ include Huntington disease, Alzheimers disease, and Creutzfeld–Jacob disease. Clinical features The main clinical consequences of alpha-1-antitrypsin deficiency are lung and liver disease. Early onset panlobular emphysema, which is severely exacerbated by smoking, asthma, bronchiectasis, and vasculitis are seen. Cirrhosis also occurs and is often present in childhood before the development of lung disease. However, only 12–15 per cent of individuals with the commonest form of alpha-1-antitrypsin deficiency will develop liver disease. Renal disease is mainly associated with severe liver disease in children and is usually due to mesangiocapillary glomerulonephritis type 1. In adults, an ANCA positive crescentic nephritis is more usual. Diagnosis and management Alpha-1-antitrypsin can be readily diagnosed biochemically and by direct mutation analysis. Activity of 10 per cent of normal is associated with disease and is seen in individuals who are homozygous for the Z variant (342Glu-Lys) of alpha-1-antitrypsin. The lung, liver, and renal disease are managed along conventional lines and organ transplantation may eventually be necessary. Stopping smoking reduces the severity of the lung disease and is the only proven preventative intervention. The role of direct enzyme replacement by intravenous infusion is currently being evaluated. Molecular genetics Alpha-1-antitrypsin deficiency is an autosomal recessive disorder affecting 1:2500 of the Caucasian population. Many naturally occurring variants exist within diverse populations but two are commonly associated with deficiency states, S and Z alpha-1-antitrypsin. The S allele (264Glu-Val) results in 60 per cent of the activity of the normal M allele and is not associated with lung disease. Being homozygous for the Z allele results in levels 10 per cent of normal and leads to lung and liver disease. The liver disease in individuals with the ZZ genotype results from the toxic effects of the abnormal protein accumulating within the endoplasmic reticulum of liver cells. This occurs because the Z mutation in alpha-1-antitrypsin results in a unique molecular interaction between two molecules, loop-sheet polymerization (Lomas et al. 1992). Wilson disease Introduction Wilson disease or hepatolenticular degeneration is an autosomal recessive disorder caused by mutations in the ATP7B gene. Its familial nature and cardinal features were first described by Kinnear Wilson in 1912. It is characterized by decreased biliary copper excretion and reduced copper incorporation into apoceruloplasmin. Total body copper is therefore dramatically elevated


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with excessive intracellular copper deposits occurring in several organ systems. The common presenting features are hepatic and neurological disease. Renal disease is rarely if ever the presenting feature. The prognosis of Wilson disease is excellent providing treatment is started early enough. Clinical features Symptoms of Wilson disease usually occur between the ages of 5 and 30. Liver disease occurs mainly in childhood whilst neurological disease occurs towards early adult life but may present as late as the third and fourth decades. These manifestations may occur alone or together. Excessive deposits of copper can be readily seen in most affected individuals at the corneal margins (Kayser–Fleischer rings) as a greenish-brown ring (Fig. 18.7). This finding is rarely seen in other forms of liver disease making it an important diagnostic sign. The liver disease may present as hepatosplenomegaly, subacute or chronic hepatitis, acute liver failure, or cirrhosis. Typical clinical features especially recurrent acute hepatitis with haemolysis or chronic liver disease of unknown cause in young patients should also suggest the diagnosis. Neurological features may be very variable. The classical description is lenticular degeneration producing a syndrome comprising of extrapyramidal features such spacticity, rigidity, and dysarthria. Tremor, athetosis, dementia, and psychiatric disturbance may also occur. Renal disease is rarely the presenting problem in Wilson disease but is a frequent finding on laboratory testing. Hypercalciuria and nephrocalcinosis are not uncommon. It is a cause of Fanconi syndrome (see Chapter 12). This is due to impaired tubular function due to copper deposition. It also responds to penicillamine therapy. The presentation of Fanconi syndrome varies with age. It is typically of later onset than in Fanconi syndrome due to other inborn errors of metabolism occurring during the second decade. In children failure to thrive, anorexia, vomiting, polyuria, polydipsia rickets, and metabolic acidosis may occur.

Fig. 18.7 A Kayser–Fleischer ring seen in the eye of a patient with Wilson disease. The sclera is also jaundiced. (See Plate 13 of the Colour Plate Section at the centre of this book.) (Picture courtesy of Dr. A.T. Moore.)

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Diagnosis and management The diagnosis may be made on clinical or biochemical findings. The most important clinical sign is the presence of Kayser–Fleischer rings which should be sought by slit-lamp examination. A low serum cauruloplasmin is highly suggestive of the diagnosis although it may be reduced in other cases of liver failure and may also be normal in 5–10 per cent of patients with Wilson disease. A low serum copper, high urine copper excretion and very high hepatic copper content all suggest the diagnosis. Liver transplantation may be necessary for acute hepatic failure and cirrhosis but Wilson disease can be effectively treated by any 1 of 3 drugs, D-penicillamine, trien, or zinc acetate. Molecular genetics The Wilson disease gene, ATP7B, at 13q14.3 encodes a copper-transporting P-type ATPase. Tissue-specific alternative splicing of exons 6–8, 12–13, and 22 of the 22 exon gene occurs with all 22 exons being expressed only in the kidney. Expression occurs in the liver, brain, kidney, and placenta. A very heterogeneous array of allelic variants and mutations in ATP7B have been described some of which are population specific. H1069Q is the commonest mutation present in 17–61 per cent of all Wilson disease chromosomes and is located in the highly conserved SEHPL sequence of the ATP binding domain encoded by exon 14 (Shah et al. 1997). Over half of other mutations occur only rarely in any population. In one study of British, Wilson disease patients 60 per cent of mutations were found in exons 8, 14, and 18 (Curtis et al. 1999). No clear genotype–phenotype correlations have been described. Routine mutation testing is not widely available and at risk siblings may be offered linkage-based screening. Mitochondrial disorders Mutations in the maternally inherited mitochondrial genome produce a wide variety of rare clinical syndromes that may present in childhood and adulthood and affected a variety of organ systems including the kidney. Mitochondrial DNA encodes ribosomal and transfer RNAs and 13 subunits of the complex respiratory chain metabolic pathway. Because it is transmitted in a random manner during cell division a cell may contain a mix of mutant and normal mitochondrial DNA (heteroplasmy). A threshold amount of mutant DNA is required for disease expression and this varies from tissue to tissue according to its oxidative metabolic rate. Thus mitochondrial disease is frequently seen in muscle and the central nervous system. Typical features of mitochondrial syndromes include encephalopathy, blindness, deafness, muscular weakness, and diabetes mellitus. Syndromes include myoclonic epilepsy, ragged red fibres (MERRF), mitochondrial encephalopathy, lactic acidosis, stroke-like episodes (MELAS), Kearns–Sayre syndrome (retinopathy, myopathy, external ophthalmoplegia, cardiac conduction defects), Leigh syndrome (subacute necrotizing encephalomyelopathy) and diabetes insipidus, diabetes mellitus, optic atrophy, and deafness (DIDMOAD) syndrome. The associated renal disease is rarely the presenting feature and often remains undiagnosed. It is typically due to abnormalities in tubular function and may present as a Fanconi syndrome (see Chapter 12). Other tubular abnormalities include renal tubular acidosis and isolated aminoaciduria. An association with glomerular disease has also been suggested. Renal disease in suspected mitochrondrial disorders should be actively sought. In addition to an assessment of GFR, tubular function should also be determined.


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References Andersson, C. and Lithner, F. (1994). Hypertension and renal disease in patients with acute intermittent porphyria. Journal of Internal Medicine, 236, 169–75. Aoki, R. Y. and Saad, S. T. (1995). Enalapril reduces the albuminuria of patients with sickle cell disease. American Journal of Medicine, 98, 432–5. Chen, H., Lun, Y., Ovchinnikov, D., Kokubo, H., Oberg, K. C., Pepicelli, C. V., et al. (1998). Limb and kidney defects in Lmx1b mutant mice suggest an involvement of LMX1B in human nail patella syndrome. Nature Genetics, 19(1), 51–5. Coogan, C. L., Mckiel, C. F., Flangan, M. J., Bormes, T. P., and Makkopv, T. G. (1998). Renal modullary carcinoma in patients with sickle cell trait. Urology, 51(6), 1049–50. Curtis, D., Durkie, M., Balac (Morris), P., Sheard, D., Goodeve, A., Peake, I., et al. (1999). A study of Wilson disease mutations in Britain. Human Mutations 14(4), 304–11. Dholakia, A. M. and Howarth, F. H. (1979). The urinary tract in haemophilia. Clinical Radiology, 30, 533–8. Dreyer, S. D., Zhou, G., Baldini, A., Winterpacht, A., Zabel, B., Cole, W., et al. (1998). Mutations in LMX1B cause abnormal skeletal patterning and renal dysplasia in nail patella syndrome. Nature Genetics, 19(1), 47–50. Falk, R. J., Scheinman, J., Phillips, G., Orringer, E., Johnson, A., and Jennette, J. C. (1992). Prevalence and pathologic features of sickle cell nephropathy and response to inhibition of angiotensin-converting enzyme. New England Journal of Medicine, 326, 910–15. Guasch, A., Cua, M., You, W., and Mitch, W. E. (1997). Sickle cell anemia causes a distinct pattern of glomerular dysfunction. Kidney International, 51, 826–33. Guasch, A., Zayas, C. F., Eckman, J. R., Muralidharan, K., Zhang, W., and Elsas, L. J. (1999). Evidence that microdeletions in the alpha globin gene protect against the development of sickle cell glomerulopathy in humans. Journal of the American Society of Nephrology, 10, 1014–19. Guidera, K. J., Satterwhite, Y., Ogden, J. A., Pugh, L., and Ganey, T. (1991). Nail patella syndrome: a review of 44 orthopaedic patients. Journal of Pediatric Orthopedics, 11(6), 737–42. Jeans, J. B., Savik, K., Gross, C. R., Weimer, M. K., Bossenmaier, I. C., Pierach, C. A., et al. (1996). Mortality in patients with acute intermittent porphyria requiring hospitalization: a United States case series. American Journal of Medical Genetics, 65, 269–73. Laiwah, A. A., Mactier, R., McColl, K. E., Moore, M. R., and Goldberg, A. (1983). Early-onset chronic renal failure as a complication of acute intermittent porphyria. Quarterly Journal of Medicine, 52, 92–8. Lichter, P. R. Richards, J. E., Downs, C. A., Stringham, H. M., Boehnke, M., and Farley, F. A. (1997). Cosegregation of open-angle glaucoma and the nail-patella syndrome. American Journal of Ophthalmology, 124(4), 506–15. Lomas, D. A., Evans, D. L., Finch, J. T., and Carrell, R. W. (1992). The mechanism of Z alpha 1-antitrypsin accumulation in the liver. Nature, 357(6379), 605–7. Looij, B. J., Jr., te Slaa, R. L., Hogewind, B. L., and van de Kamp, J. J. (1988). Genetic counselling in hereditary osteo-onychodysplasia (HOOD, nail-patella syndrome) with nephropathy. Journal of Medical Genetics, 25(10), 682–6. McIntosh, I., Dreyer, S. D., Clough, M. V., Dunston, J. A., Eyaid, W., Roig, C. M., et al. (1998). Mutation analysis of LMX1B gene in nail-patella syndrome patients. American Journal of Human Genetics, 63(6), 1651–8. Meyrier, A., Rizzo, R., and Gubler, M. C. (1990). The nail-patella syndrome. A review. Journal of Nephrology, 2, 133–40. Morello, R., Zhou, G., Dreyer, S. D., Harvey, S. J., Ninomiya, Y., Thorner, P. S., et al. (2001). Regulation of glomerular basement membrane collagen expression by LMX1B contributes to renal disease in nail patella syndrome. Nature Genetics, 27, 205–8. Nissenson, A. R. and Port, F. K. (1990). Outcome of end-stage renal disease in patients with rare causes of renal failure. Quarterly Journal of Medicine, 74, 63–74.

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Ojo, A. O., Govaerts, T. C., Schmouder, R. L., Leichtman, A. B., Leavey, S. F., Wolfe, R. A., et al. (1999). Renal transplantation in end-stage sickle cell nephropathy. Transplantation, 67, 291–5. Pegelow, C. H., Colangelo, L., Steinberg, M., Wright, E. C., Smith, J., Phillips, G., et al. (1997). Natural history of blood pressure in sickle cell disease: risks for stroke and death associated with relative hypertension in sickle cell anemia. American Journal of Medicine, 102, 171–7. Platt, O. S., Brambilla, D. J., Rosse, W. F., Milner, P. F., Castro, O., Steinberg, M. H., et al. (1994). Mortality in sickle cell disease. Life expectancy and risk factors for early death. New England Journal of Medicine, 330, 1639–44. Powars, D. R., Elliott-Mills, D. D., Chan, L., Niland, J., Hiti, A. L., Opas, L. M., et al. (1991). Chronic renal failure in sickle cell disease: risk factors, clinical course, and mortality. Annals of Internal Medicine, 115, 614–20. Roberts, G. M., Evans, K. T., Bloom, A. L., and Al-Gailani, F. (1983). Renal papillary necrosis in haemophilia and christmas disease. Clinical Radiology, 34, 201–6. Schmitt, F., Martinez, F., Brillet, G., Giatras, I., Choukroun, G., Girot, R., et al. (1998). Early glomerular dysfunction in patients with sickle cell anemia. American Journal of Kidney Diseases, 32, 208–14. Shah, A. B., Chernov, I., Zhang, H. T., Ross, B. M., Das, K., Lutsenko, S., et al. (1997). Identification and analysis of mutations in the Wilson disease gene (ATP7B): population frequencies, genotypephenotype correlation, and functional analyses. American Journal of Human Genetics, 61(2), 317–28. Small, S., Rose, P. E., McMillan, N., Belch, J. J., Rolfe, E. B., Forbes, C. D., et al. (1982). Haemophilia and the kidney: assessment after 11-year follow-up. British Journal of Medicine (Clinical Research), 285, 1609–11. Tejani, A., Phadke, K., Adamson, O., Nicastri, A., Chen, C. K., and Sen, D. (1985). Renal lesions in sickle cell nephropathy in children. Nephron, 39, 352–5. Wierenga, K. J., Pattison, J. R., Brink, N., Griffiths, M., Miller, M., Shah, D. J., et al. (1995). Glomerulonephritis after human parvovirus infection in homozygous sickle-cell disease. Lancet, 346, 475–6.

Useful WWW sites for various diseases Sickle cell disease http://sickle.bwh.harvard.edu/ http://www.emory.edu/PEDS/SICKLE/ http://www.sicklecellsociety.org/ Thalassaemia http://sickle.bwh.harvard.edu/ http://www.geocities.com/HotSprings/8730/ Haemophilia http://www.wfh.org/ http://www.infonhf.org/ Acute intermittent porphyria http://members.tripod.com/~theaipforum/ http://www.enterprise.net/apf/

19 The genetics of glomerulonephritis and systemic disorders affecting the kidney Stephen H. Powis

Introduction The last decade has brought a revolution in our understanding of the genetic basis of inherited diseases. This has arisen from major advances in the technology of DNA analysis and in the statistical approaches used to analyse family-based genetic studies. The genes responsible for many monogenic disorders have been identified and significant progress has been made in elucidating genes important in the pathogenesis of some of the common polygenic disorders, such as diabetes mellitus and psoriasis (Trembath et al. 1997; Cox et al. 2001). In renal disease, the most striking advances have also occurred in monogenic disorders, as documented in other chapters of this book. In contrast, this chapter primarily focuses on a number of disorders that almost certainly have a multi-factorial aetiology, dependent upon complex interactions between genes and the environment. These are the primary glomerulonephritides and a variety of systemic disorders affecting the kidney. Here, significantly less progress has made in understanding their genetic basis. Nevertheless, these polygenic disorders constitute the majority of clinically apparent renal disease and most commonly lead to end stage renal failure (ESRF). Evidence for genetic predisposition to renal disease is derived from many sources, including studies of familial clustering and racial predisposition. Animal models have also been particularly useful, but this chapter will concentrate on human studies. The genetic contribution to common renal disease was recently illustrated in a survey of patients with ESRF (excluding established monogenic diseases such as polycystic kidney disease and Alport syndrome) in which 351 out of 4289 patients reported a sibling with ESRF (Freedman et al. 1997). Assuming an average sibship of 2.5, this gave a sibling risk of 3 per cent compared with a 0.2 per cent population prevalence of ESRF. Risk of disease in a sibling of a patient/risk in the general population (s) was estimated to be 15, which is similar to type 1 diabetes mellitus. A variety of factors have hindered our ability to study polygenic renal disorders by the methodologies that have been successfully applied to monogenic disorders. The use of linkage analysis to identify disease genes is inherently more difficult in polygenic disorders as it requires multiply affected kindred, ideally with uniform phenotype and little pathogenetic heterogeneity. Furthermore, it is difficult to identify genes that confer low relative risk. Many renal diseases, such as the glomerulonephritides, are uncommon compared to other polygenic disorders such as diabetes or rheumatoid arthritis. It is relatively unusual to find reports of families with more than one affected individual, although there is clear evidence in disorders such as IgA nephropathy that siblings are frequently affected. Some disorders, such as membranous

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Stephen H. Powis

nephropathy and ANCA-positive vasculitis, develop in later life when it is less likely that the parents of affected individuals will be alive. Taken together, this results in difficulties in ascertaining sufficiently sized populations for linkage analysis. A further problem is that of disease heterogeneity. Whereas it is probable that type I diabetes has a common aetiology, this is much less likely for a disease such as membranous nephropathy, which is already known to develop in response to a variety of external factors such as infections, drugs, and tumours. Thus the genetic literature on disorders discussed in this chapter is predominantly based on case-control association studies. Compared to linkage analysis, these studies have limitations (Cardon and Bell 2001). They only target specific, previously identified candidate genes. They often involve small numbers, are inadequately powered, and are subject to bias through population stratification. There are often no studies of intermediate phenotype to support the study of individual polymorphisms and disease. As a result, reports sometimes yield inconsistent associations. Nevertheless, studies can be informative, especially when their findings are reproduced in independent populations. Furthermore, they can identify weakly associated genes that may not be detected by linkage analysis. A variety of candidate genes have been analysed in renal polygenic disorders, but the most common are those of the major histocompatibility complex (MHC) and the renin–angiotensin system. The human major histocompatibility complex The human MHC (HLA region) encompasses approximately 4 million base pairs on the short arm of chromosome 6 at cytogenetic location 6p21.3. The region is subdivided into three subregions. The telomeric class I region contains the genes which encode the HLA class I molecules HLA-A, -B, and -C. The centromeric class II region contains the genes encoding the HLA class II molecules HLA-DR, -DQ, and -DP. In between lies the class III region, originally identified because it contains genes encoding components of the complement pathway. The entire human MHC has recently been sequenced and each sub-region is now known to contain many other genes, a number of which have immunological functions (MHC Sequencing Consortium 1999). Both class I and class II HLA molecules present antigens to T cells through the T cell receptor, but there are fundamental differences in their function. Class I molecules primarily present endogenously derived intracellular antigens to CD8 positive cytotoxic T cells, whereas class II molecules present extra-cellular antigens to CD4 positive helper T cells. HLA molecules are highly polymorphic, a process that appears to have been driven by the evolutionary response to infectious agents (Hill et al. 1991; Powis and Geraghty 1995). A large number of studies have described associations between HLA alleles and human diseases, many of which are believed to have an immunological component (such as rheumatoid arthritis, diabetes mellitus, and multiple sclerosis). This enormous literature was initially collated by Tiwari and Terasaki (1985) and has continued to grow. However, the mechanisms underlying these associations have been harder to elucidate. Because the strongest associations are with genes encoding the class I and II HLA molecules, it seems reasonable to predict that these are directly involved in disease pathogenesis. This may result from a variety of mechanisms, extensively reviewed in Warrens and Lechler (2000), including variation in the ability of different HLA alleles to bind auto-antigens (determinant selection), differences in the T cell receptor repertoire generated by different HLA alleles and HLA-driven T cell-mediated suppression. Unfortunately, very few human auto-antigens have been identified, but one notable exception is anti-glomerular basement membrane disease, a cause of acute renal

The genetics of glomerulonephritis

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failure that is discussed later in this chapter. Because of strong linkage disequilibrium within the HLA region, it is also possible that in some diseases the true susceptibility genes are not the genes encoding the HLA molecules, but other nearby genes. Some of these, such as the TAP and LMP genes, are obvious candidate susceptibility genes for immunological disorders because of their role in the normal immune response (Powis et al. 1993). The renin–angiotensin system (RAS) The renin–angiotensin system has been strongly implicated in the pathogenesis of hypertension and cardiovascular system. Angiotensinogen (AGT) is converted by renin to angiotensinI, which is further modified by angiotensin-converting enzyme (ACE) to angiotensin II. Angiotensin II is the main effector peptide of the RAS and exerts a number of powerful effects through binding to the angiotensin II type 1 and 2 receptors (AT1R and AT2R). It is a powerful vasoconstrictor and a potent mediator of cellular proliferation and extra-cellular matrix protein synthesis. As a result, there has been much interest in the potential role of RAS polymorphisms in renal disease. Association studies have predominantly examined an insertion/deletion (I/D) polymorphism in intron 16 of ACE, the M235T polymorphism of AGT and the A1166 to C polymorphism within AT1R (Schmidt and Ritz 1997; Duncan et al. 2001). Glomerulonephritis Idiopathic nephrotic syndrome, minimal change disease (MCD), and focal segmental glomerulosclerosis Childhood nephrotic syndrome is most commonly caused by MCD and less commonly by a variety of other disorders including focal segmental glomerulosclerosis (FSGS). The clinical diagnosis of MCD is confirmed by normal renal biopsy appearances on light microscopy, but in routine clinical practice the majority of children with steroid responsive syndrome have MCD, so renal biopsy is not usually performed. Thus childhood nephrotic syndrome is also classified into steroid responsive or steroid resistant. However, this may introduce confounding factors into the interpretation of genetic studies that rely upon accurate and consistent phenotypic description. Two early large family studies of childhood nephrotic syndrome reported a sibling recurrence risk of 3.3 and 6 per cent, respectively (White 1973; Bader et al. 1974). These figures may have been inflated by the inclusion of cases of congenital nephrotic syndrome, but the majority of familial cases conformed, clinically and histologically, to the usual spectrum of idiopathic nephrotic syndrome. The reported population prevalence for these diseases is again very much lower, suggesting a s value of between 20 and 40 for childhood nephrotic syndrome. The majority of association studies in MCD have analysed HLA alleles in children with steroid sensitive nephrotic syndrome. Early studies showed that in Caucasian populations susceptibility was associated with the class I antigens HLA-B12 and -B8 (Lenhard et al. 1980; Trompeter et al. 1980), but this is due to linkage disequilibrium with the strongly associated class II region allele HLA-DR7 (de Mouzon-Cambon et al. 1981; Nunez-Roldan et al. 1982; Ruder et al. 1990) and the more weakly associated HLA-DR3 (Ruder et al. 1990; Bouissou et al. 1995). Associations with HLA-DQ2 have also been described (Clark et al. 1990; Bouissou et al. 1995), but once again this allele is in linkage disequilibrium with HLA-DR7. Associations with HLA-DP alleles have been reported, but not independently confirmed. In Japanese

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children, susceptibility is associated with HLA-DR8 and -DQ3 (Komori et al. 1983; Kobayashi et al. 1985; Abe et al. 1995). A strong association with HLA-DR7 has also been reported in a single Chinese study (Zhou et al. 1994). Of interest, HLA-DQ3 showed a strong association in this study, even although HLA-DR7 is in linkage disequilibrium with HLA-DQ2 in this population (as in Caucasians). Caucasian studies have suggested that HLA-DRB1*1501 (HLA-DR2) alleles are protective, but this is not the case in the Japanese (Bouissou et al. 1995). A comprehensive list of reported associations can be found in Phelps and Rees (2000). In some children, MCD follows a relapsing and remitting course with a small minority becoming steroid dependent. In Caucasians, there is some evidence that the HLA-DR7 association is strongest with those individuals who frequently relapse, especially when it occurs in combination with HLA-DR3 (Ruder et al. 1990; Bouissou et al. 1995; Konrad et al. 1997). In contrast, Chinese HLA-DR9 positive children appear to relapse most frequently (Zhou et al. 1994). A small number of HLA studies have been performed in adult MCD. No significant associations have been reported in Caucasians (Laurent et al. 1983, 1985), but in the Japanese, susceptibility has been associated with HLA-DR8 and -DQ3 (Naito et al. 1987; Kobayashi et al. 1995). Taken together, these observations suggest that MCD is most strongly associated with alleles of HLA-DR, but that different alleles are responsible for susceptibility and protection in different populations. This may in part reflect different underlying allele frequencies or different pathogenetic mechanisms in different populations. The exact mechanisms underlying these associations are unknown, but they clearly suggest that there is an immunological component to MCD. Evidence that associations are strongest in patients who frequently relapse suggests that HLA alleles may be more influential in disease progression than in disease initiation. Few studies have examined HLA polymorphisms in FSGS and the results are difficult to interpret. Glicklich et al. (1988) studied 57 Caucasian patients with nephrotic syndrome, the majority of whom developed ESRF, and found an increased frequency of HLA-DR5. Ruder et al. (1990) found an association with the HLA-DR7, -B8, -DR3 haplotype in 41 steroid resistant nephrotic children, an observation which was also consistent with a concomitantly studied population of steroid sensitive children. Gerbase-DeLima et al. (1998) described an HLA-DR4 association in 19 Brazilian patients. However, in a large study of 605 American renal transplant patients whose ESRF was secondary to FSGS, no association was found (Freedman et al. 1994). A small number of association studies have examined non-MHC genes in idiopathic nephrotic syndrome. Because of the long established observation that allergic episodes can trigger episodes of nephrotic syndrome, Parry et al. (1999b) studied polymorphisms in the cytokine interleukin-4 (IL-4) and the IL-4 receptor alpha chain. IL-4 is produced by T cells and mast cells and is a key cytokine in the development of atopy. No significant difference was observed between 149 Caucasian children with MCN and 79 controls. Similarly, no associations were observed with polymorphisms of Flt-1, a major receptor for vascular permeability factor (Parry et al. 1999a). Frishberg et al. (1998) found no association with polymorphisms in the RAS genes ACE, AGT, and AT1R in 47 Arab and Jewish patients with FSGS. Similarly, Burg et al. (1997) reported no association with ACE or endothelial nitric oxide synthase polymorphisms in 17 Caucasian FSGS patients. However, Hori et al. (2001) observed a higher frequency of the ACE D allele in 43 Japanese FSGS patients.


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Important advances have recently been made in elucidating the genetic basis of congenital nephrotic syndromes with a Mendelian inheritance. These include the cloning of nephrin (NPHS1) in congenital nephrotic syndrome of the Finnish type, and podocin (NPHS2) and -actinin-4 (ACTN4) in forms of familial FSGS (Salomon et al. 2000; Kaplan and Pollak 2001). These disorders are discussed in detail in Chapter 7, but it will be of great interest to determine whether polymorphisms in these genes account for non-familial forms of nephrotic syndrome. Indeed, Caridi et al. (2001) have recently reported nine Italian patients with idiopathic FSGS who have podocin mutations. Membranous nephropathy Membranous nephropathy is a common form of nephrotic syndrome in adults. In most patients, onset of the disorder is not associated with any known aetiological factors. However, a minority of patients develop membranous nephropathy as part of another process: this can include infections such as hepatitis B or C, systemic disorders such as systemic lupus erythematosus, hypersensitivity reactions to drugs such as captopril, gold or penicillamine, and certain cancers and lymphomas. Although the exact pathogenetic mechanisms underlying membranous nephropathy are unclear, there is strong evidence that it is immune mediated. The disorder is characterized by thickening of the glomerular basement membrane due to sub-epithelial deposition of immunoglobulins and complement. In rats, an almost identical disease (Heymann’s nephritis) can be caused by immunization with antigen extracted from the kidney. Finally, numerous studies have shown that susceptibility is associated with alleles of HLA region genes. In Caucasians, the strongest associations with idiopathic membranous nephropathy (IMN) have consistently been observed with HLA-DR3 (Muller et al. 1981; Roccatello et al. 1987; Vaughan et al. 1995). In contrast, Japanese populations show strong associations with HLA-DRB1*1501 which encodes the HLA molecule HLA-DR2 (Hiki et al. 1984; Tomura et al. 1984). HLA-DR3 is uncommon in the Japanese, but this does not appear to account for the lack of association in this population. Although DRB1*1501 is common in Caucasian populations, it is not associated with susceptibility. In Caucasians, associations have also been observed with HLA-DQB1*0201 and HLA-DQA1*0501, but these alleles are in strong linkage disequilibrium with HLA-DR3, as is HLA-B8 (Vaughan et al. 1995). In Japan, the haplotype HLA-DRB1*1501, -DRB5*0101, -DQA1*0102, -DQB1*0602 is associated with disease susceptibility (Ogahara et al. 1992). Several studies have attempted to determine whether particular haplotypes are associated with a worse prognosis, but there is little evidence to support this. Vaughan et al. (1998) studied HLA-DRB and -DQB1 alleles of 42 Polish children with membranous nephropathy associated with hepatitis B infection. No HLA-DR3 association was observed, but there was an increased frequency of DQB1*0303 compared to healthy controls. A complete list of reported HLA associations in IMN can be found in Phelps and Rees (2000). Like minimal change nephropathy, the evidence in IMN suggests that HLA-DR is the probable susceptibility gene. However, different HLA-DR associations are observed in different ethnic groups, suggesting that IMN may have a variety of aetiologies. An alternative interpretation is that the true susceptibility gene may not encode an HLA antigen, but may be a gene in linkage disequilibrium with HLA-DR. However reported associations with the class II TAP genes and class III region C4A and tumor necrosis factor- (TNFB) loci are probably due to linkage disequilibrium with HLA-DR3 (Chevrier et al. 1994). Furthermore, the C4A

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null allele found in Caucasians patients does not occur on the HLA-DR2 haplotype that is associated with Japanese patients (Sacks et al. 1992). Few studies have been performed on non-HLA region genes. A single study reported associations with T-cell receptor -chain and immunoglobulin heavy chain switch region polymorphisms, but this has not been repeated (Demaine et al. 1988). Burg et al. (1997) reported no association with ACE or endothelial nitric oxide synthase polymorphisms in 23 Caucasian patients. IgA Nephropathy (Berger disease) IgA nephropathy is the commonest cause of glomerulonephritis amongst patients undergoing renal biopsy (Julian et al. 1988), and is a heterogeneous disorder. The prevalence varies between different ethnic groups, suggesting shared genetic or environmental factors have a role in the aetiology, but it is likely that different biopsy strategies in different countries may also be relevant, as immunofluorescence studies are required to reveal IgA-containing mesangial deposits (Lévy and Lesavre 1998). In IgA nephropathy there are reports of strong familial clustering, particularly in in-bred communities (Julian et al. 1985; O’Connell et al. 1987; Scolari et al. 1992; Scolari 1999). Population surveys have identified a high incidence of glomerulonephritis in relatives of cases of IgA nephropathy (Levy 1993; Rambausek et al. 1993). In one survey, 9.6 per cent of patients with IgA nephropathy had at least one sibling with glomerulonephritis (Rambausek et al. 1993). In those surveys that documented renal histology in family members, the histological pattern was conserved (Julian et al. 1985; Scolari et al. 1992). In summary, the sibling risk appears greatly in excess of the reported prevalence of clinically apparent IgA nephropathy of 25–50 cases per 100,000 of the population. Even allowing for ascertainment bias and diagnostic uncertainty, the epidemiological data indicates a high value of s in IgA disease (≥ 10). There is also an increased incidence of hypertension in parents of affected individuals (Schmidt et al. 1990), particularly if the proband is hypertensive (Ho et al. 1996). Genetic studies have been difficult because of the heterogeneous nature of the disease, and no clear loci for susceptibility genes have been identified. Pharmacological blockade of ACE slows the progression of chronic glomerular renal disease in experimental models, and the value of ACE inhibitors is proven in diabetic patients with renal impairment. A 287-bp deletion polymorphism in the ACE gene has been shown to be a significant risk factor for progression to CRF in IgA nephropathy (Yoshida et al. 1995). In addition, a met235thr polymorphism of the antiotensinogen gene (AGT) is associated with a faster decline in renal function (Pei et al. 1997). Multivariate analysis detected an interaction between the AGT and ACE polymorphisms with the ACE/DD polymorphism only affecting the disease progression in patients with the AGT/MM genotype. Neither of these polymorphisms is associated with systematic hypertension, and there are likely to be several other genes which also play an important role in the aetiology and progress of this heterogeneous disorder. A variety of HLA alleles have been associated with susceptibility to IgA nephropathy, reviewed in Phelps and Rees (2000). Henoch–Schonlein purpura (HSP) nephritis Henoch-Schonlein purpura is a systemic multisystem vasculitic disorder mainly affecting the skin, joints, gastro-intestinal tract, and kidneys. Although HSP can occur at any age it is overwhelmingly a disease of childhood. Indeed it is the most common vasculitis syndrome in


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children and is thought to be immune complex mediated. The pathogenesis remains unknown but might be related to an increased production of abnormally glycosylated IgA, which is not sufficiently cleared by the liver and leads to the formation of IgA macromolecules. It has been suggested that IgA nephropathy and HSP represent two aspects of a similar disorder, the former lacking the clinical manifestations in extra-renal organs seen in HSP. These two diseases, which can be encountered consecutively in the same patient and have been described in identical twins, bear similar pathological and biological abnormalities (Davin et al. 2001). The frequency of renal involvement varies between individual studies and can range from 20 to 100 per cent (Rai et al. 1999). Renal manifestations are usually mild and transient although chronic nephropathies can occur. Overall an estimated 2 per cent of children with HSP progress to renal failure and up to 20 per cent of children who develop acute nephritis require long-term haemodialysis (Rai et al. 1999; Cameron 2001; Davin and Weening 2001). Although it can affect all ethnic groups HSP is reportedly uncommon in those of African descent, suggesting that susceptibility may have a genetic origin or specific genetic modifiers (Weiss et al. 1978; Galla 2001). Several reports suggest a deficiency of complement 4 (C4) due to deletion of C4 genes which may then predispose to IgA nephropathy in HSP nephritis (Abe et al. 1993). Japanese patients with HSP reportedly have a higher prevalence of complement C4 gene deletions than control subjects but this has not been described in other unrelated populations from Europe and the United States (Wyatt et al. 1990; Abe et al. 1993). The DQA1*301 gene has been implicated in one study (Jin et al. 1996). In another it has been demonstrated that the presence of DRB1*01 or DRB1*11 facilitate disease onset while DRB1*07 has been shown to induce resistance (Amoroso et al. 1997). The IL-1 receptor antagonist IL1RN*2 allele is also a purported genetic marker shared by patients with HSP nephritis within a group of IgA nephropathy patients presenting with recurrent gross haematuria (Liu et al. 1997, 2001). In a Swedish study the presence of HLB35 has been postulated to predispose to recurrent disease (Nathwani 1992). The influence of deletion polymorphisms at the ACE gene on the progression of nephropathy and proteinuria has been suggested but studies have shown conflicting results (Amoroso et al. 1998; Yoshioka et al. 1998). Mesangiocapillary glomerulonephritis (MCGN) Mesangiocapillary glomerulonephritis (MCGN) is a histologically defined disorder characterized by mesangial proliferation and thickening of the capillary wall. Most cases are idiopathic, although some are clearly associated with infections. In the developed world, it is an uncommon form of glomerulonephritis. MCGN has been reported in several unrelated families, and autosomal dominant and X-linked recessive patterns of inheritance have been described. A single study suggested that the HLA-B8 -DR3 haplotype was associated with development of renal failure (Welch et al. 1986). A large proportion of individuals with MCGN develop hypo-complementaemia, mainly due to low serum concentrations of the complement component C3. In some patients this may in part be due to the presence of C3 nephritic factor, an auto-antibody that prevents degradation of the normal C3 convertase. Finn and Mathieson (1993) reported an association of C3 nephritic factor with the F allotype of the C3 gene. In addition, Levy et al. (1986) has reported a family with MCGN secondary to deficiency of factor H, an important regulator of the alternative complement pathway.

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Anti-glomerular basement membrane disease Anti-glomerular basement membrane disease (Goodpasture disease) is a rare form of autoimmune disease that causes pulmonary haemorrhage and acute renal failure through the formation of antibodies directed against a specific basement membrane antigen (the Goodpasture antigen). Ethnic differences in disease incidence have been noted, with the highest frequency in white Caucasians and the lowest in Asians and Afro-Carribeans. In the United Kingdom, the disease occurs with an incidence of approximately 0.5 cases per million per year. Although it can present at any age, the disease is most likely to occur in the sixth and seventh decades of life. In common with many other autoimmune disorders, Goodpasture disease is associated with alleles of loci encoding HLA antigens. However, Goodpasture disease is of particular interest because the auto-antigen is well defined, thus offering unique opportunities to study the mechanisms underlying autoimmunity. Several studies have described associations between HLA alleles and Goodpasture disease, with the conclusion that susceptibility is most strongly associated with the class II region allele HLA-DRB1*1501 (Dunckley et al. 1991; Huey et al. 1993; Fisher et al. 1997; Phelps and Rees 1999). To examine this in more detail, a total of 139 patients from three of these studies have recently been subjected to a meta-analysis of phenotype frequencies (Phelps and Rees 1999). This confirmed a strong association with HLA-DRB1*1501 (HLADR2), but also showed a positive association with HLA-DR4 and negative associations with HLA-DR1, -DR11, and -DR13. To avoid artefactual negative associations caused by the high frequency of HLA-DR1*1501, a second analysis was performed after the removal of this allele from patient and control populations. In this analysis, positive associations were observed for HLA-DR3 and -DR4 and negative associations for HLA-DR1 and -DR7. The frequency of patients heterozygous for HLA-DRB1*1501 and either HLA-DR3 or -DR7 was not significantly different from controls, suggesting that HLA-DR3 and -DR4 may act to protect HLA-DRB1*1501 positive individuals from the development of Goodpasture disease. Analysis of HLA-DQ alleles revealed a strong association with HLA-DQB1*06 which is in linkage disequilibrium with HLA-DRB1*1501. A weaker positive association were found with HLA-DQB1*0302 and negative association with HLA-DQB1*0501. Class II HLA molecules bind peptides of approximately 12 amino acids length. The peptide is bound in an extended formation in a peptide binding groove, with 5 ‘pockets’ within the groove accommodating any projecting side chain residues from amino acids 1, 4, 6, 7, and 9 (after which the pockets are named). Different class II alleles bind different spectra of peptides because of polymorphism in their peptide binding grooves. By comparing the peptide binding grooves of disease-associated alleles, these structural differences can be exploited to gain insight into underlying molecular mechanisms. Such analyses have provided useful information in rheumatoid arthritis and celiac disease, but are potentially even more useful in Goodpasture disease because the auto-antigen is known. Detailed comparison of Goodpasture’s associated HLA-DR alleles suggests that pocket 4 is particularly important and that the Goodpasture’s HLA association is primarily due to the HLA-DRB1*1501 allele and not to other genes in linkage disequilibrium (Phelps and Rees 1999). However, the situation is complex, and structural analysis of the weakly associated HLA-DR4 suggests that this association may be best explained by linkage disequilibrium with another HLA region encoded disease modifying gene. The simplest explanation for the role of HLA-DR15 in disease pathogenesis is that it binds peptides that stimulate auto-reactive T cells. The Goodpasture antigen is the 230 amino acid


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non-collagenous domain of the 3 chain of type IV collagen (3(IV)NC1; Turner et al. 1992; Hudson et al. 1993). Analyses of overlapping peptides derived from this molecule suggest that many are able to bind to HLA-DRB15 (Phelps et al. 1998). However, when HLA-DR15 homozygous B cells were pulsed with 3(IV)NC1 in vitro, only three could be presented to T cells, suggesting that these are most likely to induce T cell tolerance in vivo (Phelps et al. 1996, 1998). It is possible, for example, that autoimmunity occurs when other high affinity HLA-DR15 binding 3(IV)NC1 peptides, not usually displayed at sufficient level to induce tolerance, are presented to T cells. HLA-DR7 could protect against disease by deleting potentially auto-reactive T cells or by competitively binding peptides which, if bound to HLA-DRB1*1501, would induce disease. Clearly there are other potential explanations for disease pathogenesis, but Goodpasture provides a unique opportunity to understand autoimmune processes. Systemic disorders affecting the kidney Essential hypertension Essential hypertension is a common condition worldwide that is responsible for as many premature deaths as tobacco smoking (Murray and Lopez 1996). It causes end organ damage in a number of organs, including the kidney. The renal pathology is characterized by segmental hyalinization of interlobular arteries and afferent arterioles and glomerulosclerosis, an appearance termed hypertensive nephrosclerosis. Up to 30 per cent of renal biopsies show evidence of nephrosclerosis and hypertension accounts for up to 25 per cent of patients entering ESRF programmes. There is compelling evidence to suggest that essential hypertension has a genetic component. Estimates of this have ranged from 30 to 60 per cent, with a s of approximately 3.5 (Mongeau 1989; Brown 1996). Several approaches have been taken to identify genes that predispose to hypertension, including linkage analysis, candidate gene association studies, and the use of animal models. Most recently, significant insights have come from the study of families with early-onset hypertension with a Mendelian mode of inheritance. The molecular basis for several of these disorders has been established, including apparent mineralocorticoid excess (AME), glucocorticoid-remediable hyperaldosteronism (GRA), and Liddle syndrome. All support the hypothesis that increased sodium re-absorption within the kidney is central to blood pressure control and the development of hypertension. These disorders, which are discussed in detail in Chapter 14, provide important candidate genes for the study of essential hypertension (Alper 2001). A number of candidate genes for essential hypertension have been subjected to linkage analysis and these are summarized in Table 19.1. However, three genome-wide linkage analysis studies of essential hypertension have recently been published. Krushkal et al. (1999) analysed 69 discordant sib pairs from Minnesota and identified four linked regions, at chromosomes 2p22.1–21, 5q33.3–34, 6q23.1–24.1, and 15q25.1–26.1. A UK based study of 169 sib pairs identified linkage on chromosome 11q (Sharma et al. 2000). Finally, a study of 564 Chinese sib pairs selected for extreme blood pressure phenotypes detected linkage at chromosome 15q (Xu et al. 1999a,b). Potential candidate genes in these regions include the estrogen receptor, the sodium-channel exchanger 1, the thiazide-sensitive NaCl co-transporter and the potassium channel ROMK gene. Nevertheless, these studies highlight the difficulties of this approach, with no loci reproduced between studies. This may represent differences in pathogenesis, variability in selection and phenotype, or reflect the polygenic nature of essential hypertension with many genes exerting a weak influence.

