University of Groningen

Genetics of syndrome Vos, Yvonne Johanna

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Download date: 26-09-2021 Genetics Ll Syndr

Yvonne Vos , '

Genetics of Ll syndrome

Yvonne Johanna Vos Vos, Yvonne J Genetics of Ll syndrome Proefschrift Groningen

ISBN: 978-90-367-4471-3

© Copyright 2010 Y.J. Vos All rights are reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmittedin any form or by any means, without permission of the author.

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Genetics of Ll syndrome

-·-····· ---· ·· -�--;. ! ' i Yvonne Vos, 13 september 2010 I \.( 11 .. ·f '.V!ed1•·,·t,.' .-1G B1�!1,••h,.1:l< ,-. Gn)ningc!n

1. De kans op het vinden van een mutatie in het LlCAMgen hangt af van het aantal klinische kenmerken bij de patient en het aantal aangedane familieleden van deze patient(dit proefschrift). 2. Mutatiesin het L1CAMgen leiden vaker dan gedacht tot een ernstig ziektebeeld bij meisjes (dit proefschrift). 3. lnformatieover de betrokkenheid van genomische variatiesbij een aandoening kan het best worden verkregen door vergelijking met de genomische sequenties van de meest aan de mens verwante dieren. 4. In het licht van stelling 3 zou het 1000 Genomes Project niet a Ileen het humane genoom van een groot aantal mensen moeten onderzoeken, maar juist ook het genoom van nauw aan de mens verwante dieren. 5. Ten onrechte wordt de invloed van silent en missense mutaties op de mRNA splicing vaak niet onderkend en daarom niet onderzocht (ditproefschrift). 6. Het vergaren van kennis over het effect van mutaties op de functionaliteit van genproducten verdient een even hoge prioriteit als het steeds sneller kunnen genereren van veel sequentiedata. 7. Vaten communiceren vaak beter dan mensen. 8. Nadenken suggereert ten onrechte dat dit achteraf dient te geschieden. 9. Voor sommige schaatscoaches is het de verkeerde baan. 10. lndien de regering onvoldoende scoort, is er feitelijk sprake van een kabinetniet.

RIJKSUNIVERSITEIT GRONINGEN

Genetics of Ll syndrome

Proefsch rift

ter verkrijging van het doctoraat in de Medische Wetenschappen aan de Rijksuniversiteit Groningen op gezag van de Rector Magnificus, dr. F. Zwarts, in het openbaar te verdedigen op maandag 13 september 2010 om 14.45 uur

Cenlrale u Mec.Jische M Bibliotheek C Groningen G

door

Yvonne Johanna Vos geboren op 15 November 1955 te Rotterdam Promoter: Prof. dr. R.M.W. Hofstra

Beoordelingscommissie: Prof. dr. C.T.R.M. Schrander-Stumpel Prof. dr. O.F. Brouwer Prof. dr. H. van Bokhoven Paranimfen: Krista van Dijk-Bos Arjan Bijlsma

Publication of this thesis was financially supported by:

Department of Genetics, University Medical Center Groningen, University of Groningen, PerkinElmer Nederland, GC biotech, Fa. A.Vos & Zoons Medische artikelen, Amsterdam (www. vosmedisch.nl), Roche Diagnostics Nederland BV, MRC-Holland, Promega Benelux, Aardworm. com Webdesign (www.aardworm.com)

Their support is gratefully acknowledged.

Aan mijnouders

CONTENTS

Chapter1 Introduction 11

Chapter 2 - correlations in Ll syndrome: a guide 25 for genetic counseling and mutationanalysis Appendix 41 Additional DNA testing results. Ll syndrome in females

Chapter3 X-linked : a novel missense in the 45 LlCAM

Chapter4 An updated and upgraded LlCAM mutation database 53

Chapter 5 RNA splicing defects caused by silent, missense and 65 intronic in the LlCAM gene can be the cause of Ll syndrome

Chapter 6 Nephrogenic diabetes insipidus in a patient with L1 77 syndrome: a new report of a contiguous gene deletion syndrome including LlCAM and AVPR2

Chapter 7 Complex pathogenesis of Hirschsprung's disease in a 87 patient with hydrocephalus, vesico-ureteral refluxand a balanced translocation t(3;17)(p12;qll)

Chapter 8 X-chromosome-specific array-CGH analysis indicates new 101 , other than the LlCAM gene, involved in Ll syndrome

Chapter 9 Discussion and future perspectives 119

Chapter10 Summary 137

Nederlandse samenvatting 143

Dankwoord/acknowledgements 149

List of publications 155

Chapter 1

Introduction ------

Chapter 1 The disease

Ll syndrome is an X-linked recessive disorder caused by mutations in the L1CAM gene[1,21• The phenotypic spectrum associated with these mutations includes four different neurological syndromes[31• The first one, X-linked hydrocephalus, also referred to as hydrocephalus due to �tenosis of the £queduct of Sylvius (HSAS, MIM # 307000), was first described in 1949 by Bickers and Adams[41• Seven males in two successive generations were born with hydrocephalus and some of them died at birth. At autopsy, structural brain malformations with a narrowing of the aqueduct of Sylvius was seen and proposed to be the primary cause of the congenital hydrocephalus. X-linked hydrocephalus is the most frequent genetic form of congenital hydrocephalus[51, with a prevalence of approximately one in 30,000 newborn boys[61• It accounts for approximately 7-15% of all congenital hydrocephalus cases[61• The diagnosis can be established in males with characteristic clinical and neuropathological findings and a family history consistent with X-linked inheritance. However, the clinical symptoms are variable. In most families, the affected boys die before, during, or soon after birth[71• Post partum, it has been observed that, in addition to the characteristics of hydrocephalus (viz. , bulging fontanelle and sunset eye phenomenon), adducted thumbs are present in at least 50% of patients (figure1)[ 51• On an MRI scan of the brain or post mortem, agenesis or dysgenesis of the , and a small brainstem can be observed in addition to ventricle dilatation[81• The bilateral absence of the pyramids (corticospinal tract) is an important and almost pathognomonic finding[91• An aqueduct stenosis is not always found to be associated with this syndrome[101• A second, less severe form of the disease was published under the acronym MASA syndrome (MIM # 303350) and originally described in 1974(111• This syndrome consists of slight to moderate mental retardation, £phasia, �huffling gait and £dducted thumbs. Brain scans of men with this syndrome usually show abnormalities such as enlargement of the ventricle system[121• Occasionally, phenotypic variation ranging from the severe X-linked hydrocephalus type to the less severe MASA syndrome can be found in one and the same family[12,131_ The third and fourth syndromes that are part of the Ll syndrome are X-linked complicated hereditary spastic type 1 (SPGl, MIM # 303350)[141 and X-linked complicated corpus callosum agenesis (X-linked ACC, MIM # 304100), although both these are seen much less frequently. The two complicated forms may be accompanied by mild to moderate mental retardation, and X-linked ACC with variable spastic paraplegia[15•161• Female carriers of an L1CAM mutation are sometimes mildly affected with slightly adducted thumbs and a cognitive level significantly lower than their non-carrier sister(s). A severe form with hydrocephalus is rarely found in girls[111• It is the clinical characteristics of each of these four syndromes that give rise to a suspicion that a patient may have a mutation in the L1CAM gene. The seven most important characteristics of Ll syndrome are

Figure 1. Adducted thumb

12 Introduction

hydrocephalus, aqueduct stenosis, mental retardation, adducted thumbs, agenesis/dysgenesis of the corpus callosum, (delayed speech development) and spastic paraplegia. Beside patients with these characteristics, we also describe rare syndromic patients in whom the L1 syndrome characteristics are only part of the phenotype. An example of a disease frequently found associated with Ll syndrome is Hirschsprung's disease (HSCR); at least 13 cases have been describedr15,is-251_ This suggests a non-random association,since the frequency of Ll syndrome is approximately 1:30,000 newborn boys and that of HSCR is 1:5,000 newborns, so by chance this coincidence would only occur in approximately 1:150,000,000 newborns. Other examples of syndromic cases with Ll syndrome comprise association with a specific 26 form of congenital idiopathic intestinal pseudo-obstruction (CIIP)r 1, with bilateral duplex 21 24 kidneys and uretersr 1, with Nephrogenic Diabetes lnsipidus (NDl)r 1, with pure lq43-qter deletionsyndrome r281 and with (chapter 2: appendix). In most of these patients a mutation in the L1CAM gene was found, sometimes in addition to another chromosomal aberration,such as the lq43-qter deletionor chromosome 21 trisomy, suggesting involvement of L1CAM in the Ll syndrome associated diseases. However, some of the syndromic cases can not be explained by L1CAM mutations.

The gene

In the 1980s different linkage studies mapped the disease locus on the long arm of the X 12 14 29 30 chromosome at Xq28r • • • 1• Later, the disease locus was narrowed down to a region of 1.5 31 mbr 1• The gene encoding the cell adhesion molecule Ll (L1CAM gene), known to be involved in 32 33 31 brain developmentr • 1, was the most obvious candidate genel 1• Shortly thereafter,mutations

34 35 in the L1CAM gene were shown to be responsible for the disorderr1, • 1_ The L1CAM gene is 16 kb in length and consists of 29 exons. The first exon of 125 bp is non­ 36 coding and was discovered laterr 1, which is why this exon, located 10 kb upstream of the ATG containing exon 1, is known as exon A instead of exon 1 as is commonly used. The L1CAM gene is primarily expressed both in the (CNS) and in the peripheral nervous system and encodes a of 1,257 amino acids, comprising a signal peptide of 19 amino acids and a functional Ll protein of 1,238 amino acids. This expression is partially controlled by the neural restrictive silencer element (NRSE) located in the first 36 intron of the genel 1• Tissue-specific expression is further regulated by homeodomain and Pax upon binding at the homeodomain and paired-domain binding (HPD) site upstream exon 1. In non-neural tissue, alternative RNA splicing generates mRNA which lacks exons 2 and 37 38 27 of the genel • 1•

Mutations in the L1CAM gene

169 different disease-causing mutations have been published: 130 of these were reviewed 39 21 22 26 0 52 in 2001r l and an additional 39 have been published more recentlyr • • A - 1 • The most are private mutationsspread over the entiregene (figure 2), comprising not only nonsense, splice site and missense mutations, but also deletions and insertions, usually resulting in a frame

53 shift. Some of these mutations have been incorporated into a databasel 1•

13 .:,...., 9

-§ p.Tfrp276Arg p.Gly480Arg pp.Thr627Met � . p.Met172t1e r r p.Leu313Pro I p.lle179Ser p.Trp335Arg p.AspSlGTyr .As 516Asn p.Arg184Gly r pp.Asn369Lys P P O p.lle645Phe r p.Arg184Gln I 0 p.Arg525His I I I p.Met187_ Val198del 1 p.Asp202Tyr p.Asn1071del p.Leu1080Gln 28

c.991+1G> t p.Arg901X �be. �.991+2T>C ) c.198-2A>T p.Tyr811X !l b p.Leu260GlyfsX45 p.Gln789X '0 p.Serl064X p.Gln66X b 645C>T c.92-2A>G p.Arg760X c.3166+2T>A p.Tyr26 p.Asn197 X l SerfsX37 t)c.1547-2A>T p.Arg739GlyfsX112 p.His978GlnfsX25 I c.76+1G>A l p.Trp662X p.Lys963GlnfsX41{2x) bc.1546+2T>A . c.1940-lG>A p.Glu974AsnfsX7 c.si3+SG>C c.1268-lG>A c.1939+1G>A p.Xl257LeufsX96 del LlCAM____ gene...... _ .....__,..,.�.... -:::;...x.:.:-.. ..,;::=-..;-----�.x -,,.;;->c:a-:;:::..- ,;;;,,�·- u m m t t• ,,.-,

lg-1 lg-2 lg-3 lg-4 lg-5 lg-6 Fnlll-1 Fnlll-2 Fnlll-3 Fnlll-4 Fnlll-5 TM Cytoplasmic domain a.a. 33-133 134-230 238-330 331-422 423-515 516-609 612-709 714-807 812-913 918-1011 1017-1107

Figure 2. Mutationsin the LlCAM gene. lg, immunoglobulin-like domain; Fn, fibronectintype Ill domain; TM, transmembrane domain Introduction

IG-1 IG-2 IG-3 IG-4 IG-5 IG-6

cyto

extracellular

Figure 3. Schematic representation of the neural cell adhesion molecule Ll (Ll protein). Cyto, cytoplasmic 119 domain; TM, transmembrane domain

·\' \ � A G B F E C D C Telokin Ll protein lg-domain 1 G B F E C D C'

Figure 4. Schematic diagrams of the folding of the telokin domain and the first lg-domain of Ll. The folds are formed by two �-sheets, shown as bold black lines. The key site residues in telokin are given with their one letter code. Horizontal lines between residues represent the main-chain hydrogen bonds that form the �-sheets. (Adapted from Bateman et al.[59)). Genotype-phenotype studies revealed truncating mutationsin the extracellular part(figure 3) of the Ll protein to be more severe than missense mutations in the same part of the protein or than mutations (truncatingas well as missense) in the cytoplasmic domain154,55l_ Since quite severe and less severe are found within the same family, the existence of modifiers is hypothesized; this is also based on indicationsreported in the literature1561•

15 Chapter 1 Although the disorder can be explained by a mutationin the L1CAM gene for only part of the group of patients, suggesting the involvement of more than one gene in this disease, so far there is no evidence of genetic heterogeneity. However, based on the mutation frequency 52 57 described for the L1CAM gene outlined in the literature, viz. 30% and 46%1 • 1, and in this thesis, it can be hypothesized that more genes are involved in Ll syndrome.

The protein

The neural cell adhesion molecule Ll (Ll protein) belongs to the immunoglobulin superfamily of cell adhesion molecules1581. It is a transmembrane glycoprotein and consists of an extracellular part of six immunoglobulin-like domains (lg-domains) and fivefibronectin type Ill domains (Fnlll­ domains), followed by a single-pass transmembrane segment, and a short cytoplasmic region (figure 3)1331• Bateman et a/. 1591 derived the outline structure of the six lg-domains and the five Fnlll-domains of the Ll protein from the known telokin structure1601, also a member of the lg superfamily, and from the known Fnlll structures of neuroglian1611, respectively. By comparing these structures and their key residues (telokin contains 35 key amino acid residues (figure4) and the first Fnlll domain from neuroglian contains 27 key residues) with Ll protein, the possible key residues in Ll protein were predicted (figure 4). The key residues mentioned determine the conformation of a protein

Heterophilic interactions: Extracellular matrix components e.g. laminin, neurocan, phosphocan, tenascin-R; lg superfamily CAMs e.g. DMl-GRASP, Fll/F3, Promote, LU! � TAG-1/axonin-1 binding \:) ----- I ril \ � Cell surface Required for neurite outgrowth Stimulation upon Ll:Ll binding

� Signaling Phosphorylation by: cascade P90rsk, CKII ERK2, CekS '111

Neurite outgrowth

Figure 5. Ll interactions. Ll interacts at the neuronal cell surface with Ll present on another surface (Ll-Ll interaction). This interaction initiates neurite outgrowth. Ll protein also interacts with other extracellular and intracellular components. Some of these may modulate Ll activity. The cytoplasmic domain interacts with the cytoskeleton16 and is subject to phosphorylation by a variety of kinases. (Adapted from Kenwrick et al.[67]) Introduction domain. They are believed to be involved in intermolecular packing and hydrogen bonding, or have the ability to take up unusual conformationsl59l_ As the key residues are crucial for the protein 59 folding, missense mutations affecting these residues are often considered to be pathogenicl 1•

The function

Ll protein is primarily expressed throughout the central and peripheral nervous systems on subsets of developing and differentiated , as well as on Schwann cells of the peripheral nervous system. It is also detected in non-neuronal cells, like melanocytes and lymphocytesl621, 63 65 intestinal crypt cells, and kidney tubule epithelial . 1• The L1 protein mediates cell-cell adhesion on differentiatedneurons by Ll-Ll interaction at regions of contact between neighboring and on the growth cones, the structures at the leading tip of axons that are responsible for sensing extracellular growth and guidance cues. Both Ll-Ll interactions and Ll-non Ll protein interactions (figure 5) play a key role in the development of the nervous system by regulating outgrowth, axon fasciculation, myelination and neuronal migration1661• These interactions trigger the activation of several intracellular signaling pathways1671 (figure 5). For instance Ll­ Ll dimerization stimulates axonal growth by activating the tyrosine kinase domains of the 68 69 fibroblast growth factor receptor1 • 1• Ll also interacts with TAG-1, triggering intracellular events important for neurite outgrowthl701 • In addition, the highly conserved cytoplasmic domain of the Ll protein has been shown to signal through Src and mitogen-activated protein kinase (MAPK1/ERK2)l71,72I, which are also important for neurite outgrowth.

Genetic screening

To date LlCAM is the only gene associated with Ll syndromel56•731. may be used to confirm a diagnosis of Ll syndrome and if a mutation is found carrier detection in the family is possible. This presents mothers who carry a mutation with the option of having prenatal testing or preimplantation genetic diagnosis (PGD). Usually the mother is a carrier, but de nova disease-causing mutationshave also been reported l74,75l_ In those cases the risk for an affected male offspring is low, but still greater than in the general population because of the possibility of germline mosaicism. Prenatal analysis can also be offered in such cases. Only a few studies report L1CAM mutation frequencies; they show quite different results depending on the composition of the target groups. In a group of patients, suspected of having Ll syndrome a frequency of 30% was reportedl571, which increased up to 74% if only patientswith at least two additional cases in the family were selected. The detection rate was 16% in cases with a negative family historyl57l_ A Japanese study1521 showed a relatively high mutation frequency of 46%: this high rate, might be due to the selection criteria used. From a group of 390 documented Ll syndrome patients, they selected 96 patients from 57 families for LlCAM mutation analysis based on the following criteria: (1) cases of hydrocephalus with X-linked inheritance and (2) sporadic cases with clinical and characteristics of X-linked hydrocephalus. A mutation was detected in 26/57 families (46%). In order to avoid the expense of DNA diagnostics as much as possible, it would be helpful to know the conditions that yield a high chance of actually finding a mutation in the LlCAM gene.

17 Chapter 1 Aims of this study

Since the discovery of the gene involved in Ll syndrome in 1992, we have learned much about the gene, the protein it encodes and the mutations involved. However several questions/ issues remain: Can we develop a tool to estimate the chance of finding a mutation based on the phenotypic characteristics and the family anamnesis? Several studies have only partly addressed this question. There is no L1CAM mutation database addressing questionson the pathogenicity of the mutations identified. The existing database is incomplete and does not contain relevant information on the clinical characteristics and family anamnesis of L1 syndrome patients. How can we determine the pathogenicity of non-truncatingmutations and can we confirm or detect more genotype-phenotype correlations? Many patients suspected of having Ll syndrome do not have a mutation in the LlCAM gene, this raises the question: might other genes be involved in the Ll syndrome? Can we explain rare syndromic patients with L1 syndrome characteristicsby mutations in the LlCAM gene alone?

In order to solve these questions/issues, we set the following aims: • To design an algorithm based on genotypical, phenotypical and family data that can predict the chance of detectingan LlCAM mutation in patientssuspected of having L1 syndrome. • To determine/confirmpotential genotype-phenotype correlations in the Ll syndrome. • To set up an easily accessible LlCAM mutationdatabase and include all the disease-causing and non-disease-causing mutations, all variants for which it is not known whether they are disease-causing (unclassified variants (UVs)), and all relevant patient data. • To determine the potential pathogenicity of some UVs due to incorrect RNA splicing by using an ex vivo RNA splicing assay. • To determine whether the clinical picture caused by mutations in the LlCAM gene is broader than generally thought by analyzing rare syndromic cases with L1 syndrome characteristics. • To try and identifynew genes potentially involved in Ll syndrome.

Outline of this thesis

Part I of the thesis deals with the mutation analysis in a large group of patients suspected of having Ll syndrome and with the gathering of the clinical characteristics and the family anamnesis of these patients. We also describe the statisticalanalysis of the acquired data. Chapter 2 summarizes the results of the mutationanalyses of the coding exons and exon-intron boundaries of the LlCAM gene in 367 referred cases. An extended analysis was performed in a selected group of patients by analyzing regulatory sequences and by looking for duplications. The mutation detection rate in the whole group as well as in a selected group of patientswas determined. Combining these results with the clinical data of the patients, revealed ways to predict the group of patients in which the chance of detecting a mutation is highest. This chapter further outlines potential genotype-phenotype correlations.

18 Introduction Chapter 3 describes in detail a case report on an l1 syndrome patient harboring a missense mutation and arguments are given for why this missense mutation should be considered disease causing. Chapter 4 reports the construction and use of an updated and upgraded LlCAM mutation database describing all the mutations we found and those reported in the literature, so that it is now easy to check whether a mutation is considered to be disease-causing, non-disease­ causing, potentiallydisease-causing, or unknown. Chapter 5 covers the use of an ex vivo RNA splicing assay to demonstrate possibly incorrect RNA splicing to determine the pathogenicity of a few unclassified variants. Part II of the thesis reports on mutation analysis in two patients with multiple congenital aberrations, including Ll syndrome. Chapter 6 describes the analysis of a patient with Ll syndrome plus Nephrogenic Diabetes Insipid us. Chapter 7 covers the analysis of a patient with Ll syndrome plus Hirschsprung's disease and describes the influence of an LlCAM mutation on HSCR disease. Part Ill of the thesis deals with the hypothesis that there are additional genes involved in Ll syndrome. Since only 20% of the patients clinically suspected of having Ll syndrome carry a mutation in the LlCAM gene, other genes are likely to be involved. In Chapter 8 a high density X-chromosome-specific array CGH analysis was used to investigate whether deletions on the X-chromosome can be found in any of 60 patients suspected of having Ll syndrome who had no mutation in the LlCAM gene. These deletions might indicate the localization of genes involved in Ll syndrome. A few of these candidate genes were analyzed to allow us to draw conclusions about potential new genes.

19 Chapter 1 References

(1) Rosenthal A, Jouet M, Kenwrick S. Aberrant splicing of neural cell adhesion molecule Ll mRNA in a family with X-linked hydrocephalus. Nat Genet 1992 Oct;2(2):107-12. (2) Jouet M, Rosenthal A, Armstrong G, MacFarlane J, Stevenson R, Paterson J, Metzenberg A, lonasescu V, Temple K, Kenwrick S. X-linked spastic paraplegia (SPGl), MASA syndrome and X-linked hydrocephalus result from mutations in the L1 gene. Nat Genet 1994 Jul;7(3):402-7. (3) Fransen E, Van CG, Vits L, Willems PJ. Ll-associated diseases: clinical geneticists divide, molecular geneticists unite. Hum Mol Genet 1997;6(10):1625-32. (4) Bickers DS, Adams RD. Hereditary stenosis of the aqueduct of Sylvius as a cause of congenital hydrocephalus. Brain 1949;72:246-262 (5) Schrander-Stumpel C, Fryns JP. Congenital hydrocephalus: nosology and guidelines for clinical approach and genetic counselling. Eur J Pediatr 1998 May;157(5):355-62. (6) Halliday J, Chow CW, Wallace D, Danks DM. X linked hydrocephalus: a survey of a 20 year period in Victoria, Australia. J Med Genet 1986 Feb;23(1):23-31. (7) Schrander-Stumpel C, Howeler C, Jones M, Sommer A, Stevens C, Tinschert S, Israel J, Fryns JP. Spectrum of X-linked hydrocephalus (HSAS), MASA syndrome, and complicated spastic paraplegia (SPGl): Clinical review with six additional families. Am J Med Genet 1995 May 22;57(1):107-16. (8) Yamasaki M, Arita N, Hiraga S, lzumoto S, Morimoto K, Nakatani S, Fujitani K, Sato N, Hayakawa T. A clinical and neuroradiological study of X-linked hydrocephalus in Japan. J Neurosurg 1995 Jul;83(1):S0-5. (9) Chow CW, Halliday JL, Anderson RM, Danks DM, Fortune DW. Congenital absence of pyramids and its significance in genetic diseases. Acta Neuropathol 1985;65(3-4):313-7. (10) Willems PJ, Brouwer OF, Dijkstra I, Wilmink J. X-linked hydrocephalus. Am J Med Genet 1987 Aug;27(4):921-8. (11) Bianchine JW, Lewis RC, Jr. The MASA syndrome: a new heritable mental retardation syndrome. Clin Genet 1974;5(4):298-306. (12) Schrander-Stumpel C, Legius E, Fryns JP, Cassiman JJ. MASA syndrome: new clinical features and linkage analysis using DNA probes. J Med Genet 1990 Nov;27(11):688-92. (13) Fryns JP, Spaepen A, Cassiman JJ, van den Berghe H. X linked complicated spastic paraplegia, MASA syndrome, and X linked hydrocephalus owing to congenital stenos is of the aqueduct of Sylvius: variable expression of the same mutationat Xq28. J Med Genet 1991 Jun;28(6):429-31. (14) Kenwrick S, lonasescu V, lonasescu G, Searby C, King A, Dubowitz M, Davies KE. Linkage studies of X-linked recessive spastic paraplegia using DNA probes. Hum Genet 1986 Jul;73(3):264-6. (15) Boyd E, Schwartz CE, Schroer RJ, May MM, Shapiro SD, Arena JF, Lubs HA, Stevenson RE. Agenesis of the corpus callosum associated with MASA syndrome. Clin Dysmorphol 1993 Oct;2(4):332-41. (16) Basel-Vanagaite L, Straussberg R, Friez MJ, lnbar D, Korenreich L, Shohat M, Schwartz CE. Expanding the phenotypic spectrum of LlCAM-associated disease. Clin Genet 2006 May;69(5):414-9. (17) Kaepernick L, Legius E, Higgins J, Kapur S. Clinical aspects of the MASA syndrome in a large family, including expressing females. Clin Genet 1994 Apr;45(4):181-5. (18) Okamoto N, Wada Y, Goto M. Hydrocephalus and Hirschsprung's disease in a patientwith a mutation of LlCAM. J Med Genet 1997 Aug;34(8):670-1. (19) Vits L, Chitayat D, Van Camp G, Holden JJ, Fransen E, Willems PJ. Evidence for somatic and germline mosaicism in CRASH syndrome. Hum Mutat 1998;Suppl 1:S284-S287. (20) Parisi MA, Kapur RP, Neilson I, Hofstra RM, Holloway LW, Michaelis RC, Leppig KA. Hydrocephalus and intestinal aganglionosis: is LlCAM a modifier gene in Hirschsprung disease? Am J Med Genet 2002 Feb 15;108(1):51-6. (21) Okamoto N, Del Maestro R, Valero R, Monros E, Poe P, Kanemura Y, Yamasaki M. Hydrocephalus and Hirschsprung's disease with a mutation of LlCAM. J Hum Genet 2004;49(6):334-7. (22) Nakakimura S, Sasaki F, Okada T, Arisue A, Cho K, Yoshino M, Kanemura Y, Yamasaki M, Todo S. Hirschsprung's disease, acrocallosal syndrome, and congenital hydrocephalus: report of 2 patients and literature review. J Pediatr Surg 2008 May;43(5):E13-El7. (23) Jackson SR, Guner VS, Woo R, Randolph LM, Ford H, Shin CE. LlCAM mutation in association with X-linked hydrocephalus and Hirschsprung's disease. Pediatr Surg Int 2009 Jul 30. (24) Tegay DH, Lane AH, Roohi J, Hatchwell E. Contiguous gene deletion involving LlCAM and AV PR2 causes X-linked hydrocephalus with nephrogenic diabetes insipidus. Am J Med Genet A 2007 Mar 15;143(6):594-8.