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Stephen H. Powis Table 19.1 Candidate gene linkage studies to human hypertension and blood pressure Study

Population

Candidate genes

Jeunemaitre et al. (1992) Wu et al. (1996) Julier et al. (1997) Svetkey et al. (1997) Cusi et al. (1997) O’Donnell et al. (1998) Fornage et al. (1998) Baima et al. (1999) Wong et al. (1999) Busjahn et al. (2000)

Utah and France Taiwan France and UK Afro-American France Framingham Minessota

AGT Lipoprotein lipase ACE b2-adrenergic receptor a-adducin ACE ACE PNMT ENaC b2-adrenergic receptor

Australia Germany

The predominant strategy to identify hypertension loci has been case-control association studies and a variety of genes have been examined. RAS genes have been a focus of much interest because of the effect this system has on the vasculature. A meta-analysis of 46 studies of the ACE I/D polymorphism in hypertension concluded that there was no association, suggesting that blood pressure is not influenced by the ACE gene (Staessen et al. 1997). An early study reporting linkage between the AGT gene and hypertension has led to many subsequent association studies (Jeunemaitre et al. 1992). Recent meta-analyses, in which Kunz et al. (1997) analysed 5493 individuals in 11 studies and Staessen et al. (1999) studied 27,906 subjects in 69 studies, have suggested that the AGT M235T allele is associated with an increased risk of hypertension. A meta-analysis of six Japanese studies also favoured an association, but there was considerable heterogeneity among the studies (Kato et al. 1999). Candidate gene linkage studies of AGT have also confirmed linkage in some populations, but not in others (Corvol et al. 1999). As mutations in the epithelial sodium channel (ENaC) are responsible for Liddle syndrome (Shimkets et al. 1994), this gene has been the subject of several studies. Baker et al. (1998) observed that the T594M mutation of the ENaC subunit conferred a higher relative risk for essential hypertension in a London based Afro-Caribbean population. However, subsequent studies of black Caribbeans and Caucasians have failed to find further associations (Melander et al. 1998; Munroe et al. 1998; Baker et al. 1999; Persu et al. 1999). Nevertheless, a recent sib pair analysis of white Australians demonstrated linkage between systolic blood pressure and the region on chromosome 16p encoding the ENaC and subunits (Wong et al. 1999). Because essential hypertension is associated with substantial abnormalities of autonomic and sympathetic function, a number of genes encoding proteins with catecholaminergic and adrenergic function have been analysed. Several studies have reported associations with the adrenergic receptor 2 (ADRB2; Kotanko et al. 1997; Bray et al. 2000b; Busjahn et al. 2000) and these are supported by the genome-wide scan of Krushkal et al. (1998, 1999) which showed linkage with the region of chromosome 5q31–34 that encodes this and other adrenergic receptors. Polymorphisms in tyrosine hydroxylase, the rate-limiting enzyme in the biosynthesis of catecholamines (Sharma et al. 1998), and the -adrenoreceptor Gs protein system (Siffert et al. 1998; Beige et al. 1999; Jia et al. 1999) have also been associated with hypertension. Several studies have reported associations between essential hypertension and


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-adducin, a renal tubular protein that may regulate ion transport through changes in the actin cytoskeleton (Bray et al. 2000a; Province et al. 2000; Schork et al. 2000). Cusi et al. (1997) have also demonstrated linkage with the -adducin region on chromosome 4p. Other genes that have recently been studied in case-control studies include kallikrien, apolipoprotein B, prostacyclin synthetase, the glucagon receptor and transforming growth factor beta (Timberlake et al. 2001). In summary, there is consistent evidence to implicate the M235T allele of AGT with an increased risk of hypertension, but other candidate genes require further study. The inconsistent findings of linkage studies are an important reminder of the limitations of this approach, even in such a common condition as essential hypertension. Diabetes mellitus Diabetic nephropathy (DN) is the commonest cause of ESRF, comprising up to 40 per cent of patients entering ESRF programmes in the developed world. Both types 1 and 2 diabetes mellitus can cause nephropathy and large-scale studies have demonstrated that the development of diabetic complications such as retinopathy and nephropathy is linked to hyperglycaemia. Improved glycaemic and blood pressure control reduces the risk of complications and slows progression if complications do develop. Nevertheless, not all patients with diabetes develop complications. Nephropathy occurs in less than one half of type 1 diabetic patients, with a peak incidence between 10 and 20 years following disease onset. Furthermore, only one quarter of patients with retinopathy or neuropathy develop nephropathy. These observations may be explained by underlying genetic influences. Additional support for a genetic basis for DN comes from family studies (Seaquist et al. 1989; Earle et al. 1992). In siblings concordant for type 1 diabetes, the cumulative risk of DN after 30 years of disease was 71.5 per cent if a diabetic sib had persistent proteinuria, but only 25.4 per cent if the sib had normoalbuminuria (Quinn et al. 1996). In type 2 diabetes, studies of Pima Indians have also shown clustering of proteinuria in families with a diabetic proband. A genome-wide linkage study of DN has recently been reported in a population of 98 Pima Indian type 2 diabetic sib pairs with nephropathy (Imperatore et al. 1998). Diabetic nephropathy was defined as the presence of overt proteinuria or macroalbuminuria. The strongest evidence for linkage was found on chromosome 7, with additional evidence for linkage on chromosomes 3, 9, and 20. In a previous, larger study of Pima Indians, this region of chromosome 7 was not linked to diabetes per se and thus seems unique to DN. However, the same regions on chromosome 3 and 9 were linked to diabetic retinopathy, suggesting the existence of loci affecting susceptibility to both these complications (Imperatore et al. 1998). The linked region of chromosome 7, 7q35, contains at least three known genes that could potentially influence the development of DM, aldose reductase (ALDR1), the T cell receptor chain and constitutive nitric oxide synthetase 3 (NOS3). Two further linkage studies have targeted regions of the genome encoding RAS genes. Yu et al. (1996) studied the region in which AT1R is encoded in 38 African–American sib pairs concordant for both type 2 diabetes and DN, but no evidence for linkage was observed. Moczulski et al. (1998) studied 66 sib pairs concordant for type 1 diabetes but discordant for DN, specifically investigating linkage at the ACE, AGT, and AT1R loci. The regions encoding the ACE and AGT loci showed no evidence of linkage, but linkage was demonstrated between DN and a 20 cM region at chromosome 3q21–25 that includes the AT1R gene. However, subsequent studies of polymorphisms

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in the AT1R gene using the transmission disequilibrium test (TDT) failed to find any significant associations. It should also be noted that Imperatore et al. (1998) failed to find linkage with chromosome 3q21–25, or indeed with the ACE and AGT regions, in their genome wide study of Pima Indians. Thus the evidence from linkage studies is currently contradictory and other large studies in independent populations, such as that recently described by Covic et al. (2001), are required. The vast majority of genetic studies in DN have been association studies of candidate susceptibility genes, and the most frequently analysed genes are those of the RAS. Unfortunately, the results of these studies have often been inconsistent. Three meta-analyses of the ACE I/D polymorphism have been undertaken in an attempt to rationalize this data. The first two concluded that the ACE D allele was associated with an increased risk of DN in general (Staessen et al. 1997; Fujisawa et al. 1998). The third analysis (Tarnow et al. 1998) found that the ACE II genotype was protective for DN in Japanese, but not Caucasian type 2 patients. In Caucasian type 1 patients, a trend towards a protective effect of the ACE II genotype was observed. A similar conclusion was reached by Kunz et al. (1998) following a systematic review of 19 studies. No evidence was found for an association between ACE I/D polymorphisms and DN in Caucasians with type 1 or 2 diabetes, but the risk of DN was increased in type 2 Asian patients with the ACE DD or ID genotype. Fewer studies have examined the AGT or AT1R genes, but there appears to be some evidence for an association between DN and the AGT M235T T allele and no evidence for an association with AT1R alleles. Only two family-base studies have been reported using the TDT, both from the same investigators. Analysis of the AGT M235T allele demonstrated that the T allele was more frequently transmitted to male offspring with DN (Rogus et al. 1998). Of particular interest, ACE I/D analysis revealed that the I allele was preferentially transmitted to DN offspring (Krolewski 1999), contradicting the trend in case control studies for the D allele to be associated with DN. Polymorphisms in many other genes thought to be involved in the pathogenesis of DN have also been studied. These include genes involved in the activation of protein kinase C, oxidative stress genes, genes influencing the development of extracellular fibrosis, glucose transport genes, cytokines, and genes involved in the formation and degradation of advanced glycation end products. Most of these are small studies and often the results are disparate (Adler et al. 2000). In summary, the evidence suggests that the ACE D allele is associated with the development of DN, although this may vary in different ethnic groups. Further family based studies are required to investigate other candidate susceptibility genes. Renovascular disease Renovascular disease is an important cause of secondary hypertension and is associated with the development of ESRF. It is defined by stenosis of the main renal artery or one of its branches. The majority of patients with renovascular disease (over 80%) have arteriosclerosis and often have evidence of atherosclerotic disease in other major vessels such as the coronary, carotid, iliac and femoral arteries. Fibromuscular dysplasia (FMD) is the next commonest cause and is the typical diagnosis in patients presenting under the age of 40. Other causes of renal artery stenosis, such as trauma, renal cysts, and arteritis, are uncommon. Several reports have documented FMD lesions in first-degree relatives of FMD patients, suggesting a genetic predisposition (Major et al. 1977; Stavenow et al. 1978; Rushton 1980; Ouchi et al. 1989; Pannier-Moreau et al. 1997; Suzuki et al. 1999). Pannier-Moreau et al.


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(1997) estimated the prevalence of familial renal artery FMD in 104 unrelated hypertensive patients with angiographic proven uni-focal or multi-focal lesions. Phenotypic screening was performed in at least one first-degree relative in 33 families, of which 11 had angiographic evidence of renal FMD in at least one sibling. This prevalence of approximately 11 per cent may be an underestimate as not all families were screened by angiography. Missouris et al. (1996) studied ACE gene polymorphisms in 56 Caucasians with atherosclerotic renal disease and 74 age, sex, and ethnically matched controls. The ACE D polymorphism occurred significantly more frequently in the patient group, consistent with studies that have found an increase in the ACE D allele in other forms of cardiovascular disease. In contrast, Bofinger et al. (2001) studied polymorphisms of the ACE, AGT, and AT1R genes in 43 patients with typical multi-focal renal artery FMD. There was a significantly higher frequency of the ACE I allele in patients compared to 89 matched controls, but no differences in the frequencies of the AT1R A1166C or AGT M235T and T174M polymorphisms. Further studies are required in independent populations to confirm these observations. However, it would be intriguing if the ACE D polymorphism was associated with atherosclerotic renovascular disease whereas the ACE-I polymorphism was associated with FMD. One possibility is that this reflects differences in disease process. In atherosclerotic renovascular disease, increased levels of ACE, and thus of angiotensin II, could promote intimal hyperplasia. In multi-focal FMD, in which the principal lesion is medial fibroplasia, reduced concentrations of angiotensin II might impair remodelling in the muscular layer of the renal arteries. Schievink et al. (1994) proposed an association between FMD and alpha-1-antitrypsin (1-AT) deficiency based on a retrospective review of 6696 autopsies performed at the Mayo clinic between 1983 and 1992. Six patients were identified with documented 1-AT deficiency, 25 patients with FMD and two with both. 1-AT is a potent protease inhibitor important in protecting connective tissue, possibly including vessel walls. Several subsequent reports have described individual FMD patients with 1-AT deficiency, a minority of whom had renal artery involvement (Schievink et al. 1996, 1998a,b; Solder et al. 1997). To determine whether renal FMD is associated with 1-AT deficiency, Bofinger et al. (2000) performed 1-AT phenotyping in 83 Australian patients with renal artery FMD and found that 10 (12%) had a deficiency phenotype. However, this did not differ from the previously reported phenotype distribution in normal Australians, suggesting that 1-AT deficiency is not an aetiological factor in the development of renal FMD. However, in concordance with previously reported individual cases, two 1-AT deficient patients in this patient population had particularly severe FMD, suggesting that the chance association of FMD and 1-AT deficiency may produce more severe manifestations of FMD. However, this observation might also be due to reporting bias. Systemic lupus erythematosus (SLE) Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder associated with the production of auto-antibodies to a variety of nuclear antigens. It causes a variety of clinical symptoms and can affect multiple organ systems. Approximately 50 per cent of SLE patients have renal involvement, which can present with a spectrum of renal abnormalities including proteinuria, haematuria, nephrotic syndrome, and acute or chronic renal impairment. Histologically, lupus nephritis is manifested as various forms of glomerulonephritis. SLE is a polygenic disorder in which it has been estimated up to 100 genes may be involved (Harley

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et al. 1998). The concordance rate in monozygotic twins ranges from 24 to 69 per cent, and first-degree relatives have a 1–3 per cent risk of developing the disease (Block et al. 1975; Deapen et al. 1992; Grennan et al. 1997). There have been six published genome-wide linkage studies of SLE, utilizing four different family collections (Gaffney et al. 1998, 2000; Moser et al. 1998; Shai et al. 1999; Gray-McGuire et al. 2000; Lindqvist et al. 2000). These have shown up to 48 possible linked loci, of which at least six appear significant. Two of these are regions encoding susceptibility genes previously identified in association studies, the Fc receptors on chromosome 1q23, and HLA genes on chromosome 6p21. The region at 1q41–42 has been replicated in several of the linkage studies, and also in another targeted study of this region (Tsao et al. 1997). The susceptibility locus is located close to the gene for poly-ADP ribosyl transferase (PARP; Tsao et al. 1999; Graham et al. 2001). Linkages at chromosome 2q37, 4p15–16, and 16q12–13 have also been identified. Chromosome 16q12–13 is of particular interest because it has also been linked with psoriasis, type I diabetes and asthma, suggesting that certain genes may dispose to several immune-mediated disorders (Wakeland et al. 2001). Because of its autoimmune aetiology, many immune system genes have been implicated in the pathogenesis of SLE. Association studies have targeted genes involved in immune complex clearance, MHC genes, and cytokines. Individuals with complete deficiencies of the early components of complement (C1, C4, and C2) have a high risk of developing lupus (Schur 1995). Deficiencies in these early complement components probably predispose to SLE through impaired immune complex clearance and defective B cell tolerance. Individuals with homozygous C1q deficiency have a particularly severe form of SLE with glomerulonephritis, and it is of note that C1q is also involved in the clearance of apoptotic cells (Korb and Ahearn 1997). Deficiencies of immune complex clearance also explain associations with the Fc receptors FcRI, FcRII, and FcRIII. Sullivan (2000) comprehensively reviews SLE association studies of genes involved in immune complex clearance. HLA genes have long been associated with the development of SLE. In Caucasians, HLADR2 and -DR3 have consistently been found at higher frequencies in patients, but in non-Caucasians a number of different HLA allele associations have been observed which are not reproducible (Sullivan 2000). In native Americans, HLA-DQ4 and -DR8 show positive associations, and HLA-B7, -DR2, and -DQ6 in Asians. Because of linkage disequilibrium within the MHC, it has been difficult to attribute susceptibility to specific HLA genes. For example, the Caucasian HLA-B8-DR3 haplotype associated with SLE also contains a C4 null allele. Interestingly, recent studies have suggested that HLA alleles are more closely associated with specific auto-antibodies or clinical features (Schur 1995). Cytokine dysregulation has consistently been described in patients with SLE and therefore studies have searched for genetic associations in cytokine expression. IL-10 is a Th2 cytokine that down-regulates immune complex clearance. IL-10 polymorphisms have been associated with SLE and with specific clinical phenotypes, including renal disease (Lazarus et al. 1997; Mehrian et al. 1998; Mok et al. 1998; Gibson et al. 2001). Of note, the IL-10 gene is encoded near a region of chromosome 1 identified as being important in SLE in linkage studies. TNF- is a pro-inflammatory cytokine that can be elevated in SLE patients (Meijer et al. 1993). Caucasian studies have shown associations between SLE and the allele TNF-308A, which is associated with high levels of TNF- expression, but this could be secondary to linkage disequilibrium with other genes on the HLA-B8 -DR3 susceptibility haplotype (Wilson et al. 1994; Danis et al. 1995b; Rudwaleit et al. 1996). However, similar associations in


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African-American populations, where this polymorphism occurs independently of HLA-B8 -DR3, suggests that TNF- does have an independent role in SLE (Sullivan et al. 1994). Associations with other TNF- polymorphisms support this, although these are not consistent in all ethnic populations (Fong et al. 1996; Sturfelt et al. 1996; Hajeer et al. 1997). Polymorphisms within the IL-1 receptor antagonist gene and IL-6 gene have also been associated with SLE (Blakemore et al. 1994; Danis et al. 1995a; Suzuki et al. 1997), but interferon- polymorphisms were found not to be associated in a recent study (Lee et al. 2001). As SLE may, in part, be mediated by defects in apoptosis, associations have been sought in genes involved in cell death. Associations have been reported with both Bcl-2 and Fasligand polymorphisms (Mehrian et al. 1998). Fas is the main genetic determinant in the MRL/lpr murine lupus model, but in humans Fas defects are associated with the Canale-Smith syndrome (Drappa et al. 1996) and polymorphisms have not been associated with SLE (Cascino et al. 1998). In summary, there is good evidence to support a role for early complement components, Fc receptors and HLA genes in SLE, but other candidate susceptibility genes require further study. Further candidate gene should be identified in linkage studies and from murine models (Sullivan 2000; Mohan 2001; Wakeland et al. 2001). Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis Small vessel vasculitis occurring in the presence of ANCA is often associated with a focal necrotizing glomerulonephritis and is the major (but not exclusive) cause of rapidly progressive glomerulonephritis (RPGN). Many patients have other organ involvement, such as upper respiratory tract inflammation and pulmonary haemorrhage, but some patients present with apparently isolated renal vasculitis. Auto-antibodies are directed against one of two neutrophil cytoplasmic antigens, myeloperoxidaes (MPO) or proteinase-3 (Pr-3). Familial occurrence has been reported in these disorders, although identical twins discordant for the disease have also been observed (Muller et al. 1984). As pathogenesis appears related to the development of auto-antibodies, a number of studies have examined possible associations with HLA region genes. Early studies were performed before ANCA could be assayed, and must therefore be interpreted with caution. Muller et al. (1984) reported an association between RPGN and HLA-DR2, although, unusually, none of the patients in this study had evidence of systemic vasculitis. Similarly, Elkon et al. (1983) found a higher frequency of HLA-DR2 in a sub-group of 17 patients with Wegener’s granulomatosis, but not in a larger group of 45 patients with vasculitis. HLA-DQB1*0301 was increased in a group of 50 patients (Spencer et al. 1992), especially when found on HLA-DR4 haplotypes, but this association did not correlate with ANCA specificity or clinical diagnosis. More recent studies failed to find significant associations (Zhang et al. 1995) or only identified a negative association with HLA-DR6 (Hagen et al. 1995). Nakamaru et al. (1996) observed an increase in HLA-DR9 in Japanese patients, but the study was extremely small. 1-antitrypsin (1-AT) is an important inhibitor of proteinase-3, and an association between Pr-3 positive vasculitis and the 1-AT deficient PiZZ phenotype has been described in several reports (Elzouki et al. 1994; Callea et al. 1997; Esnault et al. 1997). However, this alone does not appear to be sufficient to induce vasculitis (Audrain et al. 2001). A single study has suggested an association with the neutrophil integrin CD18 and ANCA, but not with other integrins (Gencik et al. 2000). Finally, several groups have failed to find associations with Fc receptor polymorphisms and the development of vasculitis (Edberg et al. 1997; Tse et al.

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1999), although a recent report has suggested an association with frequent relapse (Dijstelbloem et al. 1999). The role of non-HLA genes in ANCA-mediated vasculitis has recently been reviewed by Savage et al. (2002). Scleroderma Scleroderma (systemic sclerosis) is a connective tissue disorder, predominantly affecting females, with a spectrum of clinical manifestations from isolated skin lesions to multiple organ involvement. Major renal involvement occurs in up to 15 per cent of patients (Black and Denton 1998), but it is probable that many more patients have minor renal abnormalities. Familial clustering has provided evidence for a genetic basis to scleroderma (Arnett 1995; Manolios et al. 1995), with a recent study of 715 Australian patients producing a 1.4 per cent incidence of familial scleroderma (Englert et al. 1999), with an estimated recurrence rate in first-degree relatives (r) of 11 in 158. Until recently, association studies in scleroderma almost exclusively focused on HLA genes. Several studies have identified associations with HLA-DR5 and -DR3 haplotypes in white European and North American populations and HLA-DR2 in Japanese and Choctaw native Americans (Arnett et al. 1990; Kuwana et al. 1993; Vargas-Alarcon et al. 1995; Tan et al. 1999). Evidence also supports a role for the amino acid glutamate at position 69 of HLA-DPB1 (Briggs et al. 1993; Rihs et al. 1996; Kuwana et al. 1999; Tan et al. 1999). However, associations appear to be stronger with specific auto-antibody subsets of scleroderma. In particular, consistent associations have been reported with anti-topoisomerase I antibodies and HLA-DR5/11 haplotypes in Caucasians (Fanning et al. 1998; Frank et al. 1998; Rands et al. 2000; Vlachoyiannopoulos et al. 2000; Gilchrist et al. 2001) and HLA-DR2 (HLA-DRB1*1502) haplotypes in Japanese (Kuwana et al. 1999; Kang et al. 2001). It has been more difficult to demonstrate HLA associations with anti-RNA polymerase positive patients, who have a high incidence of renal involvement (Fanning et al. 1998; Falkner et al. 2000). Two studies have found associations with polymorphisms in the MHC class III encoded TNF- gene, but it is possible that these could be due to linkage disequilibrium with class II HLA genes (Pandey and Takeuchi 1999; Takeuchi et al. 2000). Recent studies have targeted non-MHC genes. No associations were found with the matrix metalloproteinase-1 promoter, transforming growth factor beta, or platelet-derived growth factor (Zhou et al. 2000; Johnson et al. 2001), but positive associations have been reported with type I collagen gene COL1A2 in a Japanese population (Hata et al. 2000) and with the chemokine receptor CXCR-2 and cytochrome P4501A1 (CYP1A1) gene in Caucasian populations (von Schmiedeberg et al. 1999; Renzoni et al. 2000). Associations have also been reported with micro-satellite markers close to the fibrillin 1 gene in Japanese and Choctaw native Americans (Tan et al. 1998, 2001). Sarcoidosis Sarcoidosis is a chronic immune-mediated multi-system disorder of unknown aetiology. It is almost certainly triggered in genetically susceptible individuals by an environmental agent, possibly an infectious agent. Pulmonary sarcoidosis is characterized by infiltration of macrophages and lymphocytes, the formation of non-caseating granulomas and fibrosis. Sarcoidosis can affect the kidney through hypercalcaemia and hypercalciuria, granulomatous


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interstitial nephritis or, rarely, glomerulonephritis. The exact prevalence of granulomatous renal disease is difficult to ascertain, possibly because of variation in the frequency at which renal biopsy is performed, but figures range from 15 to 40 per cent of sarcoid patients (Romer 1980; Muther et al. 1981). Evidence for a genetic basis to sarcoidosis comes from racial and ethnic variations in disease incidence and severity, and from familial clustering of the disease (Rybicki et al. 1997; Pietinalho et al. 1999b; Luisetti et al. 2000). Linkage analysis studies have not been reported in sarcoidosis, but a number of candidate susceptibility genes have been analysed in case–control association studies. Because the pathology of sarcoidosis implies an immune component, associations with HLA genes have been studied since the 1970s. Initial studies reported an increased frequency of the class I allele HLA-B8 and the HLA-DR3, -B8 haplotype (Olenchock et al. 1981; Hedfors and Lindstrom 1983), but subsequent studies identified a positive association with HLA-DR5 in German and Japanese populations (Abe et al. 1987; Nowack and Goebel 1987; Ishihara et al. 1996a). Overall, it appears that HLA-DR3, -DR5, and -DR6 are susceptibility alleles in Caucasians, and HLA-DR5, -DR6, and -DR8 in Japanese. HLA-DP alleles are of particular interest because of the observation that susceptibility to berylliosis, which produces granulomatosis lesions that are indistinguishable from those found in sarcoidosis, is associated with HLA-DPB1*0201 and in particular a Lys to Glu amino acid substitution at codon 69 (Richeldi et al. 1993). Lympany et al. (1996) found an association between HLA-DPGlu69 and sarcoidosis in a study of 41 UK patients, and similar observations were made by Schurmann et al. (1998) in a study of 17 German families. However, this association could not be reproduced in other populations (Saltini et al. 1993; Maliarik et al. 1998a; Foley et al. 1999). Other MHC encoded genes have also been studied. The class II region genes TAP1, TAP2, and LMP7 have been analysed in several Japanese studies, but any observed associations were secondary to linkage disequilibrium with HLA-DR alleles (Ishihara et al. 1996b,c, 1997a,b). However, Foley et al. (1999) reported a positive association with a TAP2 polymorphism in 117 UK and 87 Polish sarcoid patients. The TNF gene complex, located in the HLA class III region, is of interest because TNF- may be an important mediator in granuloma formation. However, recent studies of TNFA and TNFB polymorphisms have been conflicting, with no evidence of an overall association with sarcoid but some evidence of positive associations with the Lofgren syndrome subgroup and with disease progression (Seitzer et al. 1997; Somoskovi et al. 1999; Swider et al. 1999; Takashige et al. 1999; Labunski et al. 2001; Yamaguchi et al. 2001). Several studies have examined other components of the immune response. Changes in the spectrum of T cell receptor variable gene segments have been reported, suggesting that T cells are selected to interact with specific sarcoid antigens presented by HLA antigens (Grunewald et al. 1995; Moller 1998). A case control study of TCR gene polymorphism failed to find any association with sarcoidosis (Rybicki et al. 1999), but limited evidence supports a role for immunoglobulin gene polymorphisms (Martinetti et al. 2000). Chemokines and cytokines are important mediators in the inflammatory reactions associated with sarcoidosis. Hizawa et al. (1999) analysed the C–C chemokine receptor-2 (CCR2) gene in 100 Japanese patients and observed that the CCR2–64I allele conferred a lower risk for the development of sarcoidosis. Rybicki et al. (1999) observed positive associations in 105 African–American patients with markers located close to the IL-1a gene and interferon regulatory factor (IRF) genes.

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Of the non-immune genes studied in sarcoidosis, the most frequently analysed are those of the RAS. There is a strong rational for studying these genes in sarcoidosis as 50–60 per cent of sarcoid patients have elevated levels of serum ACE and there is evidence that there is a correlation between ACE levels and disease severity. Overall, there is little evidence to support an association with ACE I/D polymorphism in Caucasians or Japanese (Arbustini et al. 1996; Furuya et al. 1996; Tomita et al. 1997; Maliarik et al. 1998b; Pietinalho et al. 1999a; Papadopoulos et al. 2000; McGrath et al. 2001; Schurmann et al. 2001), but one larger study of 183 African–Americans did identify an association with the ACE DD genotype and the development of sarcoidosis (Maliarik et al. 1998b). There is also some evidence that suggests the DD genotype is associated with more severe disease. Only one study has been family based, in which TDT analysis revealed no excess transmission of ACE alleles to affected individuals (Schurmann et al. 2001). Nearly all studies agree that ACE genotype correlates with serum ACE levels in sarcoidosis, with the DD genotype associated with the highest levels and the II genotype with the lowest. A single study of AT1R and AT2R genes in Japanese patients showed no association (Takemoto et al. 1998). Vitamin D, 1,25-dihydroxyvitamin D(3) is produced at sites of granulomatous reactions in sarcoidosis. Niimi et al. (1999) observed an association between a vitamin D receptor gene polymorphism and sarcoidosis in 101 Japanese patients, although it was not a risk factor for hypercalcaemia (Niimi et al. 2000). Finally, an intriguing observation has recently been made that patients with sarcoidosis have an increased frequency of mutations in the CFTR cystic fibrosis gene, although the potential molecular basis for this is unclear (Bombieri et al. 1998, 2000). Haemolytic uraemic syndrome (HUS) Haemolytic uremic syndrome is characterized by a triad of acute renal failure, microangiopathic haemolytic anaemia (MAHA) and thrombocytopenia. It can be classified as diarrheal (d HUS) or non-diarrheal (d HUS), which in turn can be further sub-classified as sporadic or familial. d HUS is linked to gastrointestinal infection with the bacterium Eschericia coli 0157, which produces a potent verotoxin that damages endothelium (Rowe et al. 1998). E. coli 0157 infection is associated with poor food hygiene, which has led to several welldocumented outbreaks of HUS. d HUS appears to be precipitated by a number of different conditions, including some forms of cancer and following the use of the calcineurin inhibitors ciclosporin and tacrolimus. A genetic basis for d HUS has been suggested for some years by the existence of familial forms of the disorder (Thompson and Winterborn 1981; Berns et al. 1992; Pichette et al. 1994). Insight into a possible pathogenesis was also provided by observations of low levels of complement component Factor H (FH) in individuals affected by the disorder (Roodhooft et al. 1990; Pichette et al. 1994). FH is the most important plasma bound down-regulator of the alternative complement pathway activity. Warwicker et al. (1998) performed a linkage study on three familes with familial HUS and demonstrated linkage to the locus for FH on chromosome 1. Subsequent mutation analysis of FH in one of the families revealed a point mutation leading to an amino acid substitution that segregated dominantly with disease. In a second family, inheritance was recessive and caused by a 24 base-pair deletion. In addition, an exon 1-truncating mutation was found in a patient with the sporadic form of d HUS. Recently, Factor H mutations have been described in a further five patients, two with the


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inherited form of the disease and three with sporadic disease (Richards et al. 2001). All mutations appear to be clustered in exons 18–20 and possibly disrupt binding to anionic surfaces. Of interest, several affected individuals with mutations had normal serum levels of FH, suggesting that normal FH levels do not exclude HUS as a diagnosis. FH deficiency has also been reported in patients with SLE and mesangiocapillary glomerulonephritis (Levy et al. 1986; Lopez-Larrea et al. 1987). However, FH mutations have not been detected in the majority of sporadic and familial HUS patients, despite extensive screening, suggesting the involvement of other genes. Finally, it will be of interest to search for FH mutations in the E. coli 0157 associated d form of the disease. Amyloidosis Amyloidosis is a disorder in which normally soluble proteins are deposited in the extracellular space in an abnormal fibrillar form. At least 20 different proteins have been shown to form amyloid fibrils, accumulation of which causes progressive disruption of the underlying tissues and organs. In systemic amyloidosis this process often leads to death. Renal dysfunction is a frequent presenting feature of systemic amyloidosis and is usually found in three clinical settings. Reactive systemic (AA) amyloidosis occurs in a variety of chronic diseases, including inflammatory disorders such as rheumatoid arthritis, juvenile chronic arthritis, and Crohn disease; malignancies such as Hodgkin disease and renal carcinoma; and infections such as tuberculosis, leprosy, and bronchiectasis. Many of these chronic disorders have a genetic basis, and some such as diabetes and SLE are discussed in this chapter. AL amyloidosis is associated with B cell dyscrasias such as multiple myeloma and benign monoclonal gammopathy. Finally, hereditary systemic amyloidosis includes a variety of disorders such as familial renal amyloidosis and familial Mediterranean fever. The hereditary forms of amyloidosis are uncommon, but their genetic basis is best understood. The syndrome of familial renal amyloidosis (FRA) was first described by Ostertag in 1932 and is inherited in an autosomal dominant manner. Almost all patients with FRA are heterozygous for mutations in one of three genes, lysozyme (Pepys et al. 1993; Gillmore et al. 1999), apolipoprotein A1 (Soutar et al. 1992; Persey et al. 1998) or fibrinogen A alpha chain (Benson et al. 1993; Hamidi Asl et al. 1998). Most mutations give rise to single amino acid substitutions that destabilize native protein folding and promote the formation of amyloid fibrils. Only around 30 families with FRA have been described, but there is evidence to suggest that the disorder is under-reported: of 1000 UK patients with systemic amyloidosis undergoing DNA screening, 60 were found to have mutations in one of the three FRA genes (Lachmann et al. 2002). Penetrance and clinical phenotype varies between and within families (Hawkins and Lachmann 2001). Patients with the commonest form of fibrinogen A alpha chain amyloidosis, associated with the Glu526Val mutation, may not have a clear family history and can be mis-diagnosed as suffering from AL amyloidosis. Presentation is typically in late middle age with proteinuria and hypertension, progressing to ESRF within 5–10 years. Patients with amyloidosis due to lysozyme mutations often have substantial amyloid deposits within the gastrointestinal tract. The kidneys, spleen, and liver are also affected, but the disease progresses slowly. Amyloidosis secondary to apolipoprotein A1 varies significantly according to the mutation. The commonest mutation, Gly26Arg, usually presents with hypertension, proteinuria, and progressive renal impairment.

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The hereditary periodic fevers (HPFs) are a group of rare inherited disorders characterized by recurrent, self-limiting episodes of fever accompanied by synovial and serosal inflammation (McDermott and Frenkel 2001). Familial Mediterranean fever (FMF) and the rare Muckle– Wells syndrome (MWS) are frequently complicated by systemic amyloidosis, whereas it is less common in tumor necrosis factor receptor-1-associated periodic fever (TRAPS) and hyper IgD periodic fever syndrome (HIDS). FMF is in an autosomal recessive HPF caused by mutations in the MEFV gene, which is located on the short arm of chromosome 16 and encodes the protein pyrin/marenostrin (The French FMF Consortium 1997, The International FMF Consortium 1997, Booth et al. 2000). Around 30 MEFV mutations have now been identified, the vast majority of which are mis-sense mutations. In at least one-third of FMF patients, one allele of MEFV appears normal. Pyrin/marenostrin is involved in the downregulation of inflammatory mediators, a function that might be inhibited by mutations in MEFV. The cause of the autosomal dominant MWS has recently been identified as mutations in the gene CIAS1, located on the long arm of chromosome 1 (Hoffman et al. 2001). CIAS1 encodes a protein with a pyrin domain, suggesting it has a role in the regulation of inflammation. The related familial cold autoinflammatory syndrome (commonly known as familial cold urticaria) is also due to CIAS1 mutations. TRAPS (previously known as familial Hibernian fever) is caused by mutations in the gene encoding TNF receptor-1 (McDermott et al. 1999). It is postulated that these inhibit cleavage of this cell surface receptor, resulting in a diminished pool of soluble receptor available to reduce the bio-availablity of the inflammatory TNF cytokine. HIDS is caused by mutations in the gene encoding the mevalonate kinase (MVK) enzyme, an intermediate in cholesterol metabolism and isoprenoid biosynthesis (Drenth et al. 1999; Houten et al. 1999). The mechanisms through which the abnormal enzyme causes fever are not understood. Gout, hyperuricaemia, and disorders of purine metabolism Hyperuricaemia is a common metabolic abnormality that, in some individuals, can lead to gout. Hyperuricaemia may manifest itself within the kidney through a urate nephopathy or through the production of uric acid stones. Acute hyperuricaemic nephropathy is most commonly seen in patients with tumor lysis syndrome, but can also be associated with the Lesch–Nyhan syndrome of complete hypoxanthine-guanine phosphoribosyltransferase (HGPT) deficiency. Chronic ‘gouty’ nephropathy, characterized by interstitial tophi of sodium urate, is now an unusual diagnosis in primary hyperuricaemia. It is unclear why this diagnosis appears to have been more common in the past, but one explanation is that chronic renal impairment was actually due to the effects of vascular disease associated with gout. Early observations from family and twin studies suggested that hyperuricaemia has a genetic component, although evidence that this represented the effect of a major gene for uric acid has been disputed. (Gulbrandsen et al. 1979; Morton 1979; Rao et al. 1982). Two subsequent family studies concluded that hyperuricaemia is multi-factorial, representing a complex interaction between environmental factors and multiple modifying genes (Friedlander et al. 1988; Rice et al. 1992) A more recent study, which analysed serum uric acid measurements from 523 families enrolled in the National Heart, Lung and Blood Institute Family Heart Study, also provided data to support a multi-factorial, polygenic aetiology with a hereditary component estimated at approximately 40 per cent (Wilk et al. 2000).


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There are a number of monogenic inherited disorders of purine metabolism and transport that can give rise to renal abnormalities (Cameron et al. 1998). These include HGPT deficiency, phosphoribosyl pyrophosphate synthetase (PRPS) superactivity, adenine phosphoribosyltransferase (APRT) deficiency, xanthine dehydrogenase deficiency and isolated renal hypouricaemia. Of particular interest is familial juvenile hyperuricaemic nephropathy (FJHN), which often presents with renal failure in childhood or early adult life. FJHN is probably the commonest inherited cause of uric acid associated renal failure, although it is often unrecognized. Histologically, the affected kidney shows focal areas of tubular atrophy and fibrosis, with occasional inflammatory infiltrates and globally sclerosed glomeruli. Recently, Kamatani et al. (2000) localized a gene for FJHN to chromosome 16p12 through genomewide linkage analysis of a single large family with at least 20 affected members. The linked region was refined to an interval of approximately 9 cM that includes the gene encoding the epithelial sodium channel, a defect of which causes Liddle syndrome. Although PKD1, the gene responsible for autosomal dominant polycystic kidney disease, is also located on the short arm of chromosome 16, it does not lie within the candidate region and has been excluded as the FJHN gene in a previous independent study (Cameron et al. 1998).

Conclusions and future directions Progress towards identifying genes involved in polygenic renal disorders has been slow. The existing literature is littered with case–control association studies, but too often these have studied small populations and have been inadequately controlled. As a result, their conclusions must be treated with caution, and only the HLA region can confidently be said to contain susceptibility genes. There are persuasive reasons to believe that genes of the RAS, which have also been extensively studied, have a role in disorders such as diabetic nephropathy, but even here the body of evidence is still not conclusive. Relatively few genome-wide linkage studies have been performed, and when they have (such as in SLE and essential hypertension), they have typically not produced reproducible susceptibility loci. Nevertheless, there are reasons to believe that significant advances can be made. Further genome-wide linkage studies should be encouraged, but populations must be large and international collaboration may be required to achieve this (Cox et al. 2001). Better use must be made of family-based association studies, which can be better controlled through statistical approaches such as the transmission disequilibrium test (Cardon and Bell 2001). Recent advances have greatly increased the number of potential candidate genes for such studies, a trend that will continue during the next few years. First, the explosion in genomic and cDNA sequencing has meant that the sequence of most human genes is now available. Even although the function of many of these genes is currently unknown, this will change. Second, the study of monogenic renal disorders is identifying new genes that could potentially play a role in the development of polygenic renal disease, and these will increasingly become the subject of future association studies. Third, as new patho-physiological mechanisms are elucidated as a result of these discoveries, other candidate genes will be identified. It is also probable that certain susceptibility genes will have common effects in different disorders. However, the major challenge for the renal community is to establish appropriate DNA collections. With this aim, the United Kingdom’s Medical Research Council and National Kidney Research Fund has recently funded a project within the UK to establish national DNA

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20 Wilms tumour and the Wilms tumour predisposition syndromes Richard Grundy

The molecular genetics of Wilms tumour (WT) has turned out to be far more complex and interesting than originally envisaged (Knudson and Strong 1972). It is now clear that at least six separate genetic loci and loss of genomic imprinting are involved in the predisposition, development, and progression of this fascinating tumour. Epidemiology Wilms tumour is the most common primary renal tumour of childhood affecting approximately 1 in 10,000 children (Birch and Breslow 1995). Overall, there is a three- to four-fold variation in the world-wide incidence of WT, compared to 10–20-fold variation seen for adult cancers suggesting that racial/genetic factors are more important than environmental factors in the aetiology of WT (Birch and Breslow 1995). An analysis of over 1800 children with WT has revealed that these patients have a significantly higher birth weight than the general population. This has been taken to suggest an effect for prenatal growth factors on the subsequent development of WT (Leisenring et al. 1994). Most children present with unilateral WT, only 5–10 per cent have bilateral disease at diagnosis. The mean age of incidence of sporadic unilateral disease is 44 months, whilst it is 32 months for bilateral disease with 90 per cent of all WT occurring before 9 years of age (Breslow et al. 1988). The earlier age of onset of bilateral WT and the occurrence of familial WT led Knudson to propose the 2-hit mutational model (Knudson and Strong 1972). Essentially, this model proposed that WT arises from two rate-limiting mutational events, the first of which may be pre-zygotic, whilst the second is always post-zygotic. Patients carrying a germline mutation are more likely to acquire a second mutation in a time-dependent manner resulting in earlier onset and bilateral disease. In sporadic cases both mutations have to occur within the kidney cell in order to cause a WT. The tumour occurs following the loss of both alleles of a recessively acting gene whose normal function is to suppress tumour formation (Comings 1973). The 2-hit hypothesis predicts that children with an inherited predisposition to cancer would present earlier than sporadic cases and are more likely to have bilateral disease at diagnosis. Histology Although Max Wilms was not the first person to describe this tumour, his careful and prescient description has ensured that his contribution remains recognized (Wilms 1899).

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Arising in the developing kidney from mesenchymal stem cells that would normally differentiate into the epithelial components of the nephron, WT is composed of a disorganized array of blastemal, epithelial, and stromal elements (Pritchard-Jones and Fleming 1991). When all three elements are present the tumour is termed triphasic. The histological resemblance of WT to the fetal kidney provides a striking example of the relationship between abnormal embryogenesis and cancer. The presence of muscle and other elements in approximately 10 per cent of tumours suggests that WT arises from an immortalized early pluripotential stem cell. Nephrogenic rests Normal kidney development is usually complete by 36 weeks gestation, foci of abnormally persistent blastemal-derived cells are known as ‘nephrogenic rests’ (NR). These cells can be found in approximately 1 per cent of all infant post-mortems, in 30–40 per cent of kidneys removed due to WT and within the kidneys of almost all children with an inherited susceptibility to WT (Beckwith 1993). Two distinct patterns of rests are appreciated ‘intralobar’ nephrogenic rests (ILNR) located deep in the kidney and ‘perilobar’ nephrogenic rests (PLNR) which occur more peripherally. The presence of multiple, or diffuse, NR of either type occurring in one or both kidneys is termed nephroblastomatosis. NRs have a variable natural history, they may regress, persist, or hypertrophy. These lesions are thought to represent developmental insults to the kidney, the timing of which determines their location in the kidney, for example, germline mutations resulting in ILNRs (Beckwith 1993). NRs are considered to be precursors of WT although this remains to be proven. Treatment At the turn of the century WT was almost incurable. The overall cure rate for WT has now risen to over 85 per cent, a consequence of consistent improvement in treatment strategies based on the collaborative efforts of the multidisciplinary childhood cancer teams through National and International trials (D’Angio et al. 1989; Pritchard et al. 1995). Refinement of therapy, such that only those at highest risk of relapse receive intensive therapy, has minimized both the acute and long-term toxicities of treatment (Green et al. 1995). The challenge now is to improve the cure rate and further reduce late effects, best achieved by increasing our understanding of the molecular pathogenesis of WT. Cytogenetics of WT It is now generally accepted that malignant tumours are the end result of a series of accumulated genetic alterations from a wide variety of approaches that result in altered gene function. The identification and characterization of constitutional and/or tumour specific chromosomal rearrangements provide strong clues indicating the location of gene which may be involved in the malignant process. Karyotypic findings have now been reported for over 100 WTs and the results are summarized in Table 20.1. Overall, chromosomal gains are more common than losses and trisomy has been reported for chromosomes 1, 3, 7, 8, and 12 whereas losses were found on chromosomes 1p, 7p, 11p, and 16q. The most common structural chromosomal abnormality in WTs involves the short arm of chromosome 11. Of 115 WTs, this chromosomal abnormality was

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Table 20.1 Compilation of cytogenetic rearrangements reported in WT from the literature with the percentage of the different rearrangements calculated as a percentage of the whole series Chromosome

Cytogenetic rearrangements (%)

Specific aberrations (% of the whole series)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

33 8 18 6 5 20 23 19 15 17 35 35 10 10 4 30

dup 1q21–qter (16%)

17 18

16 15

Total trisomy (8%) Total trisomy (11%) Total trisomy, dup (7q) (14%) Total trisomy (15%) Total trisomy (10%) Total trisomy (9%) del (11p) (20%) Total trisomy (26%)

Total monosomy del (16q) (10%) Total trisomy (8%) Total trisomy (13%)

noted in 25 per cent of cases (Slater 1986; Slater and Mannens 1992). In all cases studied the abnormality, usually a deletion, involves the short arm of chromosome 11. Involvement of both the long and short arms of chromosome 1 have also been reported, the critical regions being 1p31–36 and 1q21. Cytogenetic abnormalities of chromosome 16 have been reported in 30 per cent of WTs and predominantly involve the long arm (Slater 1986; Austruy et al. 1995). Two regions of interest have been identified from these studies, a proximal locus at 16q13 and a more distal region involving 16q24–qter (Slater 1986; Solis et al. 1988; Kaneko et al. 1991; Matthew et al. 1996). A recent, retrospective analysis of a group of patients with WT and der (16) t (1;16) (q21;q13) has provided evidence that this particular cytogenetic abnormality does not necessarily confer an adverse prognosis (Matthew et al. 1996). Abnormalities of chromosome 7 have been reported in 23 per cent of WTs and suggest the presence of a tumour suppressor gene at 7p15 (Sawyer et al. 1993; Wilmore et al. 1994; Miozzo et al. 1996). Chromosomal breakpoints on the short arm of chromosome 17 raise the possibility of involvement of the TP53 tumour suppressor gene (Slater and Mannens 1992). Although cytogenetic studies have been invaluable in identifying the chromosomes involved in Wilms tumorigenesis, the relevant changes need to be relatively large to be detected by conventional karyotypic analysis. Comparative genomic hybridization (CGH), enables a whole genome screen based on the competitive hybridization of differently labelled tumour and constitutional DNA to normal metaphase chromosomes (Kallioniemi et al. 1992). The application of this technique to a panel of sporadic WT has shown losses of 5p and 15q and gains of chromosome 3 in addition to the findings of conventional cytogenetics (Getman et al. 1998).

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The molecular genetics of WT Loss of heterozygosity studies on the short arm of chromosome 11 Tumour-specific allelic loss has been thought to represent the second ‘hit’ resulting in the inactivation of a tumour suppressor gene and can be detected by the loss of polymorphic markers in the tumour compared to constitutional DNA (Cavenee et al. 1983). Loss of Heterozygosity (LOH) studies for 11p demonstrated allele loss in 15–20 per cent of sporadic WT consistent with the presence of a tumour suppressor gene at this locus (Reeve et al. 1989; Wadey et al. 1990). Isolation and cloning of WT1 Three lines of evidence identified 11p13 as the region likely to contain a gene involved in the development of WT, (a) the identification of WAGR deletions (Riccardi et al. 1980) (b) interstitial deletions of 11p13 in sporadic WTs (Slater 1986), and (c) loss of heterozygosity in tumour DNA for polymorphisms present in the constitutional DNA (Fearon et al. 1984; Reeve et al. 1984). WT1 was subsequently isolated from 11p13 by positional cloning (Call et al. 1990; Gessler et al. 1990). WT1 expression studies In situ hybridization studies have shown that WT1 is expressed in a very specific set of tissues during embryogenesis, namely the glomerular-precursor cells of the fetal kidney, stromal cells of the gonads and spleen and the mesothelium lining the heart, pleural and peritoneal cavities (Pritchard-Jones et al. 1990; Armstrong et al. 1993). In the developing kidney, WT1 expression is first seen in the metanephric mesenchyme increasing following reciprocal induction with the ureteric bud—WT1 expression is up-regulated as the mesenchyme condenses and undergoes transition to epithelium. WT1 expression continues in the comma and S-shaped bodies of the developing glomeruli. In the mature nephron, expression is confined to the podocytes whose role is to maintain the integrity of the glomerular filtration barrier (Pritchard-Jones et al. 1990). WT1 is expressed in most WTs reflecting aberrant differentiation, triphasic tumours express the highest levels of WT1 (Pritchard-Jones et al. 1990). The critical role of WT1 function in urogenital development was demonstrated by the WT1 ‘knockout’ mouse-model in which homozygous inactivation of WT1 causes absent kidneys and malformation of the gonads (Kreidberg et al. 1993). Mice heterozygous for WT1 deletion, mimicking patients with WAGR syndrome, showed no renal or genital abnormalities nor did they develop renal tumours (Kreidberg et al. 1993). The interaction of genes involved in renal development is complex and incompletely understood but is critical to our understanding of how disruption of specific genes gives rise to WT. The WT1 gene WT1 contains 10 exons covering approximately 50 kb and encodes a nuclear protein with four zinc fingers that binds DNA (Fig. 20.1). WT1 is thought to function as a transcription regulator (Rauscher 1993). Alternative splicing of the gene at two sites results in four different zinc finger protein isoforms with molecular weights of between 52 and 54 kDa (Haber 1991). Splicing at the first site results in either inclusion or exclusion of exon 5 encoding


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Zinc finger DNA binding domain

Proline-glutamine rich trans-regualtory region

Zn-1 C C

Zn

Zn-2 H H

C C

Zn

ZnH H

C C

Zn

Zn-4 C C

H H

Zn

H H

KTS

Alternative splice 1 (+/– 17 amino acids)

Amino terminus

Alternative splice 2 (+/– KTS)

5⬘ Carboxyl terminus Fig. 20.1 A diagrammatic representation of the WT1 protein.

17 amino acids and at the second, the inclusion or exclusion of three amino acids lysine, threonine and serine (KTS) resulting in either or KTS isoforms. This is highly conserved throughout evolution and is thought to be biologically significant. In the normal situation approximately 2– 4 times as many KTS isoforms are produced as those which lack the KTS insert (KTS). The possibility of RNA editing and the use of alternative initiation codon increases the number of potential isoforms to 16 (Breuning and Pelletier 1996). The precise ratio of the WT1 isoforms appears to be crucial for normal gene function. Accumulating evidence supports a regulatory role for WT1 in genitourinary development, though little is known about which gene cascades may be affected by its abnormal function. Numerous genes have been proposed as targets for WT1 from in vitro transfection experiments, but the physiological relevance of these observations remains unclear (Rauscher 1993). The presence of an evolutionary conserved RNA recognition motif (RRM) suggests that WT1 may also bind RNA (Kennedy et al. 1996). Furthermore, WT1 co-localizes with elements of the nuclear splicing machinery suggesting that WT1 has a role in mRNA processing (Larsson et al. 1995). These findings add another layer of complexity to WT1 function, but the physiological relevance remains to be proven. WT1 mutations in WT When WT1 was cloned in 1990 it was assumed that the molecular genetics of WT would soon be understood. However, almost a decade later we still have much to learn. Exhaustive molecular analysis has revealed that only 10–15 per cent of sporadic WT harbour WT1 mutations (Brown et al. 1993; Gessler et al. 1994; Varanasi et al. 1994), suggesting that genes other than WT1 are likely to be important in the genesis of the majority of WT in children. Germline mutations of WT1 are found in less than 5 per cent of children with WT and are essentially restricted to those with either bilateral disease or urogenital abnormalities (Li et al. 1996). It thus appears that WT1 has a number of different roles in health and disease and that its function is far more complex than simply that of a ‘tumour suppressor’ gene.

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11p15 and WT Although LOH was seen at 11p13 in a subset of WT, in the majority of these cases allele loss extended to the 11p15 region (Reeve et al. 1989; Wadey et al. 1990). In some tumours LOH occurred exclusively in 11p15 suggesting the presence of a second WT suppressor gene, WT2. Overall, approximately 40 per cent of WT show loss of 11p alleles. Somewhat surprisingly, in tumours showing allele loss for 11p15 it was the maternal allele that was preferentially lost suggesting a parent-of-origin effect (Schroeder et al. 1987). The differential expression of the maternal or paternal allele of a gene in the somatic cells of an offspring occurring due to an epigenetic modification of the gamete or zygote is known as genomic imprinting (Monk 1988). Imprinting is reversible and usually confers monoallelic expression of a specific parental gene. The mechanism of imprinting is still unknown but is thought to result from differential methylation of male and female germ cells (Reik et al. 1987). The preferential loss of the maternal allele in WTs suggested a role for imprinting and was consistent with either the loss of tumour suppressor genes or the gain of growth promoting genes. LOH for 11p15 has been shown in other embryonal and adult cancers (Fearon et al. 1985; Scrable et al. 1989; Albrecht et al. 1994), suggesting that this region contained multiple tumour suppressor genes or genes fundamental to the neoplastic process. Evidence for the existence of a tumour suppressor gene in 11p15 came from the study of sub-chromosomal transferable fragments, 2–5 Mb in size, some of which when transferred in to a rhabdomyosarcoma cell line suppressed growth (Koi et al. 1993). From these studies a putative suppressor gene was localized to a 4 Mb region on 11p15 distal to IGF2 (Koi et al. 1993).

Insulin-like growth factor II and WT Several lines of evidence implicate Insulin-like growth factor II (IGF2) in Wilms tumorigenesis. IGF2 is a potent fetal mitogen perhaps explaining the increased birth weight of children with WT and the association of the overgrowth syndromes. IGF2 mRNA is expressed in the developing kidney being over expressed in some, but not all, WT (Scott et al. 1985) and the gene for IGF2 was mapped to 11p15 consistent with the LOH findings (Koufos et al. 1989). Insulin-like growth factor II is an imprinted gene being transcribed exclusively from the paternal allele (Giannoukakis et al. 1993). In order to test whether the normally silent maternal IGF2 allele was activated in WT a series of tumours were analysed by reverse-transcriptase PCR using a transcribed polymorphism in the IGF2 gene. Over 70 per cent of tumours that retained heterozygosity for 11p15, therefore 44 per cent of all WT, expressed both IGF2 alleles presumably resulting in a double-dose of a growth promoting gene (Ogawa et al. 1993; Rainer et al. 1993). This phenomenon, which is the most frequent genetic abnormality so far discovered in WT, has been termed ‘loss of imprinting (LOI)’. However, it is not yet clear whether the allele-specific information that usually maintains the imprint is lost, overridden or altered. This finding raised the possibility that the normal function of imprinting is to repress the expression of one copy of a growth-promoting gene. Over expression of this gene, is then a crucial step on the path to tumorigenesis. A second imprinted gene, H19, maps within 200 Kb of IGF2 and is imprinted in the opposite direction being expressed only from the maternal allele (Zhang et al. 1993). Biallelic IGF2


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expression results in a marked down-regulation of H19 expression (Steenman et al. 1994). Tumours with LOH for 11p15 also showed low levels of H19 expression, consistent with the loss of maternal alleles. This observation suggested that LOI and LOH may share a ‘final common pathway’, reduction of H19 expression. In mouse studies, the paternal allele is methylated while the maternal H19 gene is not. Methylation analysis in tumours with LOI show acquisition of the paternal methylation pattern by the maternal allele (Steenman et al. 1994), consistent with the altered expression of the respective genes in the tumours with LOI. No change in methylation status was noted in those tumours that maintained the normal imprint. These findings add weight to the proposed role of imprinting in cancer and were consistent with the enhancer–competitor model in mice in which H19 and IGF2 act a functional pair (Bartolomei et al. 1993). There are, however, some findings which question a central role of IGF2 in Wilms tumorigenesis. If LOI results in tumorigenesis, the level of IGF2 expression should be considerably higher in those tumours with LOI than those tumours that maintained the normal imprint of this gene. Although an increase in IGF2 expression has been observed in tumours with LOI, increased IGF2 expression was also seen in some tumours that maintained the normal imprint (Steenman et al. 1994). The lack of evidence for increased IGF2 expression and increased IGF2 protein levels in WTs questions the role and the significance of IGF2 and LOI in Wilms tumorigenesis. Furthermore, not all WT show LOI for IGF2 or LOH for 11p15 suggesting that LOI alone is insufficient to cause cancer but that other time and tissue dependent events have to occur as well. IGF2 expression decreases as the kidney differentiates and the continued expression of IGF2 in WT is most marked in those that are blastemal predominant perhaps reflecting the continued presence of undifferentiated blastema rather than the action of permanently activated transforming gene (Brice et al. 1989; Wilkins et al. 1989). The recognition that normal tissue adjacent to WT shows LOI for IGF2 and biallelic H19 methylation suggests that the epigenetic error occurred early in development before the onset of WT and that other events are necessary for tumorigenesis (Okamoto et al. 1997). More recently the pattern of imprinting in WT has turned out to be surprisingly complex with IGF2 being expressed monoallelically from opposite parental alleles both within the tumour and in nephrogenic rests (Ohlson et al. 1999). The question of whether imprinting in WT is central to tumorigenesis or a bystander effect remains to be answered. Interestingly, imprinting at 11p15 has been reported in a number of other tumour types suggesting that LOI may be a general mechanism leading to tumorigenesis rather than a specific phenomenon in WT. Indeed, although over-expression of IGF2 in mouse models revealed an increase in the occurrence of various malignancies (Rogler et al. 1994), the long latent period and formation of diverse tumour types suggests that if IGF2 has a role in oncogenesis it is more likely to act as a ‘progression’ factor than an initiator. In summary, biallelic IGF2 expression may simply result in an increased pool of tumour precursor cells, other genetic and/or epigenetic events are then required for neoplastic development. H19 remains something of an enigma. First, there is no precedent for an untranslated RNA to act as a tumour suppressor gene or as a controller of cell growth. Second, a number of adult cancers over express H19, and mice lacking H19 do not form tumours both observations shed doubt on a tumour suppressor role for H19 (Bartolomei et al. 1991; Ariel et al. 1995; Kondo et al. 1995; Leighton et al. 1995). Final proof for a crucial role for H19 and IGF2 in tumorigenesis depends on finding specific sequence abnormalities in these genes or the genes that control these genes in WTs.

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Chromosomes 16q, 1p and WT Molecular studies have found LOH for polymorphic markers on 16q in around 20 per cent and for 1p in 12 per cent of WTs (Maw et al. 1986; Coppes et al. 1992; Grundy et al. 1994; Austruy et al. 1995; Newsham et al. 1995; Grundy et al. 1998c). A retrospective molecular analysis of patients treated on the American National WT Study Group trials 3 and 4 (NWTS 3 and 4), has shown that LOH for 16q markers confers an adverse prognosis irrespective of the stage or histological features of the tumour (Grundy et al. 1994). Although tumours with LOH for 1p were found to have an increased risk of relapse this was not statistically significant (Grundy et al. 1994). A recent molecular analysis of a series of sporadic tumours suggests the involvement of two distinct regions of 16q in Wilms tumorigenesis, a distal region (LOH 1) bounded by D16S518 in 16q23.2–qter and a proximal region (LOH 2) bounded by D16S419 in 16q13 and D16S514 in 16q21 (Grundy et al. 1998c). This finding is consistent with both cytogenetic analysis as well as studies of allelic loss in WT by other investigators (Slater et al. 1985; Maw et al. 1986; Solis et al. 1988; Coppes et al. 1992; Grundy et al. 1994; Austruy et al. 1995; Newsham et al. 1995; Matthew et al. 1996). The exact location(s) of the gene or genes involved, however, is not yet clear. The analysis of LOH for 16q and 1p therefore needs to be extended in order to determine the location of the critical genes and to clarify the relationship between molecular abnormalities and patient outcome. Chromosome 7p and WT Of sporadic WT 10–25 per cent show LOH for markers on 7p (Wilmore et al. 1994; Miozzo et al. 1996; Grundy et al. 1998b). Other supporting evidence is provided by a molecular study of a patient with a WT and bilateral nephrogenic rests (Wilmore et al. 1994) with nephrogenic rests being considered, by some, to be precursor lesions for WT (Beckwith 1993). This patient had a constitutional t(1;7) translocation interrupting the 7p15 region (Hewitt et al. 1991). Cytogenetic analysis of the tumour revealed the retention of the translocation and an isochromosome of the long arm of 7 (Wilmore et al. 1994). It is possible that the constitutional translocation breakpoint interrupts a critical gene on 7p15 representing the first hit, the remaining normal copy of the gene at 7p15 is then lost in the tumour resulting in the inactivation of both copies of a tumour suppressor gene at this locus. The most recent study identified a common region of allelic loss between loci D7S517 and D7S503, within which there was evidence for homozygous deletion in one tumour (Grundy et al. 1998b). These findings define a critical region in 7p15–21 in which a putative gene(s) involved in the genesis of WT should exist. Molecular genetics of nephrogenic rests Molecular analysis of nephrogenic rests has shown that LOH for 11p and mutations of WT1 are an early event and that allelic loss is less common in PLNR than ILNR. LOH for 16q alleles present in 17 per cent of the WT was not found in NRs consistent with the hypothesis that this genetic abnormality is a late event related to tumour progression (Charles et al. 1998). TP53 and WT The p53 tumour suppressor gene is located at chromosome 17p13 and mutations in this gene are one of the commonest somatic genetic changes identified in human adult cancers


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(Lane 1994). Co-immunoprecipitation studies have shown that WT1 interacts with p53 the ubiquitously-expressed tumour suppressor gene (Maheswaran et al. 1993) In the absence of wild-type p53, both plus and minus KTS isoforms of WT1 will activate transcription from the EGR promoter, yet, in its presence WT1 functions as a repressor. Some mutant p53 proteins associate with WT1 but do not alter its transcriptional properties (Maheswaran et al. 1993). TP53 mutations have been detected in most but not all anaplastic WTs, suggesting that TP53 mutations, are necessary but not essential for anaplasia (Bardeesy et al. 1994). The demonstration of attenuated apoptosis in anaplastic WTs with p53 mutations suggest that mutated p53 gives these clones a selective advantage resulting in drug resistance disease thereby decreasing the likelihood of cure (Bardeesy et al. 1994). Familial WT Wilms tumour affecting multiple family members is rare. Estimates of the incidence vary from 1 to 2.4 per cent depending upon the completeness of ascertainment and whether second or higher degree relatives are included (Bonaiti-Pellie et al. 1992; Breslow et al. 1996). Although inheritance is clearly dominant in some families other pedigrees suggest variable penetrance (Breslow et al. 1996). In the NWTS group survey bilateral disease was noted in 16 per cent of familial cases with median age of onset of 16 months of age, unilateral disease also presented earlier at 25 months (Breslow et al. 1996). However, another study found no increase in bilateral disease or evidence for an earlier presentation (Rahman et al. 1998). ILNR were found twice as frequently in familial tumours but the clinicopathologic stage and outcome were the same for sporadic and familial cases. Interestingly, the finding of bilateral or metastatic disease tended to cluster in families. Evidence for a Familial WT (FWT1) gene on chromosome 17q12–21 came from linkage analysis in a large French-Canadian family (Rahman et al. 1996). Analysis of further families confirmed the original findings, but also revealed genetic heterogeneity as 3 families showed evidence against linkage to FWT1, one of which carried germline mutations in WT1 (Rahman et al. 1998). A more recent analysis has revealed a second FWT gene (FWT2) at 19q13.3–q13.4, confirming the heterogeneous nature of FWT (McDonald et al. 1998). It would be interesting to see whether FWT2 is implicated in the two families reported by Rahman et al. (1998) that did not show linkage to 17q or whether other familial Wilms genes exist. Cancer-susceptibility genes usually act recessively at the cellular level requiring the loss of both alleles which can be detected by LOH analysis. LOH for 17q and 19q is rare in sporadic WT questioning the mode of action of the FWT genes. Clinical phenotypes associated with WT Although most WT are sporadic, the molecular characterization of a number of rare syndromes associated with an increased risk of developing WT has greatly increased our knowledge of the molecular pathology of this malignancy. Moreover, these studies have had a wider impact providing insight into the aetiology of cancer and of a novel epigenetic phenomenon, imprinting. Congenital anomalies are found in 13.75 per cent of patients with WT and are twice as common in children with bilateral tumours as those with unilateral tumours (Breslow and Beckwith 1982). The clinical phenotypes can be grouped into five categories shown in Table 20.2.

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Richard Grundy Table 20.2 Clinical phenotypes associated with WT Aniridia Sporadic aniridia WAGR syndrome Genitourinary Abnormalities Cryptorchidism/hypospadias Horseshoe kidney Denys–Drash syndrome (DDS) Overgrowth disorders Hemihypertrophy (HH) Beckwith–Wiedemann syndrome (BWS) Perlmann syndrome (PS) Sotos syndrome Simpson–Golabi–Behmel syndrome (SGBs) Familial Familial Wilms tumour (WT) Others Hamartomatous disorders i.e. Neurofibromatosis Trisomy 18 Turner syndrome (45,X)

Aniridia (AN) Aniridia is a congenital abnormality of the eye in which there is almost complete, bilateral absence of the iris. This condition occurs either sporadically or in a familial form with autosomal dominant inheritance but variable expressivity. Hereditary AN represents 66 per cent of all cases (Breslow and Beckwith 1982; Nelson et al. 1984). The sporadic type of AN is associated with specific congenital abnormalities and a greatly increased risk of developing WT (Miller et al. 1964). The median age at onset of WT in patients with aniridia is 28 months, considerably younger than the general WT population (Breslow et al. 1988). WAGR syndrome The congenital abnormalities associated with sporadic aniridia include mental retardation and genitourinary abnormalities, an association known as the AGR triad. These patients have a 50 per cent chance of developing WT, 20 per cent of which are bilateral (Narahara et al. 1984). The full phenotype is termed the WAGR syndrome. Cytogenetics of the WT predisposition syndromes Constitutional cytogenetic studies in children with the WAGR syndrome revealed interstitial deletions of variable length but always involving 11p13 (Riccardi et al. 1980). The location and length of the deletion determined the phenotype suggesting that the genes that control the formation of the eye, kidney, and urogenital tract lie in close proximity. Molecular pathology of the WAGR syndrome WT1 mutations were found in the majority of WT from children with the WAGR syndrome, most resulting in either frameshifts causing premature termination, or amino acid substitutions


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predicted to affect the function of the WT1 protein (Baird et al. 1992; Brown et al. 1992; Santos et al. 1993; Gessler et al. 1994; Huff et al. 1995; Nordenskjold et al. 1995). Mutations in exon 1 have also been reported (Huff et al. 1995). Congenital genitourinary abnormalities Of patients with WT 4–8 per cent have a congenital anomaly of the genitourinary system (Breslow and Beckwith 1982). The reported incidence of cryptorchidism, hypospadias, and other genitourinary anomalies in patients with WT was 27.8, 17.8, and 2.1 per 1000 cases, respectively. This represents a two-fold increase over the rate in the general population (Breslow and Beckwith 1982). The increased incidence of genitourinary abnormalities in children with WT suggests that the gene(s) for WT act(s) pleiotropically, playing a role in both the development of the gonadal and of the renal systems. Horseshoe kidney As its name implies, horseshoe kidney is a fusion of the left and right kidneys most frequently at their lower pole. This abnormality is though to arise from the union of both metanephri at around 6 weeks gestation. The incidence of horseshoe kidney among patients with WT is slightly higher than reported in the general population (Mesrobian et al. 1985). Denys–Drash syndrome (DDS) Denys–Drash syndrome is a rare congenital syndrome comprising nephropathy, ambiguous genitalia, and a predisposition to WT (Denys et al. 1967; Drash et al. 1970). Nephropathy is now generally accepted as the common denominator of this syndrome and is clinically characterized by the presence of proteinuria at an early age (Jadresic et al. 1990). In most patients the nephropathy progresses to nephrotic syndrome and to end stage renal failure by 3 years of age (Denys et al. 1967; Drash et al. 1970; Habib et al. 1985; Jadresic et al. 1990). The most consistent histological features include hypertrophy of the podocyte layer of the glomerulus and a varying degree of focal or diffuse mesangial sclerosis (Habib et al. 1985). The spectrum of intersex disorders in DDS is considerable. In children with an XY karyotype, anomalies of the external genitalia range from penoscrotal hypospadias with bilateral cryptorchidism to an enlarged ‘clitoris’ with ‘labia’ that are almost fused and the presence of a urogenital sinus. In those children with an XX karyotype, the external genitalia are usually outwardly normal but the internal reproductive organs may be affected. Here the phenotypic spectrum varies from a small atrophic vagina to severe ovarian dysgenesis or ‘streak ovaries’. Patients with a 46, XY karyotype may also have internal anomalies including dysgenetic testes (Jadresic et al. 1990). These malformations reflect an underlying defect of the processes involved in normal embryonal genital differentiation and development. The mean age of diagnosis of WT in patients with DDS is 18 months and the incidence of bilateral WT is 35 per cent, three times higher than that found among patients with sporadic disease (Jadresic et al. 1990; Coppes et al. 1993). The histological features of WT in this disorder are no different from those described in sporadic WT, though 78 per cent of patients with DDS have intralobar nephrogenic rests compared to 15 per cent of patients with sporadic unilateral disease (Beckwith 1993).

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Frasier syndrome (FS) Chronic renal failure, XY gonadal dysgenesis, streak gonads and gonadoblastoma are the main characteristics of this syndrome (Frasier et al. 1964). There are similarities between FS and DDS, and some patients may have been misdiagnosed in the past. However, both the onset and course of renal failure in FS occurs later and progresses more slowly than in DDS. Although renal histology is varied in FS diffuse mesangial sclerosis is not seen. Gonadoblastomas arising in the dysgenetic gonad have been reported. Genetics Denys – Drash syndrome A wide variety of constitutional WT1 gene mutations are seen in DDS resulting in the disruption or inactivation of DNA binding by the zinc fingers of the WT1 protein (Fig. 20.1) (Little and Wells 1997a). Nearly half the patients with DDS have missense mutations affecting a critical arginine residue in zinc finger 3 (394Arg) and collectively missense mutations in zinc fingers 2 and 3 account for 80 per cent of DDS mutations (Little and Wells 1997a). It is now recognized that these mutations affect amino acids critical for the stability of DNA binding of the zinc fingers (Rauscher 1993). As these mutations were only detected in one allele of the WT1 gene it was initially thought that DDS mutations were acting in a dominant manner. However, in a few DDS patients WT1 mutations either within or upstream of the zinc finger region produced a truncated non-functional protein suggesting that the mutant protein that results may behave in a dominant-negative fashion, somehow interfering with the function of the normal protein produced from the remaining allele (Little and Wells 1997b). In summary, the general effect of DDS mutations is to produce an abnormal protein with abnormal functions resulting in severely impaired kidney function, varying degrees of gonadal abnormality and loss of tumour suppressor capabilities. How WT1 mutations result in nephropathy characteristically found in DDS is not known. Such effects may be mediated via aberrant DNA, RNA or protein interactions perhaps during the progression of nephrogenesis or alternatively in the terminally differentiated podocytes. Molecular analysis of WTs arising in patients with DDS has revealed that most, but not all, tumours have lost the remaining normal WT1 allele, consistent with the ‘2 hit-hypothesis’. However, the same WT1 mutation results in the formation of WT in some DDS patients but not others, raising the possibility that other genetic events are necessary. Frasier syndrome By contrast to DDS, FS is caused by very specific WT1 mutations that disrupt splicing at the second alternative splice donor site (Klamt et al. 1998). Mutations are present in intron 9 of the WT1 gene and result in deficiency of the usually more abundant KTS isoforms and reversal of the normal / KTS ratio from 2 : 1 to 1 : 2 (Barbaux et al. 1997; Kikuchi et al. 1998; Klamt et al. 1998). As the WT1 protein produced in FS is normal with normal binding abilities the effect of these mutations highlights the importance of precisely balanced expression of WT1 isoforms for normal function. This has a significant impact on tumour risk as FS patients have one normal copy of WT1 and one that can only produce the KTS isoform. Mutational inactivation leads to cells that cannot produce the KTS isoform of WT1, but still produce the KTS isoform. It is interesting that in the experimental situation, the tumorogenicity of the


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G401 WT cell line in nude mice can be suppressed by both and KTS isoforms to the same extent (McMaster et al. 1995). This might explain why patients with FS do not develop WT. Of note is that the three patients reported with DDS and WT1 intron 9 mutations more commonly associated with FS did not develop WT. The high frequency of gonadoblastoma in FS may be due to the obligatory presence of dysgenetic gonads which carry a higher risk of tumorigenesis. However, there may also be differences in the stage at which gonadal development is halted in FS and DDS which may potentially increase the tumour risk. The molecular pathology of the intersex-state in DDS and FS In males, steroidogenic factor 1 (SF1) participates in sexual development by regulating expression of the polypeptide hormone Mullerian inhibiting substance (MIS). WT1 isoforms lacking a KTS insert (KTS) associate and synergize with SF1 to promote MIS expression (Little and Wells 1997b; Nachtigal et al. 1998). Furthermore, DAX-1, a gene thought to direct ovarian development, antagonizes this synergy. By contrast, if WT1 mutations characteristically seen in DDS are introduced, the resultant WT1 protein and SF1 fail to associate and synergize, functional opposition of the DAX-1 gene no longer occurs and testicular development fails. The severity of the genital phenotype is thus dependent on the degree to which WT1/SF-1 binding is disrupted. As different mutations might be expected to have different effects on the synergistic action of the protein cascade, this may explain why such a wide variety of genital phenotypes is seen in patients with DDS and a 46 XY karyotype. Interestingly, since an intact WT1 gene is not an absolute requisite for female gonadal development, individuals with DDS and a 46 XX karyotype usually have no gonadal abnormality. The molecular mechanisms underlying the intersex state in 46 XY individuals with FS is more difficult to explain than in DDS as KTS isoforms only have a limited ability to activate SF1 (Nachtigal et al. 1998). WT1 isoforms may be able to take over each other’s functions in certain situations, so a deficiency of the usually more abundant KTS isoform may in turn causes a relative deficiency of the KTS isoform while it takes over essential KTS functions. Alternatively, the KTS may participate in another pathway required for normal male urogenital development. Individuals with a 46 XX karyotype and FS nephropathy have normal gonadal development (Kikuchi et al. 1998), affirming the less critical role of WT1 in normal female gonadal development. Again the cause of the nephropathy at a molecular level in FS is the poorly understood. DDS and FS provide a model for the relevance of molecular genetics to clinical practice particularly as we move toward a taxonomy of disease based on molecular abnormality rather than phenotype (Koziel and Grundy 1999). Hemihypertrophy The association between hemihypertrophy/ hemihyperplasia (HH) and WT suggests that a growth-factor excess may be important in the pathogenesis of both conditions (Fraumeni et al. 1967; Leisenring et al. 1994). The incidence of HH among patients with WT is around 3 per cent whilst its incidence in the general population is only 0.03 per cent (Breslow and Beckwith 1982; Pastore et al. 1988). However, the age at diagnosis of WT in children with isolated HH is no different from that for unilateral sporadic disease (Breslow et al. 1988). Interestingly, the tumour does not necessarily arise on the same side of the body as the hypertrophy (Fraumeni et al. 1967).

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Beckwith–Wiedemann syndrome (BWS) Clinical features Beckwith–Wiedemann syndrome is an overgrowth syndrome characterized by multiple, congenital abnormalities and an increased risk of developing embryonal tumours in the paediatric age group (Table 20.3 and Fig. 20.2). It is a genetically heterogeneous syndrome with a wide spectrum of phenotypic expression. There is a tendency for the clinical manifestations to become less obvious with increasing age (Hunter and Allanson 1994). The incidence of BWS has been estimated to be 0.07 per 1000 births (Thorburn et al. 1970; Higuarashi et al. 1980), although there may be a degree of under-reporting (Engstrom et al. 1988). Although there are no set diagnostic criteria for BWS, three major clinical features exomphalos, macroglossia, and gigantism are generally recognized in addition to a number of minor clinical features which, in conjunction with certain laboratory or pathological findings, should make a diagnosis possible (Tables 20.3 and 20.4) (Elliot et al. 1994). BWS should be differentiated from other congenital overgrowth syndromes, including; Perlmann syndrome (Grundy et al. 1992), the Simpson–Golabi–Behmel syndrome (SGBS) (Hughes-Benzie et al. 1992) and Sotos syndrome (Cole and Hughes 1990). Major clinical features of BWS Exomphalos describes herniation of abdominal viscera covered by a thin translucent peritoneal and amniotic membrane into the base of the umbilical cord (Elliott and Maher 1994). Other, milder defects of closure of the abdominal wall including umbilical hernia and diastasis recti are recognized (Filipi and McKusick 1970). Macroglossia, the most frequent finding is present in 85–100 per cent of all patients with BWS and usually results from primary hyperplasia or hypertrophy of tongue muscle fibres (Elliott and Maher 1994b). Table 20.3 Clinical features of BWS Clinical features

Frequency (%)

Macroglossia Pre- or postnatal gigantism (growth 90th centile) Abdominal wall defect (exomphalos, umbilical hernia or diastasis recti) Ear creases or posterior helical pits Renal abnormalities (nephromegaly, multiple calyceal cysts or hydronephrosis) Facial flame naevus Hypoglycaemia Hemihypertrophy Congenital cardiac malformations Neoplasia Moderate/severe mental retardation

95 87 77 75 62 62 59 23 9 7 4

Data presented in this table is a composite from two large reviews: Elliot et al. (1994) and Pettenati et al. (1986).


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Fig. 20.2 Infant with BWS. This infant shows some of the clinical features of BWSwith macroglossia, and right sided hemihyperplasia. Although no scale is shown this boy also has somatic gigantism. He subsequently developed and was cured of a unilateral WT. Table 20.4 Major and minor clinical features of BWS* Major clinical features

Minor clinical features

Anterior abdominal wall defect Macroglossia Pre- or postnatal gigantism

Ear pits or creases Facial flame naevus Hypoglycaemia Nephromegaly Hypoglycaemia

* To fulfil the diagnosis of BWS patients should have 3 major features or 2 major features plus 3 minor features Elliott and Maher (1994).

The overgrowth or gigantism, may be somatic, limited to one side of the body—that is hemihyperplasia—or only affect certain organs or tissues. In some cases, especially in those born prematurely, the growth rate may be normal or sub-normal before growth acceleration, resulting in height and weight above the 90th centile. Growth velocity is usually above the 90th centile for at least the first 5 years of life, often persisting into adolescence, becoming

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normal thereafter (Sippel et al. 1989). Partial or complete hemihypertrophy is noted in 12.5–25 per cent of patients (Elliott and Maher 1994b), but is present in 40 per cent of the patients who subsequently develop a neoplasm (Wiedemann 1983). Minor clinical features of BWS Nephromegaly is due to excessive production of nephrons (Beckwith 1969) and is often associated with renal malformation and genitourinary–urinary abnormalities, most commonly multiple calyceal cysts and hydronephrosis. In the two large series in which all of the patients were reviewed by the authors themselves, 60 and 100 per cent of patients had renal abnormalities (Pettenati et al. 1986; Elliott and Maher 1994). Patients with BWS are reported to have both perilobular and intralobar nephrogenic rests, somewhat contrary to predicted (Beckwith 1993). Other important phenotypic abnormalities include those of the craniofacial structures including ear abnormalities, facial flame naevus, and mid-facial hypoplasia. The ear lobe abnormalities, include linear indentations of the lobe which are usually bilateral but not always symmetrical, and/or post helical pits (Elliott and Maher 1994). Occasionally, ear lobe abnormalities are the only manifestation of the syndrome. Other craniofacial abnormalities include mid-facial hypoplasia, microcephaly, and prominent occiput. Most BWS children are of normal intelligence (Hunter and Allanson 1994). BWS and malignancy Patients with BWS have an increased risk of developing a number of specific childhood tumours, both benign and malignant (Sotelo-Avila et al. 1980; Wiedemann 1983). In the largest published series of 388 affected children, 29 developed a total of 32 tumours (i.e. three with double tumours), giving a risk estimate of 7.5 per cent (Wiedemann 1983). The median age of WT at diagnosis in conjunction with BWS was 31 months compared to 41 months for unilateral sporadic disease, but there is no increase in the incidence of bilateral disease. Hemihypertrophy is found in 40 per cent of patients who develop a tumour, compared to 12.5 per cent of all patients with this disorder (Sotelo-Avila et al. 1980; Wiedemann 1983). In the absence of hemihypertrophy the overall risk of malignancy is 1 per cent. Although BWS is considered a WT predisposition syndrome, the relatively modest incidence of WT in children with this condition suggests that mutation of the gene or genes responsible for this syndrome is not the primary tumorigenic event and that mutations in other genes are necessary for tumorigenesis. Genetics of BWS The mode of inheritance of BWS is complex. The majority of cases are sporadic but, in approximately 15 per cent of patients, the condition is inherited as an autosomal dominant trait with impaired penetrance (Niikawa et al. 1986). Linkage studies mapped the BWS gene to 11p15.5 (Koufos et al. 1989) and this has subsequently been confirmed by cytogenetic and molecular studies (Weksberg et al. 1993). Interestingly, it was noted that transmission of BWS from mother to offspring was two or three times more frequent than from the father raising the possibility of genomic imprinting (Moutou et al. 1992). Two to three per cent of BWS patients carry a constitutional chromosome abnormality involving either paternally derived unbalanced duplications (which vary widely in size) or maternally derived balanced translocations involving two separate regions on 11p15 (Mannens et al. 1994; Grundy et al. 1998a).