20 Introduction

(25) Hofstra RM, Elfferich P, Osinga J, Verlind E, Fransen E, Lopez Pison J, Die-Smulders CE, Stolte-Dijkstra I, Buys CH. Hirschsprung disease and L1CAM: is the disturbed sex ratio caused by L1CAM mutations? J Med Genet 2002 Mar;39(3}:E11. (26) Bott L, Baute 0, Mention K, Vinchon M, Boman F, Gottrand F. Congenital idiopathic intestinal pseudo­ obstruction and hydrocephalus with stenosis of the aqueduct of sylvius. Am J Med Genet A 2004 Sep 15;130(1):84-7. (27) Liebau MC, Gal A, Superti-FurgaA, Omran H, Pohl M. L1CAM mutation in a boy with hydrocephalus and duplex kidneys. Pediatr Nephrol 2007 Jul;22(7):1058-61. (28) Hiraki Y, Okamoto N, Ida T, Nakata Y, Kamada M, Kanemura Y, Yamasaki M, Fujita H, Nishimura G, Kato M, Harada N, Matsumoto N. Two new cases of pure lq terminal deletion presenting with brain malformations. Am J Med Genet A 2008 May 15;146A(10):1241-7. (29) Willems PJ, Dijkstra I, Van der Auwera BJ, Vits L, Coucke P, Raeymaekers P, Van Broeckhoven C, Consalez GG, Freeman SB, Warren ST. Assignment of X-linked hydrocephalus to Xq28 by linkage analysis. Genomics 1990 Oct;8(2):367-70. (30) Winter RM, Davies KE, Bell MV, Huson SM, Patterson MN. MASA syndrome: further clinical delineation and chromosomal localisation. Hum Genet 1989 Jul;82(4):367-70. (31) Jouet M, Feldman E, Yates J, Donnai D, Paterson J, Siggers D, Kenwrick S. Refining the genetic location of the gene for X linked hydrocephalus within Xq28. J Med Genet 1993 Mar;30(3):214-7. (32) Rathjen FG, Schachner M. lmmunocytological and biochemical characterization of a new neuronal cell surface component (Ll antigen) which is involved in cell adhesion. EMBO J 1984 Jan;3(1):1-10. (33) Hlavin ML, Lemmon V. Molecular structure and functional testing of human L1CAM: an interspecies comparison. Genomics 1991 Oct;11(2):416-23. (34) Jouet M, Rosenthal A, MacFarlane J, Kenwrick S, Donnai D. A missense mutation confirms the Ll defect in X-linked hydrocephalus (HSAS). Nat Genet 1993 Aug;4(4):331. (35) Van Camp G, Vits L, Coucke P, Lyonnet S, Schrander-Stumpel C, Darby J, Holden J, Munnich A, Willems PJ. A duplication in the L1CAM gene associated with X-linked hydrocephalus. Nat Genet 1993 Aug;4(4):421-5. (36) Kallunki P, Edelman GM, Jones FS. Tissue-specific expression of the Ll cell adhesion molecule is modulated by the neural restrictive silencer element. J Cell Biol 1997 Sep 22;138(6):1343-54. (37) Jouet M, Rosenthal A, Kenwrick S. Exon 2 of the gene for neural cell adhesion molecule Ll is alternatively spliced in B cells. Brain Res Mol Brain Res 1995 Jun;30(2):378-80. (38) Coutelle 0, Nyakatura G, Taudien S, Elgar G, Brenner S, Platzer M, Drescher 8, Jouet M, Kenwrick S, Rosenthal A. The neural cell adhesion molecule Ll: genomic organisation and differential splicing is conserved between man and the pufferfish Fugu. Gene 1998 Feb 16;208(1):7-15. (39) Weller S, Gartner J. Genetic and clinical aspects of X-linked hydrocephalus (Ll disease): Mutations in the L1CAM gene. Hum Mutat 2001;18(1):1-12. (40) De Angelis E, Watkins A, Schafer M, Brummendorf T, Kenwrick S. Disease-associated mutations in Ll CAM interfere with ligand interactions and cell-surface expression. Hum Mol Genet 2002 Jan 1;11(1):1-12. (41) Simonati A, Boaretto F, Vettori A, Dabrilli P, Criscuolo L, Rizzuto N, Mostacciuolo ML. A novel missense mutation in the L1CAM gene in a boy with Ll disease. Neurol Sci 2006 Jun;27(2):114-7. (42) Senat MV, Bernard JP, Delezoide A, Saugier-Veber P, Hillion Y, Roume J, Ville Y. Prenatal diagnosis of hydrocephalus-stenosis of the aqueduct of Sylvius by in the first trimester of pregnancy. Report of two cases. Prenat Diagn 2001 Dec;21(13):1129-32. (43} Sztriha L, Vos VJ, Verlind E, Johansen J, Berg 8. X-linked hydrocephalus: a novel missense mutation in the L1CAM gene. Pediatr Neurol 2002 Oct;27(4):293-6. (44) Rodriguez CG, Perez AA, Martinez F, Vos VJ, Verlind E, Gonzalez-Meneses LA, Gomez de TS, I, Schrander-Stumpel C. X-linked hydrocephalus: another two families with an Ll mutation. Genet Couns 2003;14(1):57-65. (45) Silan F, Ozdemir I, Lissens W. A novel L1CAM mutation with Ll spectrum disorders. Prenat Diagn 2005 Jan;25(1}:57-9. (46) Hubner CA, Utermann 8, Tinschert S, Kruger G, Ressler 8, Steglich C, Schinzel A, Gal A. lntronic mutations in the L1CAM gene may cause X-linked hydrocephalus by aberrant splicing. Hum Mutat 2004 May;23(5):526. (47) Moya GE, Michaelis RC, Holloway LW, Sanchez JM. Prenatal diagnosis of Ll cell adhesion molecule mutations. Capabilities and limitations. Fetal Diagn Ther 2002 Mar;17(2):115-9.

21 ------

Chapter 1

(48) Panayi M, Gokhale D, Mansour S, Elles R. Prenatal diagnosis in a family with X-linked hydrocephalus. Prenat Diagn 2005 Oct;25{10):930-3. (49) Knops NB, Bos KK, Kerstjens M, van Dael K, Vos YJ. Nephrogenic diabetes insipidus in a patient with Ll syndrome: a new report of a contiguous gene deletion syndrome including LlCAM and AV PR2. Am J Med Genet A 2008 Jul 15;146A(14):1853-8. (SO) Griseri P, Vos Y,Giorda R, Gimelli S, Beri S, Santamaria G, Mognato G, Hofstra RM, Gimelli G, Ceccherini I. Complex pathogenesis of Hirschsprung's disease in a patient with hydrocephalus, vesico-ureteral reflux and a balanced translocation t(3;17)(p12;qll). Eur J Hum Genet 2009 Apr;17(4):483-90. (51) Wilson PL, Kartman BB, Mulvihill JJ, Li S, Wilkins J, Wagner AF, Goodman JR. Prenatal identification of a novel R937P LlCAM missense mutation. Genet Test Mol Biomarkers 2009 Aug;13(4):515-9. Nov;lOS(S (52) Kanemura Y, Okamoto N, Sakamoto H, Shofuda T, Kamiguchi H, Yamasaki M. Molecular mechanisms and neuroimaging criteria for severe Ll syndrome with X-linked hydrocephalus. J Neurosurg 2006 Suppl):403-12. {53) Van Camp G, Fransen E, Vits L, Raes G, Willems PJ. A locus-specific mutation database for the neural cell adhesion molecule LlCAM (Xq28). Hum Mutat 1996;8{4):391. (54) Yamasaki M, Thompson P, Lemmon V. CRASH syndrome: mutations in LlCAM correlate with severity of the disease. Neuropediatrics 1997 Jun;28(3):175-8. (55) Fransen E, Van Camp G, D'Hooge R, Vits L, Willems PJ. Genotype-phenotype correlation in Ll associated diseases. J Med Genet 1998 May;35{5):399-404. {56) Tapanes-Castillo A, Weaver EJ, Smith RP, Kamei Y, Caspary T, Hamilton-Nelson KL, Slifer SH, Martin ER, Bixby JL, Lemmon VP. A modifier locus on chromosome 5 contributes to Ll cell adhesion molecule X-linked hydrocephalus in mice. Neurogenetics 2009 Jun 30. (57) Finckh U, Schroder J, Ressler B, Veske A, Gal A. Spectrum and detection rate of LlCAM mutations in isolated and familial cases with clinically suspected Ll-disease. Am J Med Genet 2000 May 1;92(1):40-6. (58) Moos M, Tacke R, Scherer H, Teplow D, Fruh K, Schachner M. Neural adhesion molecule Ll as a member of the immunoglobulin superfamily with binding domains similar to fibronectin. Nature 1988 Aug 25;334(6184):701-3. {59) Bateman A, Jouet M, MacFarlane J, Du JS, Kenwrick S, Chothia C. Outline structure of the human Ll cell adhesion molecule and the sites where mutations cause neurological disorders. EMBO J 1996 Nov 15;15(22):6050-9. {60) Holden HM, Ito M, Hartshorne DJ, Rayment I. X-ray structure determination of telokin, the (-terminal domain of myosin light chain kinase, at 2.8 A resolution. J Mol Biol 1992 Oct 5;227(3):840-51. (61) Huber AH, Wang YM, Bieber AJ, Bjorkman PJ. Crystal structure of tandem type Ill fibronectin domains from Drosophila neuroglian at 2.0 A. 1994 Apr;12(4):717-31. Jun;66(6):2338-49. {62) Ta keda Y, Asou H, Murakami Y, Miura M, Kobayashi M, Uyemura K. A nonneuronal isoform of cell adhesion molecule Ll: tissue-specific expression and functional analysis. J Neurochem 1996

(63) Debiec H, Christensen El, Ronco PM. The cell adhesion molecule L1 is developmentally regulated in the renal epithelium and is involved in kidney branching morphogenesis. J Cell Biol 1998 Dec 28;143(7):2067-79. (64) Kadmon G, Altevogt P. The cell adhesion molecule Ll: species- and cell-type-dependent multiple binding mechanisms. Differentiation1997 Feb;61(3):143-50. (65) Thor G, Probstmeier R, Schachner M. Characterization of the cell adhesion molecules Ll, N-CAM and J1 in the mouse intestine. EMBO J 1987 Sep;6(9):2581-6. (66) Kamiguchi H, Lemmon V. Neural cell adhesion molecule Ll: signaling pathways and growth cone motility. J Neurosci Res 1997 Jul 1;49(1):1-8. {67) Kenwrick S, Watkins A, De Angelis E. Neural cell recognition molecule Ll: relating biological complexity to human disease mutations. Hum Mol Genet 2000 Apr 12;9(6):879-86. (68) Doherty P, Walsh FS. CAM-FGF receptor interactions: a model for axonal growth. Mol Cell Neurosci 1996;8(2-3):99-111. (69) Kulahin N, Li S, Hinsby A, Kiselyov V, Berezin V, Bock E. Fibronectin type Ill (FN3) modules of the neuronal cell adhesion molecule Ll interact directly with the fibroblast growth factor (FGF) receptor. Mal Cell Neurosci 2008 Mar;37{3):528-36. (70) Kunz S, Ziegler U, Kunz B, Sonderegger P. Intracellular signaling is changed afterclustering of the neural cell adhesion molecules axonin-1 and NgCAM during neurite fasciculation.J Cell Biol 1996 Oct;135(1):253-67. 22 Introduction

(71) Schuch U, Lohse MJ, Schachner M. Neural cell adhesion molecules influence second messenger systems. Neuron 1989 Jul;3{1):13-20. (72) lgnelzi MA, Jr., Miller DR, Soriano P, Maness PF. Impaired neurite outgrowth of src-minus cerebellar neurons on the cell adhesion molecule Ll. Neuron 1994 Apr;12{4):873-84. (73) Zhang J, Williams MA, Rigamonti D. Genetics of human hydrocephalus. J Neural 2006 Oct;253{10):1255-66. (74) Jouet M, Kenwrick S. Gene analysis of Ll neural cell adhesion molecule in prenatal diagnosis of hydrocephalus. Lancet 1995 Jan 21;345(8943):161-2. {75) Saugier-Veber P, Martin C, Le Meur N, Lyonnet S, Munnich A, David A, Henocq A, Heron D, Jonveaux P, Odent S, Manouvrier S, Monda A, Morichon N, Philip N, Satge D, Tosi M, Frebourg T. Identification of novel L1CAM mutations using fluorescence-assisted mismatch analysis. Hum Mutat 1998;12(4):259-66.

23

Chapter 2

Genotype-phenotype correlationsin Ll syndrome: a guide for geneticcouns eling and mutationanalysis

b < d

Yvonne J. Vos•, Hermien E.K. de Walle•, r Krista K. Bos•,

Jenneke8 A. Stegeman•, Anneliesh M. ten Berge•, Martijn Bruining , Merel C. van Maarle , Mariet W. Elting , Nicolette S. den Hollander", Ben Hamel , Ana Maria

0Fortuna , Lone E.M. Sunde , Irene Stolte-Dijkstra•, Connie T. R.M. Schrander-Stumpel', Robert M.W. Hofstra•.

Department of Genetics, University Medical Centre Groningen, University of Groningen, Groningen, the Netherlands blnternalMedicine, Medical Centre Leeuwarden, Leeuwarden, the Netherlands

Journalof 2010; 47: 169-1 75 Chapter 2 Abstract

Objectives To develop a comprehensive mutation analysis system with a high rate of detection, to develop a tool to predict the chance of detecting a mutation in the L1CAM gene, and to look for genotype-phenotype correlations in the X-linked recessive disorder, Ll syndrome.

Methods DNA from 367 referred subjects was analyzed for mutations in the coding sequences of the gene. A subgroup of 100 patients was also investigated for mutations in regulatory sequences and for large duplications. Clinical data for 106 patientswere collected and used for statistical analysis.

Results 68 different mutations were detected in 73 patients. In patients with three or more clinical characteristics of Ll syndrome, the mutation detection rate was 66% compared with 16% in patients with fewer characteristics. The detection rate was 51% in families with more than one affected relative, and 18% in families with one affected male. A combination of these two factors resulted in an 85% detection rate (OR 10.4, 95% Cl 3.6 to 30.1). The type of mutation affectsthe severity of Ll syndrome. Children with a truncating mutation were more likely to die before the age of 3 than those with a missense mutation (52% vs 8%; p=0.02).

Conclusions We developed a comprehensive mutation detection system with a detection rate of almost 20% in unselected patients and up to 85% in a selected group. Using the patients' clinical characteristics and family history, clinicians can accurately predict the chance of finding a mutation. A genotype-phenotype correlation was confirmed. The occurrence of (maternal) germline mosaicism was proven.

26 A guide for genetic counseling and mutationanalysis Introduction

Ll syndrome is an X-linked recessive disease caused by mutations in the L1CAM gene. The phenotypic spectrum includes X-linked hydrocephalus, also referred to as hydrocephalus due to stenosis of the aqueduct of Sylvius (HSAS, MIM 307000), MASA syndrome (mental retardation, aphasia, shufflinggait and adducted thumbs; MIM 303350), X-linked complicated hereditary spastic paraplegia type 1 (SPGl, MIM 303350) and X-linked complicated corpus callosum agenesis (X-linked ACC; MIM 304100} 11•21 • The seriousness of the disease may vary from severe hydrocephalus and prenatal death (HSAS subtype) to a mild phenotype (MASA syndrome subtype). These variations may even occur in the same family l3Al. The L1CAM gene, coding for the neural Ll cell adhesion molecule (Ll), is located on the X chromosome. The gene consists of 29 exons, the first exon being non-coding. The protein of 1257 amino acids is a transmembrane glycoprotein belonging to the immunoglobulin (lg) superfamily cell adhesion molecules151 and contains, besides a signal peptide, 13 distinct domains - that is, six lg and five fibronectin Ill-like domains at the extracellular surface, one single-pass transmembrane domain and one short cytoplasmic domain. To our knowledge, 169 different L1CAM mutations have been published; 130 of these were catalogued in 2001111 and an additional 39 were published more recently16-221• Most L1CAM mutations are unique to each family - that is, they appear to be private mutations. Only a few families harbor the same recurrent mutation. L1CAM mutation analysis is offered to all patients suspected of having Ll syndrome. Once a mutation has been established, prenatal testing can be performed in subsequent pregnancies and carriership testing can be carried out to determine the potential presence of an L1CAM mutation in female relatives.Optimization of the previously reported L1CAM mutation detection system1231 has allowed us to investigate 367 L1 syndrome referrals. The analysis resulted in a mutation frequency of almost 20%. Although this is a good score, the question remains whether this relatively low frequency is due to the inclusion of large numbers of patients who do not have Ll syndrome or whether we have missed mutations and should extend our mutation screening procedure. To investigate this, we screened additional regulatory sequences of the L1CAM gene for mutations and looked for duplications and deletions. Furthermore, we evaluated the clinical data of a large set of patients to see if this could help in predicting the mutation status. Finally, we analyzed genotype-phenotype correlations with the severity of the disease.

Patients, materials and methods

Patients All patients (n=367), from various parts of the world, analyzed in our laboratory for diagnostic L1CAM testing, were included. If no DNA from the index patient was available (n=47), the DNA of the mother (n=38) was analyzed or that of a close family member, a sister (n=6) or aunt (n=3). Of these patients, 78% were referred by clinical geneticists, 12% by (paediatric) neurologists and 10% by paediatricians. To allow investigation of the mutation detection rate for specific subsets of patients, all referral clinicians were requested to complete a questionnaire.

27 Chapter 2 Exon A lntron A El 11 E2 E3 t t 800 bp promoter region HPD NRSE

Figure 1. S'site of the LlCAM gene with regulatory regions: promoter, homodomain and paired domain binding site (HPD) and neural restrictivesilencer element (NRSE). El, Exon 1; 11, lntron 1. Exon A is 125 bp. 800 bp of the promoter region were analyzed. Mutation analysis using Denaturing Gradient Gel Electrophoresis Mutation analysis of the LlCAM gene was performed, using Denaturing Gradient Gel Electrophoresis (DGGE) analysis followed by direct sequencing of fragments showing an aberrant DGGE pattern,essentially as previously described123l_ The amplicons 1, 7, 15, 16B, 18, 21 and 25 are routinely analyzed by direct sequencing using the same primers as developed for DGGE, but with an M13 tail instead of the GC-clamp.

Sequence analysis of regulatory regions Analysis of LlCAM regulatory regions-that is, about 1000 bp of the promoter region (including the non-coding first exon of the genel24l, called exon A), the neural restrictive silencer element (NRSE) and the homeodomain and paired domain binding site (HPD) (figure1) - was performed by direct sequencing of these regions. The NRSE, within intron 1 of the gene, restricts expression of Ll to the nervous systemf24l_ The HPD is a positiveregulatory element, which induces LlCAM gene expression125l . Four primers sets were designed for the promoter region (PromA-PromD), one for the HPD region and one for the NRSE region (table 1). PCR was performed in a total reaction volume of 50 µI containing 5 µI lOxPCR buffer (Amersham Biosciences, Roosendaal, the Netherlands), 0.17 µI rTaq polymerase (SO00U/ml, Amersham Biosciences), 20 pmol of each primer (Eurogentec, Serian, Belgium), 10 pmol dNTPs (Amersham Pharmacia Biotech, Piscataway, New York) and 100 ng DNA. In addition, 5 µI dimethyl sulphoxide was added for two PCRs (PromA and PromB). For fragments PromC and Promo, the PCRx Enhancer System (lnvitrogen Life Technologies, Breda, the Netherlands) was used with a final PCRx Enhancer Solution Concentration of 3x. A standard cycling protocol of 35 cycles was used with different annealing temperatures for each fragment (table 1). The PCR products were analyzed on a 2% agarose gel; the remainder were purifiedwith ExoSAP-IT (Amersham Pharmacia Biotech) and subjected to direct sequencing using the M13 primers.

Multiplex ligation-dependent probe amplification (MLPA} analysis MLPA was performed using DNA from 98 individuals (75 males and 23 females randomly chosen from the cohort of 295 subjects not harboring a mutation in the LlCAM gene). These were randomly chosen because we started this analysis before we had evaluated the clinical data. Probes for each exon were designed in our laboratory using the 'Designing syntheticMLP A probes' protocol (SOP-CUS-DE0l VS) from MRC-Holland. Twenty-four sets were judged to be suitable for use after testing and were divided over five mixes, each mix containing three control probes from the X chromosome. MLPA was carried out using the standard protocol from MRC-Holland (MLPA DNA Detection/Quantificationprotocol V27). All probe sequences are available upon request.

28 A guide for genetic counseling and mutation analysis

AT Table 1. Primer sets regulatory regions (°C) Length Productsize PromA_FPrimer CGCCTGTSequenceGAGGT CATG(5'>3')GCClTG 22 357 53 PromA_R GCCCAGTCAGGGTCCTGTTG (bp20 ) (bp) 53 CCCAACACGCTGAGGACGAA 20 291 61.2 CAAGGGAGCCCGCTGTGAAA 20 61.2 PromB_F AGGCCGAGGCTAAGGGAGCAT 21 345 49.4 PrPromB_RomC_R GGAGAGGGAGGGAGGAGTGAGAT 23 49.4 PromC_F TGGCACCAGGGGCTAGGGT 19 318 57 GGCAGAGCGGTGTGGTGTC 19 57 HPPromD_FD_F GGCCCTTTCCCCATTCTTTCTT 22 237 53 HPDPromD_R_R CTGTGGGGATGAATCCCTGTAT 22 53 NRSE_F GCGCCTA CClTCGCCGCTAT 20 331 50.9 NRSE_R GGATGGAAGCGGAGGAGTCG 20 50.9

All primers are designed with an M13 tail: (f)CGACGTTGTAAAACGACGGCCAGT and (r)CAGGAAACAGCTATG AC. AT, annealing temperature; HPD, homeodomain and paired domain binding site; NRSE, neural restrictivesilencer element; Prom, promoter. cDNA analysis of a potential RNA splicing mutation Total RNA was extracted from peripheral blood, using the RNABee procedure (Cinna Biotecx, Friendswood, Texas, USA) and cDNA was obtained using the Ready-To-Go You-Prime First­ Stand Beads for reverse transcriptionPCR with random hexamers primers pd(N)6 (Amersham Biosciences). Primers amplifying a product from exons 5 to 8 (f)CCAAGTGGCCAAAGGAGACAG and (r)ACTGCCCAGTGAGTTCTCG were used to characterize the cDNA sequence around the c.645C>T mutation in exon 6. The primers (f)CCAAGTGGCCAAAGGAGACAG and (r) TGATGGTGGGCGTGGGAAAG were used to verify the results obtained. The PCR products were loaded on to a 2% agarose gel, purified with ExoSAP-IT (Amersham Pharmacia Biotech) and subjected to direct sequencing using the primers described to confirm the presence of an aberrant transcript.

Statistical analysis A study was carried out to determine whether, and in what way, the mutation detection rate was influenced by (a) the number of age-independent clinical characteristics (table 2), (b) the number of affectedrelatives and (c) a combination of these factors. An analysis was also performed to detect any possible genotype-phenotype correlation,notably between the severity of the disease and truncating or missense mutations. The disease was defined as severe if a child died before the age of 3. For the statistical analysis, the x2 test was carried out, except in cases of small numbers when the Fisher exact test was used. A logistic regression analysis was performed to calculate the odds ratio (OR). The likely disease-causing mutations in these analyses are considered to be disease-causing.

29 Chapter 2

Ta ble 2. Characteristics of patients (n = 338) Selected group Remnant group (n = 106) (n = 232) Characteristics n % n %

> 1 affectedrelatives 35 33 60 26 Died at young age 29 27 63 27 Age-independent characteristics

Hydrocephalus 91 86 133 57 Aqueduct stenosis 46 43 15 6 Adducted thumbs 46 43 51 22 ACC/DCC 24 23 27 12 Age-dependent characteristios 10 Mental retardation 56 53 46 20 Aphasia 370 350 4 Spastic paraplegia 36 34 49 21 No clinical data available 53 23

Characteristics of the total group of 367 patientsminus the 29 patientstested for Ll syndrome exclusion. ACC/DCC, agenesis/dysgenesis of the corpus callosum. Results

Mutation analysis by DGGE In total, 367 subjects - that is, 320 index patientsand 47 relatives, in most cases the mother - were analyzed for the presence of a mutation in the L1CAM gene. With this technique a mutation was detected in 72 of the 367 subjects (tables 3 and 4). Sixty-seven different mutations were found: three mutations were detected in two families and one mutation in three different families. Fifty-two mutations have not been published before. Five mutations have previously been reported in co-operation with us !9,10•18•19•261 and 10 mutations were also found by others I2, 27-341 (table 3). The disease-causing relevance of seven of the 67 mutations remained unclear (table 4). In additionto the 67 mutations, we also detected six variants that were probably not disease-causing (table 5). None of the variants in tables 4 and 5 match known singlenucleotide polymorphisms (SNPs).

Sequence analysis of the regulatory regions and MLPA analysis We extended our sequence analysis to the L1CAM promoter and the HPD and NRSE regions and performed an MLPA analysis to determine whether additionalmutations were present in the regulatory sequences of the gene, or whether deletions or insertions could partly explain why the remaining 80% of patients did not have a detectable coding sequence mutation. No mutation was detected in the promoter region, the non-coding sequence of the first exon, the HPD region or the NRSE site in 100 of the 295 subjects (randomly chosen) of our cohort not harboring a mutation in the LlCAM gene. The regulatory sequences of the other 195 subjects have not been analyzed. MLPA analysis identifiedone subject with a duplication from exons 2-10. So, in total, a mutation was detected in 73 of the 367 subjects.

30 A guide for genetic counseling and mutation analysis

A B

M p Contr G Bl WT

- 418 bp - 367 bp ------deletion of Sl bp MT

Figure 2. (A) cDNA analysis of patient 329. Agarose gel electrophoresis of cDNA exon 6 of the patient (P} and a control sample (Contr). Wild-type exon 6 is 418bp long. G, genomic; Bl, blank; M, marker. (B) Sequence analysis of cDNA exon 6 of patient 329. WT, wild-type; MT, mutant. RNA splicing mutation and germline mosaicism Reverse transcription PCR was carried out to determine a possible splice effect of the silent mutationc.6 45C>T. Figure 2 clearly shows that this mutation influences RNA splicing. Sequence data confirm that a new splice donor site is introduced in exon 6, causing a deletion of 51 bp at the 3' site of the exon (figure 2). In this case, a maternal germline mosaicism was also demonstrated; the mother did not carry the mutation,but a second male did.

Patients The questionnaire, covering the family anamnesis and the seven most important clinical parameters of Ll syndrome, was filled out for 135 of the 367 patients. Twenty-nine of these 135 patients were referred in order to exclude the disease. Statisticaleva luation was therefore carried out on 106 patients, 31 of whom carried a mutation. The most important clinical data have been summarized in table 2. Known data for the remaining 232 patients have also been included in this table. It is clear that this group is less well characterized. Ta ble 3 contains clinical data for patients harboring an L1CAM mutation. As many patients die before birth or at a very young age, a distinction is made in characteristics that can be observed at birth or post mortem and those that can be observed only in later stages of life (table 2). We also looked at the co-occurrence of a hydrocephalus () and a macrocephaly (head circumference of > 2SD). In the selected group, out of 91 patients with hydrocephalus, 36 also had a macrocephaly, 25 of which needed shunting. In the event of a positive family history, there is always compatibility with X-linked recessive inheritance. However, in a few families comprising only a limited number of affected family members, autosomal recessive inheritance cannot be excluded.

Statistical analysis The clinical data from 106 patients, obtained via the questionnaire were used for statistical analysis as summarized in table 6. It can be seen that patients with three or more age­ independent clinical Ll syndrome characteristics had a significantly higher percentage of mutations (66%) than patients with fewer than three characteristics (16%). This is also true for families with more than one affected family member, 51% vs 18% for families with only one

31 w -- N 9 I Ii a Table 3. Disease-causing mutations in the LlCAM gene'I i 1� �ril" I! I\.J ��•! -- _J� j_ ____J ___ ---� #affected :I Family Exon/ ID Nucleotide change lntron Consequence Clinical data family Remarks Ref. members 312 c.2T>C exon 1 p.Metl? Unknown 1 145 c.76+1G>A intron 1 RNA splicing defect HC, AT, HP 2 152 c.78T>A exon 2 p.Tyr26X HC, AT 1 248 c.92-2A>G intron 2 RNA splicing defect HC, ? 3 292 c.110T>A exon 3 p.lle37Asn HC, ? 4 106 c.196C>T** exon 3 p.Gln66X HC, AT, MR, ACC, SP, HP 2 10 14 c.198-2A>T* intron 3 RNA splicing defect HC,? 3 32 251 c.325C>T exon 4 p.Gln109X HC, AT,MR , ACC 1 63 c.397G>T exon 4 p.Glu133X HC,? 1 de nova by mother 294 c.400+5G>C intron 4 .I RNA splicing defect HC, AT 1 196 c.414G>A exon 5 p.Trp138X HC, ? 1 89 c.536T>G* exon 6 I p.lle179 Ser HC, SP >1 31 56 c.550C>G exon 6 p.Arg184Gly HC, AT, MR, Aph, SP, HP 10 235 c.551G>A* exon 6 p.Arg184Gln HC? 1 30

52 c.559_594del36 exon 6 = p.Met187_ Val198del unknown 1 169 c.588_595del8 exon 6 p.Asn197SerfsX37 HC, AT, MR, Aph 5 de nova by mother 27 c.604G>T** exon 6 p.Asp202Tyr HC, AT, MR, ACC, Aph, SP 1 26 329 c.645C>T exon 6 p. Splice defect: 51 bp deleted HC, AT 2 mosaicism by mother 164 c.761C>A exon 7 p.Ala254Asp HC, ? 4 3 C. 778_ 785del8 exon7 p.Leu260GlyfsX45 HC, AT, MR, Aph, SP 2 348 c.826T>C exon 8 p.Trp276Arg HC, AT 1 de nova by mother 35 c.938T>C exon 8 p.Leu313Pro HC, AT, ACC 1 de nova 66 c.991+1G>A intron 8 RNA splicing defect HC, AT, ? 2 163 c.991+2T>C* intron 8 RNA splicing defect HC, ? 2 de nova by mother 32 187 c.1003T>C* exon 9 p.Trp335Arg unknown 1 34 182 c.1097G>A exon 9 p.Trp366X HC, DCC 2 de nova by mother 184 c.1107C>A exon 9 p.Asn369Lys HC, MR, Aph, DCC, SP, HP 3 91 c.1243G>C** exon 10 p.Ala415Pro HC, AT, MR, Aph, ACC, SP 4 9 29 c.1267C>T* exon 10 p.Gln423X HC 3 32 305 c.1267+1G>A* intron 10 RNA splicing defect HC, AT, MR, Aph, ACC, SP 1 27 342 c.1268-lG>A intron 10 RNA splicing defect HC, AT, ? 1 36 c.1438G>A exon 12 p.Gly480Arg HC, ? 2 108 c.1546+2T>A intron 12 RNA splicing defect HC, AT,MR , SP 3 I # affectea I 1; Family I Nucleotide Exon/ Consequence Clinical data family Remarks Ref. change I! lntron 11 I• -·------�J.._ ��- members ID

222 c.1547-2A>T intron 12 RNA splicing defect HC, AT, ? 3 de novo 208 c.1933A>T exon 15 p.lle645Phe unknown 1 12 c.1939+1G>A intron 15 RNA splicing defect HC, ? 1 149 c.1940-lG>A intron 15 RNA splicing defect HC 3 92 c.1986G>A exon 16 p.Trp662X HC, ? 2 23 c.2140C>T exon 17 p.Pro714Ser HC, MR, Aph, ACC, SP 1 254 c.2215delC exon 18 p.Arg739GlyfsX112 HC, AT, HP 1 80, 1. HC,AT,MR,Aph,ACC,SP,HP 7 28 156, c.2254G>A* exon 18 p.Val752Met 2. HC,? 4 336 3. HC, ? 2 275 c.2260T>A exon 18 p.Trp754Arg HC, ? 3 368 c.2267de1C** exon18 p.Pro7561eufs95X HC, AT, MR, SP, HSCR 1 19 227 c.2278C>T exon 18 p.Arg760X HC, AT, MR, Aph, SP 2 181 c.2344_2364dup exon 18 p.Val782_Val788dup AT, MR, SP 3 252 c.2365C>T exon 18 p.Gln789X HC, AT, ? 1 116 c.2433C>G exon 19 p.Tyr811X HC, 7 2 360 c.2673C>A exon 20 p.Tyr891X HC, AT, MR, Aph, ACC, SP 1 ):,. IQ 134 c.2701C>T* exon 20 p.Arg901X HC, AT 2 de novo 30 24, 1. HC, AT, DCC 1 by mother c.2885dupG exon 22 p.Lys963GlnfsX41 60 2. HC, ? 3 C)'.... IQ 295 c.2919delC exon 22 p.Glu974AsnfsX7 unknown 2 r1) :::3 74 c.2934_2935delCA exon 22 " p.His978GlnfsX25 HC, AT, ? 2 r1) 325 c.3166+2T>A intron 23 RNA splicing defect HC 2 �- 266 c.3191C>A exon 24 p.Ser1064X HC, AT, MR, SP, HP 7 0 :::3 119 c.3211_3213delAAC exon 24 p .Asn1071del HC, ? 3 V, 162 c.3239T>A exon 24 p.Leu1080Gln HC, AT, MR, SP 2 IQ 331 c.3531-lG>A intron 26 RNA splicing defect HC, ? 1 Cl 62, c.3531-12G>A* 1.HC, AT, ? 3 29 Cl.. intron 26 RNA splicing defect de novo 126 2. AT, MR 1 de novo 3 229 c.3772dupT exon 28 p.Xl257LeufsX96 MR, ACC, SP 1 by mother ------· ·- - - . 178 c.(?_77)_(1267+_ ?)dup exons 2-10 duplication HC, AT, MR 1 0!::I'. :::3 de novo Cl 146 c.(7_-145)_(*731_ ?}del** deletion entire gene HC, AT, MR, Aph, HP 1 18 :::3 w * Previously found by other groups. w ** Previously published with our co-operation. i;j· HC, hydrocephalus; AT, adducted thumbs; MR, mental retardation; SP, spastic paraplegia; Aph, aphasia; ACC/DCC, agenesis/dysgeneis of the corpus callosum; HP, hypotonia; HSCR, Hirschsprung's disease; ?, unknown. Chapter 2

Table 4. 1 Likely disease-causing mutations lr , # affected Family Exon/ Nucleotidechange Consequence Clinical data family Remarks ID intron . members 61, 1.HC, ? 9 c.76+5G>A intron 1 ? 148 2.HC, ? 2 366 c.516G>A exon 5 p.Met172lle HC, MR, SP 2 [c.523+9_523+10CG>GA; 245 intron 5 ? HC, AT, MR, SP 6 c.523+12d elC] HC, AT, MR, Aph, de nova by 200 c.1546G>A exon 12 p.Asp516Asn 1 SP, HP mother 302 c.1546G>T exon 12 p.Asp516Tyr unknown 1 95 c.1574G>A exon 13 p.Arg525His SP 5 320 c.1880C>T exon 15 p.Thr627Met� HC, ? 1 Ta ble 5. Likely non disease-causing mutations/polymorphism # affected Family Exon/ = Nucleotide change Consequence Clinical data family Remarks ID intron members 54 c.39C>A Exon 1 p. HC 1 HC, AT, MR, 1 34 c.113C>T Exon 3 p.Thr38Met 1 = Aph, SP 2 30 c.523+6T>G lntron 5 None HC, AT, MR 1 153 c.992-32C>T intron 8 ? unknown 1 174 c.1977T>C Exon 16 p. AT, MR, Aph, SP 1 3 136, l.AT, MR, SP 1 137, c.3458-34C>T lntron 25 None 2.HC 5 144 3. HC, AT, MR 1

Remarks; 1. Also found in an other patient with a nonsense mutation 2. No influence on RNA splicing affected family member. A combination of these two characteristics - that is, patients with a positive family history for Ll syndrome and having three or more clinical characteristics - resulted in a detection rate of 85%. The OR of finding a mutation in patients with three or more clinical characteristics was 10.1 (95% confidence interval (Cl) 3.8 to 27.1). This increased slightly to 10.4 {95% Cl 3.6 to 30. 1) when these patients also had two or more affected family members. Applying the same calculations to the whole group of 338 patients (367 minus the 29 who were only been analyzed to exclude the presence of Ll syndrome), some lacking relevant clinical information (table 2), the mutation detection rate was 18% in patients with fewer than three characteristics. In patients with three or more characteristics, but no affected relatives, this rate increased to 58%, with an OR of 6. 1 {95% Cl 3.0 to12.7). In the group of patients with three or more clinical characteristics and a positive family history, the detection rate increased to 79%, with an OR of 8.1 {95% Cl 3.5 tol8.6} as compared with 85% in the selected group. These results are logical, as the second group was less well defined (unselected) and therefore showed a lower detection rate, but larger and therefore showed a smaller Cl. Interrelationships, as mentioned above, can be seen when the data of the whole group are used (figure 3). The chance of having a mutation increased when patients have more clinical characteristics,and even more when at least two family members were affected. We also determined the positive predictive value of the individual clinical characteristics in

34 A guide for genetic counseling and mutation analysis

Ta ble 6. Mutation detection rate Mutation No mutation n 84% n No of age independent clinical characteristics <3 65 12 16 �3 10 34 19 66 p<0.001 No of affectedfa mily members 1 58 82 13 18 �2 17 49 18 51 <3p<0.001 age independent clinical characteristics No of affected family members 1 50 91 5 9 �2 15 68 7 32 � p=0.013 age independent clinical characteristics No of affected family members 1 8 50 8 50 �2 2 15 11 85 p=0.050,9 0,8 �

QJ 0,7 Figure 3. +-'

0,6 Mutation C 0 .• detection rate ·.p 0,5 / versus number of QJ _,.._2ormore +-' age-independent QJ 0,4 affected family "C clinical C / , .. 0,3 members 0 / , characteristics ·.p ,· I -·•- 1 affected family 0,2 , and number of -·· member affectedfamily 0,1 ,· - members. - _____ ...- .. ---

Number of clinical characteristics relation to the presence of a mutation (table 7), showing the most predictive to be adducted thumbs (50%) and the least predictive to be mental retardation (32%).

Genotype-phenotype correlation A statistical analysis was performed on 33 patients who were carrying a mutation to detect any possible genotype-phenotype correlation. Children harboring a truncating mutation were more likely to die before the age of 3 (52%) than children with a missense mutation (8%). This indicates a relationship between the seriousness of the disease and the type of mutation. These results are statistically significant (Fisher exact p=0.02) (table 8). No significant difference was detected in the occurrence of one of the other clinical characteristics (including macrocephaly) comparing patients carrying a missense mutation with patients carrying a truncating mutation (table 9).

35 Chapter 2

Ta ble 7. Frequency of the clinical characteristics in a group of patients carrying a mutation compared with a group without a mutation in the LlCAM gene and the positive predictive value Clinical Mutationnegative Mutationpositive p* characteristic Presence of the clinical PresenceYes of the clinical Positive characteristic characteristic predictive Yes No No value % % s % n n n % n % Hydrocephalus 61 86 10 14 30 100 0 0 0.03** 33 Aqueduct stenosis 28 68 13 32 18 78 22 0.56 ACC/DCC 14 45 17 55 10 91 1 9 0.01** 42 Mental retardation 38 73 14 27 18 100 0 0 0.01 ** 32so Aphasia 23 58 17 42 14 93 1 7 0.01 ** 38 Adducted thumbs 23 37 40 63 23 85 4 15 <0.001 ** Spastic paraplegia 20 45 24 55 16 84 3 16 0.005** 44

Patients are taken from the selected group. "Fisher exact test. · ·statistical significant. ACC/DCC, agenesis/dysgeneis of the corpus callosum.

Ta ble 8. Genotype-phenotype correlation Died before the age of 3 11 No Yes Missense mutation 1 %Nu mber 92 8 11 Truncating mutation 10 %Nu mber 48 52 p=0.02 truncating mutation compared with missense mutation Ta ble - 9. Occurrence of characteristics comparing missense and truncating mutations Clinical Missense mutation - --�- - T�-;;�ti;g mutation characteristic Presence of the Presence of the I ; CJ clinical characteristic J clinical characteristic 1 -""" '"""'- Y.es No ! --= NJ� p" '--"--'-- -.::.....;::....::.�- -_,______�-�- % % % % ns n n n Hydrocephalus 10 100 0 0 20 100 0 0 Macrocephaly 6 60 4 40 9 69 4 31 0.69 .. Aqueduct stenosis 56 4 44 13 93 1 7 0.12 .. ACC/DCC 6 100 0 0 4 80 1 20 0.46 .• Mental retardation 8 100 0 0 10 100 0 0 11 Aphasia 7 78 2 22 7 100 0 0 0.48 .. Adducted thumbs 8 73 3 27 15 88 2 12 0.35•• Spastic paraplegia 8 89 1 8 73 3 27 0.59 ..

* Fisher exact test ** Statistical not significant

36 A guide fo r genetic counseling and mutation analysis

Discussion

Mutation analysis was carried out on 367 referred cases (that is 320 index patients and 47 relatives).Seventy-two patients were shown to harbor 67 different mutations. One additional mutation was found with the MLPA test, making a total of 73 patients with a disease­ causing mutation.The mutations comprise 23 missense mutations,three in-frame deletions/ duplications, 18 splice site mutations, 14 nonsense mutations, eight frame-shift mutations, one duplication of exons 2-10, and one deletion of the entire gene. This implies that about 60% of all mutations are truncating and therefore considered to be disease-causing. This is less straightforward for the 23 missense mutations. However, the identified missense mutations are mostly considered to be disease-causing, as they often occur in the so-called key residues1351 that are important for the structure of the lg domains and the fibronectin Ill-like domains of Ll protein. Basically, these conclusions relate to the findings of Holden et a/.1361, who determined the three-dimensional structure of telokin, a protein with a specific domain similar to the Ll lg domain containing 35 key residues responsible for its structure. The structure of the fibronectin lll-like3 domain and its key residues can likewise be derived from similarities to those of neuroglian1371 • Other criteria are whether or not the mutation affects a highly conserved amino acid, is de nova in the index patient, or is only found in affected males and not in unaffected male family members. Fifteen of the 23 ("'65%) disease-causing missense mutationswere found to be related to key residues, which is comparable to data reported by Michaelis et a/.1381 who detected 52 out of "' 9 78 ( 67%). In contrast with the findings of Michaelis et al. and those of Kamiguchi et a/.13 1, we found no differencein the severity of the hydrocephalus in patientswith a missense mutation 35 in key residues versus surface residues 1 1 • In our group changes in a key residue led to severe hydrocephalus in 57% of the patientsand surface changes in 50% of the patients, compared with 67% and 42%, respectively, found by Michaelis et a/.1381 and 78% and 28% determined by Kamiguchi et a/.1391, indicatingno correlation between the severity of the hydrocephalus and the locationof the missense mutation in either the key residues or the surface residues. In five of the 73 patients carrying a mutation( "'7%), the mutation was shown to be de nova or a maternal germ cell mosaicism. The latter was confirmed in one case (c.645C>T), as a second male fetus was found to have the same mutation.Germline mosaicism of L1CAM mutations 2 40 had previously been reported1 7• 1 • Du et a/.1271 described an unaffectedmale with a somatic and germline mosaicism of an L1CAM mutation and his two daughters carrying the mutation. The mutation c.645C>T proved to be a special silent mutation that influences the RNA splicing process: a splice donor site arises within exon 6 of the gene. As a consequence, 51 bp or 17 amino acids are deleted. These deleted amino acids are important for the structure of the lg2 domain and therefore this mutation is considered to be disease-causing. When no DNA material is available from the index patientthe diagnosis has to be carried out on a close relative, usually the mother. In this study, this was the case in 38 of the 367 patients. In 25 of these 38 mothers, no mutation was detected. Testingthe mother has problems because we have shown that 7% of the mutationsdetected are de nova or a germline mosaicism. Another striking finding is the very high ("'10%) occurrence of de nova mutations in mothers or a germ cell mosaicism in one of the grandparents of index patients. This and the 7% de nova mutations in the index patients means that at least one-fifth of L1 syndrome families have evolved rather recently.

37 Chapter 2 As a mutation in the UCAM-encoding DNA sequence was found in 20% of referred cases, the mutation analysis was broadened by analyzing 100 patients for potential mutations in the regulatory sequences of the LlCAM gene. This generated no extra mutations, leading to a potential detection rate in these regions of less than 1%. Screening for mutations in the 3' region was not performed because of this finding and also because too little is known about the function of specific sequences of this region. An MLPA test was used to investigate the same 100 patientsfor large duplicationsin the L1CAM gene. Only one duplication, exons 2-10, was detected, suggesting a very low prevalence of this type of mutation. The same seems to be true for large deletions, since, in 73 mutation-carrying patients, only one showed a deletion of the entire gene1181, which is in accordance with previously published results. To date, 169 different L1CAM mutations have been published: 164 small point mutations and five large deletions or duplicationsincluding one deletion of the entire gene1181, one deletion of the promoter region and exon 11171, one deletion of 2 kb at the distal part of the gene c.3543_?del2kb1411, one deletion of exons 2-5 and part of exon 61161 and one duplication of 1.3kb c.3543_?dup1.3kb1421• From these results, it can be concluded that broadening the DNA­ screening procedure to include regulatory sequences analysis is not worthwhile. Screening for deletions and duplications may be beneficial, especially in clear clinical cases, although this does not seem to greatly influence the detectionrate. In contrast with this, the combination of clinical data and the number of affected relatives, obtained via a questionnaire, shows a clear correlation with the mutation detection rate as shown in figure 3. Our findings are therefore of importance for clinicians and genetic counselors, since they may be used to predict the chance of finding a mutation. Besides, the positive predictive value showing adducted thumbs to be the most predictive may also be quite useful. Finally, it should be noted that a genotype-phenotype correlation has been identified for the LlCAM gene and its associated syndrome in our group of patients.As previously reported143A4l we also found that children with a truncating mutation were more likely to die before the age of 3, than children with a missense mutation.This illustrates that the type of mutationhas an important effect on the severity of the Ll syndrome.

Conclusions

Our study shows that the mutation detection system is efficient for detecting mutations. Using this system, we have clearly shown a correlation between the number of clinical characteristics, the number of affected family members and the chance of finding a mutation. Moreover, we found a genotype-phenotype correlationfor L1 syndrome. This study provides a comprehensive tool for genetic counseling.