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One of the BWS breakpoint cluster regions mapped close to IGF2 (BWS CR1), the other to the Haemoglobin locus (BWS CR2), excluding the possibility that interruption of a single gene resulted in the BWS phenotype. The variable size of the paternally derived duplications of 11p implicated a double dose of a paternally expressed gene. Further evidence for a role for imprinting in BWS came from a subset of cytogenetically normal children with BWS who had paternally derived uniparental disomy for 11p15.5 (Henry et al. 1991). Uniparental disomy is defined as the presence of two copies of part or all of a chromosome from the same parent. The most direct evidence for a role for imprinting in BWS has come from the demonstration of biallelic expression of IGF2 in fibroblast cultures from BWS patients (Weksberg et al. 1993). Therefore, both copies of a potent fetal mitogen are expressed, explaining some of the somatic features of this syndrome and in part explaining the predisposition to malignancy. A cluster of imprinted genes on 11p15 Chromosome 11p15.5 and mouse chromosome 7p share extensive regions of conserved synteny including a cluster of imprinted genes, IGF2, H19, p57KIP2 (CDKN1C), and KvLQT1 (KCNQ1). Cyclin dependent kinase inhibitor p57, WT and BWS The gene for p57 KIP2, a cyclin dependent kinase inhibitor whose overexpression causes G1 phase arrest, has recently been mapped to 11p15.5 and is imprinted, being expressed from the maternal allele (Matsuoka et al. 1995). This putative tumour suppressor gene maps to the 500 Kb region that suppressed malignancy when introduced into rhabdomyosarcoma cell lines (Koi et al. 1993). However, no mutations were found in WTs although some showed LOI (Matsuoka et al. 1995). Mutations in p57 KIP2 have been detected in less than 5 per cent of children with sporadic BWS (Lee et al. 1997) and approximately 40 per cent with familial BWS (Lam et al. 1999). BWS patients with p57 KIP2 mutations have a high incidence of anterior abdominal wall defects, but the risk of WT appears to be higher in patients with UPD or H19 methylation abnormalities (BWS1CR defect) than in patients with p57KIP2 mutations or KVDMR1 methylation changes (Engel et al. 2000; Maher and Reik 2000). KvLQT1 Almost all of the chromosomal breakpoints in BWSCR1 interrupt the KvLQT1 gene which encodes a voltage-gated potassium channel. This gene has two distinct isoforms, one of which is imprinted the other, expressed in the heart, is not explaining the absence of the long QT syndrome in children with BWS. LOI of an anti-sense orientated transcript LIT1 (long QT intronic transcript) or more commonly the loss of maternal specific methylation of –CpG– island upstream of LIT1, KvDMR1 are now recognized as the most common epigenetic abnormalities in sporadic BWS (Lee et al. 1999; Smilinich et al. 1999). LOI for LIT1 is not invariably linked to LOI for IGF2 (Lee et al. 1999; Smilinich et al. 1999). TSSC5 TSSC5 is expressed in fetal kidney and liver, is imprinted and although aberrant splicing has been detected in most of the WT analysed mutations are rare occurring in only 1 of 50 WTs (Lee et al. 1998). Furthermore as the same mutation was present in the maternal DNA it is not clear whether this represents a rare polymorphism or a germline mutation.

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Perlmann syndrome (PS) Perlmann Syndrome was first identified in a consanguineous Yemenite Jewish family in which the parents were second cousins. The first two of their six children presented with metanephric hamartomas and nephroblastomatosis (Liban and Kozenitzky 1970). The birth of three other similarly-affected siblings, one of whom had a WT, prompted further reports (Perlmann et al. 1973, 1974; Perlmann 1986). In 1984, two additional cases with similar features were described in a non-consanguineous family and the eponym PS was suggested (Neri et al. 1984). The spectrum of PS is now known to be much wider and a number of extra-renal manifestations are recognized (Neri et al. 1984; Greenberg et al. 1986, 1988; Hamel et al. 1989; Murata et al. 1989; Grundy et al. 1992). This condition is very rare indeed and to date only of 14 cases have been reported in the world literature. There are phenotypic similarities with BWS but the two conditions are distinct (Grundy et al. 1992). PS is characterized by fetal gigantism, distinctive facies, a high mortality rate in early life, mental retardation, and renal and genito-urinary abnormalities including a high risk of developing WT (Grundy et al. 1992). The facial features of PS—deep-set eyes, a short small nose with a depressed nasal bridge, an inverted ‘v-shaped’ upper lip with a serrated alveolar margin, low set ears and macrocephaly—are characteristic

Fig. 20.3 Facial features of PS. This figure shows the characteristic facial features of PS—deep set eyes, a small short nose with a depressed nasal bridge, an inverted ‘v-shaped’ upper lip with a serated alveolar margin, and low set ears.


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and differ subtly from those seen in BWS (Fig. 20.3). Furthermore, facial flame naevus, ear creases or pits, found widely in BWS, have not been noted in PS. Disordered growth in PS is manifest by macrocephaly, macrosomia, nephromegaly, hepatomegaly, and islet cell hyperplasia, all of which are common. Hemihypertrophy and adrenal cortical cytomegaly have not been reported in PS nor have macroglossia or omphalocele, two of the cardinal features of BWS. Mental retardation has been reported in three of the four (75%) survivors with PS, compared to an estimated 12 per cent in BWS (Elliott and Maher 1994). Only 4 patients with PS have survived the neonatal period, the cause(s) of this extremely high neonatal death rate is unknown. Wilms tumour has developed in 6 of 14 (42%) patients with PS. The mean and median age at diagnosis in PS was 8 months compared to mean ages of 44 months for sporadic unilateral disease, 32 months for bilateral disease and 31 months in BWS (Perlmann 1986; Neri et al. 1984; Greenberg et al. 1986, 1988; Grundy et al. 1992). Bilateral disease was present in 2 of the 6 cases (33%). The high incidence of WT, of bilateral disease, of nephroblastomatosis as well as the early age at onset are characteristic features of a genetic predisposition to WT. By contrast to BWS, WT is the only tumour type so far reported in children with PS. This apparently specific predisposition may reflect either a higher incidence of nephroblastomatosis—a possible precursor of WT—or a more specific genetic predisposition.

Simpson–Golabi–Behmel syndrome (SGBS) This overgrowth syndrome is characterized by macrocephaly and coarse facial features including thickened lips, a wide mouth, macroglossia, a high arched palate, malposition of the teeth and a prominent jaw. Other distinctive features include polydactyly, supernumerary nipples, cardiac defects, renal dysplasia and, in some pedigrees, mental retardation (Simpson et al. 1975; Golabi and Rosen 1984; Behmel et al. 1984). SGBS and BWS share a number of phenotypic characteristics including macrosomia, macroglossia, umbilical herniae, visceromegaly, neonatal hypoglycaemia, ear lobe creases, and phenotypic variability (Hughes-Benzie et al. 1992). There is an apparent increased risk of developing an embryonal neoplasms, although the numbers of affected children is small. WT has been reported in 3 of 27 patients with SGBS (Hughes-Benzie et al. 1994). The true incidence of SGBS is not yet known and may be masked by the overlap between this syndrome and BWS. Molecular genetics of SGBS The gene for SGBS has been mapped to Xq25-q27 by linkage analysis and from a patient previously misdiagnosed as BWS who was found to have a chromosomal translocation interrupting this region (Hughes-Benzie et al. 1994). The SGBS gene has now been cloned from Xq26, GPC3 (Pilia et al. 1996) and shows sequence homology with the glypican family (Pilia et al. 1996). GPC3 has been shown to bind to IGF2, an important fetal mitogen, and may act to modulate the action of this growth promoting gene. Deletions of GPC3 have been found in an SGBS family (Xuan et al. 1999). However, the daughter of this family, who had the SGBS phenotype and a WT, did not carry this mutation, questioning the role of GPC3 in WT and in SGBS (Xuan et al. 1999). Recently a second locus for SGBS has been mapped to Xp22 (Brzustowicz et al. 1999).

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Sotos syndrome Cerebral gigantism, or Sotos syndrome, is an overgrowth syndrome in which the affected children show post-natal gigantism and grow with excessive rapidity. Facial features include hypertelorism and a high arched palate. Large hands and feet are also characteristic. Affected children have a non-progressive mental retardation and dilatation of the cerebral ventricles is a common radiological finding (Soto et al. 1977). There is an increased risk of developing a variety of different tumour types, including hepatocellular carcinoma. Two patients with Sotos syndrome and WT have been reported, but due to the small numbers of patients with this syndrome, it is not yet certain whether this association is real or not. Screening for WT in the WT predisposition syndromes The recognition that certain syndromes predispose to the development of WT has inevitably raised the possibility of screening children in order to detect tumours at an early stage and thereby minimize the treatment required to effect a cure and reduce late effects (Evans et al. 1991; Sorenson et al. 1995). The detection of small, localized tumours also creates a possibility for limiting the surgery to partial nephrectomy, so called nephron-sparing surgery, again reducing the risk of late effects (Tullio et al. 1996). Widely accepted screening methods for WT include abdominal ultrasound and abdominal palpation by the parents and paediatrician (Green et al. 1993; Craft et al. 1995; DeBaun et al. 1998; Choyke et al. 1999). Ultrasound clearly being far more sensitive. However, all parents whose child is at risk of WT development should be taught abdominal palpation and given ‘hot-line’ referral to a paediatrician or general practitioner who has been fully informed to take the parental concerns seriously. Identification of children with sporadic aniridia at risk of WT development has been refined by developments in fluorescence in situ hybridization (FISH). Using fluorescently labelled cosmid probes that map to the WT1 gene, the aniridia gene (PAX6) and an interval locus it is possible to determine whether there is an 11p13 deletion and crucially whether there is loss of WT1 (Fig. 20.4). Children with large interstitial deletions that include WT1 are at increased risk of developing WT and should undergo screening with ultrasonography every 3 months. An alternative method involves a polymerase chain reaction-based test that can exclude 11p13 deletion involving WT1 utilizing a panel of highly polymorphic markers across the PAX6WT1 region (Gupta et al. 1998). Deletion is detectable by loss of heterozygosity. In order to prove that screening for WT is beneficial several criteria have to be met (Table 20.5). One of the difficulties has been in determining the screening interval. A unique case has enabled clinicians to follow the in vivo growth rate of a unilateral WT (Zoubek et al. 1999). Serial ultrasound examinations performed over a 7 week period enable an estimation of the doubling time of this tumour at between 11 and 13 days suggesting that the screening interval should not be greater than 77 days (Craft 1999). Early studies concluded that screening children with hemihypertrophy or BWS was not beneficial (Green et al. 1993; Craft et al. 1995). However, these studies were retrospective and the screening interval and methodology varied widely between patients. So far no prospective studies have been undertaken. A possible benefit in terms of lower stage disease at presentation compared to those diagnosed clinically was shown for aniridia (Green et al. 1993). A more recent case series analysis has shown a clinically relevant and significant statistical advantage to 4 monthly screening by ultrasonography in children with BWS or


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11p15 Cosmid probes A

B

C

AN1 11p13

Interval Locus WT1

A: Small deletion involving the Aniridia gene B: Deletion involving AN1 and the interval locus but sparing WT1. This child is at no increased risk of developing WT C: Large deletion involving all 3 loci. This child is at risk of developing WT and should be screened. Key Cosmid probe for defined locus present by FISH Cosmid probe for defined locus absent by FISH Fig. 20.4 Fluoresence in situ hybridization determination of risk of WT formation in children with aniridia.

Table 20.5 Criteria determining the value of screening for WT in the WT predisposition syndromes Improved survival in screened group Detection of earlier stage disease Reduction of intensity of chemotherapy and avoidance of doxorubucin Avoidance of radiotherapy Increased possibility of nephron sparing surgery Cost effectiveness Cost of screening ultrasonography Defined screening interval and duration ‘At risk’ population size Detection rate ‘Cost-benefit’ of managing early versus late stage disease Reduction in late effects Management ‘costs’ of false-positives Parental and family reassurance Inconvenience and indirect parental expenses

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idiopathic HH in terms of detecting early stage disease (Choyke et al. 1999). The mean tumour size in the screened group was 3.4 cm raising the possibility of partial nephrectomy (Choyke et al. 1999). However, 3 of 15 (20%) patients were found to have renal masses that were not WT, all three underwent laparotomy and two had unnecessary nephrectomies. This unacceptably high level of false positive examinations could be reduced by (a) performing CT/MRI scans on patients with intrarenal lesions (b) following suspicious lesions over a defined period of time to see if they grew and (c) the use of image guided biopsy of the suspicious lesion rather than proceeding to straight to nephrectomy. Persistent nephromegaly detected initially by ultrasound in infancy may be a strong risk factor for the development of WT in children with BWS (Choyke et al. 1999). This finding raises possibility of defining the ‘at-risk group’ thereby reducing the overall number of children screened, the cost per case of WT identified and perhaps the risk of false positive diagnosis. However, these preliminary finding need to be further examined in a larger prospective study before any evidence based recommendations can be made. In the meantime, three monthly abdominal ultrasound to age 6 would be appropriate followed by parent-led abdominal palpation to 8 years of age. Whether screening creates a survival advantage, reduces late effects or is cost effective still needs to be addressed in a large prospective International study.

Summary In summary, it now appears that WT is the end result of mutations in a number of different genes, genetic, and epigenetic pathways. Due to the degree of genetic heterogeneity seen in WTs a unifying model for Wilms tumorigenesis seems unlikely. The more likely scenario is that WT results from an accumulation of genetic and/or epigenetic changes. The distinguishing feature of WT and other childhood tumours is that these genetic alterations involve specific stages of differentiation and specific developmental lineages. These differences explain, in part, the clear presence of a ‘developmental window’ and the organ specific risk of malignancy seen in these patients. Therefore, in order to gain a clearer understanding of the molecular pathology of WT we need to better understand the genes involved in the normal development of the kidney.

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Frasier, S. D., Bashore, R. A., and Mosier, H. D. (1964). Gonadoblastoma associated with pure gonadal dysgenesis in monozygotic twins. Journal of Pediatrics, 64, 740–5. Fraumeni, J. F., Geiser, C. F., and Manning, M. D. (1967). Wilms’ tumor and congenital hemihypertrophy: report of five new cases and review of literature. Pediatrics, 40, 886–99. Gessler, M., Konig, A., Arden, K., Grundy, P., Orkin, S., Sallan, S., et al. (1994). Infrequent mutation of the WT1 gene in 77 Wilms’ Tumors. Human Mutation, 3, 212–22. Gessler, M., Poustka, A., Cavenee, W., Neve, R. L., Orkin, S. H., and Bruns, G. A. P. (1990). Homozygous deletion in Wilms tumours of a zinc-finger gene identified by chromosome jumping. Nature, 343, 774–8. Getman, M. E., Houseal, T. W., Miller, G. A., Grundy, P. E., Cowell, J. K., and Landes, G. M. (1998). Comparative genomic hybridization and its application to Wilms’ tumorigenesis. Cytogenetics and Cell Genetics, 82, 282–90. Giannoukakis, N., Deal, C., Paquette, J., Goodyer, C. G., and Polychronakos, C. (1993). Polymorphic functional imprinting of the human IGF2 gene among individuals, in blood cells, is associated with H19 expression. Nature Genetics, 4, 98–101. Golabi, M. and Rosen, L. (1984). A new X-linked mental retardation-overgrowth syndrome. American Journal of Medical Genetics, 17, 345–58. Green, D. M., Breslow, N. E., Beckwith, J. B., and Norkool, P. (1993). Screening of children with hemihypertrophy, aniridia, and Beckwith-Wiedemann syndrome in patients with Wilms tumor: a report from the National Wilms Tumor Study. Medical and Pediatric Oncology, 21, 188–92. Green, D. M., Donckerwolcke, R., Evans, A. E., and D’Angio, G. J. (eds) (1995). Late effects of treatment of Wilms tumour, W. B. Saunders, Philadelphia, PA. Greenberg, F., Copeland, K., and Gresik, M. V. (1988). Late effects of treatment for Wilms tumor. American Journal of Medical Genetics, 29, 773–6. Greenberg, F., Stein, F., Gresik, M. V., Finegold, M. J., Carpenter, R. J., Riccardi, V. M., et al. (1986). Expanding the spectrum of the Perlmann syndrome. American Journal of Medical Genetics, 24, 101–10. Grundy, P. E., Telzerow, P. E., Breslow, N., Moksness, J., Huff, V., and Paterson, M. C. (1994). The Perlmann familial nephroblastomatosis syndrome. Cancer Research, 54, 2331–3. Grundy, R. G., Aledo, R., and Cowell, J. K. (1998a). Characterization of the breakpoints in unbalanced t(5;11)(p15;p15) constitutional chromosome translocations in two patients with beckwith-wiedemann syndrome using fluorescence in situ hybridisation. International Journal of Molecular Medicine, 1, 801–8. Grundy, R. G., Pritchard, J., Baraitser, M., Risdon, M., and Robards, M. (1992). Perlmann and Wiedemann-Beckwith syndromes: two distinct conditions associated with Wilms’ tumour. European Journal of Pediatrics, 151, 895–8. Grundy, R. G., Pritchard, J., Scambler, P., and Cowell, J. K. (1998b). Loss of heterozygosity for the short arm of chromosome 7 in sporadic Wilms tumour. Oncogene, 17, 395–400. Grundy, R. G., Pritchard, J., Scambler, P., and Cowell, J. K. (1998c). Loss of heterozygosity on chromosome 16 in sporadic Wilms’ tumour. British Journal of Cancer, 78, 1181–7. Gupta, S. K., De Becker, I., Guernsey, D. L., and Neumann, P. E. (1998). Polymerase chain reactionbased risk assessment for Wilms tumor in sporadic aniridia. American Journal of Ophthalmology, 125, 687–92. Haber, D. A., Sohn, R. L., Buckler, A. J., Pelletier, J., Call, K. M., and Housman, D. E. (1991). Alternative splicing and genomic structure of the Wilms tumor gene WT1. Proceedings of the National Academy of Sciences, 88, 9618–22. Habib, R., Loirat, C., Gubler, M. C., Niaudet, P., Bensman, A., Levy, M., et al. (1985). The nephropathy associated with male pseudohermaphroditism and Wilms’ tumor (Drash syndrome): a distinctive glomerular lesion–report of 10 cases. Clinical Nephrology, 24, 269–78.

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21 Von Hippel–Lindau disease Eamonn R. Maher

Von Hippel–Lindau (VHL) disease is the most common cause of familial clear cell renal cell carcinoma (RCC). VHL disease results from germline mutations in the VHL tumour suppressor gene and is characterized by variable expression and multisite tumour susceptibility (see Table 21.1). VHL disease is named after the German ophthalmologist Eugene von Hippel who described retinal angiomas almost 100 years ago (although familial retinal angioma had been described some 20 years previously by Treacher Collins); and the Swedish pathologist Arvid Lindau who, in 1926, described the major features of VHL disease and recognized the significance of the association of retinal haemangioblastoma and cerebellar haemangioblastomas (CHB) (Melmon and Rosen 1964). Recently, the management of VHL disease has been transformed by the widespread recognition that surveillance and presymptomastic diagnosis of VHL tumours reduces morbidity and mortality in VHL disease and the isolation of the VHL gene leading to the availability of genetic testing. The effective management of VHL disease families requires a coordinated multidisciplinary team approach and increasingly nephrologists are assuming a critical role in the management of VHL disease. The VHL tumour suppressor gene The VHL gene maps to chromosome 3p25 and was isolated in 1993 (Latif et al. 1993). The VHL coding sequence is organized in three exons and encodes two proteins. The full length 213 amino acid VHL protein (VHL L) migrates with an apparent molecular weight of ~28–30 kD, but a second form of VHL protein, generated by translation initiation at an internal methionine located at codon 54, migrates with an apparent molecular weight of ~19 kD (VHLS) Table 21.1 Major manifestations of VHL disease Lesion

UK series (n 152; Maher et al. 1990a)

Literature review (n 554; Lamiell et al. 1989)

Mean age of onset: years (Maher et al. 1990a)

Retinal angioma CHB Spinal cord haemangioblastoma RCC Phaeochromocytoma

89 (59%) 89 (59%) 20 (13%) 43 (28%) 11 (7%)

317 (57%) 304 (55%) 79 (14%) 133 (24%) 106 (19%)

25.4 12.7 29.0 10.0 33.9 12.6 44.0 10.9 20.2 7.6

Mean age at onset relates to age at clinical presentation—mean onset is earlier for tumours diagnosed presymptomatically.

488

Eamonn R. Maher

(Schoenfeld et al. 1998). Although no germline or somatic VHL gene mutations have been reported in the first 53 codons and VHLS has tumour suppressor activity, evolutionary analysis of 5 VHL coding sequences suggest some functional significance (Woodward et al. 2000a). The primary sequence of VHL is not highly similar to that of any known protein and to elucidate possible VHL functions, several groups sought to identify pVHL binding proteins (see Kaelin and Maher 1998, and references within). To date, several pVHL-binding proteins have been reported (e.g. elongins B and C, Cul2, fibronectin, HIF-1 and HIF-2) (Kaelin and Maher 1998; Maxwell et al. 1999). The first VHL-interacting proteins to be identified were elongin B and C which bind to a region of the C-terminal third of the VHL protein that is frequently altered by VHL-associated mutations. Elongin B and C, when bound to elongin A, generate a transcriptional elongation complex called elongin or SIII, however there is little evidence that VHL-mediated tumour suppression is exerted by inhibiting transcriptional elongation. Subsequently the Cul2 protein, a member of the cullin protein family, was shown to bind to the pVHL/elongin C/elongin B (VCB) complex. Cullins, had been implicated in targeting cellular proteins for ubiquitinization and degradation and the similarities between the VCB-Cul2 complex and the SCF (Skp-1-Cdc53/Cul1-F box protein) class of E3 ubiquitin ligases in yeast was noted (Lonergan et al. 1998). Thereafter a series of reports provided compelling evidence that an important function of the VHL gene product is to regulate proteasomal degradation of target proteins as part of an SCF-like complex also involving elongin B and C, Cul2 and Rbx1 proteins (Kamura et al. 1999; Stebbins et al. 1999). Under this model pVHL is predicted to act as an adaptor protein to recruit specific protein targets. VHL-associated tumours are notably hypervascular and express high levels of VEGF and other hypoxia-responsive genes (Siemeister et al. 1996). The hypoxia-inducible transcription factors HIF-1 and HIF-2 (EPAS) were identified as pVHL-targets (Maxwell et al. 1999). HIF-1 and HIF-2 transcription factors plays a key role in the cellular response to hypoxia (oxygen sensing) and the regulation of genes involved in energy metabolism, angiogenesis, and apoptosis (e.g. GLUT-1 and VEGF, which have been shown to be regulated by pVHL). HIF-1 and HIF-2 subunits are normally degraded by the proteasome, but are stabilized by hypoxia, and pVHL targets these HIF sub-units for oxygen-dependent proteolysis. Accordingly, constitutively high HIF-1 and HIF-2 levels are observed with pVHL inactivation (Maxwell et al. 1999). The structure of the VHL gene product predicts two protein binding sites, the elongin C binding site in the C terminus and a second surface binding site which interact with the HIF subunits. Recent reports suggest that HIF overexpression (particularly HIF-2) is an important contributor to tumourigenesis after VHL inactivation (Kondo et al. 2002). Not all pVHL mutations interfere with HIF or elongin C binding (see later and unpublished observations) and the ability of pVHL to bind fibronectin and the failure of VHL defective cells to assemble a fibronectin matrix (Ohh et al. 1998) suggest that additional (unrelated to HIF) pVHL functions contribute to tumour suppressor activity. A notable feature of VHL disease is the complex genotype–phenotype associations. Thus large deletions and truncating mutations typically predispose to haemangioblastomas (HAB) and RCC but not phaeochromocytomas (PC) (VHL type 1) (Crossey et al. 1994; Maher et al. 1996; Zbar et al. 1996). Missense mutations may produce a type 1 phenotype, a type 2A phenotype (susceptibility to HAB and PC but not RCC), a type 2B phenotype (HAB, RCC, and PC) or, intriguingly, a phaeochromocytoma only (type 2C) phenotype (Brauch et al. 1995; Neumann et al. 1995; Woodward et al. 1997). These observations suggest that pVHL has multiple and tissue specific functions.

Von Hippel–Lindau disease

489

Statistical analysis of the age at onset of RCC and CHB in VHL and in sporadic cases was compatible with a one- and two-hit model of tumourigenesis as in familial and sporadic retinoblastoma (Maher et al. 1990b). Molecular analysis of tumours from VHL patients subsequently demonstrated somatic inactivation of the wild type VHL allele in various tumour types (Prowse et al. 1997). Furthermore somatic VHL gene inactivation occurs in most sporadic clear cell RCC and sporadic haemangioblastomas, as predicted for a classical tumour suppressor gene (Gnarra et al. 1994; Foster et al. 1994). Diagnosis of VHL disease The identification of the VHL tumour suppressor gene enabled DNA-based diagnostic and predictive testing to be offered. More than 80 per cent of germline VHL mutations can be detected by direct gene sequencing and Southern analysis. Most of the remaining mutations appear to be large deletions which may be detected by quantitative Southern or FISH analysis (Pack et al. 1999). As with many other familial cancer syndromes, predictive testing is frequently requested for at risk relatives allowing screening to be discontinued in those who are shown not to be gene carriers. Predictive testing is usually offered from age 5 years. A clinical diagnosis of VHL disease can be made in the presence of a typical VHL tumour if there is a positive family history. However isolated cases without a family history can only be identified clinically when two tumours (e.g. two haemangioblastomas or a haemangioblastoma and a visceral tumour) have developed. Molecular genetic studies provide a method by which an early diagnosis of VHL disease can be made in patients who do not satisfy the current clinically based diagnostic criteria. A diagnosis of VHL disease should be considered in all cases of retinal and central nervous system haemangioblastomas, but also patients with familial, multicentric or young onset phaeochromocytoma and RCC. Molecular genetic testing has revealed that ~50 per cent of patients with apparently isolated familial phaeochromocytoma or bilateral phaeochromocytoma have germline VHL gene mutations (Woodward et al. 1997). For patients with familial RCC, in addition to VHL disease, a diagnosis of familial papillary RCC and familial clear cell RCC which is not allelic with VHL disease should also be considered (Schmidt et al. 1997; Teh et al. 1997; Woodward et al. 2000b). Presentation and manifestations of VHL disease The age at onset of VHL disease is variable. Age dependent penetrance for clinical presentation of each of the major tumours in VHL disease is shown in Fig. 21.1. The first manifestation of VHL disease may be in childhood or old age, but most patients present in the second and third decades and penetrance is almost complete by age 60 years. The relative frequencies and mean age at diagnosis of the major complications of VHL disease are shown in Table 21.1. The data in Fig. 21.1 and Table 21.1 are based on age at clinical diagnosis and in familial cases the age at diagnosis of first manifestation will generally be earlier as most cases should be detected presymptomatically because of the widespread application of surveillance programmes to at risk relatives. The most frequent initial manifestations are retinal haemangioblastomas and CHB, but RCC is the presenting feature in ~10 per cent of cases (Lamiell et al. 1989; Maher et al. 1990a). In cross-sectional studies, retinal haemangioblastomas and CHBs are the most frequent complications of VHL disease, however the cumulative age related risk of RCC

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in VHL disease approaches that of retinal haemangioblastoma and CHB by age 60 years (see Fig. 21.1). The phenotypic expression of VHL disease is variable, but interfamilial variations in phaeochromocytoma susceptibility, and less commonly RCC, are linked to allelic heterogeneity (see above). In addition, phenotypic variability will reflect stochastic events and there is also evidence for genetic modifier effects (Webster et al. 1998).

Renal involvement in VHL disease The major renal manifestations of VHL disease are renal cystic disease and clear cell RCC. Although renal tumours were noted by Lindau, the risk of renal malignancy was generally overlooked for many years with the result that increasing numbers of VHL patients survived surgery for central nervous system haemangioblastomas to subsequently die from RCC. However in the past 10–20 years the recognition of the importance of detecting RCC presympromatically has led to a policy of annual renal imaging in VHL patients and at risk relatives. As a result of this, renal lesions in VHL disease are increasingly detected at an early asymptomatic stage, and the surgical management of RCC in VHL disease has shifted from the treatment of large symptomatic RCC, to the challenges of how to best manage small asymptomatic tumours. Computer tomography is reported to be the most sensitive method for following renal lesions (particularly in the presence of renal cysts), but MRI or ultrasound scans avoid the potential adverse effects of a large cumulative radiation exposure in cancer-predisposed

1.0

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0

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Fig. 21.1 Age related risks of CHB, retinal angioma (RA) and RCC in VHL disease (from Maher et al. 1990a). Risks relate to age at clinical presentation and detection of presymptomatic lesions will be earlier.


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individuals and are preferred for regular follow-up. The investigation of a specific lesion may, however, require multiple imaging modalities. Most small solid renal tumours enlarge slowly (mean 2 cm/year), and after establishing the growth rate, an individual lesion these can usually be scanned every 6 months (Choyke et al. 1992). The risk of distant metastasis from a solid lesion 3 cm appears to be very remote and a conservative approach is generally applied until a solid lesion reaches ~3 cm in size. It is very likely that individuals with VHL disease and RCC will ultimately develop further tumours in their remaining renal tissue as a result of new primary tumours. A conservative nephron sparing approach is now widely adopted for the management of RCC in VHL disease (Steinbach et al. 1995; Walther et al. 1995). The objective of this strategy is to maintain adequate renal function for as long as possible by avoiding the removal of normal renal tissue. Thus wherever possible solid tumours are removed by a limited partial nephrectomy or by ‘shelling out’ small encapsulated lesions. When elective renal surgery is performed, it is helpful if, in addition to removing the primary lesion, other smaller lesions are also removed when these are accessible and can be dissected without damaging normal renal tissue. Such an approach should then delay the need for further surgery. Follow-up of VHL patients managed by such a nephron-sparing approach suggests that although the risk of local recurrence (from new primary tumours) is high the risk of distant metastasis is low (Steinbach et al. 1995). In contrast, 25 per cent of VHL patients with a RCC 3 cm (treated by nephron-sparing surgery or nephrectomy) developed metastatic disease (Walther et al. 1999). As many VHL patients undergoing surgery for RCC are young, any delay in the requirement for renal replacement therapy is beneficial. Anephric VHL patients on dialysis are susceptible to fluid balance problems because of the lack of endogenous renal function. Renal transplantation is an option for a VHL patient in end stage renal failure and experience so far suggests that immunosuppression does not affect adversely the underlying course of VHL disease. It is customary to wait at least 2 years for performing a transplant in patient who have had a RCC removed, but this may prove to be unnecessary in VHL patients who have had only small tumours (3 cm or less). The identification of renal cysts in VHL patients is a frequent and expected finding. As these do not usually compromise renal function no treatment is necessary. It is known, however, that the epithelium lining the cysts in VHL kidneys is frequently atypical and may contain carcinoma in situ. If renal imaging suggests only simple cysts are present then annual follow-up is sufficient. However if complex cysts are detected these should be reviewed more frequently as they can develop into solid lesions (Choyke et al. 1992).

Retinal involvement in VHL disease Retinal angiomas are the commonest presenting feature of VHL disease, occur in most VHL patients and are multiple in many cases. Approximately 68 per cent of cases have retinal involvement with a mean 1.85 lesions. Webster et al. (1999) estimated that the cumulative risk of visual loss by age 50 years was 35 per cent in gene carriers and 55 per cent in those with retinal angiomas. Histologically retinal angiomas are benign haemangioblastomas, but untreated they enlarge progressively and may produce retinal detachment and haemorrhage resulting in visual impairment. The early detection of these tumours enables treatment by laser- or cryotherapy and reduces the risk of visual loss.

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CHB Within the central nervous system, the cerebellum is the most frequent site of haemangioblastoma followed by the spinal cord and brain stem. The incidence of supratentorial lesions is small. Approximately 30 per cent of all patients with CHB have VHL disease and the mean age at diagnosis of those with VHL disease is considerably younger than in sporadic cases (Maher et al. 1990b). About 4 per cent of isolated haemangioblastoma cases with no clinical or radiological evidence of VHL disease have a germline VHL gene mutation (Hes et al. 2000). Haemangioblastomas are benign and the results of surgery for cerebellar lesions are often excellent. However, the treatment of multiple CNS haemangioblastomas and the management of brain stem and spinal tumours is often difficult. Hence CNS haemangioblastomas remains an important cause of morbidity and mortality for VHL patients. The advent of anti-VEGF therapy may offer a medical approach to the treatment of inoperable CNS and retinal haemangioblastomas. Phaeochromocytoma Phaeochromocytoma is an important complication of VHL disease (Richard et al. 1994), although it does not occur in most VHL families (Type 1 families). However in some families (Type 2), phaeochromocytoma can be the most common manifestation of VHL disease. These interfamilial differences in phaeochromocytoma susceptibility are caused by allelic heterogeneity (see previously). Phaeochromocytomas in VHL disease may be extra-adrenal and 5 per cent or less are malignant. Compared to sporadic cases, onset of phaeochromocytoma is earlier in VHL patients (~20 years). Measurement of plasma normetanephrine levels is reported to be the most sensitive test for detecting phaeochromocytoma in VHL disease, although routine screening is usually performed by urine analysis. Pancreatic involvement The most common pancreatic manifestation of VHL disease is is the presence of multiple cysts. The incidence of pancreatic cysts is age-related with the youngest age at onset being 15 years and an ~70 per cent frequency at autopsy. Usually pancreatic cysts are asymptomatic and treatment is not required. However, rarely pancreatic insufficiency may develop. Of more concern is the presence of a solid tumour in the pancreas. These occur in 5–10 per cent of cases and are usually non-secretory islet cell tumours. A high frequency of malignancy has been reported in VHL associated islet cell tumours and early surgical intervention is generally recommended (Binkovitz et al. 1990). Recently it has been suggested surgical management might be tailored according to the size of the pancreatic tumour such that tumours 1 cm can be monitored while those 3 cm should be resected (Libutti et al. 1998). Other manifestations of VHL disease Endolymphatic sac tumours may cause hearing loss, tinnitus, and vertigo and all medical staff dealing with VHL families should be aware of this complication which can be detected by MRI scanning in up to 11 per cent of cases (Manski et al. 1997).


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Table 21.2 Example surveillance programme for VHL disease in asymptomatic affected patients and at risk relatives (adapted from Maher et al. 1990a) Affected patient 1. Annual physical examination and urine testing 2. Annual direct and indirect ophthalmoscopy 3. MRI brain scan every 3 years to age 50 and every 5 years thereafter 4. Annual renal ultrasound or MRI scan (CT scan may be required if multiple cysts renal or pancreatic cysts are present) 5. Annual 24 h urine collection for catecholamines from age 11 At risk relative 1. Annual physical examination and urine testing 2. Annual direct and indirect ophthalmoscopy from age 5 to 60 (fluoroscein angioscopy or angiography may be used from age 10 to increase sensitivity) 3. MRI brain scan every 3 years to from age 15 to 40 years and then every 5 years until age 60 years 4. Annual renal ultrasound scan or MRI scan from age 15 to 65 years 5. Annual 24 h urine collection for catecholamines from age 11

Epididymal cysts are extremely common in males with VHL disease and may, if bilateral, impair fertility. However epididymal cysts are not infrequent in the general population and their presence in an at-risk individual does not unequivocally indicate carrier status. Surveillance in VHL disease VHL disease is a multisystem disorder and the effective management of VHL patients and families requires a multidisciplinary approach. Specific complications may require expert investigations and treatment from ophthalmologists and other organ based specialists. However, there is also a need for the overall co-ordination of family ascertainment and screening. Thus when an individual with VHL disease is identified strenuous efforts should be made to identify all at risk relatives and offer them information on the inheritance and clinical manifestations of VHL disease. Patients and relatives should also be offered systematic screening to detect subclinical disease as outlined in Table 21.2. For affected individuals and individuals at high risk by DNA testing, lifelong surveillance should be offered. In the absence of DNA testing, at risk individuals without clinical or subclinical evidence of VHL should be followed up until after age 60 years (Maher et al. 1990a). Increasingly, however it is possible to modify the risk of such relatives by DNA testing and then alter the surveillance programme appropriately. Conclusion The management of RCC in VHL disease is challenging and the optimal approach is still evolving. The emphasis, however, is on preserving functioning renal tissue for as long as possible. The optimal management of VHL disease requires more than the successful treatment of specific complications such as RCC or retinal angioma. In addition, it is important that a co-ordinated system for ascertaining families and providing appropriate surveillance is available. This approach reduces morbidity and mortality from VHL disease.

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References Binkovitz, L. A., Johnson, C. D., and Stephens, D. H. (1990). Islet cell tumors in von Hippel–Lindau disease—increased prevalence and relationship to the multiple endocrine neoplasias. American Journal of Roentgenology, 155, 501–5. Brauch, H., Kishida, T., Glavac, D., Chen, F., Pausch, F., Hofler, H., et al. (1995). Von Hippel–Lindau (VHL) disease with pheochromocytoma in the Black-Forest Region of Germany—evidence for a founder effect. Human Genetics, 95, 551–6. Choyke, P. L., Glenn, G. M., Walther, M. C. M., Zbar, B., Weiss, G. H., Alexander, R. B., et al. (1992). The natural-history of renal lesions in von Hippel–Lindau disease—a serial CT study in 28 patients. American Journal of Roentgenology, 159, 1229–34. Crossey, P. A., Richards, F. M., Foster, K., Green, J. S., Prowse, A., Latif, F., et al. (1994). Identification of intragenic mutations in the von Hippel–Lindau disease tumor-suppressor gene and correlation with disease phenotype. Human Molecular Genetics, 3, 1303–8. Foster, K., Prowse, A., van den Berg, A., Fleming, S., Hulsbeek, M. M. F., Crossey, P. A., et al. (1994). Somatic mutations of the von Hippel–Lindau disease tumor suppressor gene in nonfamilial clear cell renal carcinoma. Human Molecular Genetics, 3, 2169–73. Gnarra, J. R., Tory, K., Weng, Y., Schmidt, L., Wei, M. H., Li, H., et al. (1994). Mutations of the VHL tumor-suppressor gene in renal-carcinoma. Nature Genetics, 7, 85–90. Hes, F. J., McKee, S., Taphoorn, M. J. B., Rehal, P., van der Luijt, R. B., McMahon, R., et al. (2000). Cryptic von Hippel–Lindau disease: Germline mutations in haemangioblastoma-only patients. Journal of Medical Genetics, 37, 939–43. Kaelin, W. G. and Maher, E. R. (1998). The VHL tumour suppressor gene paradigm. Trends in Genetics, 14, 423–5. Kamura, T., Koepp, D. M., Conrad, M. N., Skowyra, D., Moreland, R. J., Iliopoulos, O., et al. (1999). Rbx1, a component of the VHL tumor suppressor complex and SCF ubiquitin ligase. Science, 284, 657–61. Kondo, K., Klco, J., Nakamura, E., Lechpammer, M., and Kaelin, W. G. Jr. (2002). Inhibition of HIF is necessary for tumor suppression by the von Hippel–Lindau protein. Cancer Cell, 1, 237–46. Lamiell, J. M., Salazar, F. G., and Hsia, Y. E. (1989). von Hippel–Lindau disease affecting 43 members of a single kindred. Medicine, 68, 1–29. Latif, F., Tory, K., Gnarra, J., Yao, M., Duh, F. M., Orcutt, M. L., et al. (1993). Identification of the Vonhippel–Lindau disease tumor-suppressor gene. Science, 260, 1317–20. Libutti, S. K., Choyke, P. L., Bartlett, D. L., Vargas, H., Walther, M., Lubensky, I., et al. (1998). Pancreatic neuroendocrine tumors associated with von Hippel–Lindau disease: Diagnostic and management recommendations. Surgery, 124, 1153–9. Lonergan, K. M., Iliopoulos, O., Ohh, M., Kamura, T., Conaway, R. C., Conaway, J. W., et al. (1998). Regulation of hypoxia-inducible mRNAs by the von Hippel–Lindau tumor suppressor protein requires binding to complexes containing elongins B/C and Cul2. Molecular and Cellular Biology, 18: 732–41. Maher, E. R., Yates, J. R. W., Harries, R., Benjamin, C., Harris, R., Moore, A. T., et al. (1990a). Clinicalfeatures and natural-history of von Hippel–Lindau disease. Quarterly Journal of Medicine, 77, 1151–63. Maher, E. R., Yates, J. R. W., and Ferguson-Smith, M. A. (1990b). Statistical-analysis of the 2 stage mutation model in von Hippel–Lindau disease, and in sporadic cerebellar hemangioblastoma and renal-cell carcinoma. Journal of Medical Genetics, 27, 311–14. Maher, E. R., Iselius, L., Yates, J. R. W., Littler, M., Benjamin, C., Harris, R., et al. (1991). Von Hippel–Lindau disease: a genetic study. Journal of Medical Genetics, 28, 443–7. Maher, E. R., Webster, A. R., Richards, F. M., Green, J. S., Crossey, P. A., Payne, S. J., et al. (1996). Phenotypic expression in von Hippel–Lindau disease: correlations with germline VHL gene mutations. Journal of Medical Genetics, 33, 328–32.