Acknowledgements We thank all clinicians for filling out the questionnaires and sending us materials for DNA analysis (notably Gert Matthijs), Annet Vrieling-Kooistra for technical assistance, and Margaret Burton for editingthe manuscript.

38 A guide for genetic counseling and mutationanalysis References

(1) Weller S, Gartner J. Genetic and clinical aspects of X-linked hydrocephalus (Ll disease): Mutations in the LlCAM gene. Hum Mutat 2001;18(1):1-12. (2) Fransen E, Van CG, Vits L, Willems PJ. Ll-associated diseases: clinical geneticists divide, molecular geneticists unite. Hum Mal Genet 1997;6(10):1625-32. (3) Schrander-Stumpel C, Legius E, Fryns JP, Cassiman JJ. MASA syndrome: new clinical features and linkage analysis using DNA probes. J Med Genet 1990 Nov;27(11):688-92. (4) Fryns JP, Spaepen A, Cassiman JJ, van den Berghe H. X linked complicated spastic paraplegia, MASA syndrome, and X linked hydrocephalus owing to congenital stenosis of the aqueduct of Sylvius: variable expression of the same mutation at Xq28. J Med Genet 1991 Jun;28(6):429-31. (5) Moos M, Tacke R, Scherer H, Teplow D, Fruh K, Schachner M. Neural adhesion molecule Ll as a member of the immunoglobulin superfamily with binding domains similar to fibronectin. Nature 1988 Aug 25;334(6184):701-3. (6) De Angelis E, Watkins A, Schafer M, Brummendorf T, Kenwrick S. Disease-associated mutations in Ll CAM interfere with ligand interactions and cell-surface expression. Hum Mal Genet 2002 Jan 1;11(1):1- 12. (7) Simonati A, Boaretto F, Vettori A, Dabrilli P, Criscuolo L, Rizzuto N, Mostacciuolo ML. A novel missense mutation in the LlCAM gene in a boy with Ll disease. Neural Sci 2006 Jun;27(2):114-7. (8) Senat MV, Bernard JP, Delezoide A, Saugier-Veber P, Hillian Y, Roume J, Ville Y. Prenatal diagnosis of hydrocephalus-stenosis of the aqueduct of Sylvius by ultrasound in the first trimester of pregnancy. Report of two cases. Prenat Diagn 2001 Dec;21(13):1129-32. (9) Sztriha L, Vos YJ, Verlind E, Johansen J, Berg B. X-linked hydrocephalus: a novel missense mutation in the LlCAM gene. Pediatr Neurol 2002 Oct;27(4):293-6. (10) Rodriguez CG, Perez AA, Martinez F,Vos YJ, Verlind E, Gonzalez-Meneses LA, Gomez de TS, I, Schrander-Stumpel C. X-linked hydrocephalus: another two familieswith an Ll mutation. Genet Couns 2003;14(1):57-65. (11) Silan F, Ozdemir I, Lissens W. A novel LlCAM mutation with Ll spectrum disorders. Prenat Diagn 2005 Jan;25(1):57-9. (12) Bott L, Baute 0, Mention K, Vinchon M, Boman F, Gottrand F. Congenital idiopathic intestinal pseudo­ obstruction and hydrocephalus with stenosis of the aqueduct of sylvius. Am J Med Genet A 2004 Sep 15;130(1):84-7. (13) Okamoto N, Del MR, Valero R, Monros E, Pao P, Kanemura Y, Yamasaki M. Hydrocephalus and Hirschsprung's disease with a mutation of LlCAM. J Hum Genet 2004;49(6):334-7. (14) Hubner CA, Utermann B, Tinschert S, Kruger G, Ressler B, Steglich C, Schinzel A, Gal A. lntronic mutations in the LlCAM gene may cause X-linked hydrocephalus by aberrant splicing. Hum Mutat 2004 May;23(5):526. (15) Moya GE, Michaelis RC, Holloway LW, Sanchez JM. Prenatal diagnosis of Ll cell adhesion molecule mutations. Capabilities and limitations. Fetal Diagn Ther 2002 Mar;17(2):115-9. (16) Panayi M, Gokhale D, Mansour S, Elles R. Prenatal diagnosis in a family with X-linked hydrocephalus. Prenat Diagn 2005 Oct;25(10):930-3. (17) Tegay DH, Lane AH, Roohi J, Hatchwell E. Contiguous gene deletion involving LlCAM and AVPR2 causes X-linked hydrocephalus with nephrogenic diabetes insipidus. Am J Med Genet A 2007 Mar 15;143(6):594-8. (18) Knops NB, Bos KK, Kerstjens M, van Dael K, Vos YJ. Nephrogenic diabetes insipidus in a patient with L1 syndrome: a new report of a contiguous gene deletion syndrome including LlCAM and AVPR2. Am J Med Genet A 2008 Jul 15;146A(14):1853-8. (19) Griseri P, Vos Y, Giorda R, Gimelli S, Beri S, Santamaria G, Mognato G, Hofstra RM, Gimelli G, Ceccherini I. Complex pathogenesis of Hirschsprung's disease in a patient with hydrocephalus, vesico-ureteral reflux and a balanced translocation t(3;17)(p12;q11). Eur J Hum Genet 2009 Apr;17(4):483-90. (20) Nakakimura S, Sasaki F, Okada T, Arisue A, Cho K, Yoshino M, Kanemura Y, Yamasaki M, Toda S. Hirschsprung's disease, acrocallosal syndrome, and congenital hydrocephalus: report of 2 patients and literature review. J Pediatr Surg 2008 May;43(5):E13-E17. (21) Wilson PL, Kartman BB, Mulvihill JJ, Li S, Wilkins J, Wagner AF, Goodman JR. Prenatal identification of a novel R937P LlCAM missense mutation. Genet Test Mal Biomarkers 2009 Aug;13(4):515-9. (22) Kanemura Y, Okamoto N, Sakamoto H, Shofuda T, Kamiguchi H, Yamasaki M. Molecular mechanisms

39 Chapter 2

and neuroimaging criteria for severe Ll syndrome with X-linked hydrocephalus. J Neurosurg 2006 Nov;105(5 Suppl):403-12. (23) Hofstra RM, Elfferich P, Osinga J, Verlind E, Fransen E, Lopez Pison J, Die-Smulders CE, Stolte-Dijkstra I, Buys CH. Hirschsprung disease and LlCAM: is the disturbed sex ratio caused by LlCAM mutations? J Med Genet 2002 Mar;39(3):Ell. (24) Kallunki P, Edelman GM, Jones FS. Tissue-specific expression of the Ll cell adhesion molecule is modulated by the neural restrictive silencer element. J Cell Biol 1997 Sep 22;138(6):1343-54. (25) Meech R, Kallunki P, Edelman GM, Jones FS. A binding site for homeodomain and Pax proteins is necessary for Ll cell adhesion molecule gene expression by Pax-6 and morphogenetic proteins. Proc Natl Acad Sci US A 1999 Mar 2;96(5):2420-5. (26) Sztriha L, Frossard P, Hofstra RM, Verlind E, Nork M. Novel missense mutation in the Ll gene in a child with corpus callosum agenesis, retardation,adducted thumbs, spastic paraparesis, and hydrocephalus. J Child Neural 2000 Apr;15(4):239-43. (27) Du JS, Bason L, Woffendin H, Zackai E, Kenwrick S. Somatic and germ line mosaicism and mutation origin for a mutation in the Ll gene in a familywith X-linked hydrocephalus. Am J Med Genet 1998 Jan 13;75(2):200-2. (28) Gu SM, Orth U, Zankl M, Schroder J, Gal A. Molecular analysis of the LlCAM gene in patients with X-linked hydrocephalus demonstrates eight novel mutations and suggests non-allelic heterogeneity of the trait. Am J Med Genet 1997 Aug 22;71(3):336-40. (29) Jouet M, Mancia A, Paterson J, McKeown C, Fryer A, Carpenter N, Holmberg E, Wadelius C, Kenwrick S. New domains of neural cell-adhesion molecule Ll implicated in X-linked hydrocephalus and MASA syndrome. Am J Hum Genet 1995 Jun;56(6):1304-14. (30) MacFarlane JR, Du JS, Pepys ME, Ramsden S, Donnai D, Charlton R, Garrett C, Tolmie J, Yates JR, Berry C, Goudie D, Mancia A, Lunt P, Hodgson S, Jouet M, Kenwrick S. Nine novel Ll CAM mutations in families with X-linked hydrocephalus. Hum Mutat 1997;9(6):512-8. (31) Ruiz JC, Cuppens H, Legius E, FrynsJP , Glover T, Marynen P, Cassiman JJ. Mutations in Ll-CAM in two families with X linked complicated spastic paraplegia, MASA syndrome, and HSAS. J Med Genet 1995 Jul;32(7):549-52. (32) Finckh U, Schroder J, Ressler B, Veske A, Gal A. Spectrum and detection rate of LlCAM mutations in isolated and familial cases with clinically suspected Ll-disease. Am J Med Genet 2000 May 1;92(1):40-6. (33) Fransen E, Vits L, Van CG, Willems PJ. The clinical spectrum of mutations in Ll, a neuronal cell adhesion molecule. Am J Med Genet 1996 Jul 12;64(1):73-7. (34) Saugier-Veber P, Martin C, Le Meur N, Lyonnet S, Munnich A, David A, Henocq A, Heron D, Jonveaux P, Odent S, Manouvrier S, Monda A, Morichon N, Philip N, Satge D, Tosi M, Frebourg T. Identification of novel LlCAM mutations using fluorescence-assistedmismatch analysis. Hum Mutat 1998;12(4):259-66. (35) Bateman A, Jou et M, MacFarlane J, Du JS, Kenwrick S, Chothia C. Outline structure of the human L1 cell adhesion molecule and the sites where mutations cause neurological disorders. EMBO J 1996 Nov 15;15(22):6050-9. (36) Holden HM, Ito M, Hartshorne DJ, Rayment I. X-ray structure determination of telokin, the (-terminal domain of myosin light chain kinase, at 2.8 A resolution. J Mal Biol 1992 Oct 5;227(3):840-51. (37) Huber AH, Wang YM, Bieber AJ, Bjorkman PJ . Crystal structure of tandem type Ill fibronectin domains from Drosophila neuroglian at 2.0 A. Neuron 1994 Apr;12(4):717-31. (38) Michaelis RC, Du YZ, Schwartz CE. The site of a missense mutation in the extracellular lg or FN domains of L1CAM influences infant mortality and the severity of X linked hydrocephalus. J Med Genet 1998 Nov;35(11):901-4. (39) Kamiguchi H, Hlavin ML, Yamasaki M, Lemmon V. Adhesion molecules and inherited diseases of the human nervous system. Annu Rev Neurosci 1998;21:97-125. (40) Jouet M, Strain L, Bonthron D, Kenwrick S. Discordant segregation of Xq28 markers and a mutation in the Ll gene in a family with X linked hydrocephalus. J Med Genet 1996 Mar;33(3):248-50. (41) Vits L, Van Camp G, Coucke P, Fransen E, De Bou lie K, Reyniers E, Korn B, Poustka A, Wilson G, Schrander­ Stumpel C, . MASA syndrome is due to mutationsin the neural cell adhesion gene L1CAM. Nat Genet 1994 Jul;7(3):408-13. (42) Van Camp G, Vits L, Coucke P, Lyonnet S, Schrander-Stumpel C, Darby J, Holden J, Munnich A, Willems PJ. A duplication in the L1CAM gene associated with X-linked hydrocephalus. Nat Genet 1993 Aug;4(4):421-5. (43) Yamasaki M, Thompson P, Lemmon V. CRASH syndrome: mutations in L1CAM correlate with severity of the disease. Neuropediatrics 1997 Jun;28(3):175-8. (44) Fransen E, Van Camp G, D'Hooge R, Vits L, Willems PJ. Genotype-phenotype correlation in L1 associated 40 diseases. J Med Genet 1998 May;35(5):399-404. Appendix chapter 2 AdditionalDNA testing results

Ll syndrome in females

a b

c d a Yvonne J. Vos , Ulrike Dunkhase-Heinl , Gretel G. Oudesluijs , Carsten A. Brandt , Annelies M. ten Berge", Krista K. van Dijk-Bos , Gunnar Houge•, Robert

0M.W Hofstra•

h Department of Genetics, University Medical Centre

Groningen,c University of Groningen, the Netherlands Department of , Hospital Li/lebaelt-Kolding, dDenmark Department of Clinical Genetics, Erasmus University Medical Centre, Rotterdam, the Netherlands Department of Clinical Genetics, Hospital Lillebaelt­ Vejle, Denmark " Center for Medical Genetics and Molecular Medicine, Hauke/and University Hospital, Heise Bergen, Norway

Manuscript in preparation Appendix chapter 2 Introduction

So far, female carriers of an L1CAM mutation have been described in the literature as being sometimes mildly affected, with slightly adducted thumbs and a cognitive level significantly lower than their non-carrier sister(s). Girls with severe hydrocephalus are very rarely seen. In fact, almost everyone refers to the scientific work from 1994 by Kaepernick et a/.l11• They reported a large family with eight, more or less affected women: two showing adducted thumbs, three with mild mental retardation (two of these women were obligate carriers), and three who reportedly died because of a neonatal hydrocephalus. This diagnosis was confirmed by autopsy in one case. Thus, based on the current literature, Ll syndrome in females is believed to be very rare. This has important consequences, the major one being that in prenatal diagnostics, female carrying a mutationare considered to be healthy.

Results

Recently a girl with aqueduct stenosis, adducted thumbs and mild mental retardation was referred to our laboratory for diagnostic L1CAM testing. We detected a pathogenic mutation, c.1267C>T, p.Gln423X and were able to demonstrate skewed X-inactivation in the girl. To the best of our knowledge this is the first time that a pathogenic L1CAM mutation has been detected in an affected girl without a positive family history. In a second girl, suffering from severe hydrocephalus and down syndrome, we detected the likely disease-causing mutation c.1156C>T, p.Arg386Cys. This had been detected earlier in her brother, who also suffered from severe hydrocephalus. We were also able to demonstrate skewed X-inactivationin this girl. Furthermore, a maternal germline mosaicism was shown, as the mother did not carry the mutation. In a third girl, with spasticparaplegia, severe learning difficulties and epilepsy, we found the same UV as had been detected in her two affectedbrothers (hydrocephalus, mental retardation and spastic paraplegia): c.516G>A, p.Met17211e. In this case skewed X-inactivation could not be demonstrated.

Conclusion

Our results clearly show that female carriers do have a risk of developing Ll syndrome and this therefore has important consequences for diagnostic testing for Ll syndrome. Although we cannot yet give an accurate risk estimation of how high the chance is that a mutation carrier will be clinically affected (a figure that is important for prenatal diagnostics), it will probably be around 3-5% since we have only detected mutations in 78 patients to date. We plan to investigate the chance of having an affected girl in a family harboring a proven pathogenic mutation more precisely and we will extend our diagnostic testingfor L1CAM in girls suspected of having Ll syndrome.

42 L1 syndrome in females Reference

(1) Kaepernick L, Legius E, Higgins J, Kapur S. Clinical aspects of the MASA syndrome in a large family, including expressing females. Clin Genet 1994 Apr;45(4):181-5.

43

Chapter 3

X-linked hydrocephalus: a novel missense mutation in the L1CAM gene

b b

C d

Laszlo Sztriha•, Yvonne J. Vos , Edwin Verlind , 0 Johan Johansen , and Bertel Berg

Department of Pediatrics, Faculty of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates bDepartment of Genetics, University Medical Centre Groningen, University of Groningen, Groningen, the Netherlands 'Department of Radiology, Fa culty of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates d0epartment ofPat hology, Faculty ofMedicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates

Pediatric 2002; 27: 293-296 Chapter 3 Abstract

X-linked hydrocephalus is associated with mutations in the Ll neuronal cell adhesion molecule gene. L1 protein plays a key role in neurite outgrowth, axonal guidance, and pathfinding during the development of the nervous system. A male is described with X-linked hydrocephalus who had multiple small gyri, hypoplasia of the white matter, agenesis of the corpus callosum, and lack of cleavage of the thalami. Scanning the L1 neuronal cell adhesion molecule gene in Xq28, revealed a novel missense mutation: transition of a guanine to cytosine at position 1,243, which led to conversion of alanine to praline at position 415 in the lg4 domain of the Ll protein. It is likely that the X-linked hydrocephalus and cerebral dysgenesis are a result of the abnormal structure and function of the mutant Ll protein.

46 X-linked hydrocephalus Introduction

Cell adhesion molecules are cell surface proteins, which mediate differential adhesion, signal transduction, and physical/mechanical effectsr11• The following three major classes of cell adhesion molecules have been identified in the developing central nervous system: the integrins, the cadherins, and the immunoglobulin superfamilyf11• Ll belongs to the immunoglobulin superfamilyrz,3J_ Its encoding gene, L1CAM, is located near the telomere of the long arm of the X chromosome in the Xq28 regionf41• Mutations in the L1CAM gene are responsible for an X-linked recessive neurologic disorder that has been described as X-linked hydrocephalus (hydrocephalus as a result of stenosis of the aqueduct of Sylvius [HSAS]; MIM 307000), MASA syndrome (mental retardation, aphasia, shuffling gait, and adducted thumbs; MIM 303350), X-linked corpus callosum agenesis (ACC; MIM 304100), and spastic paraplegia type 1 (SPGl; MIM 303350)141• The varying nomenclature is a reflection of extremely variable presentation within and between families. The most consistent features in affected boys are hydrocephalus/ventriculomegaly with or without macrocephaly, abnormal gyrification, reduced white matter volume, lack of cleavage of the thalami, hypoplasia or agenesis of the corpus callosum, hypoplasia or absence of the corticospinal tract, lower limb , underdevelopment of the cerebellar vermis, mental retardation, and adducted thumbsrs-si. A single acronym, CRASH (corpus callosum hypoplasia, retardation, adducted thumbs, spastic paraplegia, hydrocephalus), was proposed for the syndrome in 1995r4l, and Ll disease has been suggested recently191. Few reports have been published on the neuroimaging and neuropathologic features of patients with L1CAM mutations15-81• We describe the magnetic resonance imaging characteristics of the cerebral dysgenesis in a male with X-linked hydrocephalus who had a missense mutation in the L1CAM gene.

Case report The proband was a male who was born at term by cesarean section to consanguineous (first cousin) Pakistani parents. His birth weight was 4,770 gm, and Apgar scores were 6 and 8 at 1 and 5 minutes, respectively. He had congenital hydrocephalus with an occipitofrontal circumference of 48.5 cm at birth. Computed tomography revealed enlarged lateral and third ventricles; the size of the fourth ventricle was normal. A ventriculoperitoneal shunt was placed shortly after birth. The mother had nine pregnancies. Of these pregnancies, three males with congenital hydrocephalus died in the neonatal period, and one female, who did not have hydrocephalus or any dysmorphic features, died during labor. Four females are healthy (figure 1).

Figure 1. The pedigree of the family.

47 Chapter 3 Axial Tl­ Parasagittal weighted (TR = 736 Tl-weighted (TR = ms, TE = 14 ms) 630 ms, TE = 15 ms) magnFigureetic 2. resonance Figuremagne tic3. reso nance image reveals multiple image reveals multiple small gyri in the small gyri mainly in occipital regions. There the parietal-occipital is a lack of cleavage areas. of the thalami in the midline, and the corpus callosum is absent.

Development of the patient was delayed. Mental retardation became more and more obvious (intelligence quotientwas approximately SO at 5 years of age). He could follow a few commands and recognize his parents but was unable to speak. On neurologic examination at 4.5 years of age his head circumference was 49 cm (twenty-fifthpercentile). Ophthalmoscopy examination revealed normal results. He had strabismus. Both thumbs were adducted at the metacarpophalangeal joints and flexed at the interphalangeal joints. He was not able to move his thumbs. Muscle tone, power, and deep tendon reflexes were normal on the upper extremities. He was able to feed himself. Severe spastic diplegia with equinovarus deformity of the feet, brisk deep tendon reflexes, and positiveBabinski reflex were observed on the lower extremities. He was not able to stand or walk unsupported. Sensation appeared normal with pain stimulus; other modalitieswere not assessed because of the mental retardation. Brain magnetic resonance imaging was performed when the child was 4.5 years of age. Sagittal

(TR = 630 ms, TE = 15 ms) and axial T1-weighted (TR = 736 ms, TE = 14 ms), coronal T1-weighted

(TR = 736 ms, TE = 14 ms) and T2-weighted (TR = 6,900 ms, TE = 90 ms), axial proton density (TR = 5,000 ms, TE= 19 ms) and T2-weighted images (TR = 5,000 ms, TE = 95 ms) were obtained. Parietal­ occipital hypoplasia with reduced white matter volume, multiplesmall gyri, and thin cortex was observed (figure 2 and figure 3). There was an unusual arborization of the cerebral mantle with widened deep sulci between fingerlike bunches of white mattercovered with gray matter in these regions. The T2-weighted images did not reveal high signal intensity in the white matter. Some of the gyri on the medial surfaces of the hemispheres had a radiating pattern. The posterior aspect of the lateral ventricles had irregular walls. The corpus callosum was absent, and the fornix was thick. The thalami lacked cleavage in the midline and the third ventricle was practically filled by the large massa intermedia (figure 2). A patent aqueduct and the superior and inferior colliculi could not be identified. The fourth ventricle was small and had a relatively high location. The cerebellum demonstrated normal structure, although it had a towering high extension of its vermis. The posterior fossa was relativelysmall. The cerebral peduncles and brainstem appeared normal. There was a small pituitary gland within a correspondingly small sella turcica. Because the clinical findingswere highly suggestiveof X-linked hydrocephalus, mutationanalysis of the LlCAM gene was performed as described previously18•101• Sequencing revealed a transition of a G (guanine) to C (cytosine) at position 1,243 (c.1243G>C}. This is a missense mutation resulting in an amino acid change of alanine to praline on position 415 (p.Ala415Pro) in the fourth immunoglobulin domain {lg4) of the Ll molecule. The same mutationwas evident in the mother of the male.

48 ------

X-linked hydrocephalus Discussion

Multiple small gyri, markedly reduced white matter volume, agenesis of the corpus callosum, and lack of cleavage of the thalami were the main magneticresonance imaging findings afterthe shunt procedure in this patient with X-linked hydrocephalus as a result of a missense mutationin the LlCAM gene. A patent aqueduct was not identified. These features demonstrate similarities to the previously published magnetic resonance imaging and neuropathologic findings [5-11• Diffuse hypoplasia of the cerebral white matter associated with ventriculomegaly, hypoplasia/ agenesis of the corpus callosum, and lack of cleavage of the thalami were common findingsin all patients.Neuropathology revealed multiplesmall gyri (polymicrogyria) similar to those described in this study [61• There were six cortical layers with sparse cells and poorly organized lamination [61• In addition, neuropathology and diffusion-weightedmagnetic resonance imaging demonstrated that the corticospinaltracts were hypo plastic or absent[6•71• The cerebral peduncles and brainstem appeared normal in our magnetic resonance imaging study; however, they were demonstrated 5 1 to be hypoplastic in other studies [ , 1, a finding that is consistent with the hypoplasia of the corticospinaltracts. The aqueduct of Sylvius was previously described as patent with or without 5 stenosis[ -11, and therefore the pathogenesis of the severe progressive hydrocephalus remained poorly understood. The outcome was poor in X-linked hydrocephalus because of the widespread cerebral abnormalitiesl5-71_ The cerebral dysgenesis, found by neuroradiologic and neuropathologic techniques among the various clinical presentations of LlCAM gene mutations or CRASH syndrome, is the result of disrupted Ll structure and function[4,111• Information processing performed by the brain is determined by an intricate network of connections between neurons [121• Neuronal connections form during embryonic development when each differentiatingneuron sends out an axon that projects through the embryonic environment to its synaptic targets. Pathfinding is directed by the coordinate action of short- and long-range attractionand short- and long-range repulsion[121• A large number of molecules have been identified that mediate the guidance mechanisms1121• Ll, a member of the immunoglobulin superfamily is the best studied among the cell adhesion molecules participatingin the development of the nervous system[2,3,121. Ll is a transmembrane glycoprotein with six immunoglobulin-like and five fibronectin type Ill domains on the extracellular surface[2·31• The extracellular part is linked to a short cytoplasmic tail by a transmembrane domain. Both the extracellular and cytoplasmic domains of the Ll molecule are involved in several interactions[31• The extracellular interactions include Ll itself (Ll-Ll homophilic binding) or other members of the lg superfamily, integrins, extracellular matrix proteins, such as laminin, and a variety of proteoglycans (heterophilic binding) [3,131, There is growing evidence that the homophilic interactions promote both neurite outgrowth and cell adhesion, which are relevant to both axon growth and fasciculation (axon bundling) [3,121• Extracellular Ll ligands are also linked to intracellular signaling pathways, and it is likely that the axonal growth is initiatedthrough the activation of tyrosine kinase-associated receptors of fibroblast growth factors13•121• The complexes formed by heterophilic interactions may also be required for the Ll-Ll-mediated regulation of neurite outgrowth131• The cytoplasmic domain interacts with the cytoskeleton. It has a binding site for ankyrin, and ankyrin-mediated tethering of Ll to the spectrin cytoskeleton can stabilize L1 within the axon[141• The LlCAM gene spans 16 kb and has 28 coding exons. Exons 3-14 encode the six immunoglobulin domains with two exons for each domain. Exons 15-24 encode the five fibronectin domains,

49 Chapter 3 exon 25 the transmembrane domain, and exons 26-28 form the cytoplasmatic domainl41. As of January 2001, 137 different L1CAM mutations have been described in 146 families (http:// dnalab-www.uia.ac.be/dnalab/Ll/index.html)l15l_ The mutations are dispersed throughout the entire L1CAM gene and include missense, nonsense, splice site mutations, deletions, and insertionsl91. The mutations can be classified into three classesr111. The first class of mutations includes mutations that affect only the cytoplasmic domain of L1 at the C-terminal end of the protein. In these cases, binding with the intracellular cytoskeleton and signal transduction might be impaired, whereas the extracellular and transmembrane domains remain intact. The second class consists of missense point mutations in the extracellular domain. In these cases the mutant protein is still an integral membrane protein with a normal transmembrane and cytoplasmic domain. However, the binding capacities of the Ll protein toward its ligands might be distorted because the structure of the extracellular domains is altered at one amino acid position. Indeed, studies on mutated L1 molecules provide evidence that missense changes in the extracellular domains have variable effects on homophilic and heterophilic binding of the moleculel131. The third class of mutant L1 proteins has a premature stop codon in the extracellular part. In these cases the transmembrane protein is absent, and the mutant protein could be secreted into the extracellular space. Obviously such a protein loses its function because its contact with the nerve cell is lostl111. A correlation between the type of mutation and the severity of the disease has been demonstrated by Yamasaki et a/. l111. Mutations that produce truncations in the extracellular domain (class 3) are more likely to produce severe hydrocephalus, grave mental retardation, or early death than point mutationsin the extracellular domain or mutations affectingonly the cytoplasmic domain of the protein. Although less severe than extracellular truncations, point mutationsin the extracellular domain (class 2) produce more severe neurologic problems than mutations in only the cytoplasmic domain (class 1). In our patient the mutation-the G to T transition at position 1,243-affects exon 10 of the L1CAM gene. This missense mutation has not been described previously and represents a form of class 2 mutation on the basis of the classification suggested by Yamasaki et at. r111. Exons 9 and 10 encode the fourth immunoglobulin domain (lg4)12I, and this mutation in exon 10 leads to a change of alanine to praline in position 415 (A415P). Alanine 415 is a key residue that is important for the conformation of the domain: change of an alanine to praline likely causes disruption of a beta sheet hydrogen bond121. The key role of alanine at position415 can explain the severe clinical manifestations of X-linked hydrocephalus in this family despite the fact that the p.Ala415Pro change is a class 2 mutation. Because the mother also carries the same mutation, it is likely that the three brothers who died of hydrocephalus in the neonatal period had the same mutation. Evidence has been provided that missense mutations in key residues tend to produce more severe hydrocephalus than mutations in surface residues121. In vitro studies confirmed that mutations in key residues of all lg domains result in the diminution of both homophilic and heterophilic binding1131. It is likely, therefore, that the abnormal binding properties of the mutated Ll in our patient led to the cerebral dysgenesis because of abnormal neurite outgrowth and pathfinding. We can conclude that further studies on the effect of normal and abnormal function of Ll and other cell adhesion molecules will allow deeper insight into the pathogenesis of a group of cerebral malformations and mental retardation.

50 X-linked hydrocephalus Acknowledgements We are grateful to Ashok Prasad, lvanna Lizarriturri, and Anne Mathew for their help.

References

(1) Bearer CF. Mechanisms of brain injury: Ll cell adhesion molecule as a target for ethanol-induced prenatal brain injury. Sem Pediatr Neural 2001; 8:100-7. (2) Bateman A, Jouet M, MacFarlane J, Du JS, Kenwrick S, Chothia C. Outline structure of the human Ll cell adhesion molecule and the sites where mutations cause neurological disorders. EMBO J 1996 Nov 15;15(22):6050-9. (3) Kenwrick S, Watkins A, De Angelis E. Neural cell recognition molecule Ll: relating biological complexity to human disease mutations. Hum Mol Genet 2000 Apr 12;9(6):879-86. (4) Fransen E, Lemmon V, Van Camp G, Vits L, Coucke P, . Willems PJ. CRASH syndrome: Clinical spectrum of corpus callosum hypoplasia, retardation, adducted thumbs, spastic paraparesis and hydrocephalus due to mutations in one single gene, Ll. Eur J Hum Genet 1995; 3: 273-84. (5) Yamasaki M, Arita N, Hiraga S, lzumoto S, Morimoto K, Nakatani S, Fujitani K, Sato N, Hayakawa T. A clinical and neuroradiological study of X-linked hydrocephalus in Japan. J Neurosurg 1995 Jul;83(1):S0- 5. (6) Graf WD, Born DE, Sarnat HB. The pachygyria-polymicrogyria spectrum of cortical dysplasia in X-linked hydrocephalus. Eur J Pediatr Surg 1998; 8 Suppl I: 10-4. (7) Graf WD, Born DE, Shaw DWW, Thomas JR, Holloway LW.Michaelis RC. Diffusion-weighted magnetic resonance imaging in boys with neural cell adhesion molecule L1 mutations and congenital hydrocephalus. Ann Neural 2000; 47:113-17 (8) Sztriha L, Frossard P, Hofstra RMW, Verlind E, Nork M. Novel missense mutation in the Ll gene in a child with corpus callosum agenesis, retardation, adducted thumbs, spastic paraparesis, and hydrocephalus. J Child Neural 2000; 15: 239-43. (9) Weller S, Gartner J. Genetic and clinical aspects of X-linked hydrocephalus {Ll disease): Mutations in the L1CAM gene. Hum Mutat 2001;18(1):1-12. (10) Hofstra RM, Landsvater RM, Ceccherini I, Stulp RP, Stelwagen T, Luo Y, Pasini B, Hoppener JW, van Amstel HK, Romeo G, . A mutation in the RET proto-oncogene associated with multiple endocrine neoplasia type 2B and sporadic medullary thyroid carcinoma. Nature 1994 Jan 27;367(6461):375-6. (11) Yamasaki M, Thompson P, Lemmon V. CRASH syndrome: mutationsin L1CAM correlate with severity of the disease. Neuropediatrics 1997 Jun;28(3):175-8. (12) Kenwrick S, Doherty P. Neural cell adhesion molecule Ll: Relating disease to function. BioEssays 1998; 20: 668-75. (13) De Angelis E, MacFarlane J, Du JS, Yeo G, Hicks R, Rathjen FG, Kenwrick S, BrummendorfT. Pathological missense mutations of neural cell adhesion molecule L1 affect homophilic and heterophilic binding activities. EMBO J 1999 Sep 1;18(17):4744-53. (14) Needham LK, Thelen K, Maness PF. Cytoplasmic domain mutations of the Ll cell adhesion molecule reduce Ll-ankyrin interactions. J Neurosci 2001 Mar 1;21(5):1490-500. (15) Van Camp G, Fransen E, Vits L, Raes G, Willems PJ. A locus-specific mutation database for the neural cell adhesion molecule L1CAM (Xq28). Hum Mutat 1996;8(4):391

51

Chapter 4

An updated and upgraded L1CAM mutation database

Y.J. Vos and R.M.W. Hofstra

Department of Genetics, University Medical Centre Groningen, University of Groningen, the Netherlands

Human Mutation2010 ; 31:E1102-E1109 Chapter 4 Abstract

The Ll syndrome is an X-linked recessive disease caused by mutationsin the L1CAM gene. To date more than 200 different mutations have been reported, scattered over the entire gene, about 35% being missense mutations. Although it is tempting to consider these missense mutations as being disease-causing, one should be careful in drawing any firm conclusions, unless there is additional supporting information. This is in contrast to truncating mutations, which are always considered to be disease-causing, unless they involve truncations close to the gene stop codon. In order to allow conclusions to be drawn on the disease-causing nature of L1CAM (missense) mutations, we have updated and upgraded our LICAM mutation database with more pathogenicity data and clinical information collected from the literature or generated by our own research. As a result, the renewed database offerscondensed scientific information, allowing conclusions to be drawn on the pathogenicity and severity of LICAM mutations based on multiplefactors. The L1CAM MutationDatabase is at: www.llcammutationdatabase.info.

54 LlCAM mutation database Introduction

Ll syndrome is an X-linked recessive disorder with an incidence of 1:30,000 newborn males. It comprises four neurological syndromes, all associated with mutations in the LlCAM gene11-31• The most severe of the four different forms, is X-linked hydrocephalus, also referred to as hydrocephalus, due to stenosis of the aqueduct of Sylvius (HSAS, MIM 307000}. This syndrome was first described in the 1940s and is the most frequent genetic form of congenital hydrocephalus 141• Other clinical characteristics are adducted thumbs, mental retardation, and lower limb spasticity. In most families, the affected boys die before, during, or soon after birth151• A less severe form of the disease has been published under the acronym MASA syndrome (MIM 303350). This syndrome consists of light to moderate mental retardation, £phasia, �huffling gait and £dducted thumbs. Occasionally, phenotypic variation ranging from the severe, X-linked hydrocephalus type, to the less severe MASA syndrome can be found in the same family !5, 71• X-linked complicated hereditary spastic paraplegia type 1 (SPGl, MIM 303350} and X-linked complicated corpus callosum agenesis (X-linked ACC, MIM 304100} are also parts of the L1 syndrome, but are seen much less frequently. The complicated forms may be combined with mild to moderate mental retardation. The LlCAM gene, coding forthe neural cell adhesion molecule Ll, is located on the X-chromosome at Xq28. The human L1CAM gene consists of 29 exons: the firstexon of 125 bp is non-coding and was only discovered later181• Therefore, in contrast to common practice, the first exon, which is located 10 kb upstream of the ATG containing exon 1, is known as exon A and not exon 1. The L1CAM gene is primarily expressed in the nervous system and encodes a protein of 1,257 amino acids, comprising a signal peptide of 19 amino acids and a functional L1 protein of 1,238 amino acids. In non-neural tissue, alternative RNA splicing generates mRNA, which lacks exons 2 and 27 of the gene 19•101 and leads to a protein that is 9 amino acids shorter. Ll is a transmembrane glycoprotein belonging to the immunoglobulin superfamily cell adhesion molecules[nJ and contains thirteen distinct domains, i.e. six immunoglobulin- (lg) and five fibronectin (Fn) Ill-like domains at the extracellular surface, one single-pass transmembrane domain, and one short cytoplasmic domain. Ll may mediate axon growth during development and axon bundling (fasciculation) [12, 131• It is also involved in interactions between Schwann cells and axons, neuronal cell migration, and neuronal cell survival[14, 151• The activity is known to be mediated by homophilic and heterophilic interactionsan d transduction of a variety of signaling events through associated proteins[151_ To date more than 200 different L1CAM mutations have been published: 130 of these were reviewed in 2001[171 and another 41 were published subsequently[1s-35l_ Moreover 52 novel 36 mutationshave been detected in our laboratory and were published recently1 1 • Most mutations have been compiled in the L1CAM Mutation Database, originally constructed by Van Camp et a/.!371 and later continued by our group. Since the amount of additional information is growing rapidly and it is important to researchers, clinicians and geneticcou nselors to have access to these new data in a fast and simple way, we decided to renew and update the L1CAM Mutation Database quite drastically. New information compiled in the upgraded database comprise: (1) elements which co-determine the possible pathogenicity of a mutation, (2) clinical data, (3) family histories, (4) reported clinical classification, and (5) effect of a mutation on molecular function and molecular interactions. The L1CAM Mutation Database is freely-accessible at: www.llcammutationdatabase.info.

55 Chopter 4 Database construction

All mutations known from the old database were transferred to the new version of the database and all new mutations, either recently found in our laboratory or published by others, were added; all were described according the nomenclature of the Human Genome Variation Society (www.hgvs.org/mutnomen)r3s. 391 and numbered according to the reference sequence (+l = A of ATG translation initiation codon), as listed in GenBank: NM_000425.3. Next we checked all the original publications for extra information related to the mutations, to upgrade the database not only with a direct link to the publicationbut, more importantly, in order to add the extra informationabout the mutations. We added informationto the truncating mutations, which are considered to be pathogenic, such as mutation history (inherited or de nova), the family history of the patient, and clinical information on the occurrence of hydrocephalus (HC), adducted thumbs (AT), mental retardation (MR), aphasia (Aph), spastic paraplegia (SP), agenesis/dysagenesis corpus callosum (ACC/DCC), or hypotonia (HP), which are the most predominant characteristics related to Ll syndrome. For missense mutations, which in general correlate much less directly to disease-causing, we have upgraded the database moreover with information on the mutation, like any published classification, i.e. disease-causing, likely disease-causing, likely non-disease-causing, polymorphism, or unknown. For this category of mutations we have also added whether the mutationis present in affectedor non-affected male relatives,as far as could be derived from the original publications. Next we investigated other parameters which could influence the mutation pathogenicity, such as (1) the difference in physical and chemical characteristics between the two amino acids, (2) the degree of conservationof the amino acid mutated, and (3) the mutation versus protein function relationship.Consequently, our new LICAM Mutation Database includes the SIFT1401 and Polyphenr411 predictions, and whether the mutations might affect a key or a surface residue, as defined by Bateman142I, because these residues are supposed to maintain the structure of the lg and Fnlll domains in the Ll protein and hence its function. As an illustration, we have summarized the results obtained with these kinds of predictionson the series of new missense mutationsdetected and studied in our laboratory in table 1 (table 1 and the LlCAM database).

Data base features

To date 240 different LlCAM mutationshave been reported, scattered over the entiregene: 204 disease-causing, 11 likely disease causing, 6 likely non disease-causing, 12 non disease-causing, and 7 of unknown pathogenicity. Most LlCAM mutationsare unique for each family, i.e. they appear to be private mutations. Out of the 215 (likely) disease-causing mutations recorded in the database, 1 appears in 6 different families (p.Val752Met), 1 appears in 4 different families (c.807-6G>A), 8 appear in three different families, and 17 appear in two families. About 58% of all mutations are truncating and therefore considered to be disease-causing. Out of the 102 remaining mutations, 66 are reported as disease-causing, 11 likely diseases-causing, 6 likely non-disease-causing, 7 unknown and 12 non-disease-causing/polymorphisms. These 102 mutations, predominantly missense mutations, also include 6 silent mutations and 15 intronic mutations.

56 LlCAM mutation database University Medical Center Groningen Departmentof Genetics - L1CAM MutationDatabase emtwt®!®IEDM:M@iil:03� Mutations Flnd Mutatlonl ------Exon/lntron:� Reportedclesslflcallon: 1 AR clessiflcetions 3 Type: �I AJ_I �types�----3 Results perpage: � 1 Search1

NB: Cilek on thecolumn headersfD change the sort order.

Deletionentire gene Disease-causing Dllalalt Dlsease-ceuslng 1 4 Likelynon disease-causing Disease-causing

2

V FigureUniversity 1. Medical Page 1Center of the Gronlngan LlCAM mutation database with 10 variants. �� Department ofGenetlcs - L1CAM Mutation Database �IE!:!l!::lel:i'lil:DE:!1:111 Mutation Details

Ml:lsansaDisease-causing Kay Not Prob8bly Highly lg 1 Tinmutation 13 de1acled In bothallec1ed mates . Themutation reduces homophlllc residue 10lernted dllmaglng (stmngly)and he!amphi&c binding (De Angelis 11199) and reduces expression Bl lhecell,surface of CHO cells (De Angalts, 2002) Patients

_1 Adductedthumb&, Aphasia, Hydmcephalus, Mental retardation, Spastic pataptegl� > 1 yrJouat et al. (19958) References

1999 DeAngellsetal. P/lthojoojg!/ml...,,e routalfomofnevralce!J•dlJnloo mojaruilUallectlpmoohWcandhe!gmphjlicbjndlngadMties ' EMBOJ. 1BNo. 17 �744-445:JI� ltllOI! ,__Ill --GiiiiL 1.t 1-11 1llZai 11995aJouet e t al. NAwdomelm at oRUcol cel�arflttwn mecule L1 lmQlcmftd In X-llnkedmtdmcAphplrns and MASA syndmmo IAm J.Hum Genet. 55 IIOOO --•• ,__Mol..--. II

Figure 2. The "details" page of the p.Gly121Ser variant. The database contains only a few large deletions or duplications that involve more than one exonl2s, 43-45l, and only one case of a deletion of the entire L1CAM genel29l. In the new L1CAM mutationdatabase, you can screen for mutations per exon or intron and select for reported classificationand type (figure 1). Mutation-specific information can be obtained by clicking the "details" button(figure 2). The L1CAM database will be updated monthly with newly published data. We will search for this, but other researchers are encouraged to submit their new mutation data via the online submission form. The submission will be checked by the curator and completed if necessary.

57 Lil 00 9 ---,r- 1::i Ii �.... Table 1 (a). Disease-causing missense mutationsin the L1CAM gene -1:,. ,11i Family 'Nucleotide # Affected I Exon Consequence j • Domain Remarks In silico datae, ID change J re1 atives II Poly- Bateman SIFT ** Conserved �· h en JI 292 c.110T>A 3 p.lle37Asn 4 lg1 Not detected in one unaffected Key residue Not tolerated 1 Highly male relative 89 c.536T>G* 6 p.lle179 Ser >1 lg2 Previously, linkage analysis Key residue Not tolerated 1 Highly confirmed a locus on Xq28 56 c.550C>G 6 p.Arg184Gly 10 lg2 Key residue Not tolerated 1 Highly 235 c.551G>A* 6 p.Arg184Gln 1 lg2 Previously described as pathogenic Key residue Not tolerated 1 Highly 27 c.604G>T 6 p.Asp202Tyr 1 lg2 Surface Not tolerated 1 Highly 164 c.761C>A 7 p.Ala254Asp 4 lg3 Present in two affectedmales Key residue Not tolerated 2 Highly Absent in one unaffected male 348 c.826T>C 8 p.Trp276Arg 1 lg3 De novo in mother of index patient Key residue Not tolerated 1 Highly 35 c.938T>C 8 p.Leu313Pro 1 lg3 De nova Surface Not tolerated 2 Moderately 187 c.1003T>C* 9 p.Trp335Arg 1 lg4 Previously described as pathogenic Key residue Not tolerated 1 Highly 184 c.1107C>A 9 p.Asn369Lys 3 lg4 Index patient with an affected Key residue Not tolerated 1 Highly brother and nephew 10 91 c.1243G>C p.Ala415Pro 4 lg4 Index patient with three affected Key residue Not tolerated 1 Highly uncles (maternal line) 36 c.1438G>A 12 p.Gly480Arg 2 lgS Two affected brothers, their Key residue Not tolerated 1 Highly mother is a carrier 208 c.1933A>T 15 p.lle645Phe 1 Fnl De nova Key residue Not tolerated 1 Highly 23 c.2140C>T 17 p.Pro714Ser 1 Fn2 Key residue Not tolerated 1 Highly 80,156, c.2254G>A* 18 p.Val752Met 7, 4,2 Fn2 3 families Key residue Not tolerated 2 Highly 336 Previously described as pathogenic 275 c.2260T>A 18 p.Trp754Arg 3 Fn2 Familial history Key residue Not tolerated 1 Highly

162 c.3239T>A 24 p.Leu1080Gln 2 FnS The mutation was detected in two Key residue Not tolerated 1 Highly affected brothers

* Previously found by other groups, ** 1 probably damaging, 2 possibly damaging, 3 benign, 4 unknown -- - ,_ #Affected Table l(b). Likely disease-causing mutationsin the LlCAM gene Family Nucleotide II 11 Exon Consequence Domain Remarks I In sillco data ID change relatives I J "'- - Poly- Bateman SIFT *"', Conserved peh n

The mutation was detected in 366 c.516G>A 5 p.Metl 72IIe 3 lg2 two affectedbrothers and one Surface Not tolerated 1 Highly 1 affectedsister Low De nova in mother 200 c.1546G>A 12 p.Asp516Asn lg6 Surface Tolerated 3 (in frog Affects RNA splicing most likely Asn516) 3 302 c.1546G>T 12 p.Asp516Tyr 1 lg6 Affects RNA splicing most likely Surface Not tolerated 1 Low

320 c.1880C>T 15 p.Thr627Met 1 Fnl Surface Not tolerated Low

* Previously found by other groups, ** 1 probably damaging, 2 possibly damaging, 3 benign, 4 unknown

r­ N � s: 3 a­ g. §­ a- u, \.0 Chapter4 Discussion

Recently the contribution of modifiers to the severity of X-linked hydrocephalus in mouse 46 models has been describedl 1 • Furthermore, also in animal models, some 10 genes causing 45 47 hydrocephalus have been detectedl , 1, but to date in humans only the L1CAM gene has been identified to be associated with the Ll syndrome. Therefore, and since hardly any polymorphisms occur in the L1CAM gene, it is tempting to consider the missense mutations, detected in the group of patients suffering from Ll syndrome, as disease-causing. However, without any further supporting information, we should be careful in drawing any firm conclusions about the missense mutations, in terms of their being disease-causing. This is in contrast to the truncating mutations, which, in general due to their nature, are always likely to be disease-causing unless they involve truncations close to the gene stop codon. In order to allow firmer conclusions as to the pathogenicity of missense mutations, we have upgraded our LICAM mutation database with additional information collected from the literature. As a result, we have been better able to indicate the disease-causing nature of many of the missense mutations in the L1CAM gene, thereby providing researchers, clinicians and counselors with a more useful tool for drawing conclusions. Next, by adding information to the database, like the live span of patients suffering from Ll syndrome (i.e. age at early death), the database offers background information that allows the correlation of to phenotypes, i.e. links the severity of disease to the type of mutation, essentially according to Fransen et a/.1481 and Yamasaki et a/.1491 and more recently Vos et al. l35J_ In conclusion, this updated and upgraded version of the L1CAM mutation database offers more scientific information to enable conclusions to be drawn on the pathogenicity and severity of L1CAM mutations based on multiple factors.

Acknowledgements We thank Jan Herman Veldkamp (www.aardworm.com) for his assistance in developing the website and Jackie Senior for editing the manuscript. The long-term curation of the database is financially supported by the Department of Genetics, University Medical Centre Groningen, Groningen, the Netherlands.