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Manski, T. J., Heffner, D. K., Glenn, G. M., Patronas, N. J., Pikus, A. T., Katz, D., et al. (1997). Endolymphatic sac tumors—a source of morbid hearing loss in von Hippel–Lindau disease. Journal of the American Medical Association, 277, 1461–6. Maxwell, P. H., Wiesener, M. S., Chang, G. W., Clifford, S. C., Vaux, E. C., Cockman, M. E., et al. (1999). The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygendependent proteolysis. Nature, 399, 271–5. Melmon, K. L. and Rosen, S. W. (1964). Lindau’s disease. American Journal of Medicine, 36, 595–617. Neumann, H. P. H., Eng, C., Mulligan, L., Glavac, D., Ponder, B. A. J., Crossey, P. A., et al. (1995). Consequences of direct genetic testing for germline mutations in the clinical management of families with multiple endocrine neoplasia type 2. Journal of the American Medical Association, 274, 1149–51. Ohh, M., Yauch, R. L., Lonergan, K. M., Whaley, J. M., StemmerRachamimov, A. O., Louis, D. N., et al. (1998). The von Hippel–Lindau tumor suppressor protein is required for proper assembly of an extracellular fibronectin matrix. Molecular Cell, 1, 959–68. Pack, S. D., Zbar, B., Pak, E., Ault, D. O., Humphrey, J. S., Pham, T., et al. (1999). Constitutional von Hippel–Lindau (VHL) gene deletions detected in VHL families by fluorescence in situ hybridization. Cancer Research, 59, 5560–4. Prowse, A. H., Webster, A. R., Richards, F. M., Richard, S., Olschwang, S., Resche, F., et al. (1997). Somatic inactivation of the VHL gene in von Hippel–Lindau disease tumors. American Journal of Human Genetics, 60, 765–71. Richard, S., Chauveau, D., Chretien, Y., Beigelman, C., Denys, A., Fendler, J. P., et al. (1994). Renal lesions and pheochromocytoma in von Hippel–Lindau disease. Advances in Nephrology, 23, 1–27. Schmidt, L., Duh, F. M., Chen, F., Kishida, T., Glenn, G., Choyke, P., et al. (1997). Germline and somatic mutations in the tyrosine kinase domain of the MET proto-oncogene in papillary renal carcinomas. Nature Genetics, 16, 68–73. Schoenfeld, A., Davidowitz, E. J., and Burk, R. D. (1998). A second major native von Hippel–Lindau gene product, initiated from an internal translation start site, functions as a tumor suppressor. Proceedings of the National Academy of Sciences, USA, 95, 8817–22. Siemeister, G., Weindel, K., Mohrs, K., Barleon, B., MartinyBaron, G., and Marme, D. (1996). Reversion of deregulated expression of vascular endothelial growth factor in human renal carcinoma cells by von Hippel–Lindau tumor suppressor protein. Cancer Research, 56, 2299–301. Stebbins, C. E., Kaelin, W. G., and Pavletich, N. P. (1999). Structure of the VHL-ElonginC–ElonginB complex: implications for VHL tumor suppressor function. Science, 284, 455–61. Steinbach, F., Novick, A. C., Zincke, H., Miller, D. P., Williams, R. D., Lund, G., et al. (1995). Treatment of renal-cell carcinoma in von Hippel–Lindau disease—a multicenter study. Journal of Urology, 153, 1812–16. Teh, B. T., Giraud, S., Sari, N. F., Hii, S. I., Bergerat, J. P., Larsson, C., et al. (1997). Familial non-VHL non-papillary clear-cell renal cancer. Lancet, 349, 848–9. Walther, M. M., Lubensky, I. A., Venzon, D., Zbar, B., and Linehan, W. M. (1995). Prevalence of microscopic lesions in grossly normal renal parenchyma from patients with von Hippel–Lindau disease, sporadic renal cell carcinoma and no renal disease: clinical implications. Journal of Urology, 154, 2010–4. Walther, M. M., Choyke, P. L., Glenn, G., Lyne, J. C., Rayford, W., Venzon, D., et al. (1999). Renal cancer in families with hereditary renal cancer: prospective analysis of a tumor size threshold for renal parenchymal sparing surgery. Journal of Urology, 161, 1475–9. Webster, A. R., Maher, E. R., and Moore, A. T. (1999). Clinical characteristics of ocular angiomatosis in von Hippel–Lindau disease and correlation with germline mutation. Archives of Ophthalmology, 117, 371–8. Webster, A. R., Richards, F. M., MacRonald, F. E., Moore, A. T., and Maher, E. R. (1998). An analysis of phenotypic variation in the familial cancer syndrome von Hippel–Lindau disease: evidence for modifier effects. American Journal of Human Genetics, 63, 1025–35.

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Woodward, E. R., Eng, C., McMahon, R., Voutilainen, R., Affara, N. A., Ponder, B. A. J., et al. (1997). Genetic predisposition to phaeochromocytoma: analysis of candidate genes GDNF, RET and VHL. Human Molecular Genetics, 6, 1051–6. Woodward, E. R., Hurst, L., Clifford, S. C., Affara, N. A., and Maher, E. R. (2000a). Comparative sequence analysis of the VHL tumour suppressor gene. Genomics, 65: 253–65. Woodward, E. R., Clifford, S. C., Astuti, D., Affara, N. A., and Maher, E. R. (2000b). Familial clear cell renal cell carcinoma (FCRC): clinical features and mutation analysis of the VHL, MET, and CUL2 candidate genes. Journal of Medical Genetics, 37, 348–53. Zbar, B., Kishida, T., Chen, F., Schmidt, L., Maher, E. R., Richards, F. M., et al. (1996). Germline mutations in the von Hippel–Lindau disease (VHL) gene in families from North America, Europe, and Japan. Human Mutation, 8, 348–57.

22 Inherited predispositions to kidney cancer Berton Zbar, Gladys Glenn, Maria Turner, Gregor Weirich, Peter Choyke, Stephen Hewitt, Michael Nickerson, Noboru Nakaigawa, W. Marston Linehan, and Laura Schmidt

There are a number of diseases characterized by an inherited predisposition to develop epithelial tumors of the kidney (Table 22. 1). This chapter will focus on hereditary papillary renal carcinoma type 1 (HPRC type 1), familial renal oncocytoma (FRO)/Birt–Hogg–Dubé (BHD) syndrome, renal carcinoma associated with constitutional translocations involving chromosome 3p, and familial clear cell renal carcinoma. Histologic appearance as the phenotype in inherited renal epithelial tumors One of the generalizations that has emerged from the study of inherited epithelial tumors of the kidney is that each inherited form of renal cancer has a distinct histologic appearance, that is, distinct histologic phenotype (Zbar and Lerman 1998). This conclusion is based on studies that have shown that each of the multiple renal tumors in a patient with hereditary renal carcinoma has the same histologic appearance, and that all affected family members have renal tumors with the same histologic appearance (Zbar and Lerman 1998). Renal tumors that occur in Von Hippel–Lindau (VHL) syndrome, and renal cancers that occur associated with constitutional translocations involving chromosome 3 are clear cell renal carcinomas (Zbar and Lerman 1998). The renal tumors that occur in patients affected

Table 22.1 Diseases characterized by an inherited predisposition to develop renal epithelial tumors 1. VHL disease 2. Renal cancer associated with a constitutional translocation involving chromosome 3 3. HPRC type 1 4. FRO 5. The BHD syndrome 6. Renal carcinoma associated with supernumerary nipples 7. Renal cell carcinoma (type 2 papillary) associated with familial leiomyomatosis (Kiurce et al. 2001)

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with HPRC type 1 are papillary renal carcinomas of a particular subtype, type 1 (Lubensky et al. 1999). Families with FRO have been described (Weirich et al. 1998). A corollary of these observations is that the finding of multiple renal tumors with the same histologic appearance in a patient who has relatives with renal tumors of the same histologic appearance, is presumptive evidence of a germline mutation in a renal carcinoma gene. Hereditary papillary renal carcinoma type 1 Hereditary papillay renal carcinoma type 1 is a recently described inherited disorder characterized by a predisposition to develop bilateral, multiple, type 1 papillary renal carcinomas (Zbar et al. 1994, 1995; Zbar and Lerman 1998; Lubensky et al. 1999). The disorder is inherited as an autosomal dominant Mendelian trait and is caused by activating mutations in the tyrosine kinase (TK) domain of the MET proto-oncogene (Jeffers et al. 1997, 1998a,b; Bardelli et al. 1998; Schmidt et al. 1997, 1998, 1999). Information gathered and research studies in the last few years have provided estimates of the incidence of HPRC type 1, documentation of founder effects (Schmidt et al. 1998), estimates of penetrance (Schmidt et al. 1998), details of the pathogenesis of HPRC tumors (Fischer et al. 1998; Zhuang et al. 1998), and identification of germline mutations in the MET gene in certain kidney cancer families previously described in the literature (Schmidt, L., unpubl. data). Incidence of HPRC type 1 In a US and Canadian population of about 300 million persons, we have observed 28 individuals with MET germline mutations at the National Institutes of Health over a 5-year period. One estimate of the incidence of MET germline mutations in the US and Canada would be 1 in 10 million. Figure 22.1 is a map of the US, Canada, and Europe showing the country (state) of origin of families with HPRC studied at the National Cancer Institute, Frederick, Maryland. So far, 27 families with HPRC type 1 have been identified in the world. Founder effects observed in families with HPRC Schmidt described two large North American families (families 150 and 160) with HPRC and the identical germline mutation in the MET proto-oncogene (H1112R) (Schmidt et al. 1998). Ancestors of both families could be traced to a common geographic area. Haplotype studies showed that affected members of families 150 and 160 shared a common haplotype within and immediately distal to the MET proto-oncogene. These two families were the first HPRC type 1 families described with a founder effect. Since this report, our laboratory has identified two other probable examples of founder effects in HPRC families. A large HPRC family in Spain was found to have a germline mutation in MET (V1238I); a nuclear family in France was found to have HPRC and the identical germline MET mutation; ancestors of the French HPRC family were reported to have come to France from Spain (Richard, S. and Schmidt, L., unpubl. data). We have identified a Northern Italian HPRC family with a V1110I mutation in the MET proto-oncogene (Schmidt, unpublished). A Northern Italian HPRC family with the V1110I MET mutation has been reported (Olivero et al. 1999).

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Fig. 22.1 Map of the US, Canada, and Europe showing the location of known HPRC type 1 families. The black circular symbols illustrate the location of a family affected with HPRC type 1.

Age dependent penetrance of MET mutation H1112R—relation to founder effect Schmidt et al. (1998) determined the age dependent penetrance of the MET H1112R mutation. The authors determined the frequency of renal tumors, as measured by abdominal CAT scan, in carriers of the MET H1112R mutation (Schmidt et al. 1998). Of particular interest, the penetrance of the MET H1112R mutation was found to be low. By age 50, only 30 per cent of carriers of the MET H1112R mutation had a renal tumor as detected clinically, or by abdominal CAT scan (Choyke et al. 1997). It is likely that some carriers of germline MET mutations do not develop symptomatic papillary renal tumors during their lifetimes, and that the carrier state does not compromise their reproductive ability. Pathology of HPRC type 1 tumors The recent Heidelberg pathologic classification of epithelial tumors of the human kidney recognized a type of tumor referred to as ‘papillary renal carcinoma’ (Kovacs et al. 1997). More recent studies by Delahunt and Eble (1997) suggest that papillary carcinoma of the kidney can be divided, based on pathologic criteria, into two types. Lubensky et al. (1999) found that all renal tumors of patients with germline mutations in the MET proto-oncogene were of the papillary type 1 category. This remarkable observation suggests that the pathologic subtype of papillary renal carcinoma can be used as a clue to search for germline and somatic mutations in the MET proto-oncogene, and reemphasizes the concept of a specific histologic phenotype associated with germline mutations in the MET proto-oncogene.

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Papillary type 2 renal tumors are usually single, of higher nuclear grade (3–4) and have poorer prognosis than the papillary type 1 renal tumors which are usually multiple, lower nuclear grade, less aggressive tumors.

Pathogenesis of HPRC tumors: duplication of the chromosome bearing the mutant MET allele in papillary renal carcinomas from patients with HPRC type 1 Studies by Zhuang et al. (1998) and Fischer et al. (1998) have demonstrated non-random duplication of chromosome 7 in renal tumors from patients with HPRC type 1. Using polymorphic markers that identified wild type and mutant alleles of the MET gene, these researchers demonstrated that there was consistent duplication of the chromosome 7 bearing the mutant MET allele in renal tumors. In contrast to the nonrandom duplication of chromosome 7 in HPRC tumors, Fischer et al. (1998) found that the duplication of chromosome 17 in HPRC tumors was random. This is the first example of the duplication of a human chromosome carrying a mutated oncogene in the cells of a solid tumor. Duplication of the chromosome bearing the mutant MET oncogene is the second step in the pathogenesis of HPRC tumors.

Genetic events underlying the development of HPRC type 1 tumors The evidence suggests that papillary renal carcinomas develop in HPRC patients by a multistep process (Fig. 22.2). (1) The first step in the process is the inheritance of an activating mutation in the MET TK domain. (2) In the second step, there is selection of renal cells with duplication of the chromosome 7 bearing the mutated MET oncogene. These two events lead to proliferation of a clone of renal proximal tubule cells that can be recognized as a microscopic area of cell dysplasia. Subsequent steps required for the formation of HPRC tumors may include the duplication of chromosomes 17, 12, 20, and 16 (Kovacs 1993). The genes whose expressions are altered by these trisomies are not known. One hypothesis is that the critical genes on the trisomic chromosomes are oncogenes, perhaps other receptor TK and/or their ligands.

MET mutation

Renal tubule cell

Nonrandom trisomy 7

Trisomy 17

Dysplastic renal cell

Trisomy 16 or 20

Renal adenoma

Renal carcinoma

Fig. 22.2 Diagram illustrating the stages in the development of HPRC type 1 renal carcinomas.


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Mutations in the TK domain of the MET proto-oncogene cause HPRC All mutations identified in the MET proto-oncogene to date have been missense mutations located in the TK domain of the MET proto-oncogene (Schmidt et al. 1997, 1998, 1999; Olivero et al. 1999). These mutations were restricted to 10 codons within the TK domain (Schmidt et al. 1999). Of particular interest is the observation that several of the mutations detected in the MET TK domain were located in positions identical to those of other receptor TKs mutated in human disease (Schmidt et al. 1997, 1999). Mutations were identified in the MET TK domain at a position that corresponds to a mutation in the RET proto-oncogene which causes multiple endocrine neoplasia type 2B, and at positions that correspond to mutations in the KIT receptor TK that produce mastocytosis with an hematologic disorder. These observations highlight the prominence of several codons in receptor TKs as potential mutation sites predisposing to the development of human disease.

Mutations in the MET TK domain convert the proto-oncogene to an oncogene When the mutations detected in patients with HPRC were introduced into mouse MET cDNA, these mutant mouse MET genes transformed NIH 3T3 cells, caused constitutive phosphorylation of the MET protein, and elevated kinase activity (Jeffers et al. 1997, 1998a,b; Bardelli et al. 1998). Downstream signal transducing pathways were constitutively activated in NIH 3T3 cells transformed by mutant MET genes. The adaptor protein Grb-2 was constitutively bound to MET proteins in 3T3 cells transformed by mutant MET genes (Bardelli et al. 1998; Nakaigawa, unpubl. data). PLC- was constitutively phosphorylated in extracts of 3T3 cells transformed by mutant MET genes (N. Nakaigawa, unpubl. data). In order to predict how these disease-associated mutations activated the MET TK, Schmidt et al. (1999) prepared a three-dimensional (3D) model of the MET TK domain based on the structure of the insulin growth factor receptor. When wild-type amino acids in the TK domain model were replaced with the disease-causing mutant amino acids, the mutant amino acids were predicted to interfere with the intrasteric mechanism of TK autoinhibition, which would facilitate the transition from the inactive to the active form of the kinase.

Signaling pathways used by mutant MET proteins Considerable interest has focused on the discovery of the MET mutations in HPRC because this observation provided proof of a direct connection between the MET gene and human cancer. Two groups of investigators have tried to identify the pathways triggered by constitutively active, mutant MET proteins (Jeffers et al. 1997, 1998a,b; Bardelli et al. 1998). A great deal of effort has gone into studying the pathways used by mutant mouse MET genes to transform NIH 3T3 cells. These experiments can be divided into those studying the effect of additional mutations on the ability of mutant mouse MET to transform NIH 3T3 cells, and those designed to directly evaluate signal transduction pathways.

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In a series of papers Jeffers and coworkers studied the signaling requirements for mutant MET proteins (Jeffers et al. 1997, 1998a,b). Previously these workers had found that mutant MET proteins were active in the absence of ligand. The authors set out to determine how mutant MET molecules signal, what pathways are activated, and particularly, what amino acid residues of the mutant MET protein are required for focus formation in NIH 3T3 cells and tumor growth in nude mice. In two series of experiments, the authors studied mutant MET molecules with a single mutation in the TK domain, identical to the mutations observed in patients with HPRC. In a second set of experiments the authors studied mutant MET molecules with two mutations in the TK domain. The authors dissected the signaling requirements by focusing on tyrosines Y8, 9 (autophosphorylation site) and Y14, 15 (SH2-docking site). The authors found that mutant MET molecules with a single mutation in the TK domain required intact Y8, 9 and Y14, 15 for focus formation; mutant MET molecules with two mutations in the TK domain were largely independent of the requirement of intact Y8, 9 and Y14, 15. The experiments performed with a single mutation in the TK domain of the mouse MET protein more closely reflect the situation observed in patients with HPRC and thus are more relevant to the pathogenesis of human disease. Bardelli et al. (1998) asked similar questions with mutant MET constructs in 3T3 cells and found that tyrosines Y14, Y15 were essential to focus formation in 3T3 cells transfected with mutant MET. Inhibition of transformation could be demonstrated by replacement of tyrosines Y14, 15 with phenylalanines F14, 15 and also by the addition of a soluble peptide inhibitor of the SH2-docking site (Bardelli et al. 1998). Specific inhibition of signaling molecules involved in pathways utilized by mutant MET The studies performed by Jeffers and Bardelli have introduced additional mutations into mouse MET cDNAs containing activating mutations in the TK domain. In most experiments, tyrosine residues in the catalytic region were replaced with phenylalanines to assess the role of these tyrosines in mediating the biologic effects of the activating mutations. Another approach that has been used to study mechanisms of transformation by MET has been to inhibit specific members of the signal transduction pathway. Nakaigawa (Nakaigawa, N., unpubl. data) obtained evidence suggesting that c-Src participates in the process whereby mutant MET molecules produce cellular transformation of NIH 3T3 cells. Transfection of a clonal NIH 3T3 cell line (F4) transformed by MET mutant M1268T with dominant negative c-Src genes returned the growth properties of this clonal cell line toward those of nontransformed cells. The presence of dominant negative c-Src markedly reduced the size of tumors induced by injection of 3T3 cells carrying the M1268T mutation into nude mice. Conclusions Hereditary papillary renal carcinoma type 1, a disease characterized by a predisposition to develop multiple papillary renal tumors, is caused by activating mutations in the tyrosine kinase domain of the MET proto-oncogene. Nonrandom duplication of the chromosome 7 bearing the mutant MET allele may act as the second event leading to the development of


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papillary renal tumors. 3D modeling experiments predict that the mutations interfere with the intrasteric mechanism of autoinhibition of the MET TK, transitioning the enzymatically inactive form of the kinase to the active form. FRO and the BHD syndrome FRO is a recently described disorder characterized by two or more family members affected with single or multiple, unilateral, or bilateral renal oncocytomas (Weirich et al. 1998). In the report by Weirich et al. (1998), five families were described. Renal tumors were usually detected incidentally in asymptomatic individuals by ultrasound examinations, or by CAT scans of the abdomen. The FRO families were usually small nuclear families with a maximum of four affected individuals. The major problem in recruiting additional FRO families was probably a consequence of the fact that renal oncocytomas often do not produce symptoms. FRO families might be detected by screening the population for renal tumors. FRO appears to be distinct from other forms of inherited renal neoplasia. No germline mutations in VHL, MET, or PTEN genes were detected in members of FRO families (Weirich et al. 1998; Toro et al. 1999). Some FRO family members were found to have a rare hamartomatous tumor of the hair follicle, termed a Fibrofolliculoma (FF) (Toro et al. 1999), previously identified as the phenotypic feature of the BHD syndrome (Birt et al. 1977). These FF occurred in 3 FRO families (families 166, 167, 168) and one family (family 171) with an unusual type of papillary renal carcinoma (histologically distinct from papillary renal carcinoma type 1) (Fig. 22.3). Examination of families with other forms of inherited renal carcinoma (VHL disease, HPRC Spontaneous pneumothorax/ pulmonary cysts

Renal oncocytoma/ papillary renal tumor

166, 171

167, 168, 172

Fibrofolliculoma Fig. 22.3 Venn diagram illustrating National Cancer Institute’s (NCI) renal cancer families with FF and spontaneous pneumothorax/ pulmonary cysts. Note that members of families 168, 167, and 172 had members affected with renal tumors, FF, and spontaneous pneumothorax. Families 166 and 171 had members affected with renal tumors and FF, without evidence of pneumothorax. Family 172 was originally reported by Birt et al. (1977).

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type 1, familial clear cell renal carcinoma) did not detect any instances of patients with FF (Toro et al. 1999). Members of FRO families 167 and 168 were also found to have pulmonary cysts and spontaneous pneumothorax (Toro et al. 1999). These results suggest the presence in families 166, 167, 168, and 171 of the manifestations of two inherited diseases, FRO, and the BHD syndrome. There are several possible explanations of the occurrence of FRO and BHD in these four families. (1) Members of these four families have two disease genes segregating together. (2) Members of these families have a single disease gene segregating that produces manifestations of both FRO and BHD. (3) Members of these families have a disease gene that is interacting with an environmental factor. The BHD syndrome The BHD syndrome is a rare inherited disorder of the skin first described in 1977 (Birt et al. 1977). The disorder, inherited as an autosomal dominant trait, is characterized by tumors of the hair follicle. These hair follicle tumors are hamartomas with proliferation of mesenchymal and epithelial elements of the hair follicle. Typical features of the hair follicle tumors are long, thin strands of epithelial cells radiating from a distorted hair follicle. The hair follicle tumors, termed FF, occur on the face, nose, cheeks, forehead, ear lobes, neck and upper chest. The FF do not hurt, itch, invade or metastasize. FF usually develop after age 20. The papules, 1–3 mm in diameter, vary considerably in number in affected individuals from less than 10 lesions to hundreds, or thousands of lesions. FF may be analogous to the thousands of polyps that form in the colons of people with hereditary polyposis. It is not known whether these lesions are clonal; based on other disorders, these FF are likely to be clonal. Since the report of Birt et al. in 1977, a number of case reports have appeared describing families with this disorder (Hornstein and Knickenberg 1975; Hornstein 1976; Hornstein et al. 1976; Binet et al. 1986; Rongioletti et al. 1989; Roth et al. 1993; Chung et al. 1996; Schachtsschabel et al. 1996; Le Guyadec et al. 1998; Schulz and Hartschuh 1999). BHD appears to be more common than HPRC. Some of the clinical reports suggested that renal tumors, spontaneous pneumothorax, lipomas and colonic adenomas might be associated with BHD.

Renal tumors in the BHD syndrome When the frequency of renal tumors in FF positive FRO family members was compared to the frequency of renal tumors in FF negative family members, the renal tumors were more common in FF positive compared with FF negative family members, but the differences were not statistically significant.

Synopsis of observations on FRO and the BHD syndrome The observations presented by Toro et al. (1999) raise the possibility that the predisposition to renal tumors, FF, spontaneous pneumothorax and colonic tumors may be a consequence of


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the mutation of a single gene. The idea predicts that if patients/families are recruited on the basis of a diagnosis of the BHD syndrome, families recruited on this basis would have members affected with renal tumors; furthermore, one would predict that the renal tumors should occur in family members affected with FF (disease gene carriers). This hypothesis can be tested by determining the location of the BHD gene by linkage analysis, cloning the BHD gene and testing for BHD gene mutations in FRO families and in families with an unusual type of papillary renal carcinoma. Recently the BHD gene was mapped to 17p11.2 providing a basis for these studies (Khoo et al. 2001; Schmidt et al. 2001). The gene responsible for the Birt–Hogg–Dube syndrome has since been cloned (Nickerson et al. 2002).

Clear cell renal carcinomas associated with constitutional translocations involving chromosome 3 In 1979, Cohen et al. described a large Italian American family with an inherited predisposition to develop bilateral multiple clear cell renal carcinomas associated with a constitutional translocation between the short arm of chromosome 3 and the long arm of chromosome 8 [t(3;8) (p14;q24)]. The incidence of RCC among translocation carriers was 67 per cent. Initial attempts to find additional renal carcinoma-associated translocation families were unsuccessful. However, several new translocation families with chromosome 3 involvement and incidence of renal carcinoma have recently been reported (Kovacs and Hoene 1988; Kovacs et al. 1989; Bodmer et al. 1998; Geurts van Kessel et al. 1999). Kovacs et al. (1989) reported a family which inherited a constitutional t(3;6)(p13;q25) and in which one member has developed bilateral multiple RCC. A new familial case was described by Bodmer et al. (1998) with a constitutional translocation between 2q35 and 3q21. Four patients over three generations which carry the translocation have developed clear cell renal carcinoma (33 per cent of translocation carriers) and one developed bladder cancer. Studies of the molecular genetics of renal tumors formed in the t(3;8) family of Li were performed by Schmidt et al. (1995; Knudson 1995) and in the t(2;3) family by Bodmer (Bodmer et al. 1998). The results of these studies may be summarized as follows: (1) Renal tumors from these translocation patients showed a consistent loss of the derivative chromosome carrying the translocated chromosome 3p. (2) Renal tumors from family members carrying the translocation were found to contain different somatic mutations in the VHL tumor suppressor gene on 3p25. The frequency of developing RCC in chromosome 3 families is high but the individual age of onset varies considerably. This wide range in penetrance may reflect the degree of somatic mosaicism with which loss of the derivative chromosome occurs in the kidney epithelial cells (Bodmer et al. 1998). The location of the fragile site (breakpoint) may also play a role in the frequency of RCC development in the translocation families (i.e. familial translocations in at-risk families exhibit pericentromeric breakpoints in the proximal 3p and 3q arms) (Geurts van Kessel et al. 1999). Schmidt (1995; Knudson 1995), Bodmer et al. (1998), and Kovacs et al. (1989) have proposed similar models for multistep tumorigenesis in familial translocation-associated RCC which include the following steps: (1) inheritance of the translocation involving chromosome 3, (2) loss of the translocation-derived chromosome 3 by random nondisjunction (somatic mosaicism), and (3) somatic mutation of the remaining copy of the VHL gene leading to loss of tumor suppression and initiation of tumorigenesis (Fig. 22.4).

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VHL

VHL

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Point mutation of VHL gene on normal chromosome 3

Loss of derivative 8 8q24 3

der 3

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VHL der 8 Inheritance of a balanced chromosome (3;8) translocation

Fig. 22.4 Steps in the development of clear cell renal carcinoma associated with the chromosome 3;8 translocation. Events wihin a single renal epithelial cell are shown. In the order shown, loss of the derivative chromosome 8 precedes mutation of the VHL gene. Mutation of the VHL gene could also precede the loss of the derivative 8. VHL, wild-type VHL gene; mutVHL, mutated VHL gene. (Reprinted with permission from Schmidt et al. (1995). All rights reserved.)

Both Schmidt et al. (1995) and Bodmer et al. (1998) report renal tumors from these translocation families which did not harbor a VHL mutation. Schmidt et al. (1995) found no hypermethylation of the 5 end of the VHL gene in renal tumors that did not have mutational inactivation of VHL. Mutations of the 3UTR or promoter region were not examined and therefore cannot be ruled out. Another explanation is the possibility that another tumor suppressor gene on chromosome 3 is responsible for the development of RCC in these particular tumors. Chromosome 3p14 and 3p21 are proposed sites of tumor suppressor genes. Familial clear cell renal carcinoma (nonVHL, nonpapillary renal carcinoma) Familial clear cell renal carcinoma is characterized by two or more family members affected with clear cell renal carcinoma. In these families renal tumors usually occur in older individuals; tumors are usually single, and unilateral. VHL mutation testing in the germline is negative. (There have been no studies of VHL mutations in the renal tumors.) The number of affected individuals ranged from 2 to 5. The cause of these familial aggregations of clear cell renal carcinoma is not known. Some of these cases of clear cell RCC may occur by chance (Teh et al. 1997). Other possible inherited predispositions to renal cancer The possibility remains that there are some forms of inherited epithelial renal neoplasia that remain to be discovered. One family with multiple members affected with papillary renal carcinoma type 2 has been seen at the NCI (Linehan Zbar, unpubl. data).


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Screening for tumours in familial renal carcinoma Annual screening by renal ultrasound or MRI scanning is recommended for at risk relatives as in VHL disease (see Chapter 21). However, the age at starting screening may be later depending on the family history. Acknowledgements This project has been funded in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. NO1-CO-56000. References Bardelli, A., Longati, P., Gramaglia, D., Basilico, C., Tamagnone, L., Giordano, S., et al. (1998). Uncoupling signal transducers from oncogenic MET mutants abrogates cell transformation and inhibits invasive growth. Proceedings of the National Academy of Sciences, USA, 95, 14379–83. Binet, O., Robin, J., Vicart, M., Ventura, G., and Beltzer-Garelly, E. (1986). Fibromes perifolliculaires polypose colique familaile pneumothorax spontanes familiaux. Annals Dermatology and Venereology, 113, 928–30. Birt, A. R., Hogg, G. R., and Dubé W. J. (1977). Hereditary multiple fibrofolliculomas and trichodiscomas and acrochordons. Archives of Dermatology, 113, 1674–7. Bodmer, D., Elevled, M. J., Ligtenberg, M. J. L., Weterman, M. A., Janssen, B. A. P., Smeets, D. F. C. M., et al. (1998). An alternative route for multistep tumorigenesis in a novel case of hereditary renal cancer and a t(2;3) (q35;q21) chromosome translocation. American Journal of Human Genetics, 62, 1475–83. Choyke, P., Walther, M., Glenn, G., Wagner, J., Venzon, D., Lubensky, I., et al. (1997). Imaging features of hereditary papillary renal carcinoma. Journal of Computer Assisted Tomography, 21, 737–41. Chung, J. Y., Ramos-Caro, F. A., Ford, M. J., and Flowers, F. (1996). Multiple lipomas, angiolipomas, and parathyroid adenomas in a patient with Birt–Hogg–Dube syndrome. International Journal of Dermatology, 35, 365–7. Cohen, A. J., Li, F. P., Berg, S., Marchetto, D. J., Tsai, S., Jacobs, S. C., et al. (1979). Hereditary renalcell carcinoma associated with a chromosomal translocation. New England Journal of Medicine, 301, 592–5. Delahunt, B. and Eble, J. (1997). Papillary renal carcinoma: a clinicopathologic and immunohistochemical study of 105 tumors. Modern Pathology, 10, 537–44. Fischer, J., Palmedo, G., von Knobloch, R., Bugert, P., Prayer-Galetti, T., Pagano, F., et al. (1998). Duplication and overexpression of the mutant allele of the MET proto-oncogene in multiple papillary renal cell tumors. Oncogene, 17, 733–40. Geurts van Kessel, A., Wijnhoven, H., Bodmer, D., Eleveld, M., Kiemeney, L., Mulders, P., et al. (1999). Renal cell cancer: chromosome 3 translocations as risk factors. Journal of the National Cancer Institute, 91, 99–100. Hornstein, O. P. (1976). Generalized dermal perifollicular fibroma with polyps of the colon. Human Genetics, 33, 193–7. Hornstein, O. P. and Knickenberg, M. (1975). Perifollicular cutis with polyps of the colon—a cutaneointestinal syndrome sui generis. Archives of Dermatological Research, 253, 161–75. Hornstein, O. P., Knickenberg, M., and Morel, M. (1976). Multiple dermal fibromas with polyps of the colon—report of peculiar clinical synndrome. Acta Hepatitis Gastroenterology, 23, 53–8.

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Jeffers, M., Fiscella, M., Webb, C. P., Anver, M., Koochekpour, S., and Vande Woude, G. (1998a). The mutationally activated Met receptor mediates motility and metastasis. Proceedings of the National Academy of Sciences, USA, 95, 14417–22. Jeffers, M., Koochekpour, S., Fiscella, M., Sathyanarayana, B. K., and Vande Woude, G. (1998b). Signaling requirements for oncogeneic forms of the Met tyrosine kinase receptor. Oncogene, 17, 2691–700. Jeffers, M., Schmidt, L., Nakaigawa, N., Webb, C., Weirich, G., Kishida, T., et al. (1997). Activating mutations for the Met tyrosine kinase receptor in human cancer. Proceedings of the National Academy of Sciences, USA, 94, 11445–50. Khoo, S. K., Bradley, M., Wong, F. K., Hedblad, M. A., Nordenskjold, M., and Teh, B. T. (2001). Brit–Hogg–Dube Syndrome: mapping of a novel hereditary neoplasia gene to chromosome 17 p12-911.2. Oncogene, 20(37), 5239–42. Kiurce, M., Launonen, V., Hietala, M., Aittomaki, K., Vierimaa, O., Salovaava, R., et al. (2001). Familiar cutaneous leiomyomatosis is a two-hit condition associated with renal cell cancer of characteristic histopathology. American Journal of Pathology, 159(3), 825–9. Knudson, A. G. (1995). VHL gene mutation and clear cell renal carcinomas. The Cancer Journal from Scientific American, 1, 180–1. Kovacs, G. (1993). Molecular cytogenetics of renal cell tumors. Advances in Cancer Research, 62, 89–124. Kovacs, G., Akhtar, M., Beckwith, B. J., Bujert, P., Cooper, C. S., Delahunt, B., et al. (1997). The Heidelberg classification of renal cell tumours. Journal of Pathology, 183, 131–3. Kovacs, G., Brusa, P., and DeRiese, W. (1989). Tissue-specific repression of a constitutional 3;6 translocation development of multiple bilateral renal-cell carcinoma. International Journal of Cancer, 43, 422–7. Kovacs, G. and Hoene, E. (1988). Loss of der(3) in renal carcinoma cell of a patient with constitutional t(3;12). Human Genetics, 78, 148–50. Le Guyadec, T., Dufau, J.-P., Poulain, J.-F., Vaylet, F., Frossin, M., and Lanternier, G. (1998). Trichodiscomes multiples associes a une polypose colique. 125, 717–19. Lubensky, I. A., Schmidt, L., Zhuang, Z. P., Weirich, G., Pack, S., Zambrano, N., et al. (1999). Hereditary and sporadic papillary renal carcinomas with c-MET mutations share a distinct morphologic phenotype. American Journal of Pathology, 155, 517–26. Nickerson, M. L., Warren, M. B., Toro, J. R., Matrosova, V., Glenn, G., Turner, M. L., et al. (2002). Mutations in a novel gene lead to kidney tumors, lung wall defects, and benign tumors of the hair follicle in patients with the Birt–Hogg–Dube’ syndrome. Cancer Cell, 2, 157–64. Olivero, M., Valente, G., Bardelli, A., Longati, P., Ferrero, N., Cracco, C., et al. (1999). Novel mutation in the ATP-binding site of the MET oncogene tyrosine kinase in a HPRCC family. International Journal of Cancer, 82, 640–3. Rongioletti, F., Hazini, R., Gianotti, G., and Rebora, A. (1989). Fibrofolliculomas, trichdiscomes and acrochordons (Birt–Hogg–Dube) associated with intestinal polyposis. Clinical and Experimental Dermatology, 14, 72–4. Roth, J. S., Rabinsowitz, A. D., Benson, M., and Grossman, M. E. (1993). Bilateral renal cell carcinoma in the Birt–Hogg–Dube syndrome. Journal of the American Academy of Dermatology, 29, 1055–6. Schachtsschabel, A. A., Kuster, W., and Happle, R. (1996). Perifollikulare fibrome der haut und kolonpolypen: Hornstein–Knickenberg syndrom. Hautarzt, 47, 304–6. Schmidt, L., Duh, F. M., Chen, F., Kishida, T., Glenn, G., Choyke, P., et al. (1997). Germline and somatic mutations in the tyrosine kinase domain of the MET proto-oncogene in papillary renal carcinomas. Nature Genetics, 16, 68–73. Schmidt, L., Junker, K., Nakaigawa, N., Kinjerski, T., Weirich, G., Miller, M., et al. (1999). Novel mutations in the MET proto-oncogene in papillary renal carcinomas. Oncogene, 18, 2343–50.


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Schmidt, L., Junker, K., Weirich, G., Glenn, G., Choyke, P., Lubensky, I., et al. (1998). Two North American families with hereditary papillary renal carcinoma and identical novel mutations in the MET proto-oncogene. Cancer Research, 58, 1719–22. Schmidt, L., Li, F., Brown, R. S., Berg, S., Chen, F., Wei, M.-H., et al. (1995). Mechanism of tumorigenesis of renal carcinomas associated with the constitutional chromosome 3;8 translocation. The Cancer Journal from Scientific American, 1, 191–5. Schmidt, L. S., Warven, M. B., Nickerson, M. L., Weirich, G., Matrosova, V., Toro, J. R., et al. (2001). Brit–Hogg–Dube syndrome, a genodermatosis associated with spontaneous pneumothorax and kidney neoplasia, maps to chromosome 17p11.2. American Journal of Human Genetics, 69(4), 876–82. Schulz, T. and Hartschuh, W. (1999). Birt–Hogg–Dubé syndrome and Hornstein-Knickenbergsyndrome are the same. Different sectioning technique as the cause of different histology. Journal of Cutaneous Pathology, 26, 55–61. Teh, B. T., Giraud, S., Sari, N. F., Hii, S. I., Bergerat, J. P., Larsson, C., et al. (1997). Familial non-VHL, non-papillary clear-cell renal cancer. Lancet, 344, 848–9. Toro, J., Duray, P., Glenn, G., Darling, T., Weirich, G., Zbar, B., et al. (1999). Birt–Hogg–Dube syndrome: a novel marker of renal neoplasia. Archives in Dermatology, (in press). Weirich, G., Glenn, G., Junker, K., Merino, M., Störkel, S., Lubensky, I., et al. (1998). Familial renal oncocytoma: clinicopathologic study of 5 families. Journal of Urology, 160, 335–40. Zbar, B., Glenn, G., Lubensky, I., Choyke, P., Walther, M. M., Magnusson, G., et al. (1995). Hereditary papillary renal cell carcinoma: clinical studies in 10 families. Journal of Urology, 153, 907–12. Zbar, B. and Lerman, M. I. (1998). Inherited carcinomas of the kidney. Advances in Cancer Research, 75, 164–201. Zbar, B., Tory, K., Merino, M., Schmidt, L., Glenn, G., Choyke, P., et al. (1994). Hereditary papillary renal cell carcinoma. Journal of Urology, 151, 561–6. Zhuang, Z., Park, W. S., Pack, S., Schmidt, L., Vortmeyer, A. O., Pak, E., et al. (1998). Trisomy 7-harboring non-random duplication of the mutant c-met proto-oncogene allele in hereditary papillary renal carcinomas. Nature Genetics, 20, 66–9.


23 Gene therapy in renal disease Anand K. Saggar-Malik

Molecular biological intervention therapy has come of age and generates tremendous excitement. Gene therapy has the distinct potential to treat disease at the most fundamental level. However, therapy of renal diseases is at present still too immature for clinical application and significant barriers remain before successful organ-specific molecular therapy can be applied to the kidney. By the end of 1998, 389 patients had been treated with gene therapy according to 367 protocols although most methods were doomed to fail (Imai 2001). Gene therapy requires the successful transfer of nucleic acids either as oligonucleotides or genetic constructs into specific renal structures. The main reasons for failure result from technical issues such as imperfect vectors and limited availability of robust and adequate systems for gene delivery into the kidney. Another problem which has been identified is the shutdown of transgene expression after successful gene transfer. New and substantially improved vector systems and related technologies are undergoing development: many have shown promise in animal studies, and some are now being used in clinical trials (Imai 2001; Kaneda 2001). Viral vectors, principally adenoviruses which have the most potential, all have limitations for clinical use either because of toxicity or immunogenicity. From the practical point of view, two promising strategies for the therapy of renal diseases are skeletal muscle targeted gene therapy and kidney transplantation. These offer favourable approaches to gene delivery with sustained gene expression. Skeletal muscle targeted gene therapy is an alternative strategy for the treatment of kidney disease given that the systemic delivery of a functional protein may be adequate for treatment (Imai and Isaka 1999). Skeletal muscle targeting gene therapy is relatively easy to perform, repeatable and highly efficient in generating the secreting protein. Similarly the transplanted kidney is an ideal target as genes can be delivered during reperfusion prior to transplantation. The feasibility of delivering therapeutic genes ex-vivo, has been shown to reduce the rejection rate of transplanted kidneys in an animal model. Zeigler et al. (1997), using an adenovirus vector, reported the first trial of ex-vivo gene transfer into the isolated human kidney under the conditions of organ preservation. Perfusion of the adenovirus vector into the renal artery for twelve hours resulted in an intense expression of the reporter gene in 85 per cent of the glomeruli. In future, gene therapy to the transplant kidney may potentially improve graft outcome by reducing acute and chronic rejection. The kidney has an advantage over some other solid organs since it is accessible by many routes, including intrarenal artery infusion, retrogradely through the bladder and ureters, and ex-vivo before transplantation. Through the renal circulation, exogenous genes can be targeted to the vasculature and glomerulus and possibly to the proximal tubules, while access to the collecting ducts can be gained by the retrograde approach and implantation of genetically modified cells under the capsule of the kidney allows access to the interstitium.