60 LlCAM mutation database References

(1) Fransen E, Van Camp G, Vits L, Willems PJ. Ll-associated diseases: clinical geneticists divide, molecular geneticists unite. Hum Mol Genet 1997;6(10):1625-32. (2) Rosenthal A, Jouet M, Kenwrick S. Aberrant splicing of neural cell adhesion molecule Ll mRNA in a family with X-linked hydrocephalus. Nat Genet 1992 Oct;2(2):107-12. (3) Jouet M, Rosenthal A, Armstrong G, MacFarlane J, Stevenson R, Paterson J, Metzenberg A, lonasescu V, Temple K, Kenwrick S. X-linked spasticparaplegia (SPGl), MASA syndrome and X-linked hydrocephalus 1949;72: result from mutationsin the Ll gene. Nat Genet 1994 Jul;7(3):402-7. (4) Bickers DS, Adams RD. Hereditary stenosis of the aqueduct of Sylvius as a cause of congenital hydrocephalus. Brain 246-62. (5) Schrander-Stumpel C, Howeler C, Jones M, Sommer A, Stevens C, Tinschert S, Israel J, Fryns JP. Spectrum of X-linked hydrocephalus (HSAS), MASA syndrome, and complicated spastic paraplegia (SPGl): Clinical review with six additional families. Am J Med Genet 1995 May 22;57(1):107-16. (6) Fryns JP, Spaepen A, Cassiman JJ, van den Berghe H. X linked complicated spastic paraplegia, MASA syndrome, and X linked hydrocephalus owing to congenital stenos is of the aqueduct of Sylvius: variable expression of the same mutation at Xq28. J Med Genet 1991 Jun;28(6):429-31. (7) Schrander-Stumpel C, Legius E, Fryns JP, Cassiman JJ. MASA syndrome: new clinical features and linkage analysis using DNA probes. J Med Genet 1990 Nov;27(11):688-92. (8) Kallunki P, Edelman GM, Jones FS. Tissue-specific expression of the Ll cell adhesion molecule is modulated by the neural restrictive silencer element. J Cell Biol 1997 Sep 22;138(6):1343-54. (9) Jouet M, Rosenthal A, Kenwrick S. Exon 2 of the gene for neural cell adhesion molecule Ll is alternatively spliced in B cells. Brain Res Mol Brain Res 1995 Jun;30(2):378-80. (10) Coutelle 0, Nyakatura G, Taudien S, Elgar G, Brenner S, Platzer M, Drescher B, Jouet M, Kenwrick S, Rosenthal A. The neural cell adhesion molecule Ll: genomic organisation and differential splicing is conserved between man and the pufferfishFugu. Gene 1998 Feb 16;208(1):7-15. (11) Moos M, Tacke R, Scherer H, Teplow D, Fruh K, Schachner M. Neural adhesion molecule Ll as a member of the immunoglobulin superfamily with binding domains similar to fibronectin.Nature 1988 Aug 25;334(6184):701-3. (12) Grumet M, Edelman GM. Neuron-glia cell adhesion molecule interacts with neurons and astroglia via differentbinding mechanisms. J Cell Biol 1988 Feb;106(2):487-503. (13) Lemmon V, Farr KL, Lagenaur C. Ll-mediated axon outgrowth occurs via a homophilic binding mechanism. Neuron 1989 Jun;2(6):1597-603. (14) Haney CA, Sahenk Z, Li C, Lemmon VP, Roder J, Trapp BD. Heterophilic binding of Ll on unmyelinated sensory axons mediates Schwann cell adhesion and is required for axonal survival. J Cell Biol 1999 Sep 6;146(5):1173-84. (15) Lindner J, Rathjen FG, Schachner M. Ll mono- and polyclonal antibodies modify cell migration in early postnatal mouse cerebellum. Nature 1983 Sep 29;305(5933):427-30. (16) Kenwrick S, Watkins A, De Angelis E. Neural cell recognition molecule Ll: relating biological complexity to human disease mutations. Hum Mol Genet 2000 Apr 12;9(6):879-86. (17) Weller S, Gartner J. Genetic and clinical aspects of X-linked hydrocephalus (Ll disease): Mutations in the LlCAM gene. Hum Mutat 2001;18(1):1-12. (18) Simonati A, Boaretto F, Vettori A, Dabrilli P, Criscuolo L, Rizzuto N, Mostacciuolo ML. A novel missense mutation in the LlCAM gene in a boy with Ll disease. Neurol Sci 2006 Jun;27(2):114-7. (19) Senat MV, Bernard JP, Delezoide A, Saugier-Veber P, Hillion Y, Roume J, Ville Y. Prenatal diagnosis of hydrocephalus-stenosis of the aqueduct of Sylvius by ultrasound in the first trimester of pregnancy. Report of two cases. Prenat Diagn 2001 Dec;21(13):1129-32. (20) Sztriha L, Vos YJ, Verlind E, Johansen J, Berg B. X-linked hydrocephalus: a novel missense mutation in the LlCAM gene. Pediatr Neurol 2002 Oct;27(4):293-6. (21) Rodriguez CG, Perez AA, Martinez F, Vos YJ, Verlind E, Gonzalez-Meneses LA, Gomez de TS, I, Schrander-Stumpel C. X-linked hydrocephalus: another two families with an Ll mutation.Gene t Couns 2003;14(1):57-65.

61 Chapter 4

{22) Silan F, Ozdemir I, Lissens W. A novel LlCAM mutation with L1 spectrum disorders. Prenat Diagn 2005 Jan;25(1):57-9. (23) Bott L, Boute 0, Mention K, Vinchon M, Boman F, Gottrand F. Congenital idiopathic intestinal pseudo­ obstruction and hydrocephalus with stenosis of the aqueduct of sylvius. Am J Med Genet A 2004 Sep 15;130(1):84-7. (24) Okamoto N, Del MR, Valero R, Monros E, Pao P, Kanemura Y, Yamasaki M. Hydrocephalus and Hirschsprung's disease with a mutation of LlCAM. J Hum Genet 2004;49(6):334-7. (25) Hubner CA, Utermann B, Tinschert S, Kruger G, Ressler B, Steglich C, Schinzel A, Gal A. lntronic mutations in the LlCAM gene may cause X-linked hydrocephalus by aberrant splicing. Hum Mutat 2004 May;23(5):526. (26) Moya GE, Michaelis RC, Holloway LW, Sanchez JM. Prenatal diagnosis of Ll cell adhesion molecule mutations.Capabilities and limitations. Fetal Diagn Ther 2002 Mar;17(2):115-9. (27) Panayi M, Gokhale D, Mansour S, Elles R. Prenatal diagnosis in a family with X-linked hydrocephalus. Prenat Diagn 2005 Oct;25(10):930-3. (28) Tegay DH, Lane AH, Roohi J, Hatchwell E. Contiguous gene deletion involving LlCAM and AVPR2 causes X-linked hydrocephalus with nephrogenic diabetes insipidus. Am J Med Genet A 2007 Mar 15;143(6):594-8. (29) Knops NB, Bos KK, Kerstjens M, van Dael K, Vos VJ. Nephrogenic diabetes insipidus in a patient with Ll syndrome: a new report of a contiguous gene deletion syndrome including LlCAM and AVPR2. Am J Med Genet A 2008 Jul 15;146A(14):1853-8. (30) Griseri P, Vos Y, Giorda R, Gimelli S, Beri S, Santamaria G, Mognato G, Hofstra RM, Gimelli G, Ceccherini I. Complex pathogenesis of Hirschsprung's disease in a patient with hydrocephalus, vesico-ureteral reflux and a balanced translocation t(3;17)(p12;qll). Eur J Hum Genet 2009 Apr;17(4):483-90. (31) Nakakimura S, Sasaki F, Okada T, Arisue A, Cho K, Yoshino M, Kanemura Y, Yamasaki M, Todo S. Hirschsprung's disease, acrocallosal syndrome, and congenital hydrocephalus: report of 2 patients and literature review. J Pediatr Surg 2008 May;43(5):E13-E17. (32) Wilson PL, Kattman BB, Mulvihill JJ, Li S, Wilkins J, Wagner AF, Goodman JR. Prenatal identification of a novel R937P LlCAM missense mutation. Genet Test Mol Biomarkers 2009 Aug;13(4):515-9. (33) Kanemura Y, Okamoto N, Sakamoto H, Shofuda T, Kamiguchi H, Yamasaki M. Molecular mechanisms and neuroimaging criteria for severe Ll syndrome with X-linked hydrocephalus. J Neurosurg 2006 Nov;105(5 Suppl):403-12. (34) De Angel is E, Watkins A, Schafer M, Brummendorf T, Kenwrick S. Disease-associated mutations in Ll CAM interfere with ligand interactions and cell-surface expression. Hum Mal Genet 2002 Jan 1;11(1):1-12. (35) Piccione M, Matina F, Fichera M, Lo Giudice M, Damiani G, Jakil MC, Corsello G. A novel LlCAM mutation in a fetus detected by prenatal diagnosis. Eur J Pediatr 2009 Aug 16. (36) Vos VJ, de Walle HE, Bos KK, Stegeman JA, Ten Berge AM, Bruining M, van Maarle MC, Elting MW, den Hollander NS, Hamel B, Fortuna AM, Sunde LE, Stolte-Dijkstra I, Schrander-Stumpel CT, Hofstra RM. Genotype-phenotype correlations in Ll syndrome: a guide for genetic counselling and mutation analysis. J Med Genet 2010 Mar;47(3):169-75. (37) Van Camp G, Fransen E, Vits L, Raes G, Willems PJ. A locus-specific mutation database for the neural cell adhesion molecule LlCAM (Xq28). Hum Mutat 1996;8(4):391. (38) den Dunnen JT, Antonarakis SE. Mutation nomenclature extensions and suggestions to describe complex mutations:a discussion. Hum Mutat 2000;15(1):7-12. (39) den Dunnen JT, Paalman MH. Standardizing mutation nomenclature: why bother? Hum Mutat 2003 Sep;22(3):181-2. (40) Ng PC, Henikoff S. Predicting deleterious amino acid substitutions. Genome Res 2001 May;l1(5):863- 74. (41) Ramensky V, Bork P, Sunyaev S. Human non-synonymous SNPs: server and survey. Nucleic Acids Res 2002 Sep 1;30(17):3894-900. (42) Bateman A, Jouet M, MacFarlane J, Du JS, Kenwrick S, Chothia C. Outline structure of the human Ll cell adhesion molecule and the sites where mutations cause neurological disorders. EMBO J 1996 Nov 15;15(22):6050-9. (43) Panayi M, Gokhale D, Mansour S, Elles R. Prenatal diagnosis in a family with X-linked hydrocephalus. Prenat Diagn 2005 Oct;25(10):930-3.

62 LlCAM mutation database

(44) Vits L, Van Camp G, Coucke P, Fransen E, De Bou lie K, Reyniers E, Korn B, Poustka A, Wilson G, Schrander­ Stumpel C, . MASA syndrome is due to mutations in the neural cell adhesion gene LlCAM. Nat Genet 1994 Jul;7{3):408-13. (45) Van Camp G, Vits L, Coucke P, Lyonnet S, Schrander-Stumpel C, Darby J, Holden J, Munnich A, Willems PJ. A duplication in the LlCAM gene associated with X-linked hydrocephalus. Nat Genet 1993 Aug;4(4):421-5. (46) Tapanes-Castillo A, Weaver EJ, Smith RP, Kamei Y, Caspary T, Hamilton-Nelson KL, Slifer SH, MartinER , Bixby JL, Lemmon VP. A modifier locus on chromosome 5 contributes to Ll cell adhesion molecule X-linked hydrocephalus in mice. Neurogenetics 2009 Jun 30. (47) ZhangJ, Williams MA, Rigamonti D. Genetics of human hydrocephalus. J Neurol 2006 Oct;253(10):1255- 66. (48) Fransen E, Van Camp G, D'Hooge R, Vits L, Willems PJ. Genotype-phenotype correlationin Ll associated diseases. J Med Genet 1998 May;35(5):399-404. (49) Yamasaki M, Thompson P, Lemmon V. CRASH syndrome: mutations in LlCAM correlate with severity of the disease. Neuropediatrics 1997 Jun;28(3):175-8.

63

Chapter 5

RNA splicing defects caused by silent, missense and intronic mutationsin the L1CAM gene can be the cause of Ll syndrome

Yvonne J. Vos, Krista K. van Dijk-Bos, Christine S. van der Werf and Robert M.W. Hofstra

Department of Genetics, University Medical Centre Groningen, University of Groningen, the Netherlands Chapter 5 Abstract

Ll syndrome, characterized by hydrocephalus, aqueduct stenosis, mental retardation, adducted thumbs, agenesis/dysgenesis of the corpus callosum, aphasia and spasticparaplegia is caused by mutations in the LlCAM gene. We analyzed 367 patients suspected of having Ll syndrome and referred to our laboratory for diagnostic testing. Of these, 73 patients were found to harbor one of 68 different mutations, 61 of which are considered to be disease­ causing and seven of which are unclassified variants (UVs). For some of these UVs we have in silica evidence that they might be involved in aberrant splicing of the pre-mRNA. In order to prove an effect on splicing for three of these UVs, c.76+5G>A, c.1546G>A (Asp516Asn) and c.1546G>T (p.Asp516Tyr), we used an ex vivo RNA splicing assay and were able to show that all three were pathogenic. A previously proven splice mutation, c.645C>T (p.=), was used as a positivecontrol in the assay. The number of disease-causing DNA variants increased from 61 to 64. Thus our analysis showed that not only is this number higher than previously thought, but also that UVs might well cause RNA splicing problems and should therefore be studied carefully.

66 Ex vivo RNA splicing assay Introduction

Ll syndrome is an X-linked recessive neurological disorder caused by mutations in the L1CAM gene[1, 21, a gene coding for the neural cell adhesion molecule Ll. The phenotypic spectrum associated with these mutations includes four different neurological syndromes: X-linked hydrocephalus, also referred to as hydrocephalus due to stenosis of the aqueduct of Sylvius (HSAS; MIM 30700), the MASA syndrome (MIM 303350), standing for mental retardation, §.phasia,�huffling gait and §.dductedthumbs, X-linked complicated hereditary spastic paraplegia type 1 (SPGl, MIM 303350) and X-linked complicated corpus callosum agenesis (X-linked ACC, MIM 304100)131• The seven most important clinical characteristics of Ll syndrome are hydrocephalus, aqueduct stenosis, mental retardation, adducted thumbs, agenesis/dysgenesis of the corpus callosum, aphasia (delayed speech development) and spastic paraplegia. To date more than 200 different LlCAM mutations have been published!41• We detected 68 of these in a large group of patients (n=367) who were referred to our laboratory for diagnostic testing. These 68 different mutations were found in 73 patients[51, 61 of the mutations are considered to be disease-causing and seven variants have an unknown pathogenic status and are therefore called unclassified variants (UVs). These seven UVs consist of five missense mutationsand two intronic mutations(table 1). Based on in silica data these UVs are believed

Ta ble 1. Unclassifiedvari ants Nucleotiaechange Exon7 In sllico data intron RNA splicing Polyphen Coi,served prediction c.76+5G>A intron 1 ? Splice donor site of intron 1 is very weakened - c.516G>A exon 5 p.Met172lle 1* Not Probably Highly tolerated damaging [c.523+9_523+10delinsGA; intron 5 ? Splice donor c.523+ 12delC] 2** site of intron 5 is weakened c.1546G>A exon 12 p.Asp516Asn Splice donor To lerated Benign Low site of intron (in frog 12 is very Asn516) weakened c.1546G>T exon 12 p.Asp516Tyr Splice donor Not Probably Low site of intron tolerated damaging 12 is very weakened c.1574G>A exon 13 p.Arg525His To lerated Benign Moderately c.1880C>T exon 15 p.Thr627Met Not Benign Low tolerated

*1. The mutationwas detected in two affected brothers and one affectedsister **2. De nova in mother

67 Chapter 5 Exon + mutation � Figure 1. \ J 0/ Principle of ex vivo splicing assay. In the pSPL3 vector two (non-Ll) exons have been cloned separated by an intron with a multiple cloning site. PCR-amplified SD SA SD SA wild-type and mutant exons from the LlCAM gene, including flanking intronic sequences, are cloned into this multiple cloning site. After transfection into HEK293 cells total RNA is extracted and amplified © pSPL3 © to synthesize cDNA. cDNA is amplified using pSPL3 specific primers and the PCR products are analyzed I: Mutant II: Wild type by gel electrophoresis and direct sequencing. If a wild-type exon and flanking intronic sequences are cDNA cloned into the vector, the L1 exon will be present in the synthesized cDNA (lower right site of the figure). The Ll exon (or part of it) will be absent when a PCR splice site affectingmutation is present in the cloned product sequence (lower left part of the figure).

to be "likely disease causing" (table 1), but further research is needed to determine their status accurately. Four out of the seven likely disease-causing mutations most probably influence correct pre­ mRNA splicing. These comprise a mutationin intron 1, a mutationin intron 5 and two mutations in exon 12. Both mutations in exon 12 affect the last nucleotide of the exon and according to three out of four splice site prediction programs tested, the splice site at the beginning of intron 12 is not, or almost not, being recognized. Due to the mutations in intrans 1 and 5, it is predicted that the original splice donor sites of intrans 1 and 5 will be weakened. To prove that these possibly splice mutations are indeed disease causing, we decided to investigate three mutations in terms of their involvement in RNA splicing. Since it was not possible to isolate and analyze RNA from the patients, we used an ex vivo RNA splicing assay (figure 1). The c.645C>T mutation in exon 6 of the LlCAM gene, a silent mutation we have proved to be pathogenic by influencing the RNA splicing[51, was used as a positive control. The mutationin intron 5 has not been examined yet.

Materials and methods

Constructs for ex vivo splicing assay Wild-type and mutant LlCAM exons 1, 6 and 11 and 12, including 60-200 bp of 5'- and 3' flankingintronic sequences, were amplified from control and patient genomic DNA (table 2: primers). The PCR was carried out using Phusion High-Fidelity DNA polymerase (NEB) in the following steps: denaturation at 98°C for 30 seconds, followed by 35 cycles of denaturation at 98°C for 10 seconds, annealing at 63°C for 30 seconds, elongationat 72°C for 30 seconds, and a final step at 72°C for 10 minutes. The products were analyzed on, and extracted from, an agarose gel (Qiagen kit). To obtain 3' A-overhangs, 7 µI of purified PCR product, 1 µI Taq

68 Ex vivo RNA splicing assay

Sequence Table 2. Primer sequences Name Exon LCl_patientl_F CTTA-EcoRl,5' -CACTAACGTCCTTCCGTTCTC-3' 1 LCl_Patientl_R CTAT- Ba m H 1,5'-TGTCCCTGGTG CTGAAATCG-3' 1 LCl_Patient 2&3_F CTTA-EcoRl,5' -GGGAGCAGGGAAGCAAGAT-3' 11 & 12 LCl_Patient 2&3_R CTAT- BamH 1,5'-TGCGTGGGGAGTC ACTCT-3' 11 & 12 LCl_PositiveControl_F CTTA-EcoRl,5' -CTCGTGGGGTCGGGGTGTTA-3' 6 LCl_PositiveControl_R CTTA-BamH 1,5'-GAAGGGGTCTGGGCAGG GTA-3' 6

polymerase, 1 µI l0xRB and 1 µI dNTPs were incubated at 72°C for 15 minutes and put on ice for 2 minutes. 4 µI of this product was added to 1 µI pCR2.1 TOPO plasmid (lnvitrogen) and 1 µI salt and incubated for 5 minutes at room temperature. The pCR2.1 TOPO constructs were then transformed to DH Sa competent cells, according to the manufacturer's protocol for "Transforming One Shot Machl-TlCompetent Cells". 40 µI of X-gal (20 mg/ml) was spread over ampicillin-containing agar plates. Then the transformed DHSa cells were plated and incubated for 16 hours at 37°C. White colonies were picked and grown in Low Broth medium for 16 hours at 37°C and 225 rpm. The DNA constructs isolated from these cells using the Machery-Nagel kit were checked by BamHI and EcoRI restriction enzyme analysis and direct sequencing. Then the correct plasmids and the exon trapping vector pSPL3 {lnvitrogen) were digested, loaded on and extracted from an agarose gel (Qiagen). 100 ng pSPL3 and 50 ng insert were put together with 3 µI lOx ligationbuffer (Biolabs) and 1 µI T4 Ligase (Biolabs) in a total volume of 30 µI. Ligation was performed overnight at 16°C and 2 µI of the product was transformed to DHSa competent cells as described. The final DNA constructs were checked by restriction enzyme analysis and direct sequencing.

Trans/ection of HEK 293 cells and RT-PCR Human embryonic kidney (HEK) 293 cells were plated in 6-well plates containing 6 x 105 cells/well and cultured in DEMEM supplemented with glutamine, 10% foetal bovine serum (FBS), 1% penicillin/streptomycin (penicillin 10000 U/ml, streptomycin 10000 µg/ml) and ° • incubated at 37 C in air with 5% CO2 After 24 hours the cells were transfected with 1 µg plasmid DNA using polyethylenimine according the manufacturer's instructions (Polyscience INC}. The pSPL3 plasmid containing the wild-type UCAM sequences and the pSPL3 vector, as such (without an LlCAM insert), were used as controls. After 48 hours the cells were lysed and RNA was isolated according the manufacturer's instructions (Qiagen). 4.5 µg total RNA was used as a template to synthesize cDNA using the cDNA primers pd(N)6 (GE Healthcare). Then PCR was performed using the primers (SD6) 5'-CTGAGTCACCTGGACAACC-3' and (SA2) 5'-ATCTCAGTGGTATTTGTGAGC-3' and the following amplificationprogram: 5 minutes at 94°C, followed by 35 cycles of 1 minute at 94°C, 1 minute at 60°C and 5 minutes 72°C, and a final elongation time of 10 minutes at 72°C. The PCR products were analyzed by gel electrophoresis and direct sequencing.

69 Chapter 5 Splice site prediction The Alamut Splicing prediction module from Interactive Biosoftware was used to predict the influence of a mutation on the pre-mRNA splicing. This module integrates four different predictionmethods: SpliceSiteFinder, MaxEntScan, NNSplice and GeneSplicer. (www.interactive-biosoftware.com).

Results

Silent mutation c.645C>T The c.645C>T mutation in exon 6 was used as a positive control. Previously research on RNA isolated from patient's blood has demonstrated that a new splice donor site created by the mutation is recognized rather than the original splice donor site at the beginning of intron 6, resulting in a 51 bp deletion at the 3' site of exon 6 (figure 2A). The c.645C>T mutation gave in the ex vivo RNA splicing assay exactly the same result (figure 2B). The cDNA fragment obtained is 51 bp shorter than that of the wild type, which could be confirmed by sequence analysis of the fragments recovered from the agarose gel.

A

TCCCAGGTACCAGGACCATCATTCAGAAGGAACCCATTGACCTCCGGGTCAAGCCCc.645C>T Deletion of 51 bp gtgagt exon 6 intron 6 650 660 670 680 694

Splice donor site mutant splice donor site wild type

B cDNA fragments

pSPL3 exon 6 pPSPL3

Pos 383 bp

pSPL3 263 bp

Figure 2. (B) (A) Sequence of the 3' site of exon 6 of the L1CAM gene. A new splice donor site is created due to the c.645C>T mutation. Analysis of the PCR products of the different cDNA samples: B = no template control, M = marker, WT = wild type: pSPL3 containing wild type exon 6, Pas = positive control: pSPL3 containing mutated exon 6 (c.645C>T), pPSPL3:70 pSPL3 plasmid without an L1CAM insert. Ex vivo RNA splicing assay A 59 base pairs c.76+5G>A

20 30 40 50 60 76 t CTGCGGTACGTGTGGCCTCTCCTCCTCTGCAGCCCCTGCCTGCITATCCAGATCCCCGAGGAAT gtgaatagc

Potentialsplice donor site Original splice donor site

B cDNA fragments

pSPL3 exon 1 pPSPL3

Patl 388 bp

pSPL3 263 bp

LlCAM

Figure 3. (B) (A) Sequence of the 3' site of exon 1 of the gene. The potential 5' splice donor site in exon 1 is used instead of the original splice site due to the c. 76+5G>A mutation. Analysis of the PCR products of the different cDNA samples: B = no template control, M = marker, LlCAM WT = wild type: p5PL3 containing wild type exon/intronl, Patl = patientl, p5PL3 containing mutated exon/intronl (c.75+5G>A), lntronpPSPL3: p5l PLvariant3 plasmid c. without 76+5G>A an insert. The c.76+5G>A variant in intron 1 of the LlCAM gene caused a shorter cDNA fragment than generated from the wild-type construct (figure 3B). Sequence analysis of the recovered fragments showed a deletion of 59 bp at the 3' site of exon 1. The original splice donor site was weakened by the mutation at +5 such that the potential splice donor site present in exon 1 of the LlCAM gene was being used rather than the weakened original splice donor site (figure 3A). This was also predicted by at least three prediction programs. The cDNA sample of patient 1 (Patl) contains a second fragment (figure 3B) that turned out to be pSPL3 without an insert. Probably the splice donor site of pSPL3 itself competes with the splice donor site in exon 1.

Exon 12 variants p.Asp516Asn and p.Asp516Tyr (c.1546G>A and c.1546G>T) Both the c.1546G>A (present in patient 2) and the c.1546G>T (present in patient 3) variants at the end of exon 12 completely eliminated the splice donor site, such that RNA splicing had to occur at the splice donor site of exon 11, thus inducing complete skipping of exon 12 (figure 4A, Pat2 and Pat3). Since this was predicted by various prediction programs, we cloned a fragment containing exon 11 and exon 12, with and without one of the variants, in pSPL3. Sequence analysis of the largest cDNA fragment of the wild type (figure 4A) confirmed the presence of exons 11 and 12, whereas the analysis of the largest fragment of Pat2 and Pat3

71 Chapter 5 A cDNA fragments

pSPL3 exon 11 exon 12 pSPL3

exon 11 WT542 bp

Pat 2 + 3 375 bp

pSPL3 263 bp B

------tgs ------200 ------205 ------210------zt t i<• 0 ·"f O ·"f· T <; -A· O ·T· • 0 · · T · -0,1 · I- - - 1· J...,.vir_ i\J_ �,�Af · \J.....L:I �· 11�;_ 1. ��V-:... ��.. 1 ...... I_A\ . · /<, \ J • · 0• \ Ai� O ·A, � rl� A � tAC ·"f � Exon 11 +-----Jj_.�: i-\ h :-;JI oS PL3 �.. �l.,,.,... •

Figure 4. cDNA analysis of the c.1546G>A (p.Asp516Asn) and c.1546G>T (p.Asp516Tyr) variants. (A) Analysis of the PCR products of the different cDNA samples: B = no template control, M = marker, WT = wild-type: pSPL3 containing wild-type exonll-intronll-exonl2, Pat2 = patient2, pSPL3 containing the c.1546G>A variant (B) Pat3 = patient3, pSPL3 containing the c.1546G>T variant pPSPL3: pSPL3 plasmid without an LlCAM insert. Sequence analysis of the cDNA fragment obtained with the c.1546G>A variant. (figure 4A) confirmed the absence of exon 12 (figure 4B). As can be concluded from figure 4A, the 5' splice site at the beginning of intron 12 is rather weak anyway, since the wild-type cDNA also showed some cDNA lacking exon 12. Probably the splice donor site of pSPL3 itself competes with the other splice donor sites, since the cDNA fragment of pSPL3 was detected in all the cDNA samples.

Conclusion

Ex vivo RNA splicing analysis is a fast and reliable method to determine the effectof mutations on RNA splicing, as demonstrated with a comparable system for mutations in MLHl and M5H2f61 and BRCAl and BRCA2171• Here we confirmthe reliability of our ex vivo RNA splicing assay with the LlCAM c.645C>T mutation, since it generated the same result as found in vivo in the RNA of the patient151• Also, as we assumed from the in silica data, the other three variants analyzed by this method, c.76+5G>A, c.1546G>A (Asp516Asn) and c.1546G>T (p.Asp516Tyr), were shown to disturb the correct splicing. These UVs are therefore considered as disease causing. Now we have shown the three UVs to be pathogenic, the number of disease-causing mutations

72 Ex vivo RNA splicing assay has increased from 61 to 64 in the group of 68 mutations found in 367 patients. Consequently the number of UVs judged to be likely disease-causing drops from 7 to 4. In conclusion, our analysis shows that unclassified variants (intronic variants as well as silent and missense mutations) might well cause RNA splicing problems and should therefore be studied carefully. We suggest pre-screening via splice site prediction methods such as used by Alamut (Interactive Biosoftware), followed by ex vivo RNA splicing assaying.

Acknowledgements We thank Jackie Senior for editing the manuscript.

73 Chapter 5 References

(1) Rosenthal A, Jouet M, Kenwrick S. Aberrant splicing of neural cell adhesion molecule Ll mRNA in a family with X-linked hydrocephalus. Nat Genet 1992 Oct;2(2):107-12. (2) Weller S, Gartner J. Genetic and clinical aspects of X-linked hydrocephalus (Ll disease): Mutations in the LlCAM gene. Hum Mutat 2001;18(1):1-12. (3) Fransen E, Van Camp G, Vits L, Willems PJ . Ll-associated diseases: clinical geneticists divide, molecular geneticists unite. Hum Mal Genet 1997;6(10):1625-32. (4) Vos VJ, Hofstra RM. An updated and upgraded LlCAM mutation database. Hum Mutat 2010 Jan;31(1):E1102-E1109. (5) Vos VJ, de Walle HE, Bos KK, Stegeman JA, Ten Berge AM, Bruining M, van Maarle MC, Elting MW, den Hollander NS, Hamel B, Fortuna AM, Sunde LE, Stolte-Dijkstra I, Schrander-Stumpel CT, Hofstra RM. Genotype-phenotype correlations in Ll syndrome: a guide for genetic counselling and mutation analysis. J Med Genet 2010 Mar;47(3):169-75. (6) Tournier I, Vezain M, Martins A, Charbonnier F, Baert-Desurmont S, Olschwang S, Wang Q, Buisine MP, Soret J, Tazi J, Frebourg T, Tosi M. A large fraction of unclassified variants of the mismatch repair genes MLHl and MSH2 is associated with splicing defects. Hum Mutat 2008 Dec;29(12):1412-24. (7) Bonnet C, Krieger S, Vezain M, Rousselin A, Tournier I, Martins A, Berthet P, Chevrier A, Dugast C, Layet V, Rossi A, Lidereau R, Frebourg T, Hardouin A, Tosi M. Screening BRCAl and BRCA2 unclassified variants for splicing mutations using reverse transcription PCR on patient RNA and an ex vivo assay based on a splicing reporter minigene. J Med Genet 2008 Jul;45(7):438-46.

74

Chapter 6 Nephrogenic diabetes insipidus in a patientwith Ll syndrome: A new report of a contiguous gene deletion syndrome including L1CAM and AVPR2

1 2 , , 2 2

Noel B.B. Knops Krista K. Bos Mieke W.S. Kerstjens­ 1Frederikse , Karin van Dael 3, Yvonne J. Vos

Department of Pediatric Nephrology, Wilhelmina 2Children's Hospital, University Medical Centre Utrecht, Utrecht, the Netherlands Department of Genetics, University Medical Centre 3Groningen, University of Groningen, Groningen, the Netherlands Department of Pediatric Nephrology, Beatrix Children's Hospital, University Medical Centre Groningen, University of Groningen, Groningen, the Netherlands

American Journalof Medical Genetics Part A 2008; 146A: 1853-1858 Chapter 6 Abstract

We report on an infant boy with congenital hydrocephalus due to Ll syndrome and polyuria due to diabetes insipidus. We initially believed his excessive urine loss was from central diabetes insipidus and that the cerebral malformation caused a secondary insufficient pituitary vasopressin release. However, he failed to respond to treatment with a vasopressin analogue, which pointed to nephrogenic diabetes insipidus (NDI). Ll syndrome and X-linked NDI are distinct clinical disorders caused by mutations in the L1CAM and AVPR2 genes, respectively, located in adjacent positions in Xq28. In this boy we found a deletion of 61,577 basepairs encompassing the entireL1CAM and AVPR2 genes and extending into intron 7 of the ARHGAP4 gene. To our knowledge this is the first description of a patient with a deletion of these three genes. He is the second patient to be described with L1 syndrome and NOi. During follow-up he manifested complicationsfrom the hydrocephalus and NOi including global developmental delay and growth failure with low IGF-1 and hypothyroidism.

78 Nephrogenic diabetes insipidus/Ll syndrome Introduction

Ll syndrome is a spectrum of X-linked syndromes caused by mutations in the L1CAM gene. The LlCAM protein plays an important role in the central nervous system development. One manifestation of Ll syndrome is congenital hydrocephalus with aqueduct stenosis. Approximately half of affected children show adductionand flexion of the thumbs, probably reflecting corticospinal tract compression. Other, often milder, clinical forms of Ll syndrome have been described as MASA syndrome (mental retardation, aphasia, shuffling gate and adducted thumbs; OMIM 303350), Complicated SpasticParaplegia type 1 (SP-1), and Agenesis of the corpus callosum (ACC). Nephrogenic diabetes insipidus (NDI) is characterized by the excessive loss of dilute urine caused by the inability of the renal collectingtubules to respond to vasopressin. Mutations in the vasopressin receptor gene (AVPR2) are responsible for 90% of patients with NDI. The genes for these disorders are adjacent on Xq28. Here we report a patient with a contiguous gene deletion leading to Ll syndrome and NOi.

Clinical report

The baby's mother was 35 years old and she had one healthy 2-year-old daughter. She had no history of or abortion. Family history revealed no specificabnormalities. The current pregnancy was without complications and a fetal sonogram in the third trimester showed no structural defects. Labor began after 40 weeks of pregnancy, a caesarean was performed for cephalopelvic disproportion. A boy was born with APGAR scores of 7 and 9 after 1 and 5 min, respectively. He weighed 3,470 g (+1.3 SD) and had a head circumference of 46 cm (+11.1 SD). Physical examination revealed bulging fontanelles with separated sutures, sunset phenomenon, bilateral adducted thumbs with single palmar creases (figure 1), hypertonia of the legs, and glandular hypospadias. Cranial MR imaging showed hydrocephalus with a thin layer of cortex and a relatively small fourth ventricle due to an aqueduct stenosis (figure 1). Initially, a third ventriculostomy was performed, but due to persisting increased , a ventriculoperitoneal shunt was inserted. Ll syndrome was suspected due to the combination of hydrocephalus with adducted thumbs in a boy. In the third week he became less active and lost weight, despite an adequate intake for his age. Laboratory investigation revealed a hypernatremia of 158 mmol/L and increased serum osmolality of 330 mOsm/kg. A relative polyuria was found with urinary osmolality of 89 mOsm/kg. An ultrasound of the kidneys and urinary tract was normal. The initial working diagnosis was diabetes insipid us, probably of central origin due to the cerebral malformations and insufficient vasopressin release. Treatment with extra fluid intake and desmopressin was initiated. However, the baby persistently needed a high fluidintake, which led to the suspicion of NOi. A diagnostic challenge with desmopressin had no effect on urine or serum osmolality and confirmed the diagnosis of NOi (maximum urinary osmolality 200 mOsm/kg). Diuretic treatment (hydrochlorothiazide and amiloride) was started at age 5 months, after which he required less fluid. Further evaluation revealed secondary/tertiaryhypothyroidism (at age 2 months: FT4 9.2 pmol/L; TSH 0.86 mE/L), but MRI revealed no structural abnormalities of the pituitary gland or hypothalamus. Thyroxine supplements were started. At age 2 years a

79 Chapter 6

Figure 1. A: Adduction and flexion of thumb. B: Magnetic resonance image of the brain showing hydrocephalus with aqueduct stenosis (arrow).

percutaneous gastrostomy was installed because of feeding difficulties and stunting (height 80 cm (-3.8 SD); target height: 164 cm (-0.5 SD); weight 11.2 kg (weight for height: +0.2 SD)). Height SD scores did not improve despite adequate nutrition and fluid intake. A stimulation test demonstrated a growth hormone deficiency and supplements were started. During fo llow­ up the boy suffered from global developmental delay: smiling responsively at 6 months, able to sit unsupported at 2 years, walking at 3 years. He is now 5 years old and is just able to make two word combinations. The combination of Ll syndrome and NOi, with the corresponding genes LlCAM and AVPR2 located close to each other on the long arm of the X chromosome, led to the hypothesis of a contiguous gene deletion syndrome and genetic analysis was performed.

Materials and methods

DNA was isolated from peripheral blood by the high salt/chloroform extraction method 111• To analyze the LlCAM and AVPR2 gene, the first and last exons of both genes were amplified using a single PCR program and the products were analyzed on a 2% agarose gel. The MLPA kit "Salsa MLPA POlSc MECP2 kit" from MRC-Holland, containing one probe for exon 4 of the LlCAM gene (personal communication and product information from the MRC-Holland website www. mrc-holland.com), was used to determine the number of LlCAM alleles in the DNA of the boy's mother. The analysis was performed according to the manufacturer's instructions. To prove that the mother is the boy's biological parent, we performed microsatellite analysis with five polymorphic markers. Primers around the LlCAM and AVPR2 gene were designed to map the deletion breakpoints by the presence or absence of fragments (table 1). Amplification was carried out using a standard PCR protocol at the annealing temperature given in table 1. Finally, the deletion junctions were characterized by sequence analysis of the PCR product obtained after amplification, by using primer pair LlCAMdel2 F (4F) and lntron7C R (11R). Sequence analysis was perfo rmed using the ABI Big Dye Te rminator Kit and the ABI Prism 3100 Genetic Analyzer.

80 Ta ble Nephrogenic diabetes insipidus/Ll syndrome Primer name Primer sequence Location PCRproduct 1. Primers to map the deletion breakpoints in proband # AT 58 LlCAMlocus {GenBank U52112.2) lF Exon 1 F GTGGCTGTGCTGCGCGGTGC Around exon 1 No lR Exon 1 R {40GC)CATAGCGGCGAAGGTAGGCG LlCAM gene 2F Exon 28 F {40GC,8T)TTCATTGGCCAGTACAGTG Exon 28 58 No 2R Exon 28 R GAGAGGGAGGGGCCTGGATC LlCAM gene 3F LlCAMdell F TTGCTGTTCCGCCATCCTCAACG 11967-11989 57 No 3R LlCAMdell R TGAGATGGAGTCTCGCTCGATCAC 12344-12367 57 4F LlCAMdel2 F AGGGCTAACAGTGCTAAATC 9447-9466 51 No 4R LlCAMdel2 R GGGAGTTAGGACTCTTCAAACA 9934-9955 51 SF DXS7065 F GGGATGGATATAACATTGG 9160-9178 60 Yes SR DXS7065 R ATAGGCTGTTAGAGTCAC 9069-9085 60 AVPR2 gene {GenBank NC_000023.9) 6F Exon 1-2 P AGTCCGCACATCACCTCCA Exon 1 and 2 65 No 6R Exon 1-2 R TGTCCAGTGGCCTCTCCTG 65 7F Exon 3 P TCTATGTGCTGTGCTGGGCA lntron 2 59 No 7R Exon 3 R CTGAGCTTCTCAAAGCCTCT Exon 3 57 ARHGAP4 gene {GenBank NC_000023.9) Exons 10 and SF Exon 10-11 F CAAGACACCTCCTGCTCCTCCTTGT 68 No 11• SR Exon 10-11 R CAGGAGTATCTGAGTGGACGGAGCAT 68 9F lntron 7A F CTGTACTCCAGCCTGGACAAC lntron 7 53 No 9R lntron 7A R GGGCTTGGTCCTGGATTGGATG 53 10 F lntron 7B F TGTAGTCCCAGCTACTTG lntron 7 50 No 10 R lntron 7B R TCTCTTGCCTCAGGAATG 50 11 F lntron 7C F CAGCTGTGACGCCATCGG lntron 7 54 Yes ll R lntron 7C R CCGCAATGGCCAAGGAGG 12 F Exon 6-7 F TCGAGAACCTTGGCTTTGTC Exons 6 and 7 55 Yes 12 R Exon 6-7 R TCAACGCTGCTGTCAGTAAC 55

• See Demura et al. [10] Results

The first and last exons of the LlCAM gene and the first and last exons of the AVPR2 gene were amplified and the PCR products were analyzed by agarose gel electrophoresis. None of the four fragments could be detected, indicating that both genes could have been deleted. In addition, several X chromosomal polymorphic markers were amplified. In all cases clear PCR products were seen, indicating the good quality of the DNA sample. To determine the exact size of the deletion, different primer pairs at both sites of the genes were used to amplify the DNA. We obtained clear fragments with the primers 5, 11, and 12 (see table 1 and figure 2) and none with the other primer pairs. In additionto the LlCAM and AVPR2 genes, part of the ARHGAP4 gene (figure2) was found to be deleted. The breakpoint was sequenced with the 4F and llR primers. The deletionappears to span 61,577 basepairs and an insertionof one basepair, a Thymidine, was also identified.We showed that the boy's mother does not carry the deletionby using the MLPA technique (see figure 3). Polymorphic marker analysis had proved that the DNA was of the biological mother of our patient.

81 Chapter 6 Chromosome Xq28 Prlmerpalr 5 4 9 10 ]] 12 ! ! ii

l l l WT R LlCAM l ! ! !! ....

WT L: TGC'!'A'l'CAGCCA A'!'C'I'GA'l'CAGG aagtagaaagg gacagctcctccc MT Junction: TGCTATCAGCCAGG ,ATCTGATCAGGtGGGGTGGGTTAI GGGAGCAGCTGTGA WT R: tctgggggtgggagctgggaacggga GGGGTGGGTTA GGGAGCAGCTGTGA

Figure 2. Determination of the deletion. Solid arrows indicate primer pairs (1, 2, 3, 4, 6, 7, 8, 9, 10) that did not amplify. Broken arrows indicate primer pairs (5, 11, 12) that did amplify. WT L: Wild-type sequence downstream the LlCAM gene, WT R: Part of the ARHGAP4 sequence around the breakpoint site. MT junction: The breakpoint sequence of our patient.

C C C C C

Patient cl'

Mother

Control i

Control cl'

Figure 3. MLPA analysis. The DNA was amplified with the specific probes and the fragments obtained were analyzed on an ABI 377 sequencer. The peak area is a measure for the number of alleles. C: control probes for two alleles. Cx control probes located on the X-chromosome. 1. LlCAM probe (arrow). 2. MECP2 probe. No signal was obtained with the LlCAM probe and the patient's DNA, as expected. The control male gave a peak half as high as those in a control female and the boy's mother.

82 Nephrogenicdi abetes insipidus/Ll syndrome Discussion

Ll syndrome and NOi are two distinct clinical disorders caused by mutations in genes located in the same area of the X-chromosome. In the present patient, we found a deletion of 61,577 basepairs encompassing the entireL1CAM and AVPR2 genes and extending into the ARHGAP4 gene, located telomeric to AVPR2. To our knowledge, this is the second patient to be described with Ll syndrome and X-linked NOi. Te gay et a!Y1 described an infant boy with the same combination and found a 32.7 kb deletion from L1CAM intron 1 to AVPR2 exon 2. The boy also sufferedfrom Hirschsprung's disease. The child died at 89 days. His asymptomatic mother was found to carry the same microdeletion. Hirschsprung's disease has been reported in patients with L1CAM mutationsl31. In contrast, the present patient has a larger deletion but a milder phenotype and does not show any signs of Hirschsprung's disease. Patients with NDI need a high fluid intake, and often suffer from vomitingand feeding difficultiesl41• The combination with Hirschsprung's disease could further compromise intake and uptake of adequate amounts of nutrition. There is one other report of a patient with the possible combination of Ll syndrome and NDI, a 15-week-old girl with a combination of congenital hydrocephalus and NDll51• No follow up data or family history were reported and it was not possible to detect mutations in the L1CAM and AVPR2 genes at that time. Symptomatic female heterozygotes have been described for both AVPR2 and L1CAM mutations. The X-chromosome with the normal allele was found to be preferentially inactivated by methylation in the women with the disease phenotypel6·81. To our knowledge, the present patient is the only person described with a deletion of both L1CAM and AVPR2 with part of the ARHGAP4 gene. The ARHGAP4 protein is a signaling protein, involved in the regulation of GTP-binding proteins of the RAS superfamily. It is predominantly expressed in hematopoietic cells, but also in the placenta and lung and during fetal development. Contiguous gene deletions of AVPR2 and the ARHGAP4 have been described, but these patients only showed signs of NDI and not hematopoietic disorders or other symptoms19·111• A boy has been described with a 34.4 kb deletion encompassing the AVPR2 and ARHGAP4 gene, extending further into a highly conserved DNA segment between ARHGAP4 and AR01A1121.This chromosomal segment is believed to contain regulatory elements for hematopoietic cells. That patient also suffered from severe combined immunodeficiency. Similar to the patients described by Schoneberg et af.l91 and Demura et a/. 1101 the patient in the present report showed no hematologic abnormalities. In addition to neural tissue, the L1CAM adhesion protein is expressed in the urogenital tract. Ll activity has been found in epithelial cells of differentregions including the epididymal tail, deferent duct, ejaculatory duct, seminal vesicles, bladder, and urethra1131• The Ll protein is also expressed throughout collecting duct development in the mesonephric duct and mesonephros. Mice with a targeted deletion of the L1CAM gene show various renal malformations including duplex kidneys and malformed or an incorrectly positioned outer medullaf141• In humans, only one patient with Ll syndrome has been described with congenital abnormalities of the kidney and urinary tract (i.e., duplex kidneys)I151_ The present patient showed normal renal anatomy on ultrasound but had mild hypospadias. We observed secondary/tertiary hypothyroidism, low IGF-1 and growth retardation in the patient reported here. We believe this to result from both the NDI and the treatment of hydrocephalus. These endocrine abnormalities are common in patients with hydrocephalus

83 Chapter 6 after third ventriculostomy and are possibly related to the perforation of the third ventricle floor (part of the hypothalamic-pituitary neuronal network) or unphysiological intracranial pressure profiles developed after a shunt procedurel16• 171• These patients have a higher incidence of growth failure1181, which has also been described in NDl[41. We did not find any possible mutationalmechanisms at the breakpoints of deletiondelineated in this report. Chromosome rearrangement breakpoints predominate in pericentromeric and subtelomeric regions and low copy repeats (LCR), Alu elements, AT-rich palindromes and pericentromeric repeats are often found at these sites[191 • Te gay et a/Y1 identifiedshort direct repeat homology between the junctional insertionfragments and the flanking segments at the breakpoints in their patient with Ll syndrome and NDI. As they state, a common predisposing mechanism for this particularcontiguous gene syndrome seems unlikely, given the rarity of the genomic disorder described. In conclusion, we here report a second patient with contiguousgene deletion syndrome with Ll syndrome and NDI due to haploinsufficiencyof the LlCAM and AVPR2 genes. In contrast to the patient described previously[21 the patient reported here displays a milder clinical course, possibly due to the absence of Hirschsprung's disease. In addition to the typical features of Ll syndrome and NDI, he demonstrates severe growth failure with growth hormone deficiency and hypothyroidism. We regard these as secondary features of the hydrocephalus. Glandular hypospadias could be related to the LlCAM mutation. The deletion discovered in this patient was found to extend into a third gene: ARHGAP4. However, mutations in this gene are not associated with a clearly defined clinical phenotype.

Acknowledgements We thank the patient's family for their cooperation. Furthermore, we are grateful to Jackie Senior for revising the manuscript.

84 Nephrogenic diabetes insipidus/Ll syndrome

References

(1) Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988 Feb 11;16(3):1215. (2) Tegay DH, Lane AH, Roohi J, Hatchwell E. Contiguous gene deletion involving LlCAM and AVPR2 causes X-linked hydrocephalus with nephrogenic diabetes insipidus. Am J Med Genet A 2007 Mar 15;143(6):594-8. (3) Okamoto N, Del Maestro R, Valero R, Monros E, Poo P, Kanemura Y, Yamasaki M. Hydrocephalus and Hirschsprung's disease with a mutation of LlCAM. J Hum Genet 2004;49(6):334-7. (4) van Lieburg AF, Knoers NV, Monnens LA. Clinical presentation and follow-up of 30 patients with congenital nephrogenic diabetes insipidus. J Am Soc Nephrol 1999 Sep;10(9):1958-64. (5) Aggarwal R, Janakiramen N, Luken J, Kumar S. Nephrogenic diabetes insipidus in a female infant with hydrocephalus. Am J Dis Child 1986 Nov;140(11):1095-6. (6) Kaepernick L, Legius E, Higgins J, Kapur S. Clinical aspects of the MASA syndrome in a large family, including expressing females. Clin Genet 1994 Apr;45(4):181-5. (7) Nomura Y, Onigata K, Nagashima T, Yutani S, Mochizuki H, Nagashima K, Morikawa A. Detection of skewed X-inactivation in two female carriers of vasopressin type 2 receptor gene mutation. J Clin Endocrinol Metab 1997 Oct;82(10):3434-7. (8) Kinoshita K, Miura Y, Nagasaki H, Murase T, Banda Y, Oiso Y. A novel deletion mutation in the arginine vasopressin receptor 2 gene and skewed X chromosome inactivation in a female patient with congenital nephrogenic diabetes insipidus. J Endocrinol Invest 2004 Feb;27(2):167-70. (9) Schoneberg T, Pasel K, von Baehr V, Schulz A, Volk HD, Gudermann T, Filler G. Compound deletion of the rhoGAP Cl and V2 vasopressin receptor genes in a patient with nephrogenic diabetes insipidus. Hum Mutat 1999;14(2):163-74. (10) Demura M, Takeda Y, Yoneda T, Furukawa K, Usukura M, Itch Y, Mabuchi H. Two novel types of contiguous gene deletion of the AVPR2 and ARHGAP4 genes in unrelated Japanese kindreds with nephrogenic diabetes insipidus. Hum Mutat 2002 Jan;19(1):23-9. (11) Dong Y, Sheng H, Chen X, Yin J, Su Q. Deletion of the V2 vasopressin receptor gene in two Chinese patients with nephrogenic diabetes insipidus. BMC Genet 2006;7:53. (12) Broides A, Ault BH, Arthus MF, Bichet DG, Conley ME. Severe combined immunodeficiency associated with nephrogenic diabetes insipidus and a deletion in the Xq28 region. Clin Immune! 2006 Aug;120(2):147-55. (13) Kujat R, Miragall F, Krause D, Dermietzel R, Wrobel KH. lmmunolocalization of the neural cell adhesion molecule Ll in non-proliferating epithelial cells of the male urogenital tract. Histochem Cell Biol 1995 Apr;103(4):311-21. (14) Debiec H, Kutsche M, Schachner M, Ronco P. Abnormal renal phenotype in Ll knockout mice: a novel cause of CAKUT. Nephrol Dial Transplant 2002;17 Suppl 9:42-4. (15) Liebau MC, Gal A, Superti-Furga A, Omran H, Pohl M. LlCAM mutation in a boy with hydrocephalus and duplex kidneys. Pediatr Nephrol 2007 Jul;22(7):1058-61. (16) Leppanen T, Paakko E, Laitinen J, Saukkonen AL, Serio W, Tapanainen P, Ruokonen A, Pirttiniemi P, Poikela A, Knip M. Pituitary size and function in children and adolescents with shunted hydrocephalus. Clin Endocrine! (Oxf) 1997 Jun;46(6):691-9. (17) Fritsch MJ, Bauer M, Partsch CJ, Sippel! WG, Mehdorn HM. Endocrine evaluation after endoscopic third ventriculostomy (ETV) in children. Childs Nerv Syst 2007 Jun;23(6):627-31. (18) Leppanen T, Saukkonen AL, Serio W, Tapanainen P, Ruokonen A, Knip M. Reduced levels of growth hormone, insulin-like growth factor-I and binding protein-3 in patients with shunted hydrocephalus. Arch Dis Child 1997 Jul;77(1):32-7. (19) Shaw CJ, Lupski JR. Implications of human genome architecture for rearrangement-based disorders: the genomic basis of disease. Hum Mol Genet 2004 Apr 1;13 Spec No 1:R57-R64.