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Anand K. Saggar-Malik Table 23.1 Summary of different methods for gene therapy delivery In-vivo gene transfer Viral Retrovirus Adenovirus Adeno-associated virus Non-viral vectors Direct intravenous injection Liposome mediated Haemagglutinating virus of Japan (HJV)-liposome Electroporation Ex-vivo transfer Transfusion of genetically modified cells Transplanted kidney Adapted from Imai (2001).

At the experimental level, several approaches regarding renal gene therapy exist. Recent advances have led to pre-clinical tests of gene-therapeutic approaches in the treatment of renal diseases and hypertension (Kone 2000). Azuma et al. (1999) reported that fibrotic changes in an animal model of chronic kidney rejection could be attenuated by the administration of the anti-fibrotic hepatocyte growth factor. These results suggest that prevention of early fibrosis by gene therapy is possible in the treatment of chronic rejection. Another exciting application of gene therapy technology and research is within the field of renal replacement therapy. Dialysis neglects the resorptive, homeostatic, metabolic, and hormonal functions of the kidney. The main focus has been on replacement, albeit partially, of the kidneys filtration properties. An area of great interest is the development of ‘intelligent dialysis’ systems. Identification of the growth factors capable of directing tissue development, coupled with techniques for delivering these factors, has increased such that it is now possible to develop engineered human tissue. Application of tissue-engineering techniques within artificial dialysis devices herald improvements in many aspects of renal function replacement. The combination of tissue-engineering strategies with gene therapy might allow the transfection of diseased tissues. To date, such devices are only experimental (for a more detailed review see Amiel et al. 2000). In a truly futuristic vision, biological and diagnostic components could be combined according to the specific needs of the individual patient. Given that gene therapy cannot alter basic renal structure, such treatment is limited to preservation of renal structure in progressive conditions. Growth factors and cytokines play a crucial role in the progression of renal diseases. Evidence from experimental studies suggests that manipulation of the activity of growth factors and cytokines is a potential therapeutic approach for renal diseases. This may be achieved by inhibition of apoptosis of renal intrinsic cells and/or decreasing the fibrotic signal. Inhibition of transforming growth factor (TGF-), platelet-derived growth factor, interleukin-1 and tumor necrosis factor , and supplementation of hepatocyte growth factor, vascular endothelial growth factor and bone morphogenic protein-7 are suggested approaches. Targeting signal transduction molecules and their co-factors and regulators is another possibility because the signals from various growth factors use a common pathway. The role of

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targeting growth factors and cytokines in renal diseases as a therapeutic approach has recently been reviewed by Imai and Isaka (2002). Gene therapy for the treatment of experimental glomerulonephritis is ongoing. Inhibition of the TGF- action by antisense oligonucleotides can suppress the development of the experimental glomerulonephritis (Imai et al. 1998). Finally, transplantation of embryonic metanephric tissues may provide another route for genetic manipulation. Furthermore, utilization of fertilized eggs or embryonic stem cells would enable the creation of ‘transgenic kidneys’ or ‘gene knockout kidneys’ (Imai 2001; Kitamura and Fine 1997). While the manufacture and widespread use of gene therapy products as conventional pharmaceuticals for renal diseases and hypertension remains a goal for the future, the much needed technology and intellectual resources are rapidly becoming available. Gene therapy of well-selected kidney diseases will certainly become feasible in the future. Obvious targets for therapy are: polycystin in dominant polycystic kidney disease; human immunodeficiency virus (HIV) type 1 in HIV-associated nephropathy; -galactosidase A in Fabry disease; and the collagen IV chains in Alport syndrome. In addition, several potential mediators of progressive renal disease may be amenable to molecular therapeutic strategies, such as TGF-, interleukin-6, and basic fibroblast growth factor (bFGF). However, perhaps the greatest benefit to date is found in the treatment of renal malignancies. Some protocols against renal cancer have shown possible inhibition of growth in certain cancer types. It is for this reason that the following chapter focuses upon gene therapy for renal cancers. References Amiel, G. E., Yoo, J. J., and Atala, A. (2000). Renal therapy using tissue-engineered constructs and gene delivery. World Journal of Urology 18, 71–9. Azuma, H., Takahara, S., Kitamura, M., Wang, J. D., Wega, A., Sayegh, M. H., et al. (1999). Effect of hepatocyte growth factor on chronic rejection in rat renal allografts. Transplant Proceedings 31, 854–5. Imai, E. (2001). Gene therapy approach in renal disease in the 21st century. Nephrology, Dialysis, Transplantation 16, 26–34. Imai, E. and Isaka, Y. (1999). New paradigm of gene therapy: skeletal-muscle-targeting gene therapy for kidney disease. Nephron 83, 296–300. Imai, E. and Isaka, Y. (2002). Targeting growth factors to the kidney: myth or reality? Current Opinion in Nephrology and Hypertension 11, 49–57. Imai, E., Isaka, Y., Akagi, Y., Kaneda, Y., and Hori, M. (1998). Gene transfer and kidney disease. Journal of Nephrology 11, 16–19. Kaneda, Y. (2001). Improvements in gene therapy technologies. Molecular Urology 5, 85–9. Kitamura, M. and Fine, L. G. (1997). Genetic manipulation of the kidney. Pediatric Nephrology 11, 773–7. Kone, B. C. (2000). How will gene therapy apply to the kidney in the 21st century? Seminars in Nephrology 20, 47–59. Zeigler, S. T., Kerby, J. D., and Thompson, J. A. (1997). Molecular conjugate-mediated gene transfer into isolated human and transplanted rat kidneys. Experimental Nephrology 5, 508–13.


24 Gene therapy for renal cancer Michael J. Gough and Richard G. Vile

Introduction This chapter looks at the basic principles of gene therapy in the kidney and in particular the potential for the treatment of renal malignancies. Despite greater understanding of the molecular mechanisms of renal cancer, there remains a pressing need for a novel therapy (Marshall et al. 1997). Survival rates have improved little over the last 30 years (Robson et al. 1969), largely as a result of the failure of conventional therapies such as chemotherapy and radiotherapy, such that surgery remains the most effective form of treatment for patients with kidney cancer (Marshall et al. 1997). Yagoda et al. (1995) reviewed the literature and found no definitive evidence that therapy has favorably affected survival of patients with advanced local and distant disease over a 10-year period. A potential reason for the poor response to cytotoxic agents is intrinsic drug resistance (Fojo et al. 1987; Volm et al. 1992), coupled with a long doubling time and low growth fraction of renal cell carcinoma that reduces susceptibility to chemotherapeutic agents. Despite these problems, spontaneous regressions have been reported in patients with renal cancer (Kallmeyer and Dittrich 1992; Marcus et al. 1993; Edwards et al. 1996). Renal carcinoma tissue contains clonal expansions of cytotoxic T cells (Caignard et al. 1996), and these exhibit specific anti-tumor reactivity in vitro (Bernhard et al. 1994; Brouwenstijn et al. 1996; Angevin et al. 1997). Thus renal cell carcinoma may present an attractive target for immunotherapies. Systemic cytokine therapies have been associated with clinical responses in renal cell carcinoma, and responses where present are durable (Rosenberg et al. 1994; Bukowski 1997; Negrier et al. 1998). An alternative is the use of more specific immunotherapies, utilizing tumor antigens. The majority of tumor antigen research to-date has focused on melanoma as an accessible, easily cultured, and immune-responsive tumor. However, there are a number of shared and unique tumor antigens that are expressed by renal cells (Gaugler et al. 1996; Tureci et al. 1996, 1998a,b; Tannapfel et al. 1997; Zhang et al. 1997) recognizable by T cells (Kim et al. 1990; Bernhard et al. 1994; Brouwenstijn et al. 1996), and antibodies (Oosterwijk et al. 1986; Luiten et al. 1996; Steffens et al. 1997). These represent both differentiated antigens expressed in the normal kidney, as well as apparently renal cell carcinoma-restricted antigens. Thus renal cell carcinoma may be a suitable candidate for specific anti-tumor immunotherapy. Transfection of renal cells with cytokine genes (Golumbek et al. 1991; Pardoll et al. 1996) or co-stimulatory molecules (Kerkmann-Tucek et al. 1998) has provided good results in animal models, and clinical studies in humans are ongoing at this point (Simons et al. 1997; Veelken et al. 1997). Therapy with antigen-pulsed dendritic cells is a very powerful therapy for many cancers in animal models (Porgador et al. 1996; Specht et al. 1997), and loading of dendritic cells with autologous tumor lysates has generated responses to renal cells in vitro (Mulders et al. 1999) and in vivo

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(Holtl et al. 1999). Furthermore treatment of patients with electrofused allogeneic dendritic cells and autologous tumor cells has been associated with complete responses in renal cell carcinoma (Kugler et al. 2000). It is in this context of renal cell carcinoma as a prominent malignancy with limited effective therapeutic options, but with significant potential for use of immune mechanisms to treat the disease, that gene therapy should be considered. The malignant phenotype is likely to be due to a combination of activated oncogenes, inactivated tumor suppressor genes, and enhanced genomic instability that are all frequently observed in tumor cells and associated with cancer progression. Potentially, each of these facets of malignancy can form targets for gene therapy. However, the importance of individual mutations in the overall process of tumorigenesis is uncertain, and the relative importance of these different genetic components in tumorigenesis is still under debate (Prehn 1994; Jackson and Loeb 1998; Tomlinson and Bodmer 1999). Thus unlike monogenic genetic diseases such as cystic fibrosis, the multiple genes that may be involved in tumor growth provide a very difficult target to correct. In addition, the oligoclonal nature of tumors, imply that single gene targeting strategies may be inappropriate within a specific tumor type, or even an individual patient. We believe that the principle aim of cancer therapies is to destroy the tumor mass, rather than ‘cure’ individual cells of their genetic defects, and thus we concentrate on strategies resulting in cytotoxicity. For these reasons, in this chapter, we will discuss gene therapy strategies that are likely to be effective against tumors regardless of the genetic make-up, and which are likely to be broadly applicable to a wide range of human cancers. Though there are wide ranges of cancer gene therapy options in development and in clinical trials, only small subsets have been applied to renal cell cancer. For this reason we will discuss the applicability of these therapies, before discussing their use in therapy of renal cell cancer. Vector systems Arrays of vector systems are available for in vivo gene delivery and a greater number are in development. However, currently each is associated with significant limitations, and the choice of delivery system is highly dependent on the intended application (Verma and Somia 1997; Jane et al. 1998; Vile et al. 2000). Gene therapy to repair genetic defects may require long-term expression and repeated administration, whereas gene therapy designed to stimulate immune responses or kill target cells may require only short-term expression. Cancer gene therapy may require a combination of these approaches. Thus long-term expression of anti-angiogenic therapies would be intended to control the angiogenic switch in growing tumors and inhibit metastasis of established tumors, while targeted, short-term expression of cytotoxic genes and immune-related molecules would be intended to aggressively counter more established tumor masses. The most relevant available options and potential vector systems in development will be discussed below. Viral vectors Retrovirus C-type retroviral vectors are used in the majority of approved protocols for gene therapy (Roth and Cristiano 1997) (Fig. 24.1). The retrovirus life cycle has been extensively investigated, and

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Patients by vector Retrovirus (n = 1751) 51% Adenovirus (n = 620) 18.0% Lipofection (n = 169) 18.0% Naked/Plasmid DNA (n = 93) 2.7% Pox virus (n = 88) 2.6% Adeno-associated virus (n = 36) 1.0% RNA transfer (n = 30) 0.9% Gene gun (n = 35) 1.0% Herpes simplex virus (n = 21) 0.6% N/C (n = 143) 4.2% Protocols by vector Retrovirus (n = 204) 38.3% Adenovirus (n = 136) 25.6% Lipofection (n = 68) 12.8% Naked/Plasmid DNA (n = 45) 8.5% Pox virus (n = 34) 6.4% Adeno-associated virus (n = 10) 1.9% RNA transfer (n = 5) 0.9% Gene gun (n = 5) 0.9% Herpes simplex virus (n = 3) 0.6% N/C (n = 22) 4.1% Fig. 24.1 Vector systems. (Reproduced with permission from The Journal of Gene Medicine, © John Wiley & Sons 2001 www.wiley.co.uk/genmed.)

this has permitted extensive re-engineering of retroviral vectors for gene therapy, with the majority of such vectors based around Moloney murine leukemia virus (Vile and Russell 1995). Retroviral entry is mediated via the product of the env gene, which binds to cellular receptors and mediates endocytosis followed by membrane fusion to access the cytoplasm. Here the RNA viral genome is reverse transcribed into single-stranded DNA (using proteins encoded in the viral pol gene) and through a complex series of priming and polymerization events is transformed into a double-stranded DNA molecule. At this point, in dividing cells where the nuclear membrane is degraded, the viral DNA is randomly integrated into the host genome through the action of the viral integrase gene (encoded in pol). Once integrated the viral 5 long terminal repeat (LTR) drives expression of a full length viral RNA that can be packaged into new virions, along with spliced forms that generate the necessary proteins for packaging, assembly and the next cycle of viral infection once in a new host cell. Excluding the replication-competent vector systems described later, C-type retroviral vectors

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are made replication-incompetent through deletion of key viral genes, usually including most of gag and pol. For assembly of infectious virions, it is necessary to use viral packaging or producer cell lines that complement the requisite viral genes in trans through stable expression of gag, pol, and env. These cells cannot themselves make infectious virus since the viral genes are integrated from separate DNA sequences and they lack the requisite packaging sequence () for RNA incorporation into the virus particle. As will be discussed later, the env gene encodes the initial entry capacity of the virus, and this is the focus of re-engineering strategies to target viral entry to specific cell types. Since C-type retroviruses require nuclear membrane breakdown to enter the nucleus and integrate into the host genome, this makes effective infection and gene expression using these vectors dependent on cell proliferation (Miller et al. 1990). Though this limits the cells which can be infected with retroviruses, as will be discussed later this property may be a targeting advantage for tumors. The integration event itself is both a positive and negative feature of C-type retroviral vectors. Thus a major advantage of these vectors is that encoded genes are stably integrated into the host cell genome, such that subsequent progeny retain copies of the transgene. Though this feature is a great advantage for stable, long-term expression of transgenes, long-term expression may not be an important consideration in cancer gene therapy. Furthermore the integration process is essentially random (Craigie 1992), and a major concern with the use of retroviral vectors is the possibility of insertional mutagenesis, where the retrovirus inserts to disrupt, derepress, or transcriptionally activate an endogenous proto-oncogene. There is conflicting data in primate models where attenuated retroviral vectors have been reported as safe (Cornetta et al. 1991), and as inducing T cell lymphomas on long-term follow-up (Donahue et al. 1992). However, if retroviral vectors for tumor gene therapy are engineered to express cytotoxic genes to destroy infected cells, insertional mutagenesis is less of an issue. Although integration occurs within transcriptionally active chromatin domains these genes can be transcriptionally inactivated in vivo (Palmer et al. 1991; Ramesh et al. 1993). One significant limiting factor in the use of retroviral vectors for large-scale gene therapy applications is the titer of infectious virus achievable. Even with optimal producer cells and efficient concentration procedures, titers are limited to around 109 infectious virus particles per milliliter (Burns et al. 1993). When this titer is compared to the potential number of tumor cells in a clinically relevant tumor mass, and especially considering the effective dilution that will occur on systemic delivery, it is clear that such titers are not sufficient for systemic gene therapy to human tumors. A final limiting factor in the use of retroviral vectors is that despite the deletion of the majority of viral structural genes, retroviral vectors are limited to delivery of transgenes of around 8 kb in size. An alternative class of vectors utilizes the additional properties of the lentivirus genus of retroviruses. Although the human immunodeficiency virus (HIV)-1 vector system has been most studied, alternative systems have been developed, in part due to safety concerns, based around HIV-2 (Poeschla et al. 1998a), simian immunodeficiency virus (SIV) (White et al. 1999) and feline immunodeficiency virus (FIV) (Poeschla et al. 1998b). The HIV-2 virus is less pathogenic in humans, and like SIV is amenable to studies in primate models. The FIV virus is not pathogenic in humans, can be pseudotyped with alternate env genes to permit infection of human cells, and engineered to incorporate a modified LTR for efficient gene expression in humans cells (Johnston and Power 1999). Such viruses have a more complex genome than C-type retroviruses, still based around gag, pol, and env, but with additional genes that are critical for specific portions of the lentiviral life cycle. The product of the tat


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gene activates the viral LTR for more efficient gene expression, but also functions as a transcription factor in a number of host promoters, altering the gene expression profile on infection and potentially contributing to in vivo pathogenicity (Ott et al. 1997, 1998). Importantly the product of the vpr gene appears to bind to the nuclear pore complex (Vodicka et al. 1998), and in combination with other viral genes, can allow the viral genome to pass into the host nucleus in the absence of nuclear membrane breakdown. Thus lentiviral vectors are able to productively infect non-dividing cells (Lewis and Emerman 1994). This expanded range of infection makes lentiviral vectors attractive for gene therapy. The ability to infect nondividing cells is an especially relevant for renal cell cancer due to the long doubling time and low growth fraction of this tumor type. However, lentiviral vectors are still party to many of the same limitations as C-type retroviral vectors, namely risk of insertional mutagenesis, transcriptional inactivation, limited transgene size, and perhaps most significantly, titer. Due to the toxicity of various lentiviral proteins, stable packaging cell lines have only recently been used in the production of lentiviral vectors, through regulated expression systems (Kafri et al. 1999; Pacchia et al. 2001). Instead investigators have applied simultaneous transfection with a number of plasmids each providing key genes in trans to package a functional virus, whilst reducing the risk of recombination events assembling a fully replication-competent lentivirus. Adenovirus After retroviral vectors, adenoviral vectors are the next most common viral gene delivery systems applied in vivo (Fig. 24.1). Adenoviruses are composed of 30–35 kb of doublestranded, linear DNA contained in an icosahedral protein capsid, which incorporates entry structures consisting of a trimer of penton bases each terminating in a knob protein. The knob protein mediates binding to the coxsackie and adenovirus receptor (CAR), followed by a further interaction with v3 or v5 integrins for viral entry into endosomes and escape into the cytoplasm. Once cytoplasmic, the adenoviral capsid translocates to the nucleus under the influence of nuclear localization signals in the capsid proteins, enabling adenoviral vectors to productively infect non-dividing cells. The adenovirus has a large number of genes designed to interact with host cellular processes to subvert them to virus production, while preventing antiviral mechanisms and pro-apoptotic pathways that would detect virus replication and interrupt the viral life cycle. The genome of the more commonly used adenoviral serotypes has been extensively studied, and a number of viral vectors have been constructed with deletions in key genes that permit viral replication and adversely interfere with host cellular functions. The most common deletion in adenoviral vectors for gene therapy removes the function of E1. The proteins encoded in E1 are required for viral replication; thus E1-deleted adenoviral vectors are amplified in engineered cell lines such as 293 that express the E1 gene products in trans (Graham and Prevec 1995). Commonly adenoviral infection in vivo results in a local inflammatory response, leading to immune-mediated destruction of infected cells. Additional adenoviral genes have been deleted to minimize host cell responses, and thus reduce subsequent immune responses, commonly including deletions in specific genes in the E2 and E4 regions. However deletion of the majority of viral genes minimizes (Chen et al. 1997) but does not eliminate the immune destruction of cells infected with adenovirus (Kafri et al. 1998). Restricting gene expression using tissue specific promoters in gutless vectors can significantly prolong in vivo gene expression from adenoviral vectors in both mouse (Pastore et al. 1999) and primate (Morral et al. 1999) models. Despite these data,

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currently immunogenicity is a major factor preventing repeated administration and long-term expression of genes via viral vectors, and this is especially true of adenoviral vectors. Adenoviral genes either do not, or very inefficiently incorporate into the host cell genome (Harui et al. 1999). The result is transient expression, limited passage of genes to cellular progeny, and minimal risk of insertional mutagenesis. Thus as a result of host immune responses and lack of incorporation, adenoviral vectors provide only transient gene expression in vivo, typically over a 1–3 week period. This is a significant disadvantage for the use of adenoviral vectors for applications requiring long-term stable gene expression. However, for cancer gene therapy where the desired result may be death of the infected cell, transient expression and immune activation may not be disadvantageous. A major advantage of adenoviral vectors is their large capacity for incorporated DNA, especially in later generation vectors where many deleted host genes can be replaced with therapeutic transgenes (Parks et al. 1996; Chen et al. 1997). Typically transgene size is limited to around 8 kb, however in the ‘gutted’ adenoviral vectors where the majority of host genes have been deleted, insert sizes up to a theoretical 30 kb size are achievable. Importantly, as mentioned above, adenoviral vectors have the ability to efficiently infect both dividing and non-dividing cells, and additionally have the practical advantages of high viral titers (up to 1013 infectious units per milliliter) and stability. Adenoviruses are essentially non-pathogenic in humans, however the majority of the population have pre-existing neutralizing antibodies that accelerate clearance of vectors even during the first administration of an adenoviral vector. Systemic intravenous application of adenoviral vectors results in preferential infection of liver cells (Li et al. 1993; Huard et al. 1995), though many other tissues are also infected to a lesser extent (Huard et al. 1995), and liver clearance of adenovirus is a major limiting factor for systemic adenovirus delivery (Alemany et al. 2000). Nevertheless, aortic injection of microsphere-conjugated adenoviral vectors has resulted in efficient transduction of rat glomerular cells in the normal kidney (Nahman et al. 2000). Herpesvirus Herpesvirus-based vectors are increasingly common gene delivery systems, especially in the area of neuronal gene delivery and oncolytic replication-competent viruses, as will be discussed later. Many of the herpesvirus vectors for gene therapy are based around Herpes Simplex Virus (HSV)-1, and although wild-type HSV-1 is known to cause destructive encephalitis, the virus is well studied and many of the genes associated with neurovirulence have been identified. HSV-1 is an enveloped virus, with the lipid bilayer incorporating 10–12 glycoproteins that mediate virus binding and entry into target cells. The tegument layer, between the envelope and the capsid contains a further 12 or so proteins; some involved in transactivation of viral genes, but some with unclear functions at this point. The capsid itself is an icosahedral structure and contains the viral genome. Entry into cells is mediated via binding of viral gB and gD glycoproteins to heparin sulfate residues on the cell surface, and subsequent fusion event releases the viral capsid into the cytoplasm along with the tegument proteins. The viral DNA exists in the nucleus as a circular element, where one of the tegument proteins, VP16, acts together with cellular factors to induce expression of the HSV-1 immediate early genes. These are primarily transactivators of the subsequent early genes that include many of the enzymes required for replication of the viral genome (Lehman and Boehmer 1999). Over the later part of viral DNA synthesis, the late genes are expressed, and


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these constitute many of the structural proteins of the virus. HSV vectors are prone to latency, with minimal gene expression in the intervening latent state. An exception is the use of vectors where the latency promoter regulates transgene expression (Marconi et al. 1996). In the wild-type virus, on infection of neuronal cells the products of the latency promoter turn off expression of all other viral genes, ensuring minimal visibility of the virus to host detection and clearance mechanisms, and prolonged virus survival in infected hosts. HSV-1 based gene therapy vectors have been developed with therapeutic transgenes under the control of the latency promoter, and have demonstrated prolonged, stable transgene expression in vivo, with therapeutic effect (Goins et al. 1999). Herpesvirus vectors do not integrate, and will remain episomal even after long periods of latency. One attractive feature of herpesvirus is the large genome size. Of the approximately 150 kb of DNA, theoretically more than 30 kb of genes associated with neurovirulence can be removed and replaced with therapeutic transgenes, while still retaining viral replication and thus oncolysis. Using standard procedure for production of gene therapy vectors, the herpesvirus genome has been deleted for a number of genes in combination that renders the virus infectious, yet replication-deficient, thus requiring complementation for vector production (Marconi et al. 1996; Goins et al. 1999). The alternative is the use of HSV-1 amplicon vectors. Amplicon vectors avoid some potential safety issues in the use of herpesvirus vectors, since the majority of viral genomic structure has been removed. HSV amplicons were initially identified as contaminating defective genomes containing multiple concatamers of partial HSV sequence. Importantly these sequences consistently contain at least one origin of replication and DNA packaging signal (Sena-Esteves et al. 2000). Thus amplicon vectors have a potential packaging capacity of approximately 150 kb, and will package multiple concatamers of smaller genomes into the viral particles. The majority of gene delivery approaches using HSV-1 based vectors have targeted the brain as the major target of wild-type infection in vivo. However studies have applied selectively replication-competent HSV-1 based vectors for a range of other targets, including experimental models of prostate carcinoma (Walker et al. 1999) and melanoma (Randazzo et al. 1997). Importantly these vectors demonstrate good safety in HSV-sensitive primate models (Hunter et al. 1999). A conditionally replicating HSV-derived vector has been applied in mouse models of human renal cancer, achieving significant delay in tumor growth (Oyama et al. 2001). Thus herpesvirusderived vectors may be useful in gene therapy for renal cancer. Adeno-associated virus (AAV) Adeno-associated virus was originally identified as a defective satellite virus contaminating adenoviral stocks. The virus is defective in genes required for infection, and these genes must be provided in trans by a helper virus, such as adenovirus. Of the six primary isolates of AAV known thus far, the AAV-2 serotype has been most studied for gene therapy applications. Initially AAV-2 virions bind to cells on heparin sulfate proteoglycans (Summerford and Samulski 1998) then access the cytoplasm using v5 integrin as a co-receptor (Summerford et al. 1999). Since the different serotypes of AAV differ in the capsid region, it is likely that alternate serotypes will have alternate in vivo tropisms. The fate of AAV infection depends on the presence of helper virus. In the presence of helper virus, the 4.7 kb AAV genome is replicated and a lytic cycle proceeds. In the absence of helper virus, the single-stranded AAV DNA is converted to a double-stranded template and the products of the Rep (replication) and Cap (capsid) genes are produced. Under the action of the Rep proteins the AAV genome is integrated into the host

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genome in a site-specific manner. AAV is the only known eukaryotic virus to integrate predominantly into a specific location, which in the natural human host is chromosome 19q13.3–qter. This defined integration reduces the risk of random insertional mutagenesis, however Rep proteins are crucial for this specificity. The limited available space in the AAV genome means that both Rep and Cap are generally deleted in gene therapy vectors, with an associated loss of integration-specificity. The integrated AAV continues to produce AAV genes, and in the presence of helper virus the wild-type AAV is reactivated and will produce a lytic phase of virus production. The most attractive features of AAV as a gene therapy vector is their ability to efficiently infect non-dividing cells, and to stably integrate with persistent, long-term gene expression in vivo. The latter feature is achieved in part through an apparent lack of immunogenicity of AAV infection. AAV is reportedly entirely non-pathogenic in vivo, and delivery of high doses of AAV vectors to the lung or skeletal muscle have shown no toxicity and stable transgene expression in animal models (Conrad et al. 1996; Fisher et al. 1997). Importantly AAV vectors have been shown to sustain therapeutic systemic levels of transgene expression in animal models over a 6-month period following intramuscular virus administration (Herzog et al. 1997). As mentioned above, the packaging constraint of AAV vectors is a clear limitation for delivery of larger transgenes or smaller genes with more complex regulatory systems. AAV vectors may be most suitable for gene therapy applications requiring stable, long-term gene expression, such as anti-angiogenic therapy, as discussed below. AAV is capable of infecting a range of primary renal cell types in vitro, and direct intra-parenchymal injection has demonstrated a predominantly tubular epithelial infection pattern in mice in vivo (Lipkowitz et al. 1999). Thus, in addition to distant, long-term gene expression, for example via intramuscular injection of AAV; AAV could be a candidate for direct gene expression in renal cell tumors. Non-viral vectors Non-viral gene delivery is the ever-improving standard against which viral delivery systems must be compared. Non-viral gene delivery varies from injections of ‘naked’ DNA, through to vehicles incorporating advanced stability, cellular delivery, and more effective gene expression. Despite the multiple developments in non-viral vectors, in vivo targeting and the efficiency of transgene expression following infection limit this approach. Nevertheless, nonviral vectors are superior to the vectors described above in a number of features, such as the scalability of the approach and that they lack certain safety concerns inherent in genetically modified potential pathogens. The intrinsic targeting capacity of non-viral gene transfer has been successfully applied with in vivo kidney gene delivery models. Intravenous injection of ‘naked’ radiolabelled oligonucleotides resulted in accumulation of label in the kidney and liver (Rappaport et al. 1995), with particular uptake by proximal tubular epithelial cells (Oberbauer et al. 1995) and delivery of antisense oligonucleotides by this method was able to influence proximal tubular cell function in vivo (Oberbauer et al. 1996). The rapid destruction of oligonucleotides limits the usefulness of this technique to generate transgene expression in vivo, however liposome encapsulated DNA has resulted in gene expression in the kidney by both renal artery injection and renal pelvic injection (Lien and Lai 1997). Liposome-mediated gene transfer is currently limited by the efficiency of gene expression in transduced cells, since the majority of transferred DNA is degraded in the cytoplasm without causing transgene expression (Orrantia and Chang 1990). However a recent study by Tsujie et al. (2001) applied liposomes


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coated with hemagglutinating virus Japan (HVJ) fusion proteins for efficient cell entry, and DNA bound to DNA-binding non-histone chromosomal proteins for enhanced DNA translocation to the nucleus. Such liposomes have resulted in efficient gene delivery to the kidney via renal pelvic (Tsujie et al. 2000) and renal artery injection (Tsujie et al. 2001). Additionally, sustained gene expression could be achieved in the non-dividing transduced cell populations through incorporation of the Epstein Barr Virus (EBV) nuclear antigen gene and its relevant origin of replication in the transferred DNA (Tsujie et al. 2001). Liposome coating of adenoviral vectors has resulted in gene expression in glomeruli and not in the liver following system intra-arterial in vivo gene delivery (Nahman et al. 2000). These intrinsic features of the renal glomerulus and tubular epithelia are greatly advantageous for systemic gene delivery (where these cells are the target) (Imai and Isaka 1998). However, the large-scale disruption of renal architecture where tumors are present entirely alters the prospect for intrinsic targeting to kidney tumors. Additionally, since the aim may be to destroy tumors while maintaining normal kidney function, gene delivery to normal cells of the kidney is a great disadvantage. Nevertheless, the highly vascular nature of the most common renal cancers and the efficiency with which non-viral delivery methods such as liposomes can cross tumor vasculature to locally deliver encapsulated contents (Gabizon et al. 1994), suggests non-viral delivery could be a viable option for renal cancer. One of the few phase I/II clinical trials of gene therapy in renal cell carcinoma applied non-viral gene delivery of IL-2 to overcome the toxicities associated with systemic delivery of recombinant protein (Galanis et al. 1999). PCR studies demonstrated transfer of the IL-2 gene to tumor deposits and infiltrates of CD8 T cells (Galanis et al. 1999). Out of fourteen patients with three dose levels, 20 per cent achieved partial responses and 20 per cent stable disease, each time in the high dose groups (Daniels and Galanis 2001). Despite these indications of successful gene delivery, it is likely that future experimental gene therapies for renal cancer will apply some of the currently available viral vector systems for increased efficiency, level and duration of in vivo gene expression. Vector targeting The requirement for gene targeting very much depends on the application. For gene replacement therapies, it is of course necessary to deliver vectors to each target cell. For antiangiogenic therapies discussed below, it may be sufficient to achieve a desired level of the active agent in the blood, without any requirement for kidney- or tumor-specific expression. For cytotoxic gene therapy for cancer the therapeutic targets are unlikely to be affected by delivery of genes to irrelevant cells and tissues. With the additional consideration of toxicity where such genes are broadly expressed, targeting of gene delivery is an important issue in the gene therapy of cancer (Fig. 24.2). Intrinsic targeting Certain gene delivery systems have intrinsic targeting action. As described earlier, C-type retroviruses are only able to productively infect dividing cells. As cancer cells may make up the majority of dividing cells in most therapeutic circumstances, retroviruses have been used to specifically target proliferating tumors following systemic delivery in murine models (Culver et al. 1992; Vile et al. 1994). However certain other cell types, for example, those of

Intrinsic targeting Gene expression Liver targeting

i.v. adenovirus

Uses natural viral tropisms, for example, innate mechanisms result in preferential infection of the liver on i.v. adenovirus administration

Regulated expression

Targeted infection Virus only productively infects in target cells

Tumor

i.v. retrovirus envelope modified

Targeted expression Virus infects non-target cells, but gene expression only in target

i.v. vector with transcriptional control

Vectors are modified to preferentially infect cells bearing target receptors, for example, EGF receptor that is commonly overexpressed in tumors

Vectors are modified so that the transgene expression is controlled by a tissue- or tumor-specific promoter element

Gene modulation

Cellular delivery

Delivery of regulator

Cells target to neovasculature

Vector delivery

i.v. endothelial progenitor cells

Uses regulated promoter elements to modulate gene expression following vector delivery, for example, oral delivery of rapalog

Oral delivery leads to systemic levels of rapalog sufficient to assemble transcription complex and induce gene expression in infected cells

Fig. 24.2 Vector targeting.

Progenitor cells modified to produce therapeutic transgenes are systemically delivered and home to target sites. Once in place gene expression occurs.


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the haematopoietic lineage, actively proliferate under normal conditions. Tumors are heterogeneous and many tumor types may have only a small fraction of cells dividing at any one time, limiting the efficacy of replication targeting in vivo. In contrast to retroviral infection, systemic intravenous application of adenoviral vectors results in preferential infection of liver cells (Li et al. 1993; Huard et al. 1995), though many other tissues are also infected to a lesser extent (Huard et al. 1995). In a similar manner HSV-1 is neurotropic in vivo, and EBV preferentially infects B-lymphocytes. However these tropisms may be insufficient for safe tumor targeting, and do not include the cell types involved in most cancers, including renal cancer. Thus a number of artificial targeting strategies have been developed (Peng 1999). Targeted gene delivery can be seen to consist of three components: targeting gene delivery; restricting gene expression to specific cells; and regulating the timing and degree of gene expression. Targeted infection One complication in designing targeting strategies is the difference between in vitro mechanisms of viral infection, compared to true in vivo behavior. For example, although measles virus is capable of infecting human cells expressing the ubiquitous CD46 molecule or non-human cells engineered to express CD46 (Dorig et al. 1993), in vivo measles virus is only found to infect macrophages (Mrkic et al. 2000). Thus targeting strategies must take into account factors only relevant in vivo, such as route of administration (Huard et al. 1995) and the influence of innate clearance mechanism (Wolff et al. 1997). The broad specificity of many viral entry mechanisms requires that the majority of strategies for retargeting first prevent entry via the natural receptor. For example, adenoviral infection can be associated with significant liver toxicity. Much of this is due to innate immune mechanisms and host inflammation, and can be reduced by deleting key viral genes (Chen et al. 1997; Christ et al. 2000). However the toxicity may relate directly to the adenoviral particle itself. Receptor-mediated entry of adenovirus into cells depends on both the fiber and penton base coat proteins. The fiber causes primary attachment to the CAR protein on host cells (Bergelson et al. 1997), while the penton base protein binds to integrins that mediate internalization (Wickham et al. 1993). There is evidence that adenovirus binding to integrin structures is itself sufficient to cause an inflammatory response in the host cell (Bruder and Kovesdi 1997; Nemerow and Steward 1999). Since the relevant integrins are commonly highly expressed on immune cells such as macrophages, these data may explain the innate inflammatory response associated with adenoviral infection and viral clearance (Wolff et al. 1997). For this reason, in addition to effective viral retargeting, there may be important reasons for re-engineering viral attachment to enhance specificity and efficacy, while reducing any toxicity associated with gene therapy. Currently two major techniques are used to target adenoviral entry: bind additional molecules to the virus that block CAR interaction and redirect binding to an alternative molecule; or re-engineer the adenovirus coat proteins for modified tropism. The former system has widely made use of bi-specific antibodies to specifically block CAR-binding regions of the fiber knob, while providing alternate specificity (Wickham 2000). In this way adenovirus infection has been retargeted to cells overexpressing molecules such as fibroblast growth factor (FGF) receptor (Goldman et al. 1997) and epidermal growth factor (EGF) receptor (Watkins et al. 1997). Initial difficulties in the genetic retargeting of adenoviral coat proteins related to difficulties in identifying the CAR-binding portion of the fiber proteins,

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and developing strategies to successfully grow recombinant adenovirus in the absence of CAR binding. However, recent studies have identified the critical CAR-binding structure (Roelvink et al. 1999), and replacements of this region with retargeting entities have permitted construction of genetically retargeted adenoviral vectors specific for haemagglutinin (Einfeld et al. 1999; Roelvink et al. 1999), and EGF receptor (Dmitriev et al. 2000). Retargeting with minimal modification of the adenovirus fiber has additionally been achieved through insertion of small peptides, identified from phage targeting studies, such as a consensus motif for binding the human transferrin receptor (Xia et al. 2000). Related strategies have been applied to retarget many other viruses, including C-type retroviruses and lentiviruses. However the more detailed understanding of retroviral envelope proteins and their greater amenability to modification and re-engineering means that most retroviral retargeting strategies involve stable expression of receptor fusion proteins. Retroviral vectors have provided a model in which many vector-targeting strategies have been developed. The env gene expressed on the surface of retroviral vectors is commonly substituted for another with more suitable properties, such as greater range of infectivity or greater particle stability. For example, it is advantageous to use a concentration step in order to enhance the titer of lentiviral vectors for gene therapy. However, many viral particles are destroyed by centrifugation. Pseudotyping the lentivirus with a glycoprotein encoded by the env gene of vesicular stomatitis virus (VSV-G) is known to form sufficiently stable particles for concentration techniques such as centrifugation (Akkina et al. 1996), allowing much higher titer virus production for in vivo gene delivery (Diaz et al. 2000). The range of strategies currently in use for retroviral retargeting has been reviewed (Cosset and Russell 1996; Buchholz et al. 1999; Russell and Cosset 1999). However, recent studies have demonstrated that there may be very significant obstacles to the goal of retroviral targeted infection. Very high levels of non-specific adhesion of viral particles are observed on the surface of cells before the specific envelope–receptor interaction takes place (Pizzato et al. 1999). Thus where retroviral particles are adsorbed onto random cell surfaces in vivo, despite envelope targeting the effective local titer achievable would be greatly reduced. Nevertheless, there are reports of highly efficient in vivo retrovirus retargeting in the literature (Hall et al. 2000). Renal cell carcinoma cells are known to express surface molecules such as the target of the G250 antibody, that have been used for in vivo targeting studies (Luiten et al. 1996; Steffens et al. 1997). Conjugation of G250 monoclonal antibodies to plasmid DNA results in gene expression in renal cell carcinoma cells in vitro (Durrbach et al. 1999), though G250 has not been applied as a retargeting moiety for specific infection of renal carcinoma cells in vivo to date. Targeted delivery The normal kidney provides an attractive target for gene delivery since many structures including the tubular epithelium and glomerulus can be accessed externally via the urogenital tract. Similarly, the filtration system of the glomerulus can allow access of vector material beyond initial delivery via the vasculature. However, the target areas of renal cancer do not share these features and the techniques for targeted delivery to renal cancer have much in common with other cancer types. Targeted expression In view of difficulties in achieving targeted delivery, restricting gene expression to specific cell types is a valuable technique to target gene therapy. Broad ranges of promoter and


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enhancer systems that provide tissue specificity have been characterized (Miller and Vile 1995; Vile et al. 1998). Importantly, tissue specificity is not the same as tumor-specificity, since although there are circumstances where tissue-restricted expression is valuable in cancer therapy, it is generally more desirable to target expression to tumor cells and avoid expression in surrounding normal tissues. For example, prostate-specific antigen (PSA) promoter regions have been demonstrated to provide some specificity for prostate tissue in vitro (Lee et al. 1996) and in vivo (Martiniello-Wilks et al. 1998). In these circumstances, destruction of normal prostate tissues may be an acceptable consequence of destruction of the tumor mass. However, for example in the case of brain tumors, the brain-specific transferrin promoter (Bowman et al. 1995) may be unsatisfactory due to expression of transgenes in this critical organ. Nevertheless, tissue specific promoters may be of great value in targeting of metastases. For example, higher-grade colorectal carcinoma is commonly associated with liver metastasis. Thus, local delivery of genes to the liver under the control of colorectal-specific promoter elements (Richards et al. 1995) could result in specific targeting of metastases with lesser risk of damage to normal tissues. With the view of the kidney as an essential organ, gene therapy for renal cancer would thus be limited to use of tissue-specific promoters to target metastatic deposits. A number of renal tissue-specific promoter elements have been identified. The multiple distinct cell types that make up normal renal architecture have resulted in identification of a number of cell-type-specific promoter regions. For example, a collecting duct-specific promoter element has been identified that can confer specificity to collecting-duct-derived cells, but not other renal cell lines (Calmont et al. 2000). Recently a number of genes that appear to be selectively expressed in the kidney have been identified, the majority of which encode membrane transporters, and promoter elements from these genes can cause kidney-specific gene expression (Igarashi et al. 1996; Sepulveda et al. 1997). An alternative class of gene associated with kidney-specific promoter elements is the kidney androgen-regulated protein. This mRNA of this gene is highly expressed in proximal tubular cells, and gene expression is induced by androgen treatment (Catterall et al. 1986). Transgenic mice constructed with the human angiotensinogen gene under the control of this promoter demonstrated specific gene expression in proximal tubular epithelial cells in the renal cortex associated with human angiotensinogen secreted into the urine (Ding et al. 1997). Interestingly, androgen stimulation of these animals resulted in 100-fold elevated transgene expression compared to 4-fold induction of the endogenous kidney androgen-regulated protein, presumably due to action of additional positive or negative regulatory elements either present or absent from the transgene, respectively. An applicable option for renal cancer could also be the kidney-specific cadherin (cadherin 16) promoter. Elements of this promoter have shown kidney-specific expression in renal epithelial cell lines (Whyte et al. 1999), and have maintained specificity in transgenic mouse models (Igarashi et al. 1999). Importantly transformed renal epithelial cell lines maintain promoter activity (Whyte et al. 1999) thus this promoter could have significant value for gene therapy to renal tumors. Cancer-specific regulatory elements are less common than their tissue-specific counterparts. Candidates include promoter regions from carcinoembryonic antigen (CEA) (Richards et al. 1995) and -fetoprotein (AFP) (Kaneko et al. 1995), however these are not strictly tumor-specific. These promoters work well in experimental models using transplanted tumors and tumor xenografts, though their value in more physiological in vivo studies remains to be proven. An alternative approach in the goal of tumor-specificity is to use the promoters of genes that are related to the higher proliferation rate in tumor cells than normal cells. For example, telomerase activity is broadly elevated in tumor cells (Buys 2000), and the

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telomerase reverse transcriptase promoter has shown tumor specificity with therapeutic effect (Koga et al. 2000). A further approach is the development of promoters specific to the environment commonly seen in tumors. An example here is the use of the promoters of hypoxiaresponsive genes, since hypoxia is a common feature of tumors and associated with pressure for neovascularization. A defined genetic region responsible for binding of a key hypoxiaregulated transcription factor has been shown to cause hypoxia-specific gene expression in vivo (Binley et al. 1999). Similarly a region of the VEGF promoter responsible for hypoxiaresponsiveness has caused therapeutic in vivo expression of suicide genes in tumors (Koshikawa et al. 2000). Thus the development of tumor-specific promoters and their combination with related promoters for tissue and environmental specificity will significantly enhance the selectivity and reduce any toxicity of gene therapy to renal cancer. Regulated expression One feature of gene therapy not addressed thus far is the ability to actively regulate the level and duration of gene expression in vivo. An increasing number of approaches now use regulated promoters for inducible or repressible gene expression. The most advanced of these systems use chimeric transcription factors that combine sequence-specific promoter binding, with small-molecule inducible transcriptional activity. Two such examples are in common use in gene therapy models, the tetracycline- and rapamycin-inducible expression systems. The tetracycline (Tet)-regulated system makes use of a bacterial transcriptional repressor that responds to the presence of tetracycline to induce conformational change and inhibit gene expression. Fusion of the Tet-responsive domain to the HSV VP16 transcriptional activator was shown to generate a transcription factor that could selectively modulate gene expression from a promoter region bearing a Tet operon (Gossen et al. 1995). A range of mutants have been constructed that can respond to the presence of tetracycline or its analog doxocycline with activation or suppression of gene expression (Gossen et al. 1995), and such systems have been used for regulated expression of therapeutic transgenes in animal models (Yu et al. 1996). The rapamycin (Rapa)-regulated system makes use of the ability of Rapa to bind the cellular proteins FK506 binding protein (FKBP) and FKBP-rapamycin associated protein (FRAP). The key domains of FKBP and FRAP have been fused to a designed, sequence-specific DNA binding domain and a transcriptional activation domain, respectively, to construct a multicomponent transcription factor that is only united, and therefore active, in the presence of rapamycin or its analogs (Rivera et al. 1996). One advantage of this system over the Tetregulated expression system is that all proteins are of human origin, potentially minimizing immune recognition of bacterial peptide sequences. This system has been shown to cause regulated gene expression over prolonged periods in mouse and primate models (Ye et al. 1999). It is likely that regulated gene expression will play an increasingly important role in gene therapy applications. With regard to immune mechanisms, altering the timing and degree of gene expression can have opposite effects. Combination of regulated expression systems with other gene therapy systems will be a potent method of controlling the efficacy and toxicity of anti-tumor therapies in vivo. Cell-based delivery An alternative and relatively novel method of targeting involves the rapidly expanding field of cell-based delivery. A large portion of this approach arises from stem cell research


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(Prockop 1997; Pittenger et al. 1999). Following ex vivo maturation of isolated bone marrow or specific tissue stem cell populations, these cells can be re-administered and can show migration to specific tissues (Pereira et al. 1995; Gomez-Navarro et al. 2000). Of most relevance to tumor therapy currently is the application of endothelial progenitor cells, since these cells have been shown to populate sites of active neo-angiogenesis, such as tumors (Gomez-Navarro et al. 2000). Migrating cells could thus be used as ‘Trojan horses’ for delivery of transgenes or even as virus producer cells for specific virus production in the tumor site (Herrlinger et al. 2000). In brain tumor models, direct administration of neural progenitor cells engineered to express the IL-4 gene has resulted in therapy of established tumors (Benedetti et al. 2000). Cellular delivery has been demonstrated in kidney models, with established mesangial cells engineered to express a transgene successfully populating glomerular capillaries following renal artery injection (Kitamura et al. 1994). As described above, since the normal architecture is disrupted in kidney tumors, this particular technique may have limited applicability in therapy of kidney cancer, yet in principal a relatively local focus of virus production could be of great therapeutic value. The general angiogenic nature of renal cancer makes modified endothelial stem cells populating renal tissues a potential strategy for cell-based delivery to renal cancer. One mode of cellular delivery that may be very applicable to renal cancer is the use of macrophages. Renal cell carcinoma is frequently associated with large areas of necrosis, and macrophages have a well-established natural targeting capacity for areas of necrosis associated with hypoxia (Negus et al. 1998; Turner et al. 1999). Thus macrophages are good candidates for delivery of transgenes to renal tumor tissue. However, although macrophages may accumulate in tumor tissue, macrophages are not selective in their traffic to these sites. For this reason it is advantageous to add a further layer of selectivity to the tumor sites. Griffiths et al. (2000) have demonstrated macrophages incorporating a suicide gene transcriptionally controlled by the hypoxia response element (HRE) were able to infiltrate into tumor spheroids and specifically express the gene under hypoxic conditions (Griffiths et al. 2000). Preliminary in vivo studies have been performed in a murine model where a transformed macrophage cell line itself formed a tumor mass (Pastorino et al. 2001). Macrophages were engineered to produce retrovirus bearing the GFP gene, and tumor-infiltrating lymphocytes were shown to express GFP, demonstrating effective gene transfer to host cells (Pastorino et al. 2001). It is likely that if associated with sufficient layers of targeting and transcription control, cell-based carriers would greatly enhance the systemic application of gene therapies for cancer.

Candidate genes for tumor therapy The desired end result of cancer gene therapy is death of tumor cells. This common goal can be achieved by multiple means, principally represented by direct cytotoxicity, activation of endogenous immune-mediated cytotoxicity, and removal of tumor growth requirements through targeting tumor angiogenesis (Fig. 24.3). These strategies are not mutually exclusive, but it is possible these can be applied in combination to optimally destroy tumors. As described earlier, a major limitation in gene therapy is the efficiency of gene delivery to tumor deposits, such that current gene delivery systems may cause gene expression in only a small fraction of the tumor cell population. For this reason it is necessary to select candidate genes that will overcome this limitation and, through significant bystander effects, ensure that each infected cell will cause destruction of multiple neighbors.

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Michael J. Gough and Richard G. Vile Enzyme-prodrug

Fusogenic protein

NaI symporter

Initial transgene expression I131

Prodrug Bystander effect

Transgene expression activates prodrug to its toxic metabolite. The metabolite can pass to neighboring cells and there cause toxicity without gene expression

Target cell express fusogenic proteins on their surface. Interaction with receptors on neighboring cells causes cell fusion and results in cell death

Transgene expression of NIS results in accumulation of I131 in target cells. Radioactive decay of I131 is toxic to those cells and local neighbors

Fig. 24.3 Cytotoxic gene therapy.

Cytotoxic genes For cancer gene therapy, an attractive option is delivery of a cytotoxic gene, or ‘suicide gene’ to tumor cells. The most commonly applied and best-characterized cytotoxic genes are enzymes that convert relatively harmless prodrugs to toxic metabolites. Thus systemic delivery of the prodrug results in selective toxicity of cells engineered to express the relevant enzyme (Moolten 1994; Springer and Niculescu-Duvaz 2000). The most common enzyme applied thus far is the Herpes Simplex Virus-1 thymidine kinase (HSVtk). HSVtk phosphorylates purine analogs (such as ganciclovir and acyclovir) approximately 1000 times more efficiently than mammalian enzymes (Springer and Niculescu-Duvaz 2000). Cellular enzymes further modify the prodrug to the triphosphate form, which is incorporated into DNA during S-phase, resulting in chain termination and cell death. Though the toxic metabolites cannot diffuse across cell membranes, critically HSVtk-mediated cytotoxicity incorporates a bystander effect (Freeman et al. 1993). Thus neighboring cells not expressing HSVtk are also killed, with a mechanism apparently involving gap junction communication (Touraine et al. 1998). HSVtk has been applied in a number of clinical trials, with moderate to low toxicity, but limited clinical efficacy (Smythe 2000). At present, no clinical trials are described in the literature using HSVtk-mediated suicide gene therapy for renal cell cancer. In a number of alternate tumor types early phase clinical trials of HSVtk delivery to tumors using replicationdeficient viral vectors have demonstrated clinical responses, improved survival and little to no toxicity (Herman et al. 1999; Alvarez et al. 2000). However, these response rates do not improve on currently available options, thus further development is required to make HSVtk gene therapy a clinically relevant therapeutic option (Rainov 2000). Efforts are underway to


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improve the potency of HSVtk for cancer gene therapy by screening libraries of mutants (Encell et al. 1999), therefore further therapeutic benefits can be expected from the HSVtk/GCV system in vivo. A number of alternative suicide gene-prodrug combinations have been developed (Encell et al. 1999; Springer and Niculescu-Duvaz 2000). The intrinsic features of these enzymes and toxic metabolites results in characteristic modes of cytotoxicity, effective prodrug concentration range and bystander cytotoxicity. Investigators have been able to modify the characteristics of these enzymes through re-engineering of the active site and generation of novel prodrugs with alternative characteristics (Encell et al. 1999; Springer and Niculescu-Duvaz 2000). Since the majority of suicide gene-prodrug combinations have been isolated from viral and bacterial genomes, it is likely that additional, more potent combinations will be identified with continued sequencing and advances in functional genomics. One novel approach for cancer gene therapy involves the envelope proteins of a number of viruses that are required for viral infection. These proteins, known as fusogenic membrane glycoproteins (FMG), function by causing fusion of the viral envelope with the cell membrane, permitting entry of the viral core to the cytoplasm (Weiss and Chetankuma 1995; Klasse et al. 1998). A number of classes of virus are known to form syncytia on infection in vitro and in vivo (Klasse et al. 1998), and syncytia formation can be reproduced through expression of the FMG alone in target cells. Syncytia formation due to FMG expression results in death of the incorporated cells (Bateman et al. 2000; Higuchi et al. 2000), and importantly a single cell expressing FMGs can incorporate large numbers of non-expressing neighboring cells into a growing syncytia, resulting in a significant bystander effect (Bateman et al. 2000). One example of a FMG is the Gibbon Ape Leukemia Virus envelope protein (GALV). A hyperfusogenic GALV, modified to remove the regulatory R-peptide from its cytoplasmic domain, exhibits at least 1 log greater bystander effect than HSVtk/GCVmediated killing (Bateman et al. 2000). Preliminary experiments indicate FMG is an effective cytotoxic gene in vivo (Diaz et al. 2000), though the species specificity of GALV has limited studies of toxicity thus far. FMG are attractive cytotoxic genes since they exhibit potent bystander cytotoxicity, potentially overcoming limited efficacy of gene delivery in vivo. An alternate class of potential cytotoxic gene is represented by the sodium/iodide symporter (NIS). This gene mediates active iodine transport, such that administration of radioactive iodine (I131) will radiolabel cells expressing NIS, or at higher doses cause radiation-mediated cell death. This effect has long been applied for visualization and treatment of thyroid malignancies, but gene delivery of NIS permits the application of this gene in other malignancies. One of the advantages of NIS-based therapy is that initial radioiodine accumulation will have bystander effects on neighboring uninfected cells, potentially offsetting poor in vivo transfection efficiencies. Adenoviral vectors encoding NIS have been used to cause accumulation of I131 in tumors and reduction in tumor growth in mouse models (Spitzweg et al. 2000; Boland et al. 2000). Combination of NIS with a prostate-specific promoter element results in prostate cell line-specific iodine accumulation, and therapeutic efficacy in nude mouse models of human tumors (Spitzweg et al. 2000). Interestingly, a form of NIS has been identified in normal human lactating mammary glands, and mammary tumors, but not non-lactating mammary tissue (Tazebay et al. 2000), with the implication that I131 could be applied therapeutically in diagnosis and treatment of breast cancer. Of relevance to treatment of renal cancer, NIS mRNA expression has been identified in normal human kidney tissue, with protein expression and iodide uptake specific to tubular epithelial cells

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(Spitzweg et al. 2001). Further studies must identify whether NIS expression is maintained in renal tumors, and any effects on kidney function following NIS gene delivery to renal tumors and I131 therapy. The development of increasingly toxic genes for in vivo gene therapy will become dependent on specific targeting of gene expression to tumor cells in vivo. Though existing targeting options discussed above may be sufficient for less potent genes where low level or ‘leaky’ targeting results in gene expression below effective doses, this ‘leakiness’ may be enough to cause undesired toxicity with more potent genes. This principle has an example in the use of the tissue-specific tyrosinase promoter. Restricted elements of this promoter have been shown to exhibit tissue-specific expression of therapeutic genes such as cytokines and HSVtk in vitro and in vivo (Vile et al. 1994; Vile and Hart 1994). However the same promoter element was shown to allow sufficient expression of GALV protein in non-melanoma cell lines to cause delayed, but significant syncytia formation, resulting in death of the nonmelanoma cells (Gough et al. 2001). Further promoter analysis identified a 300 bp element providing expression apparently ‘on’ in melanoma cells and ‘off ’ in non-melanoma cells by sensitive nested RT-PCR analysis, however this element was unable to cause sufficient gene expression in melanoma cells for therapeutic effect. It was necessary to construct a transcriptional positive feedback loop by incorporating a transcription factor 3 of the toxic gene and the relevant factor-responsive element 5 of the 300 bp tyrosinase promoter, resulting in highly tissue specific and high level gene expression (Emiliusen et al. 2001). For truly specific gene expression it is likely to be necessary to incorporate a number of such regulatory controls in order to ensure minimal toxicity of highly potent genes. Therefore vectors with complex regulatory controls will be key elements of cancer gene therapies. Immune activation Another option to amplify the effect of the transduced gene and provide bystander cytotoxicity is to utilize the immune system. Immune-based therapies for cancer have advanced apace with emerging theories on the organization and role of the immune system. It is likely that immune mechanisms already play a significant role in the in vivo effects of many therapeutic strategies, including suicide gene therapy (Vile et al. 1994), showing that immunotherapies may be most potent in combination. As described above, there are a number of tumor antigens identified as expressed in renal cell cancer (Oosterwijk et al. 1986; Kim et al. 1990; Bernhard et al. 1994; Brouwenstijn et al. 1996; Gaugler et al. 1996; Luiten et al. 1996; Tureci et al. 1996, 1998a,b; Steffens et al. 1997; Tannapfel et al. 1997; Zhang et al. 1997), thus antigen-specific therapies can be designed for this disease. However, one advantage of in vivo gene therapy approaches is the potential to perform antigen-independent treatments that will activate host immune responses regardless of the host antigen and MHC profile. Additionally, non-specific immune mechanisms may be of equal or greater value in immunotherapy of cancer. For example, one of the more important actions of IL-12 in vivo is the effect on tumor vasculature (Bergers et al. 1999), independent of immunomodulatory and inflammatory responses. The majority of immunotherapy options tested in tumor models in vivo involve delivery of cytokine genes. Initial experiments centered on cytokines known to activate T cells, such as IL-2. IL-2 therapy has shown varying promise for renal cell carcinoma, with high systemic doses of recombinant cytokine achieving objective responses in clinical trials (Rosenberg et al. 1994; Bukowski 1997; Negrier et al. 1998). Systemic IL-2 therapy is


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associated with significant dose-limiting toxicities, thus gene therapy provides an attractive option for such molecules (Galanis et al. 1999; Daniels and Galanis 2001). Engineering tumor cells to express IL-2 in animal models caused tumor regression and immunity (Fearon et al. 1990), and clinical trials have used a mixture of autologous tumor cells plus allogeneic IL-2 secreting normal cells (Veelken et al. 1997). However the mechanism of IL-2 action in these models raises questions as to whether the immune response initiated will generate long-lasting memory responses (Fearon et al. 1990). For example, although engineering tumor cells to express the costimulatory molecule B7 results in tumor regression in vivo (Chen et al. 1992), the mechanism of action may be indirect (Huang et al. 1996). In a comparison of 10 different cytokine genes introduced into tumor cells via retroviral vectors, those directly acting on the effector phase of immune action, such as IL-2 and IL-4 were less effective in generating protective immune responses than the cytokine GM-CSF (Dranoff et al. 1993). GM-CSF is an important growth factor for cells of the monocyte/macrophage lineage, and one of the key events that is thought to be responsible for the action of GM-CSF in vivo is the accumulation of professional antigen presenting cells in the tumor site (Lee et al. 1997). For this reason clinical studies have applied autologous renal tumor cells retrovirally transduced ex vivo to express GM-CSF, and demonstrated similar vaccination site infiltrates to those described in animal models (Simons et al. 1997). Transgenic models have demonstrated that even in the presence of large numbers of effector cells recognizing tumor antigens, generation of anti-tumor immunity required delivery of antigen-loaded dendritic cells (Hermans et al. 1998). Thus effective antigen presentation appears to be the key to initiating therapeutic anti-tumor immune responses in vivo. These data are extended by further studies showing that tumor antigens are constitutively presented in the draining lymph nodes of tumors (Marzo et al. 1999), yet in a variety of models either pathogenic infection or dendritic cell vaccination are required to initiate effective anti-tumor immune responses (Speiser et al. 1997; Hermans et al. 1998; Limmer et al. 1998). These observations are in agreement with the predictions of the ‘danger’ theory developed by Matzinger (1994). Thus in addition to the need for antigen and associated specific immune cells, immune responses will not proceed unless cells are stimulated in the correct manner, and in the correct context. Matzinger proposes that the key event is provision of recognizable ‘danger’ signals, such that the immune system perceives some pathological event worthy of initiating potentially damaging immune responses. One example is cell debris, which under normal circumstances would not be present since the physiological process of apoptosis neatly packages cell contents for efficient phagocytosis (Savill 1998; Ren and Savill 1998; Savill and Fadok 2000). Thus Gallucci et al. (1999) demonstrated that dendritic cells are activated in the presence of cell fragments. Similarly Melcher et al. (1998) demonstrated that tumor cells induced to die in vivo via apoptotic mechanisms poorly generated therapeutic immunity when compared to tumor cells induced to die via non-apoptotic mechanisms, which protected against subsequent tumor challenge. Co-delivery of dendritic cells with tumor cells has been demonstrated to produce therapeutic immunity in vivo (Melcher et al. 1999), and host bone marrowderived cells have been shown to be key for presentation of tumor antigens in vivo (Huang et al. 1994). Dendritic cell-based therapy has been applied in renal cell carcinoma. Autologous peripheral blood-derived dendritic cells loaded with autologous tumor lysates were able to activate T cell anti-tumor activity in vitro (Mulders et al. 1999) and successfully generated antigen-specific immune responses in vivo (Holtl et al. 1999). One of the more novel approaches involved electrofusion of allogeneic dendritic cells with autologous renal cell carcinoma cells,

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with 4 out of 17 patients achieving complete responses (Kugler et al. 2000). These data suggest that optimized vaccination-based treatments for renal cell cancer could be a valuable therapeutic advance. Despite these data, vaccination-based treatments remain labor-intensive and commonly rely on successful culture of autologous tumor cells. It is in this scenario that gene therapy for cancer could achieve significant benefits in the ability to achieve many of these processes in vivo at the tumor site. Unfortunately this aim remains hypothetical, yet according to current knowledge many of the elements required are in place. Cytokines such as GM-CSF that are known to cause dendritic cell accumulation, in combination with cytotoxic therapies such as HSVtk plus GCV, would both attract and load dendritic cells with tumor antigens in vivo. Further immune strategies could be utilized to ensure the tumor site was associated with ‘danger’ signals, whether those signals are appropriate inflammatory cytokines or cell contents released via HSVtk/GCV killing, to result in optimal immune activation. Tumor cells commonly undergo immune evasion (Bodmer et al. 1993; Restifo et al. 1993; Ishida et al. 1998), therefore it would be anticipated that since combination therapies include direct anti-tumor cytotoxicity, tumor debulking along with potent immune activation would be preferable to relying on immune mechanisms alone. Anti-angiogenic therapy Anti-angiogenic gene therapy (Fig. 24.4) may be a particularly applicable option for renal cancer. Renal cell carcinoma is known to be a particularly angiogenic tumor type, and metastases have been found more frequently in those patients with a highly vascularized primary tumor (Blath et al. 1976). Endothelium provides an attractive target for tumor therapy, since as normal tissue it is not subject to the selection pressures on tumor tissue that commonly renders tumor cells resistant to cell death and immune recognition (Boehm et al. 1997). Though angiogenesis is most active during the development of tumors (Hanahan et al. 1996), even in large, established tumors anti-angiogenic therapy may be critical to prevent the development of metastasis. Anti-angiogenic therapy will prevent the growth of tumors, and destruction of tumor vasculature will kill tumor cells starved of nutrients (Hanahan et al. 1996). Importantly, in the adult physiological angiogenesis is limited to pregnancy and the female reproductive cycle, limiting the risk of toxicity in other systems. Since angiogenesis is primarily active during an early stage of tumor development, it could be hypothesized that anti-angiogenic therapies are less relevant for the large solid tumors commonly found in renal cancer patients. Thus one argument states that such therapies should be combined with screening programs for risk factors to catch tumor development before the ‘angiogenic switch’ (Boggio et al. 2000). However in a mouse model of spontaneous tumor development, although a number of angiogenic agents were able to prevent activation of the angiogenic switch, they also caused regression of late-stage tumors and extended survival (Bergers et al. 1999). These data have a great deal of relevance for renal cancers. The hereditary cancer syndrome Von Hippel–Lindau (VHL) disease is associated with mutations of the VHL gene (Latif et al. 1993), and VHL is inactivated in the majority of non-hereditary clear cell renal carcinomas (Gnarra et al. 1994; Herman et al. 1994). VHL has been shown to inhibit the expression of the important pro-angiogenic protein VEGF post-transcriptionally (Gnarra et al. 1996), and there is an inverse relationship between VHL expression and VEGF levels in renal tumors (Brieger et al. 1999). Although VEGF is expressed in the


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Increased provision of nutrients and oxygenation permits further tumor growth

Hypoxic signals in small tumor deposit stimulates growth of local endothelial cells

Angiogenesis Angiogenesis

Blocking neoangiogenesis with agonists of pro-angiogenic factors or blocking endothelial cell proliferation will inhibit tumor growth and prevent establishment of metastatic deposits

Fig. 24.4 Anti-angiogenic therapy.

normal kidney, the level of VEGF protein can be used as a prognostic marker in renal cancer (Jacobsen et al. 2000). In the last 5 years a wide variety of angiogenic regulatory factors have been identified, and applied for tumor therapy. A number of these novel agents are associated with the regulation of blood clotting and are cleavage fragments of the haemostatic cascade (Browder et al. 2000). One such example is angiostatin, a fragment of plasminogen capable of inhibiting endothelial cell growth in vitro, and reducing in vivo angiogenesis and tumor growth (O’Reilly et al. 1996). A similar strategy was used to identify endostatin as an anti-angiogenic fragment of collagen XVIII capable of inhibiting tumor growth in vivo (O’Reilly et al. 1997), and it is anticipated that further regulators of angiogenesis will be identified and applied to renal cancer. As described above, VEGF is a potent angiogenic protein that may be important in the pathogenesis of renal cancer. Blocking VEGF action using a soluble form of its receptor, FLT-1 (sFLT-1) resulted in significantly delayed tumor growth of human tumor cells in a nude mouse model, and reduced the implantation and growth in a metastasis model (Goldman et al. 1998). Similarly, specific inhibitors of VEGF receptor tyrosine kinase activity have shown efficacy in animal models of renal cancer (Drevs et al. 2000). Gene therapy offers a number of potential advantages over conventional systemic delivery of recombinant proteins and drugs for anti-angiogenic therapy. Specifically, since angiogenic therapy is likely to require long-term treatment to control tumor neo-angiogenesis and reduce metastasis, gene therapy is an attractive option for sustained delivery with a single application. Additionally, since many of the anti-angiogenic proteins have a short half-life, in vivo production of these factors will maximize in vivo efficacy. Adenoviral vectors have been used to deliver endostatin in mouse models, reducing angiogenesis and tumor growth

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in vivo (Chen et al. 2000), and completely prevented metastasis formation (Sauter et al. 2000). A comparison of anti-angiogenic proteins delivered via adenoviral gene therapy demonstrated that the VEGF inhibitors Flk-1 and Flt-1 were more potent at inhibiting in vivo tumor growth compared to either endostatin or angiostatin (Kuo et al. 2001). Importantly from a gene delivery perspective, anti-angiogenic therapy does not require targeting of the transgene to tumor cells, thus more permissive cell populations can be used to manipulate systemic levels of the inhibitor (Feldman et al. 2000). Intramuscular administration of an endostatin expression plasmid in a polymer formulation resulted in therapeutic inhibition of tumor growth, with very low systemic drug levels detectable (Blezinger et al. 1999). In this way the vector characteristics required for anti-angiogenic gene therapy involve chronic and regulated long-term gene expression, rather than the acute and targeted gene expression described above for cytotoxic and immune-related cancer gene therapies. The majority of anti-angiogenic therapies applied thus far in vivo have utilized adenoviral vectors, despite the limited duration of gene expression achievable with this vector system (Chen et al. 2000; Feldman and Libutti 2000; Sauter et al. 2000; Kuo et al. 2001). Producer cells packaging a retrovirus encoding a dominant negative form of Flt-1, implanted along with glioma cells subcutaneously (Millauer et al. 1994) or intracerebrally (Machein 1999), inhibited growth or prolonged survival, respectively. However, no studies of in vivo gene therapy published to date have applied recognized long-term gene expression strategies such as intramuscular injection of AAV to control tumor development. In view of the angiogenic nature of renal cell carcinoma and the potential for delayed tumor development and reduced metastatic potential associated with anti-angiogenic therapy, it would be anticipated that this approach will have significant benefits in treatment of this tumor type. Vector cytotoxicity—replicating viral vectors An alternative strategy to achieve cytotoxicity within tumor sites is the use of replicationcompetent viral vectors (Fig. 24.5). This strategy is not novel, with multiple trials during the 1950s–1970s achieving limited success (Pennisi 1998; Sinkovics and Horvath 2000). However, greater understanding of viral lifecycles and the ability to manipulate specific desired viral properties means this is still a viable approach for cancer gene therapy. Beyond vector-induced cytotoxicity, retaining the ability of viruses to replicate will overcome a number of current limitations in gene therapy, namely poor efficiency of in vivo gene delivery and poor access of virus beyond the initial site of infection. This latter limitation is clearly seen in a number of experimental models, where marker gene expression is limited to needle tracks through solid tumor tissue. In contrast, it might be expected that a replication-competent virus could generate an on-going ‘front’ of infection from the initial site as each infected cell becomes a producer cell for productive infection of neighboring cells. Thus a recent clinical trial demonstrated replication-defective adenovirus delivered to the pleural space resulted in gene delivery only to superficial cell layers (Sterman et al. 1998). In contrast replicationcompetent herpesvirus has been shown to penetrate deeply into solid tumors in an animal model (Coukos et al. 1999), the virus apparently advancing into the tumor and leaving behind areas of necrosis. Nevertheless, recent experiments with murine models using human tumor xenografts with the selectively replication-competent ONYX-015 virus have shown that multiple injections cause more efficient distribution of virus than single injections (Heise et al. 1999). These data suggest that viral spread may be limited despite replication

Gene therapy for renal cancer Tumor

Tumor cells

Intra-tumoral injection of selectively replicationcompetent virus

Normal cells

Infected cell

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Virus replication

Oncolysis

No viral replication and thus no lysis in normal cells Fig. 24.5 Selectively replication-competent virus.

competency, and the multiple-injection approach has been used in subsequent clinical trials (Khuri et al. 2000). Thus viral penetration through tumor tissues remains an issue. One problem with the use of adenoviral vectors may be low levels of CAR expression on tumor cells (Hemmi et al. 1998; Li et al. 1999). For this reason some of the vector retargeting strategies described above may be important in replication-competent adenoviral vectors. Addition of poly-lysine regions to adenovirus fiber protein, to enhance binding to heparin sulfate and certain cell-surface receptors, has been shown to enhance the virus’s ability to penetrate tumors in vivo (Shinoura et al. 1999). Additionally the normal course of tumor cell lysis by replication-competent adenoviruses does not necessarily produce optimal titers or rate of viral spread. Replication-competent viruses have had commonly deleted viral genes selectively restored to enhance tumor cell death to encourage earlier release of virus and faster spread (Yu et al. 1999; Sauthoff et al. 2000). Since great effort has been made to remove the replication-competency of viral vectors for gene therapy for safety issues, it seems perverse to return full virulence to these vectors. Replication-competent viruses bearing immune-modulatory genes or cytotoxic genes may modify host responses and turn previously non-pathogenic viruses into highly pathogenic agents (Jackson et al. 2001). Additionally one of the primary benefits of gene therapy when compared to alternate therapies, such as systemic recombinant protein, is the principally local effect associated with reduced toxicity. Thus it would be extremely advantageous to selectively target replication-competency to tumor cells. A number of strategies have been applied to target replication-competency in vivo. To this end, the knowledge of adenoviral proteins and their interaction with cellular processes such as apoptosis and the cell cycle has greatly

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assisted in generating adenoviral vectors with tumor-specific replication competency. One of the first systems generating a conditional replicating adenovirus used an E1b 55 kDa-deleted adenovirus. The E1b 55 kDa protein binds and inactivates p53 in cells to permit virus replication; such that only in transformed cells bearing an inactivating p53 mutation will viral replication take place (Bischoff et al. 1996). Clinical trials applying this virus have demonstrated a lack of toxicity, such that large doses of up to 2 1013 virus particles can be administered, though no objective responses were reported (Heise et al. 1997). Responses were enhanced through combination therapy with chemotherapy (Heise et al. 1997), radiotherapy (Rogulski et al. 2000) or addition of suicide genes (Rogulski et al. 2000). Questions have been raised however as to the specificity of this adenovirus, since papers subsequent to the virus’s initial characterization have found no correlation between p53 functionality and virus replication (Rothmann et al. 1998). An alternative vector system exploits a mutation in E1a such that it cannot bind the retinoblastoma gene product (Fueyo et al. 2000). In non-dividing cells viral replication cannot proceed, whereas in transformed cells the adenovirus completes its lytic cycle. A different approach to generate selectively replication-competent viruses uses tumor- or tissue-specific promoters to drive expression of key adenoviral genes. A range of different promoter systems has been applied, such as the tissue-specific PSA and kallikrein promoters (Rodriguez et al. 1997; Yu et al. 1999), or the tumor-specific promoters of -fetoprotein or MUC-1 (Hallenbeck et al. 1999; Kurihara et al. 2000). These approaches are subject to the same limitations described above for all tissue or tumor-specific promoters, though it is likely that a combination of such approaches could greatly enhance the tumor and tissue specificity of a replicating adenoviral vector system. Of course adenovirus is not the only vector system with application as a selectively replicating gene therapy reagent, with a large number of studies applying modified forms of HSV-1. Initial studies applied mutants lacking genes such as thymidine kinase or ribonuclease reductase that are required for viral DNA synthesis in non-dividing cells (Martuza et al. 1991; Mineta et al. 1994). Another class of vectors utilize mutations in key genes that prevent the apoptosis related to premature shut-off of host protein synthesis in host cells (Poon and Roizman 1997). Experiments with such mutated HSV vectors have demonstrated good safety, with the mutated virus entirely avirulent in nude mice compared to lethal effects of the wildtype HSV-1 (Kucharczuk et al. 1997). Further HSV mutants have been developed with multiple modifications to enhance specificity and reduce toxicity in vivo (Advani et al. 1999; Toda et al. 1999). An alternative approach to continued replication competency has been employed with the modified form of HSV known as the DISC virus. In this case a deletion of the essential glycoprotein H ensures that the virus is capable of undergoing a single lytic burst upon infection, but all viral particles produced are non-infectious (Dilloo et al. 1997). This virus has been shown to be efficacious in vitro and in vivo using murine models (Todryk et al. 1999). The principal target of selectively replication competent HSV vectors has been gliomas, to utilize the neurotropic features of herpes virus, however HSV-1 vectors have also been applied in tumors of other cell types (Randazzo et al. 1997; Walker et al. 1999). As mentioned above, a conditionally replicating HSV-derived vector has been applied in mouse models of human renal cancer, resulting in a significant delay in tumor growth (Oyama et al. 2001). Despite the very promising and interesting results with selectively replicating viral vectors, as pointed out by Alemany et al. (2000), few replication-selective vectors have been compared to wild-type virus in their ability to cause lysis of tumor versus normal cells.


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Normal cells may be less permissive to lysis while virus replication is nonetheless proceeding, or similarly normal cells may simply demonstrate a delayed cytopathic effect, leading to skewed results at shorter time-points. The amplification effect of replication competency can additionally overcome the limited titers achievable with C-type retroviral and lentiviral vectors, since only low levels of initial infection will be required assuming in vivo replication occurs. This widens the potential applications for these viruses (Logg et al. 2001). However, both retroviral sub-types will be required to carry additional genes since they are not naturally lytic viruses. Thus, the majority of replication-competent viruses in clinical development are based on adenovirus or herpesvirus vectors. A number of additional viral classes possess interesting properties that make them attractive candidates for replication competent viral gene therapy studies. For example, human Reovirus has been shown to require an activated Ras signaling pathway for infection (Strong et al. 1998), and since activating mutations of Ras are present in 30 per cent of all human tumors, reovirus would appear to have tumor-specific replication-competency. Initial experiments with injection of replication-competent reovirus into tumors in nude and immunocompetent mice demonstrated tumor regression, with no evidence of viral proteins in the underlying skeletal muscle subsequent to tumor regression (Coffey et al. 1998). One of the oldest oncolytic viruses in use in tumor therapy is Newcastle Disease Virus (NDV) (Sinkovics and Horvath 2000) and is undergoing something of a revival with renewed interest in replication-competent viruses (Nelson 1999). Clinical trials have been performed in melanoma using NDV as an adjuvant along with tumor lysates following tumor resection, generating greater than 60 per cent survival at 10 years, compared with historical controls ranging between 5 and 33 per cent (Cassel and Murray 1992). Similar studies have been performed in renal cell carcinoma patients, in combination with low-dose recombinant IL-2 and IFN- (Kirchner et al. 1995). Again patients achieved improved disease-free survival over historical controls, however detailed analysis of patient cellular and humoral immune responses to the vaccine demonstrated specific responses to the virus, but not the tumor component of the vaccine (Zorn et al. 1997). Though NDV has been described as a selectively oncolytic agent (Reichard et al. 1992; Schirrmacher et al. 1999), trials of direct viral injection into tumors or systemic administration of virus are ongoing and not yet published. Initial trials with replication-competent viruses demonstrated an inverse association between increased levels of neutralizing antibodies to the virus and therapeutic responses. Such issues may not be present in many animal models of human cancer, since pre-existing neutralizing antibodies to the most commonly used adenoviral serotypes do not exist in mice. The mechanism of in vivo antiviral responses to gene therapy vectors in humans is not clearly defined. CD8 T cell responses inhibit long-term gene expression in replication-incompetent adenoviral-infected cells (Yang et al. 1994) and deletion of all viral genes enhances persistence of gene expression (Chen et al. 1997), suggesting viral proteins are responsible for the CD8 T cell activity. However, Kafri et al. (1998) demonstrated that cellular immune responses to infected cells do not require expression of adenoviral proteins. Ikeda et al. (1999) demonstrated that neutralization of viral activity in vivo occurred through innate and cell-mediated mechanisms, and that immunosuppression via cyclophosphamide resulted in enhanced in vivo viral propagation and tumor regression. These requirements create a paradox, where immune mechanisms may be suppressed in order to achieve viral spread within the tumor, while many, if not all, approaches require immune activation in order to contribute to control of tumor growth. Additionally immune control is frequently cited as a key response to concerns about the safety of replication-competent viruses. Furthermore, although viruses

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such as adenovirus are commonly cited as being without pathogenicity in human cells, adenoviral genes can act in combination as oncogenes to cooperatively transform human and murine cells (Nevels et al. 2001). Thus despite the attractive potentials of replicationcompetent viruses for gene therapy, further trials are required. Conclusions A range of vectors, targeting strategies and therapeutic transgenes are currently available for gene therapy of cancer. A great deal of success has been demonstrated in animal models and a selection of therapeutics has progressed to early-stage clinical trials. Translation to clinical trials has identified many of the difficulties inherit in gene therapy with the current levels of vectorology and technology. Although relevant therapeutic endpoints have been identified, such as systemic levels of transgene expression and activation of immune mechanisms, clinical outcomes in terms of disease regression and patient survival have been lacking. The major problems have been efficiency of gene delivery, and transgene efficacy in the tumor site. Few tumor cells are transduced even with direct application of vector to the tumor site, and currently available transgenes have been unable to overcome this limited expression with sufficiently potent bystander effects. These limitations in gene delivery technology have been clear for a number of years. For this reason there has been a trend back from the ‘bedside’ and back towards the ‘bench,’ for redevelopment of vectors and identification of novel transgenes for therapeutic effect. Of the different vector systems and therapeutic approaches described above, many have already been applied to models of renal cancer. Nevertheless few clinical trials to date have applied gene delivery to tumor deposits in vivo (Galanis et al. 1999; Daniels and Galanis 2001), with the majority of approaching involving variations of tumor vaccination using gene modified autologous or allogeneic vaccines. Application of gene therapy to a primary tumor deposit does not fit with current clinical practice, where the first line of therapy may be surgical removal (Yagoda et al. 1995; Marshall et al. 1997). However, gene therapy could be applied to the surgical margins to target any remaining tumor material, or perhaps more likely, to target metastasis in distant sites. Targeting distant metastasis has certain advantages over targeting the primary tumor. A metastatic deposit of renal cell cancer in the lung represents a colony of transformed kidney cells on a field of normal cells. Use of a kidney-specific promoter could effectively restrict gene expression to the tumor and exclude lung expression. This would contrast with the primary tumor site where gene expression would also be expected to occur in uninvolved normal renal tissues. As alluded to previously, the ideal gene therapy vector and transgene combination have not been identified. The direction of gene therapy research is increasingly moving towards hybrid vector forms that combine suitable features of different gene delivery vehicles for prolonged, high level gene expression in a safe, high titer vector form. For example, a modified retroviral genome has been incorporated into an adenoviral vector such that following adenoviral infection the retroviral genome is excised and stably integrated into the host cell (Zheng et al. 2000). In this way a high titer adenovirus can achieve long-term, stable expression in target cells. In a related manner, vectors have been engineered to be selectively replication competent to achieve oncolysis without killing normal cells (Bischoff et al. 1996; Rodriguez et al. 1997; Hallenbeck et al. 1999; Yu et al. 1999; Fueyo et al. 2000; Kurihara et al. 2000).


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Such strategies to re-engineer vectors can potentially overcome the current limitations in efficiency of gene delivery and penetration of vector though the tumor. The use of replication-competent lytic viruses adds an additional element to the therapy through interaction with immune mechanisms. Viral infection, replication and cell lysis are all relevant indicators of pathogenic infection, and highly conserved pathways are responsible for detecting and responding to such infections. These ‘signs’ of pathogenicity (Matzinger 1998; Gallucci et al. 1999) are likely to be present when using replication-competent vectors; therefore in addition to oncolysis potent activation of endogenous, innate immune processes will occur (Guidotti and Chisari 2001). Such overlap can already be seen in the commonly used suicide gene therapies, for example, HSVtk/GCV, such that a significant portion of the anti-tumor efficacy of this combination is due to immune activation (Vile et al. 1994). This immune activation relates to the manner in which tumor cells are killed in vivo (Melcher et al. 1998), with direct relevance to the design of cytotoxic gene therapies. For these reasons, in future the design of cytotoxic cancer gene therapies is likely to take into account not just that cells are killed, but also the manner in which cells are killed for optimal activation of endogenous immune mechanisms. Therefore it is likely that gene therapy for renal cell cancer will develop to use a combination of strategies, including long-term control of tumor angiogenesis combined with aggressive, multi-targeted cytotoxic gene therapies. The therapies could be designed for optimal efficiency of gene delivery through cellular delivery to target sites along with targeted transgene expression and selective replication-competency. Finally, the cytotoxic therapy may be designed to additionally activate endogenous immune mechanisms and additionally express immuno-modulatory genes to predictably control the outcome of immune responses. References Advani, S. J., Chung, S. M., Yan, S. Y., Gillespie, G. Y., Markert, J. M., Whitley, R. J., et al. (1999). Replication-competent, nonneuroinvasive genetically engineered herpes virus is highly effective in the treatment of therapy-resistant experimental human tumors. Cancer Research, 59, 2055–8. Akkina, R. K., Walton, R. M., Chen, M. L., Li, Q. X., Planelles, V., and Chen, I. S. (1996). Highefficiency gene transfer into CD34 cells with a human immunodeficiency virus type 1-based retroviral vector pseudotyped with vesicular stomatitis virus envelope glycoprotein G. Journal of Virology, 70, 2581–5. Alemany, R., Balague, C., and Curiel, D. T. (2000). Replicative adenoviruses for cancer therapy. National Biotechnology, 18, 723–7. Alemany, R., Suzuki, K., and Curiel, D. T. (2000). Blood clearance rates of adenovirus type 5 in mice. Journal of General Virology, 81(Pt 11), 2605–9. Alvarez, R. D., Gomez-Navarro, J., Wang, M., Barnes, M. N., Strong, T. V., Arani, R. B., et al. (2000). Adenoviral-mediated suicide gene therapy for ovarian cancer. Molecular Therapy, 2, 524–30. Angevin, E., Kremer, F., Gaudin, C., Hercend, T., and Triebel, F. (1997). Analysis of T-cell immune response in renal cell carcinoma: polarization to type 1-like differentiation pattern, clonal T-cell expansion and tumor-specific cytotoxicity. International Journal of Cancer, 72, 431–40. Bateman, A., Bullough, F., Murphy, S., Emiliusen, L., Lavillette, D., Cosset, F. L., et al. (2000). Fusogenic membrane glycoproteins as a novel class of genes for the local and immune-mediated control of tumor growth. Cancer Research, 60, 1492–7. Benedetti, S., Pirola, B., Pollo, B., Magrassi, L., Bruzzone, M. G., Rigamonti, D., et al. (2000). Gene therapy of experimental brain tumors using neural progenitor cells. Nature Medicine, 6, 447–50.

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Bergelson, J. M., Cunningham, J. A., Droguett, G., Kurt-Jones, E. A., Krithivas, A., Hong, J. S., et al. (1997). Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5. Science, 275, 1320–3. Bergers, G., Javaherian, K., Lo, K. M., Folkman, J., and Hanahan, D. (1999). Effects of angiogenesis inhibitors on multistage carcinogenesis in mice. Science, 284, 808–12. Bernhard, H., Karbach, J., Wolfel, T., Busch, P., Storkel, S., Stockle, M., et al. (1994). Cellular immune response to human renal-cell carcinomas: definition of a common antigen recognized by HLAA2-restricted cytotoxic T-lymphocyte (CTL) clones. International Journal of Cancer, 59, 837–42. Binley, K., Iqball, S., Kingsman, A., Kingsman, S., and Naylor, S. (1999). An adenoviral vector regulated by hypoxia for the treatment of ischaemic disease and cancer. Gene Therapy, 6, 1721–7. Bischoff, J. R., Kirn, D. H., Williams, A., Heise, C., Horn, S., Muna, M., et al. (1996). An adenovirus mutant that replicates selectively in p53-deficient human tumor cells. Science, 274, 373–6. Blath, R. A., Mancilla-Jimenez, R., and Stanley, R. J. (1976). Clinical comparison between vascular and avascular renal cell carcinoma. Journal of Urology, 115, 514–19. Blezinger, P., Wang, J., Gondo, M., Quezada, A., Mehrens, D., French, M., et al. (1999). Systemic inhibition of tumor growth and tumor metastases by intramuscular administration of the endostatin gene. National Biotechnology, 17, 343–8. Bodmer, W. F., Browning, M. J., Krausa, P., Rowan, A., Bicknell, D. C., and Bodmer, J. G. (1993). Tumor escape from immune response by variation in HLA expression and other mechanisms. Annals of the New York Academy of Sciences, 690, 42–9. Boehm, T., Folkman, J., Browder, T., and O’Reilly, M. S. (1997). Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance [see comments]. Nature, 390, 404–7. Boggio, K., Di Carlo, E., Rovero, S., Cavallo, F., Quaglino, E., Lollini, P. L., et al. (2000). Ability of systemic interleukin-12 to hamper progressive stages of mammary carcinogenesis in HER2/neu transgenic mice. Cancer Research, 60, 359–64. Boland, A., Ricard, M., Opolon, P., Bidart, J. M., Yeh, P., Filetti, S., et al. (2000). Adenovirus-mediated transfer of the thyroid sodium/iodide symporter gene into tumors for a targeted radiotherapy. Cancer Research, 60, 3484–92. Bowman, B. H., Jansen, L., Yang, F., Adrian, G. S., Zhao, M., Atherton, S. S., et al. (1995). Discovery of a brain promoter from the human transferrin gene and its utilization for development of transgenic mice that express human apolipoprotein E alleles. Proceedings of the National Academy of Sciences, USA, 92, 12115–9. Brieger, J., Weidt, E. J., Schirmacher, P., Storkel, S., Huber, C., and Decker, H. J. (1999). Inverse regulation of vascular endothelial growth factor and VHL tumor suppressor gene in sporadic renal cell carcinomas is correlated with vascular growth: an in vivo study on 29 tumors. Journal of Molecular Medicine, 77, 505–10. Brouwenstijn, N., Gaugler, B., Kruse, K. M., van der Spek, C. W., Mulder, A., Osanto, S., et al. (1996). Renal-cell carcinoma-specific lysis by cytotoxic T-lymphocyte clones isolated from peripheral blood lymphocytes and tumor-infiltrating lymphocytes. International Journal of Cancer, 68, 177–82. Browder, T., Folkman, J., and Pirie-Shepherd, S. (2000). The hemostatic system as a regulator of angiogenesis. Journal of Biological Chemistry, 275, 1521–4. Bruder, J. T. and Kovesdi, I. (1997). Adenovirus infection stimulates the Raf/MAPK signaling pathway and induces interleukin-8 expression. Journal of Virology, 71, 398–404. Buchholz, C. J., Stitz, J., and Cichutek, K. (1999). Retroviral cell targeting vectors. Current Opinion in Molecular Therapeutics, 1, 613–21. Bukowski, R. M. (1997). Natural history and therapy of metastatic renal cell carcinoma: the role of interleukin-2. Cancer, 80, 1198–220. Burns, J. C., Friedmann, T., Driever, W., Burrascano, M., and Yee, J. K. (1993). Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proceedings of the National Academy of Science, USA, 90, 8033–7.


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Index of genes 11beta HSD2 312 ACE 420, 422, 426–79, 434 ACTN4 174, 178, 421 ADRB2 426 AGT 420, 422, 426, 427, 429 AGXT 283, 287 ALDR1 427 ALMS1 380 AQP2 323 AT1, AT2 100–2 ATP5V1B1 325, 327 ATP6V0A4 325, 327 ATP7B 412–14 AT[U]1[u]R 427–9 AVPR2 322 BBS1-7 362–7 BCL2 66, 75, 92, 97, 431 BF2 72, 92 BMP 74, 92 BSND 320 C3 423 CA2 325, 328 CaSR 283–4 CCR-2 433 CDKN1c 75–6 CFTR 434 CIAS1 436 ClCKB 318 ClCN5 283, 294, 295, 325 ClCNKB 308, 320 COL1A2 432 COL4(1–6) 186, 190–5 COX2 78, 92 CTNS 263–7 CXORFS 157 CYP1A1 432 CYP11B2/B1 308 DHCR7 273 DMD 10 DQA1*301 423 DRB1*07 423 EGF 73, 92

EGR1 71 EMX2 69, 92 ENaC 310, 315–16, 426 EYA1 71, 92, 95 FAH 268 Fas 431 FGF3 293 FGFR2 72, 95 FH 434–5 FHJN 437 fibrillin-1 432 FLT1 535 FRA 435 G6PT 270 GA-IIA 257 GDNF/RET 72–3 GHRPR 283, 288 GLA 271 GLUT2 270 GPC3 95, 473 H19 460–1, 471 HbSS 401 HGF 73 HGF/MET 73 HIF1/2 488 HLA complex 418–33 HMCS 283, 296–7 HNF1beta 95 HOX11 71, 92 HPH2 293 IGF2 460–1, 471 IL-1a 433 IL-2 523, 532–3 IL-10 430 IRF 433 jagged-1 149 KAL 95–9 KCNJ1 308, 320 KCNQ1 471

558

Index of Genes

LCAT 272 LIM1 69, 92 LMP7 433 LMX1B 69, 92, 95, 409, 411 MCKD1/2 256–7 MEFV 436 MET 73, 136, 498–502 MOCS1/2 297 MR 311 MUT 273–4 MVK 436 MYC 71–2, 92 nephrin 170, 171 NF1 350–5 NOS3 427 NPH1/2/3 255 NPHS1 167–71, 421 NPHS2 178, 421 NPHS 173–4, 178 NR3C2 308, 320 OCRL1 154 p53 75, 462 p57-KIP2 75–6, 95, 471 PARP 430 PAX 69–70, 73–4 PAX2 66, 69–71, 92, 95–7 PAX3 96 PAX6 96, 474 PAX8 69 PCLN1 283, 290 PDGF 72, 92 PKD1 437 PKD1/2 10, 204, 220–5, 337 PKHD1 240

PNMT 426 POD1 69 RAR 78, 92 RAS 420, 427, 434 RET 72–3 ROMK 425 SALL1 159 SCNN1 308 SCNN1A,B.G 320 SF1 467 SGBS 473 SLC3A1 283, 291 SLC4A4 325, 328 SLC7A9 283, 291–2 SLC7A10 283, 292 SLC12A1/3 308, 320 SOX9 69, 95 TAP1/2 433 TAP 421 TCR 433 TNFbeta 421 TP53 457, 462–3 TSC1/2 337–9, 345 TSSC5 471–2 V2R 323 VDR 282–3 VEGF 534–5 VHL 136, 487–93, 534 WAGR 458 WNK1/4 308, 312–13 WNT 74–5, 92 WT1 66, 70–1, 92, 95, 178, 458–63 XDH 283, 296

Index NB: Page numbers in bold denote tables abortion, spontaneous, symbols for denoting 16 acid–base homeostasis 33 acidopathies, primary 324–9 acute interstitial nephritis 47–9 adenine phosphoribosyl transferase (APRT) deficiency 437 adeno-associated virus vector, gene therapy 521–2 adenovirus vector, gene therapy 519–20, 535–6, 540 adrenergic receptor beta-2 426 AGR triad 464 Alagille syndrome 149 aldosteronism familial hyperaldosteronism type-2 314 glucocorticoid-remediable 308, 313–14 pseudohypoaldosteronism type-1 (PHA1) 308, 315–17 type-2 (PHA2) 312–13 alpha-1-antitrypsin deficiency 411–12, 429, 431–2 alpha-adducin 427 alphafetoprotein in CNF 170–1 promoter region 527 Alport syndrome 183–201 animal models 194 autosomal dominant 191–4 autosomal recessive 191 benign familial haematuria (BFH) 194–5 classic X-linked 183–90 deafness 188–9 ophthalmic signs 186–8 gene therapy 194 mitochondrial mutations 193 mosaicism 193 Alström syndrome 379–91 investigations 386–8 management 388–91 vs Bardet-Biedl syndrome 399 aminoacidurias 290–3 amniocentesis 21 amyloidosis, glomerulonephritis 435–6 aneuploidy, and pyelectasis 132–4 aneurysms, intracranial, ADPKD 214–15 angiogenesis, VEGF 534 angiogenic switch 534–6 angiomyolipomata 340–2

angiotensin converting-enzyme (ACE) 422 ACE inhibitors, hypertension in pregnancy, teratogenesis 93 angiotensin II receptor-2 100–1 animal models ADPKD 205–6 nephrogenesis 91–3 aniridia 464, 474 FISH, risk determination 475 antiangiogenic therapy, candidate genes 534–6 antigen presentation, cell debris 533 antiglomerular basement membrane disease 424–5 antineutrophil cytoplasmic antibody (ANCA)associated, vasculitis 431–2 Apert syndrome 95 apoptosis 68 apparent mineralocorticoid excess (AME), syndrome of 311–12, 425 aquaporin-1 deficiency 323 asphyxiating thoracic dystrophy 153–4 atherosclerosis, and renal artery stenosis 41 autosomal inheritance dominance 4–5 recessive 5–6 Bardet-Biedl syndrome 95, 361–79 investigations 386–8 management 388–91 vs Alström syndrome 399 Bartter syndrome 308, 317–21 Beckwith–Wiedemann syndrome 75–6, 95, 128, 468–71 Bellini, duct of 59 benign familial haematuria (BFH) 194–5 Berger disease 46–7, 422 berylliosis 433 Birt–Hogg–Dubé (BHD) syndrome 503–5 bladder cancer 138 bone morphogenetic proteins (BMPs) 74, 92 branchio-oto-renal syndrome 71, 95, 127 “Bright disease” 184 C1q deficiency 430 calcium stones, hypercalciuric disorders 282–4, 294–5

560

Index

campomelic dysplasia 95 candidate genes, gene therapy 529–36 CAR protein 525–6, 537 carcinoembryonic antigen (CEA) promoter 527 cardiomyopathy Alström syndrome 385–6 Bardet-Biedl syndrome 389 carnitine palmitoyltransferase II deficiency 95 Caroli disease 242, 244 carriers, pedigree symbols 16 cat-eye syndrome 130 caudal regression 94 CD2-associated protein 174 cell adhesion molecules 76–8, 92 galectin-3 77 integrins 77 laminin 76 NCAMs 76 cell debris, antigen presentation 533 cell lineage, nephrogenesis 78–9 cell proliferation, nephrogenesis 67 cerebral haemangioblastoma 492 cerebro-oculo-facial-skeletal syndrome 150–1 CHARGE association 95, 150 chloride channel (CLCN-5) gene 294 cholesterol synthesis defects 272–3 chondrodysplasia syndromes 247 chondroitin sulphate 77 chorionic villus sampling (CVS) 21–2 chromosomal disorders 117–45 categories 117, 119 deletion 125–30 duplication 125–30 effect on kidney 118–20 fetal ultrasonography 132–6 rearrangement syndromes 125–30 sex chromosomes 124–5 triplication 125–30 triploidy 123–4 trisomies 119, 121–4 uniparental disomy (UPD) 117–18, 130–2, 471 chromosome(s) abnormalities 3 karyotyping 2 map 121 notation 2–3 structure 1–2 chronic interstitial nephritis 49, 50 clear cell carcinoma 136–7, 490–1, 505–6 familial 506 cloning functional cloning 13 positional cloning 12 Cockayne syndrome 150–1

collagen type IV 175 in Alport syndrome 183 genes 185–7 collecting duct 32, 59–60 coloboma CHARGE association 95, 150 renal–coloboma syndrome 95, 96–7 computer databases 13 congenital adrenal hyperplasia (CAH) 314 consultand, pedigree symbols 16 creatinine clearance, procedures compared 36–7 crescentic (rapidly progressive) glomerulonephritis 45–6 cri-du-chat syndrome 126 cryptophthalmos syndrome 151–2 cyclin dependent kinase inhibitor 471 cyclo-oxygenase-2 (COX2), nephrogenesis 78, 92 cystinosis 261–8 mutation analysis 265–7 prenatal diagnosis 267 cystinuria 290–2 types I/II 283 cysts 253–60 classification 253 familial juvenile nephronophthisis 255–6 glomerular cystic kidney disease 246–77, 254 glutaricaciduria type IIA 257–8 medullary cystic kidney disease 254–5 multicystic kidneys 135–6 renal dysplasia 254 syndromes 147, 148 trisomies 119 tuberous sclerosis complex 342–3 see also medullary cystic kidney disease; polycystic kidney disease cytogenetic abnormalities, mapping 10 cytotoxic genes cancer gene therapy 530–2 vector cytotoxicity 536–40 deafness Alström syndrome 389–90 Bardet-Biedl syndrome 377 deletion syndromes 125–30 (4p) Wolf–Hirschhorn syndrome 125–6 (5p) cri-du-chat syndrome 126 (7q) Williams syndrome 126–7 (9p) syndrome 127 (11p13) WAGR syndrome 71, 127, 458, 464 (15q11) Prader–Willi syndrome 128–9 (17p11.2) Smith–Magenis syndrome 129–30 (17p13.3) Miller–Dieker syndrome 129 (18q) 130 (22q11) velocardiofacial syndrome 130 Wilms tumour 457

Index

561

Dent disease (X-linked hypercalciuric nephropathy) 294–5 Denys–Drash syndrome 95, 173, 465–7 diabetes insipidus (NDI) 321–4 diabetes mellitus glomerulonephritis 427–8 maturity-onset/renal malformation syndrome 95 and teratogenesis 93 dialysis, historical aspects 51–2 DIDMOAD syndrome 414 diet, treatment of renal disease 38–9 diffuse mesangial sclerosis (DMS) 171–3 diffuse proliferative glomerulonephritis 45 DiGeorge syndrome 95, 130 distal tubule 30, 31 structure 58–9 dominant inheritance autosomal 4–5 carriers, offspring risks 18 X-linked 8 Down syndrome 122–3 drug-induced nephritis 48, 49 Duchenne muscular dystrophy (DMD) 10 duplex kidney 90, 119 duplication syndromes 125–30 Beckwith–Wiedemann syndrome 75–6, 95, 128 dup (3q) 125 dup (4p) 126 dup (6p) 126 dup (20p) 130 dup/rea (11p) 128 dysmorphic syndromes 147–65

familial juvenile hyperuricaemic nephropathy 437 familial juvenile nephronophthisis 255–6 familial Mediterranean fever 436 familial renal amyloidosis 435–6 familial renal oncocytoma (FRO) 503–4 familial steroid-resistant nephrotic syndrome 173–4 Fanconi anaemia 95 Fanconi syndrome cystinosis 261–8 Dent disease 294–5 Wilson disease 413 Fanconi–Bickel syndrome 269–70 Fechtner syndrome 191 fetal ultrasonography 20 detection of chromosomal disorders 132–6 fibroblast growth factor (FGF) 72 fibromuscular dysplasia 428–30 treatment 39–40 fibronectin 488 FK506 binding protein 528 fluorescent in situ hybridization (FISH) 11 high resolution 12 risk determination 475 focal proliferative glomerulonephritis 45 focal and segmental glomerulosclerosis (FSGS) 44–5, 419–21 formin 92 Frasier syndrome 151–2, 466–7 fumaryl acetoacetase 268 functional cloning 13 fusogenic membrane proteins (FMGs), syncytia 531

ellipsocytosis 192 elongin/SIII 48 end-stage renal failure, treatment 51–3 endocrine abnormalities, ALS and BBS 382, 390–1 endolymphatic sac tumours 492 epidermal growth factor (EGF) 73, 92 Epstein anomaly 191 erythropoietin, production 32 ethical issues confidentiality 24 genetic counselling 23–5 inadvertent disclosure 24 informed consent 23 paternity issues 24 predictive testing 24–5 presymptomatic testing 23 termination of pregnancy 22–3

alpha-galactosidase A deficiency 270–1 galectin-3 77–8, 97 gene therapy 511–47 Alport syndrome 194 candidate genes 529–36 antiangiogenic therapy 534–6 cytotoxic genes 530–2 immune activation 532–4 methods kidney transplantation 511 skeletal muscle protein 511 summary 512 replication-competent viral vectors 536–40 vector cytotoxicity 536–40 vector systems 516–23 adeno-associated virus 521–2 adenovirus 519–20, 535–6 herpesvirus 520–1 lentivirus 518–19 non-viral vectors 522–3 retrovirus 516–19 summary 517

Fabry disease 270–1 familial focal segmental glomerulosclerosis 174 familial haematuria, benign (BFH) 194–5 familial hyperaldosteronism type-2 314

562

Index

gene therapy (cont.) vector targeting 523–9 cell-based delivery 528–9 intrinsic targeting 523–5 regulated expression 528 summary 524 targeted delivery 526 targeted expression 526–8 targeted infection 525–6 genetic counselling 14–25 ethical issues 23–5 indications for referral 17 services 14–19 see also prenatal diagnosis genitourinary abnormalities 465–6 genomic imprinting 470 germinal mosaicism 5 Gitelman syndrome 308, 319–21 glial cell-derived neurotrophic factor (GDNF) 72, 92 glomerular disease, histopathological types 44–7 glomerular filtrate renal function 27–33 volume 27 glomerular filtration barrier, model 177 glomerular filtration rate, cut-off point: disease 37 glomerular structure 58–60 glomerulocystic kidney disease 246–7, 254 glomerulonephritis 417–54 acute 41–2 amyloidosis 435–6 chronic 42 crescentic (rapidly progressive) 45–6 diabetes mellitus 427–8 diffuse proliferative 45 focal proliferative 45 gout 436–7 haemolytic uraemic syndrome 434–5 IgA nephropathy 422 membranoproliferative 46 membranous 46 mesangiocapillary 423 minimal change 44 rapidly progressive (RPGN) 42, 45–6, 431 renovascular disease 428–9 sarcoidosis 432–4 scleroderma 432 systemic lupus erythematosus 429–31 glomerulosclerosis, idiopathic focal and segmental (FSGS) 44–5, 419–21 glucocorticoid-remediable aldosteronism (GRA) 308, 313–14, 425 glucose-6-phospatase deficiency 269, 285 glutaricaciduria type II 95 type IIA 257–8

glycogen storage diseases 269–70 GM-CSF 533 Goldenhar syndrome 152–3 gonadal mosaicism 5 Goodpasture (anti-GBM) antibodies 184, 424 gout 436–7 growth factors 72–5, 92 haemolytic uraemic syndrome (HUS) 434–5 haemophilia 406–7, 416 Hartnup disease 292–3 hearing loss Alström syndrome 389–90 Bardet-Biedl syndrome 377 hemifacial microsomia 152 hemihypertrophy 467 Henoch–Schonlein nephritis 422–3 heparan sulphate 77 hepatic cysts, ADPKD 213–14 hepatic disease Alström syndrome 383 hepatic fibrosis Bardet-Biedl syndrome 378–9 congenital, CHF-associated disease 247 hepatocyte growth factor 73 hereditary papillary renal carcinoma-1 (HPRC-1) 498–502 herpesvirus vector gene therapy 520–1, 538 HSVtk 530 heteromeric amino acid transporter (HAT) 291 Heymann nephritis 421 Hirschsprung disease Bardet-Biedl syndrome 379 HLA anti-glomerular basement membrane disease 424–5 focal and segmental glomerulosclerosis 419–21 Goodpasture-associated alleles 424 major histocompatibility complex 418–19 membranous nephropathy 421–2 SLE 430 homozygote 5 horseshoe kidney 90, 119, 122, 465 syndromes 147, 148 hydronephrosis 90, 119 syndromes 147, 148 25-hydroxycholecalciferol, production 33 11-beta- and 17-alpha hydroxylase deficiencies 314 hyper IgD periodic fever 436 hypercalciuric disorders 282–4 X-linked nephropathy 294–5 hyperkalaemia, sickle cell disease 402 hyperoxaluria 283, 286–8 hypertension, candidate genes 426 hypertension, essential 425–7

Index hypertensive disorders 308–14 ADPKD 211–13 in BBS 378 essential hypertension 425–7 exacerbated by pregnancy 311 monogenic 308 NF1 352 phaeochromocytoma 353 pregnancy-associated 353–4 renin-angiotensin system 419 renovascular hypertension 352–3 hyperuricaemia 436–7 hyperuricosuria 283, 284–5 hypocitraturia 288–9 hypogenitalism, Bardet-Biedl syndrome 372–3 hypogonadism, Bardet-Biedl syndrome 373 hypomagnesaemia 289–90 hypoxanthine–guanine phosphoribosyl transferase (HGPT) deficiency 285, 436 hypoxia response element (HRA) 529 hypoxia-inducing transducible factors 488 IgA nephropathy 46–7 imaging procedures compared 34–7 renal configuration 34 renal size 34 immune activation, candidate genes 532–4 immunoglobulin A nephropathy 46–7 imprinting genomic 470 loss of (LOI) 460–1 in situ hybridization 11 informed consent 23 insulin-like growth factor 72 IGFII and WT 460–2 integrins 77, 92, 431 interleukins IL-2 gene delivery 523, 532–3 IL-10 polymorphisms in SLE 430 intracranial aneurysms, ADPKD 214–15 inulin clearance, procedures compared 36–7 Jeune syndrome 153–4 juxtaglomerular apparatus 60 Kalmann syndrome 95, 97–9 karyotyping 2 kidney stones see nephrolithiasis Klinefelter syndrome 124–5 Knudson two-hit mutational model 455, 466 laminin 76, 175 Laurence–Moon-Biedl see Bardet-Biedl syndrome Leber amaurosis, and tapetoretinal degeneration 255–6

563

lecithin–cholesteryl acyl transferase (LCAT) deficiency 272 lentiviruses, vector systems 518–19 Lesch–Nyhan syndrome 285, 436 Liddle syndrome 308–11, 425, 437 limb abnormalities, in BBS 371 linkage analysis 9–10 Lofgren syndrome 433 logarithm of the odds (LOD) score 10 loop of Henle 59 loss of heterozygosity (LOH) 458, 460–2 loss of imprinting (LOI) 460–1 Lowe oculocerebrorenal syndrome 154 lower urinary tract malformations 90–101 Lyonization 6–7 McKusick–Kaufman syndrome (MKKS) 363–5 macroglossia 468 magnesium, hypomagnesaemia 289–90 major histocompatibility complex (MHC) 418–19 see also HLA mapping 9–14 candidate genes 12 computer databases 13 cytogenetic abnormalities 10 functional cloning 13 high-resolution 11–12 linkage analysis 9–10 low-resolution 10–11 mutational analysis 13–14 positional cloning 12 pulsed-field gel electrophoresis 12 restriction enzymes, DNA cleavage 9 in situ hybridization 11 YACs 12 Meckel syndrome 95, 154–5 medullary cystic kidney disease 254–5 autosomal dominant (ADMCKD) 256–7 MELAS syndrome 193 membranoproliferative glomerulonephritis (MCGN) 46 membranous nephropathy 421–2 Mendelian disorders 3–8 mesangiocapillary glomerulonephritis (MCGN) 46, 423 mesenchyme, differentiation 66 mesoblastic nephroma, Wilms tumour 137 mesonephric duct 63 mesonephros 62–3 metabolic acidosis, sickle cell disease 404 metabolic diseases of kidney 261–80 list 262 metanephros 64–6, 79 methylmalonic acidaemia 273–4 mevalonate kinase (MVK) 436

564

Index

Miller–Dieker syndrome 129 minimal change disease (MCD) 44, 419–21 mitochondrial disorders MERFF 414 nephropathy-associated mutations 193 mitogen activated protein kinase cascade (MAPK) 171 molecular biology, nephrogenesis 68–79 mosaic tetrasomy 127 mosaicism, germinal/gonadal 5 Muckle–Wells syndrome 436 Mullerian duct, renal and cervicothoracic abnormalities 155–6 Mullerian inhibiting substance 467 multicystic kidneys, fetal ultrasonography 135–6 MURCS association 155–6 nail–patella syndrome 95, 408–11 nephrin cloning 421 and SD 177 nephrin zipper 170, 171 nephritis acute interstitial 47–9 chronic interstitial 49, 50 drug-induced 48, 49 Henoch–Schonlein 422–3 Heymann 421 syndromes 147, 148 nephrogenesis 60–79, 91–3 anatomy 62–6 animal models 91–3 cell lineage 78–9 investigations 60–2 mechanisms 66–8 molecular biology 68–79, 92 cell adhesion molecules 76–8, 92 cyclo-oxygenase-2 78 growth factors 72–5 renin–angiotensin system 78 retinoic acid receptors 78, 92 survival/proliferation factors 75–6 transcription factors 68–75 timing of events 65 vascular development 79 nephrogenic diabetes insipidus 321–4 autosomal dominant NDI 324 autosomal recessive NDI 323–4 test for 387 X-linked NDI 322–3 nephrogenic rests 456, 462 nephrolithiasis 281–308 aminoacidurias 290–3 candidate genes 283 hypercalciuric disorders 282–4

hyperoxaluria 283, 286–8 hypocitraturia 288–9 hypomagnesaemia 289–90 primary hyperuricosuria 283, 284–5 X-linked hypercalciuric nephropathy 294–5 xanthinuria 283, 296–7 nephromegaly 470 nephronophthisis (NPH) 255–6 Leber amaurosis and tapetoretinl degeneration 255–6 nephrons number 90 structure 58–60 nephropathies associated, mitochondrial mutations 193–4 familial juvenile hyperuricaemic 437 IgA nephropathy 422 membranous 421–2 numbers of syndromes 147, 148 primary hereditary 167–82 see also Alport syndrome sickle cell disease 402 nephrotic syndrome characteristics 42–4 congenital, of Finnish type (CNF) 167–71 familial focal segmental glomerulosclerosis 174 familial steroid-resistant 173–4 minimal change disease (MCD) 419 neurofibromas 355–6 plexiform 355–6 neurofibromatosis 349–60, 354–6 clinical presentation 350–1 complications 351–4 genetics 351 renal function 28 tumours 354–6 rhabdomyosarcoma 354–6 Wilms 354 type-1 349–54 see also Wilms tumour 354 neurturin 73 Newcastle disease virus (NDV) 539 nidogen/entactin 176 NIH 3T3, transformation by MET 501 NIS (sodium/iodide symporter) 531–2 2-(2-nitro-4-trifluoromethylbenzoyl)1,3cyclohexanedione (NTBC) 268 nocturnal enuresis 102 Norum disease 272 NTBC, in tyrosinaemia type I 268 obesity in Bardet-Biedl syndrome 371 in Bardet-Biedl syndrome and Alström syndrome 389

Index obstructive uropathy 119–20 causes 50–1 fetal ultrasonography 134–5 oculoauriculovertebral syndrome 152–3 oculocerebrorenal syndrome 154 oncocytoma, familial renal (FRO) 503–4 oncogenes, conversion from proto-oncogene 501 ONYX-015 536 ophthalmic signs Alport syndrome 186–8 Alström syndrome 382, 388 Bardet-Biedl syndrome 388 oral–facial–digital syndrome 156–7 osteo-onychyodysplasia (nail–patella syndrome) 95, 408–11 oxalate, hyperoxaluria 283, 286–8 P-glycoprotein Pallister–Killian syndrome, mosaic tetrasomy 128 pancreatic cysts/tumours, von Hippel–Lindau disease 492 papillary renal carcinoma, hereditary (HPRC-1) 498–502 paracellin, PCLN1 283 Patau syndrome 122 paternity issues, ethics 24 pedigree symbols 16 penetrance, variable 5 periodic fevers 436 perlecan 175 Perlmann syndrome 472 persephin 73 phaeochromocytoma 353, 492 phosphoribosyl pyrophosphate synthetase overactivity 285 platelet-derived growth factor (PDGF) 72, 92 podocin 174, 177, 421 polycystic hepatic disease 213–14 polycystic kidney disease autosomal dominant (ADPKD) 203–38 animal models 205–6 clinical aspects/diagnosis 206–11 genetics 220–5 hepatobiliary aspects 213–14 hypertension 211–13 intracranial aneurysms 214–20 autosomal recessive (ARPKD) 239–52 clinical aspects/diagnosis 241–7 differential diagnosis 247 management 247–8 genetics 239–40 prognosis 248 see also cysts; medullary cystic kidney disease polycystins 221

porphyria, acute intermittent 407–8, 416 positional cloning 12 post-renal disease, causes 50–1 posterior urethral valves 90 Potter sequence/syndrome 91, 122, 125, 241 Prader–Willi syndrome 128–9 pre-renal disease, treatment 39 predictive testing, ethical issues 24–5 predisposition syndromes, and screening 475 pregnancy, exacerbating hypertension 311 pregnancy-associated hypertension 353–4 prenatal diagnosis 19–23 indications 19–20 non-invasive/invasive techniques 20–2 termination of pregnancy 22–3 presymptomatic testing, ethical issues 23 proband, pedigree symbols 16 proliferation factors 75–6 pronephros 62 proteinuria nephrotic syndrome 42–4 sickle cell disease 402 proteoglycans 77 proto-oncogene, conversion to oncogene 501 proximal tubule 31–2 structure 58–9 pseudohypoaldosteronism type-1 (PHA1) 308, 315–17 type-2 (PHA2) 312–13 pulsed-field gel electrophoresis 12 purine metabolism disorders 436–7 pyelectasis 132–4, 136 pyrin/marenostrin 436 rapidly progressive glomerulonephritis (RPGN) 42, 45–6, 431 rearrangement syndromes 125–30 (8q) branchio-oto-renal syndrome 71, 95, 127 recessive inheritance autosomal 6–8 carriers, offspring risks 18 X-linked 6–8 renal abnormalities 147, 148 renal agenesis 147, 148 fetal ultrasonography 136 renal anatomy 57–60 size 57 renal angiomyolipoma 138 renal artery stenosis, and atherosclerosis 41 renal calculi see nephrolithiasis renal calyces, development 65–6 renal cancer 136–8 Birt–Hogg–Dubé (BHD) syndrome 504–5 epithelial histology 497–8 familial clear cell renal carcinoma 506

565

566

Index

renal cancer (cont.) familial renal oncocytoma (FRO) 503–4 hereditary papillary renal carcinoma-1 (HPRC-1) 498–502 renal cell carcinoma 136–7, 490–1, 505–6 targeted immunotherapy 515–16 see also gene therapy renal cystic disease dysplasia 254 VHL disease 490–1 renal development 60–79, 89–90 see also nephrogenesis renal disease causes 40 management 37–53 pre-renal disease 39 renal dysplasia 90, 120, in Bardet-Biedl syndrome 373–6 syndromes 147, 148 renal ectopia 147, 148 renal failure, acute and sickle cell disease 402 renal failure, chronic and sickle cell disease 401 renal failure, end stage treatment 51–3 renal function 27–33 non-excretory 32–3 renal function tests, procedures compared 36–7 renal hypoplasia 90, 119 renal lithiasis see nephrolithiasis renal malformations 90 renal obstruction see obstructive uropathy renal oncocytoma 137 renal pelvis, development 65–6 renal physiology 28–33 renal pyelectasis 132–4 renal replacement therapy 405–6 intelligent systems 512 renal size abnormal, syndromes 147, 148 imaging 34 renal syndromes, numbers with specific abnormalities 147, 148 renal transplantation 51–3 gene therapy 511 renal tubular acidosis 324–8 distal RTA 325–8 mixed RTA 328 proximal RTA 328 renal–coloboma syndrome 95, 96–7 renin–angiotensin system 419 nephrogenesis 78 renovascular disease, glomerulonephritis 428–9 replication-competent viral vectors 536–40 restriction enzymes, DNA cleavage 9 retinal angiomas, VHL disease 491

retinal degeneration, in BBS 368–70 retinoic acid derivatives, teratogenesis 93–4 retinoic acid receptors (RAR), nephrogenesis 78, 92 retrovirus vector, gene therapy 516–19 rhabdomyosarcoma, neurofibromatosis 354–6 RNA recognition motif (RRM) 459 Rokitansky malformation 155 Russell–Silver syndrome 131 salt homeostasis disorders 307–21 salt-wasting 309, 314–21 sarcoidosis, glomerulonephritis 432–4 SCF box protein 488 scleroderma, glomerulonephritis 432 screening, WT predisposition syndromes 475 Senior–Loken syndrome, nephronophthisis, Leber amaurosis and tapetoretinl degeneration 255–6 serpins 411 services, genetic counselling, organization 14–19 sex chromosomal disorders 124–5 sickle cell disease 399–406, 416 signalling molecules, specific inhibition by MET 502 Simpson–Golabi–Behmel syndrome 95, 473 skeletal muscle gene therapy 511 slit diaphragm (SD) 176–8 podocin 174 SD-associated diseases 178 Smith–Lemli–Opitz syndrome 95, 157–8, 272–3 Smith–Magenis syndrome 129–30 sodium/iodide symporter (NIS) 531–2 somatic cell hybridization 10–11 Sotos syndrome 474 steroidogenic factor-1 (SF1) 467 stillbirth, pedigree symbols 16 stone formation see nephrolithiasis suicide (cytotoxic) genes 530–2 survival/proliferation factors 75–6 syncytia, fusogenic membrane proteins (FMGs) 531 systemic lupus erythematosus, glomerulonephritis 429–31 Takayasu syndrome 41 teratogenesis 93–4 termination of pregnancy 22–3 pedigree symbols 16 tetrasomy 9p 127 (22pter-q11), cat-eye syndrome 130 mosaic 128 thalassaemia 416 tongue, macroglossia 468 Townes–Brocks syndrome 158–9

Index transcription factors 69–72, 92 winged helix 72 transforming growth factors TGF alpha 72, 73, 430, 432 TGF beta 73–4 transmission disequilibrium test (TDT) 428 triplication syndromes 125–30 mosaic tetrasomy 127 tetrasomy (9p) 127 tetrasomy (22pter-q11) cat-eye syndrome 130 Trp (2q) 125 triploidy 123–4 trisomies 119, 121–4, 126 age-related risk for trisomy-21 133 cysts 119 Wilms tumour 137, 456, 457 tuberous sclerosis complex 337–48, 343–5 genetic counselling 345 genetics 337–9 renal disease 339–45 tubular transport disorders 307–36 renal tubular acidosis 324–8 salt homeostasis disorders 307–21 water balance disorders 321–4 tubulointerstitial disease 403 acute 47–9 drug-induced 48, 49 tumour necrosis factor receptor-1-associated periodic fever (TRAPS) 436 tumours see renal cancer Turner syndrome 124, 137 twinning, pedigree symbols 16 two-hit mutational model 455, 466 tyrosinaemia type I 268–9 uniparental disomy (UPD) 117–18, 130–2, 471 ureteric bud development 64–5 and WT1 expression 458 ureters, duplication 90 urethral cancer 138 uric acid FHJN 437 hyperuricaemia 436–7 hyperuricosuria 283, 284–5 urinary tract chromosomal disorders 117–45 malformations 90–101 chromosome-6 100 non-syndromal 99–100 vesico-ureteric reflux 101–2 see also lower urinary tract; renal urothelial cancer 138

vaccination-based treatments 532–4 VACTERL/VATER association 95, 159–61 vascular development, nephrogenesis 79 vascular endothelial growth factor (VEGF) 72 angiogenesis 534 promoter 528 vasculitis, ANCA-associated 431–2 vector systems 516–23 adeno-associated virus 521–2 adenovirus 519–20, 535–6 herpesvirus 520–1 lentiviruses 518–19 non-viral vectors 522–3 replicating viral vectors 536–40 retrovirus 516–19 summary 517 vector targeting, gene therapy 523–9 velocardiofacial/DiGeorge syndrome 130 vesico-ureteric reflux, primary 90, 101–2 viral vectors see vector systems vitamin A, retinoic acid derivatives, teratogenesis 93–4 vitamin D receptor (VDR) 282–3 and sarcoidosis 434 von Hippel–Lindau disease 487–96, 534–5 cerebral haemangioblastoma 492 clinical presentation 489–90 diagnosis 489 major forms 487 pancreatic cysts/tumours 492 renal manifestations 490–1 retinal disease 491 surveillance programme 493 WAGR syndrome 71, 127, 458, 464 water balance disorders 321–4 websites, mapping, computer databases 13 Williams syndrome 126–7 FISH 11 Wilms tumour 70–1, 120, 124, 137, 455–86, 457 clinical phenotypes (predisposition to WT) 463–5 WAGR syndrome 71, 127, 458, 464 congenital genitourinary abnormalities 465–6 cytogenetics 456–7 epidemiology 455 histology 455–6 and insulin-like growth factor-2 460–2 molecular genetics 458–63 nephrogenic rests 456, 462 and neurofibromatosis 354–6 treatment 456

567

568

Index

Wilson disease 412–14 Wolf–Hirschhorn syndrome 125–6 WT1 protein 459 X-linked dominant inheritance 8 X-linked hypercalciuric nephropathy 294–5 X-linked recessive inheritance 6–8 carriers, offspring risks 18 X-inactivation, skewed 6–7

xanthinuria 283, 296–7 XYY syndrome 125 Y-linked inheritance 8 yeast artificial chromosomes (YACs), mapping 12, 263 Zellweger syndrome 95, 161 zonula occludens-1 protein (ZO-1) 176

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