85

Chapter 7

Complex pathogenesis of Hirschsprung's disease in a patient with hydrocephalus, vesico-ureteral reflux and a balanced translocation t(3;17)(p12;qll)

1 2

4 3 1 5 2 1 , 3 Paola Griseri , Yvonne Vos1 , Roberto Giorda , Stefania Gimelli , Silvana Beri , Giuseppe Santamaria , Guendalina Mognato , Robert Hofstra Giorgio Gimelli and Isabella Ceccherini 1

2 Laboratory Molecular Geneticsand Cytogenetics, /st. G. Gaslini, Genova, Italy Department of Genetics, University Medical Centre 31 4Groningen, University of Groningen, Groningen, the Netherlands E. Medea' Scientific Institute, Bosisio Parini, Lecco, Italy

5Dipartimento di Patologia Umana ed Ereditaria, Biologia Generate e Genetica Medico, Universita di Pavia, Pavia, Italy Clinica Chirurgica Pediatrica, Dipartimento di Pediatria, Universita di Padova, Padova, Italy

European Journalof Human Genetics 2009; 17: 483-490 7

Chapter Abstract

Hirschsprung's disease (HSCR), a congenital complex disorder of intestinalinnervation, is often associated with other inherited syndromes. Identifying genes involved in syndromic HSCR cases will not only help understanding the specificunderlying diseases, but it will also give an insight into the development of the most frequent isolated HSCR. The association between hydrocephalus and HSCR is not surprising as a large number of patients have been reported to show the same clinical association, most of them showing mutations in the L1CAM gene, encoding a neural adhesion molecule often involved in isolated X-linked hydrocephalus. L1 defects are believed to be necessary but not sufficient for the occurrence of the intestinal phenotype in syndromic cases. In this paper, we have carried out the molecular characterization of a patient affected with Hirschsprung's disease and X-linked hydrocephalus, with a de nova reciprocal balanced translocation t(3;17)(p12;q21). In particular, we have taken advantage of this chromosomal defect to gain access to the predisposing background possibly leading to Hirschsprung's disease. Detailed analysis of the RET and L1CAM genes, and molecular characterization of MY018A and TIAF1, the genes involved in the balanced translocation, allowed us to identify, besides the Ll mutation c.2265delC, different additional factors related to RfT-dependent and -independent pathways which may have contributed to the genesis of enteric phenotype in the present patient.

88 HSCR disease in a patientwith an LlCAM mutation Introduction

Hirschsprung's disease is the major congenital defect affecting the enteric nervous system (ENS). It is characterized by the absence of enteric ganglion cells along a variable length of the intestine. Mutations of the RET proto-oncogene, encoding a tyrosine kinase receptor mainly involved in neural crest cells development, were identified in 1994 as being responsible for the disease, and are still considered as the most frequent cause of intestinal aganglionosis (7-35% of sporadic patients)r11• Besides RET, nine other HSCR susceptibilitygenes have been identified, most of them found by studying pedigrees with the recurrence of intestinal aganglionosis in association with other clinical signs (syndromic HSCR forms)r2-41• The greatest proportion of these genes has been shown or supposed to be related to three specific pathways that are involved in different cellular programmes crucial for the normal development of the enteric nervous system: the RET pathway (RET, GDNF, NTN, SOXlO, PHOX28), the endothelin pathway (EDN3, EDNRB, SOXlO} and, to a lesser extent, the TGF-6 signalling pathways {ZFHXl B). However, the great proportion of unexplained cases, incomplete penetrance and the variable expressivity suggest that the disease is the result of complex interactions between known and/ or unknown modifier genes. Additional evidence supporting this model is the existence of other genetic syndromes such as trisomy 21, Smith-Nemli-Opitz, and cartilage-hair hypoplasia which show an association with HSCR to a variable extent. All these data suggest that studying syndromic families showing an association of HSCR with other congenital abnormalities might be worthwhile. In this paper, we have carried out the molecular characterization of a patient affected with HSCR disease, X-linked hydrocephalus, and vesico-ureteral reflux showing a de nova structural chromosome anomaly t(3;17)(p12.11;q11.21). X-linked hydrocephalus has an incidence of 1/30 000 male births and it is characterized by severe mental retardation, spastic tetraplegia and bilateral adducted thumbs, with major additional phenotypes including agenesis of corpus callosum and/or corticospinal tract. The great proportion of cases is ascribed to loss of function mutations of LlCAM, a neural cell adhesion molecule located in Xq28 and involved in the development of the , axonal tracts and the cerebellumrsi . The association between hydrocephalus and HSCR is not surprising as brain development is largely controlled by the same neural growth factors acting in the ENS161• Until now, mutations of the LlCAM gene have been found in seven out of eight patients reported to show association of X-linked hydrocephalus with HSCR diseaser7-9l_ These results suggest that mutations in LlCAM may be involved in HSCR development in association with a predisposing genetic background. Ta king advantage of the chromosomal rearrangement t(3;17)(p12.11;q11.21) detected in our patient, we have identified genes encompassing the breakpoints and evaluated their possible involvement as additional genetic factors which may predispose to syndromic HSCR pathogenesis.

89 7

Chapter Materials and methods

Cytogenetics investigations Fluorescent in situ hybridization (FISH) was carried out using genomic BAC clones spanning the chromosomal 3p12 and 17qll, two regions selected from the human library RPCl-11 according to the UCSC Human Genome Assembly (March 2006 freeze). Array-CGH was performed using the Agilent Human Genome CGH Microarray Kit 44B (Agilent Technologies)r101 •

Gene analysis and profile expression Screening of LlCAM and RET coding regions was performed as already describedl6,111• The expression profile of Myosin XVIIIA (MY018A} and TGF-81-induced antiapoptotic factor l (TIAF1) in human tissues was performed on a human cDNA commercial panel (Origene technology Inc., Rockville, MD, USA; www.origene.com). Sequence primers are available under request.

Gene expression levels Analysis of SNP rs2320786, corresponding to the synonymous substitution p.A1470A of the MY018A gene, was performed on the patient's genomic DNA and corresponding cDNA. Quantitative determination of MY018A, TIAF1 and RET expression levels was performed using the ABI Prism 7500 Sequence Detection System in combinationwith TaqMan chemistry (SYBR Green PCR Master Mix. PE Biosystems), according to the manufacturer's guidelines. PCR conditions and primer sequences are available under request.

Results

Clinical report The boy (NF, 10 years) is the first child of healthy non-consanguineous parents. He was electively delivered at 38 weeks of gestationalage by cesarean section, with a birth weight of 3620 g, length of 52 cm, and macrocephaly (OFC=39.5 cm). Ultrasound examination revealed marked ventriculomegaly with reduction of the brain cortex and a shunt was inserted at 8 days after birth. At 16 months he was evaluated for chronic constipation and abdominal distention. Rectal suctionbiopsy and histological examination confirmed the diagnosis of a short form of HSCR with absence of ganglion cells in the rectosigmoid colon. At 20 months, he underwent a laparoscopic colectomy for short-segment disease. At the age of 9 years, bilateral spastic paraplegia, adducted thumbs and mental retardation were present. He interacts exclusively with a social smile and eye contact, whereas speech is limited to a few words. The patient was also diagnosed with a severe vesico-ureteral reflux (VUR) associated with recurrent urinary infections. For the last 2 years the patient has been treated with continuous low-dose antibiotic prophylaxis.

Karyotype analysis Standard karyotype performed on peripheral blood lymphocytes from the patientshowed a de nova reciprocal balanced translocation 46,XY, t(3;17)(pl2.l;qll.21} (figure 1). The karyotype of both parents was normal.

90 HSCRdisease in a patient with an LlCAM mutation

Figure 1. Cytogenetics and FISH results obtained from the patient's lymphocytes. (a) Cut-out and (b) ideogram of the normal

dor(3) der(l7) 17 and derivative chromosomes 3 and 17. (c) FISH with BAC RP11-331K15 (black signals) shows the chromosome 3 breakpoint on d the short arm. Arrowhead indicates the b (d) signal on normal chromosome 3 and the arrows indicate the der(3) and der(17) ii ii derivative chromosomes. FISH with ...ii ...; BAC RP11-321Al 7 demonstrates that the ...... � ! chromosome 17 breakpoint is within this !; clone. Signals on normal chromosome 17 =;; is indicated by arrowhead whereas der(3) � der(3) der(17) l7 and der(l 7) derivative chromosomes are indicated by arrows .

Figure 2. Sequence analysis of patient's DNA, showing the c.2265delC (p.Pro756fs) mutation in the LlCAM gene. The sequence of the parental DNAs demonstrates that the mutation is inherited from the heterozygous mother. Molecular screening of L1CAM and RET genes LlCAM is a cell adhesion molecule responsible for X-linked hydrocephalus affected males. Molecular testing of the gene revealed a single nucleotide deletion in exon 18 in the patient's DNA, inherited from the healthy mother, which leads to a premature stop codon causing the protein truncation at the level of the third fibronectin type Ill domain (c.226SdelC, p.Pro756Leufs9SX) (figure 2). This kind of mutation is commonly associated with the most severe phenotype of X-linked hydrocephalus151 • In the attempt to explain the intestinal phenotype of the patient, we performed the mutationanalysis of the RET gene. Analysis of the 21 exons of the gene did not reveal any mutation in the coding region or in the intron-exon junctions. Interestingly, the patient showed a heterozygous result for the RET predisposing

91 7

Chapter haplotype, a specific combination of genetic variants located between the promoter and the second exon of the gene, largely reported to be over-represented in HSCR patients with respect to controls1121• This haplotype has been associated with reduced gene expression and, accordingly, demonstrated in strong linkage disequilibrium with an intronic nucleotide substitution which has been shown to impair an RfTtranscriptionalenhancer in vitro1131• The molecular analysis of LlCAM and RET genes is fully in agreement with the previous screening performed on eight patients with the association of hydrocephalus and HSCR disease, most of which showed severe LlCAM defects but no RET coding mutations17-91• The presence of an LlCAM mutation fully accounts for the neurological phenotype, whereas the molecular defect causing HSCR disease remains unknown. The main hypothesis proposed to explain the co-occurrence of the two diseases is that the LlCAM mutation itself may confer a predisposing background on which additional factors may act causing the disease phenotype.

Breakpoint characterization To define the translocationat the molecular level, we constructed two genomic contigs with BACs covering the region containing the breakpoints on both chromosomes. For chromosome 3p12.1, the breakpoint mapped to the RP11-331K15 clone, giving signals on normal chromosome 3, on derivative der(3), and on derivative der(17). Using a similar strategy, we were also able to map the chromosome 17q11.2 breakpoint to the BAC clone RP11-321A17 (AC024619). Comparativegenomic hybridization (CGH) array analysis of patient DNA allowed us to exclude the presence of further deletions or duplications elsewhere in the genome with a resolution of about 75 Kb. Somatic cell hybrids from the patient's lymphoblasts were generated to separate the normal chromosomes 3 and 17 and the two derivative chromosomes der(3) and der(17). DNA from these hybrids was used in a PCR-based strategy with selected non-polymorphic markers spanning the region defined by BAC clones (not shown). We therefore localized both breakpoints, amplified and sequenced both junction fragments thus providing the exact molecular definition of the translocation (figure 3). The breakpoint on der{3) lacks one nucleotide from each parental chromosome, whereas the der(17) breakpoint contains seven additional bases, probably derived from a partial duplication of a nearby sequence (figure 3). There is no apparent homology between the two sequences, and no repeats are involved The chromosome 3 breakpoint is located at 390 kb far from the 3'UTR of the CADM2{/GSF4D) gene and 480 kb far from the 3' terminal of the VGLL3 gene. Comparative genomic analysis performed with the Genome Vista browser ((http://pipeline.lbl.gov/index.html) did not reveal any significant homology with the mouse sequence, thus making unlikely the presence of a regulatory region in this genomic fragment. On chromosome 17 the breakpoint occurred in a region containing two genes, MY018A and TI AFl. Myosin XVIIIA is encoded by 40 exons with the production of two alternative isoforms. The protein has recently been demonstrated as a high affinityreceptor (SP-R210) for surfactant protein A {SP-A), a highly versatile immune molecule, which contributes significantly to surfactant homeostasis and pulmonary immunityl141_ TGF-81-induced antiapoptoticfactor 1 (TIAFl) is an intronless gene, which resides within the 3' untranslated region of the MY018A gene. Identified and cloned in 1998, TIAFl was shown to participate in the transforming growth factor Bl-mediated growth regulation by both increasing the expression of p53, Cip1/p21, and Smad proteins, suppressing ERK phosphorylation, and

92 HSCR disease in a patientwith an LlCAM mutation 88500000 am i em i WPi 86500000 ) 87000000 I 87500000 88000000 t 3p11.2 f f f chr3 CGGBPI CGGBP1 L VGLL31 CGGSPHTR1F4I BC0'".,()34.41 C3ori38) CHMP2BI POU1F1I civ-11 I ZNF654I 17q11.2

I PIPOX H t I I TAOKI ...... g!�=� 24300000 24400000 I 24500000 I 24600000 24100000 TAOl<1 ...... ,. C17orl� --« CRYBA1 1 MY018ATIAFI L.11'"111A1"� BC0566621 I MY016A1-11111111---+-t NUFlP21t-H ERA!.1' , I MY018AHllfffl..,...... ,._"'f-1 ERA!.1 "'91> OI-IRS13UN0419 oogc J FLOT2 BC033W7 _. AX795351OKFZp547N203 ..- Chrom. Position FLJ00295� Sequence 17 24488584 - 2448864, ATAGTGAACTCAATGCCTGGGT TTGCAGG G AGGGCAAGGACACTGaaattgcttagtagc 3 (rev) 86590820 - 86590761 TTTCATTACCATTTATCTTTTTATTCTTAA C ACCTATAATAGCATATTTCCTAAAAGATA der(3) TTTCATTACCATTTATCTTTTTATTCTTAA AGGGCAAGGACACTGaaattgcttagtagc der(17) ATAGTGAACTCAATGCCTGGGT TTGCAGG CATTGCA ACCTATAATAGCATATTTCCTAAAAGATA

Figure 3. Map of the tran51ocated regions. The breakpoint on chromosome 3 is located between the CADM2(1GSF4D) and VEGL3 genes. The breakpoint on chromosome 17 interrupts the MY018A and TIAFl genes. The sequences at the breakpoints are reported in the table at the bottom. 15 15 altering TGF-81-mediated Smad2/3 phosphorylation[ , J_ It has also been shown that TI AFl and p53 functionallyinteract in regulating apoptosis, and TIAFlis likely to participatein the nuclear translocation of activated pS3[17J_ The role of TIAFl in preventing cellular apoptosis is very intriguing in the light of the patient's HSCR phenotype as it has been hypothesized that one of the causes of HSCR pathogenesis is a defect in normal survival of enteric neurons and/or premature apoptosis[1l. The expression profile of the MY018A and TIAF genes performed on a panel of 24 human tissues is in agreement with literature data, showing a quite ubiquitous presence of the two genes with a preferential expression in the muscle (data not shown). Considering the role of TIAFl as an antiapoptotic factor, we decided to carry out a genetic analysis of this gene to determine if it may affect the survival of enteric neurons, playing a role in the normal development of the intestine. To this end, we screened the gene in four additional patients showing the associationof X-linked hydrocephalus with HSCR disease. An analysis of 1256 bp of the MY018A 3'UTR of gene, containing the whole coding region of TIAFl, did not show any alterationin these individuals, with the exception of a silent polymorphism (rs1049848) in one patient. To further investigate the role of TIAFl in favouring the intestinal phenotype in syndromic HSCR, we analysed a selected group of 17 HSCR patients showing aganglionosis in association with other clinical symptoms (six multiple malformations, five limb defects, three dismorphisms, one renal hypoplasia, one mental retardation, and one cardiopathy). We did not find any mutation or polymorphic variant of the TIAFl gene in these patients, a circumstance which does not support the effectof mutations of this gene as a major event in HSCR disease.

93 7

Chapter MYO18A Genomic DNA MYO18A cDNA

G C G C a

r

b MYO18A TIAFl 12 6 -� eC: 1 f � 0.8 � ,Q 0.6 <( � z C1J 0.4 E C1J� 0.2 u...-< "O <( 0 �� control patient

Figure 4. Expression profile of the patient. (a) Monoallelic expression at an informative polymorphic marker locus of the MY018A gene, determined by comparing the sequence of the patient's genomic DNA with his corresponding lymphoblasts cDNA. (b) Quantitative determination of the expression levels of MY018A and TIAFl in patient's lymphoblast mRNA. Data are normalized on the control's gene expression levels. e .Q § � � 0.8 Figure 5. RET expression in lymphoblastoid cells. RET O...Q � l/l 0.6 expression levels are reduced in the patient carrying <( � one copy of the predisposing haplotype compared � � 0.4 E a> with a normal wild-type control. f- :!2 0.2 UJ 0 a::u.. - 0 CONTROL PATIENT

Expression profile of the patient After investigatingthe presence of informative alleles of single nucleotide polymorphisms (SNPs) of the MY018A and TIAF1 genes among those reported in the SN P database (http://www.ncbi. nlm.nih.gov/SNP/), the patient showed a heterozygous result CIT for rs2320786 (p.A1470A) in the coding region of MY018A. RT-PCR performed on patient lymphoblasts mRNA could confirmthe presence of the only C allele at the SNP rs2320786 locus, suggesting a monoallelic expression of the MY018A gene as a likely result of the translocation (figure 4a). To exclude a physiological monoallelic expression of this gene, we checked if the MY018AITIAF1 gene(s) and the 17q21 region have previously been implicated in imprinting(http://igc .otago.ac.nz/home. html), finding negative results. Moreover, we also performed Real-time RT-PCR for the two genes involved in the translocation, testing the gene expression level in the patient compared with a normal control individual (figure 4b). MY018A and TIAF1 show a reductionin their expression of approximately SO% in patient's mRNA with respect to the double expression dose detected in the control, thus confirming a monoallelic expression because of the translocation.

94 HSCR disease in a patient with an LlCAM mutation It has been proposed that the presence of a RET predisposing haplotype can increase the risk of aganglionosis above the threshold. For this reason, we performed real-time RT-PCR on the RET gene using lymphoblasts' mRNA of our patient, who carries one copy of the RET predisposing S'haplotype, and compared results to that obtained using mRNA from a control homozygote for a wild-type version of the RET gene. Our patient showed a marked reduction of RET mRNA (58 vs 100% of the control, P-value=0.019) (figure 5), a finding in agreement with previous data showing 18 an impaired RET expression associated with this haplotype1 1• Our data support the hypothesis of a major role played by the association of L1CAM mutation and the RET predisposing haplotype in the occurrence of the two phenotypes, as previously reported in eight patientsl7-9l_ Moreover, based on present results, the hypothesis that the impaired expression of the MY018AITIAF1 genes represents an additional risk factor, acting together with LlCAM and RET defects in our patientto the occurrence of the Hirschsprung's phenotype, cannot be excluded.

Discussion

In this paper we describe a patient associated with HSCR and X-linked hydrocephalus, who shows a mutation in the L1CAM gene, a decreased RET expression and haploinsufficiency of two genes involved in a balanced translocation (3;17)(p12;q21). Of eight patients reported so far with co-occurrence of HSCR and hydrocephalus, seven showed mutations in the L1CAM gene, which was proposed as a candidate HSCR gene. L1CAM may be implicated in HSCR for several reasons. First of all, the 4:1 male/female bias in the incidence of HSCR has suggested, for a long time, a role of an X-linked gene in the disease aetiology; however, linkage studies have failed to yield any X chromosome locus and, to date, L1CAM is the only X-linked gene known to associate with HSCR.l191.Second, L1CAM expression has been shown to be under tight regulation in mouse ret k-/k- intestine t201 and a selective deficit in L1CAM immunostaining has been reported in sectionsof bowel from patients with HSCR1211• Third, L1CAM is expressed exclusively by neural crest-derived cells in the developing mouse gut and L1CAM-deficient mice showed a small but significant reduction in enteric 22 neural crest cell migration throughout the developing gut1 1 • Although these observations suggest an important role for L1CAM in the migration of neural crest cells in the developing gut, the low incidence of HSCR disease in patientswith L1CAM mutations (about 3%) and the absence of L1CAM mutations in males affected with isolated HSCR suggest that L1CAM is not an HSCR causative gene but provides a predisposing genetic background, likely acting as an X-linked modifier gene for the development of HSCR17-91• Additional factors may act on this background promoting the occurrence of Hirschsprung's disease, among which the RET predisposing haplotype, found in our infant affectedwith HSCR and hydrocephalus and carrying a L1CAM mutation. In agreement with the presence ofone copy of this low-expressed RET haplotype, we found a marked decrease of the RET gene expression in our patient compared with a normal control. Though still in need of further validation in a larger number of patients, these data seem fully in agreement with the model proposed for other syndromes also showing association with HSCR disease, such as the congenital central hypoventilation syndrome (CCHS), Bardet-Biedl Syndrome and Down syndrome, and sharing the RET predisposing haplotype as a risk factor for the intestinal phenotype1231•

95 7

Chapter WILD - TYPE PATIENT • •

EDNRB RET TGFB EDNRB RET TGFB Signalling Signalling Signalling Signalling Signalling Signalling +++ +++++ ++ +++ +++ + l l l l l D l EDNRB­ RET­ TGFbeta- EDNRB- RET­ TGFbeta­ dependent dependent dependent dependent dependent dependent genes genes: �n� �n� genes: genes LlCAM � �---v--- � �---�----==---v -:::::___ / Normal development of enteric nervous system �

Figure 6. Hypothetical model of Hirschsprung pathogenesis in our patient. The three major pathways involved in the normal development of the enteric nervous system are shown. RET decreased expression and TIAF haploinsufficiencycan concur together with LlCAM mutationto HSCR pathogenesis. With respect to the de nova balanced translocation carried by the patient, we considered two different scenarios: (i) the de nova balanced translocation can be non-pathogenetic and only by chance associated with the disease phenotype; ii) the genes involved in the translocation may act as modifier genes in HSCR and/or in other phenotypes observed in the patient. MY018A, encoding for the receptor of collectin SP-A, seems not to be involved in HSCR disease but may play a pivotal role in the immunity response of the lung. However, with the exceptionof a hospitalization for bronchiolite at 9 months of age, untilnow our patient does not show any susceptibilityto recurrent pulmonary infectionsor asthma and allergic reactions. More interestingly, the second gene involved in the translocation, TIAF1, may play a role as predisposing HSCR factor by acting in the TGF-8-signalling pathway as an antiapoptotic factor. One of the known HSCR genes, ZFHX1B, is also involved in the TGF-8/BMP/Smad-mediated signalling cascade as a transcriptional repressor of smad proteins. Indeed, mutations of the ZFHX1B gene results in Mowat-Wilson syndrome, a multiple congenital anomaly characterized by mental retardation,microcephaly and, at variable extent, Hirschsprung's disease, congenital heart disease, genitourinary anomalies and hypospadiasl51. The patient described here shows haploinsufficiency for T/AF1, but further molecular analyses allowed us to detect mutations neither in four patients showing hydrocephalus and HSCR disease nor in 17 additional HSCR patients characterized by different syndromic

96 HSCR disease in a patient with an LlCAM mutation associations;therefore, no definitive conclusion can be drawn regarding the role of TIAF1 in HSCR. Nonetheless, in the present patient, we hypothesize that TIAF1 haploinsufficiency may have perturbed an alternative, RET-independent HSCR pathway, namely the TGF-8-signalling pathway, thus increasing not only the risk of developing intestinal aganglionosis but also, based on the control that TGF-8 variants seem to exert on the pathogenesis of urinary tract infection and vesico-ureteral refluxl241,the risk of developing VUR. In particular, we propose a complex mechanism in which one copy of the RETpredisposing haplotype and the L1CAM mutation play a critical role affecting the RET-mediated cellular program, whereas TIAF haploinsufficiency may concur to HSCR disease and VUR pathogenesis by altering a RET-independent-signalling pathway (figure 6). However, the pathogenetic mechanism we have postulated in the present patient, while confirming the complex genetic etiology of syndromic HSCR, cannot be easily generalized to other cases. Interestingly, both the pathways mediated by TGF-8 and Ret may concur to the development of the VUR observed in our patient. VUR has often been reported to be associated with TGF-8 signal-dependent Mowat-Wilson syndrome (38%) and TGF-81 polymorphisms have been recently shown to be associated with the susceptibility to VURls, 241_ On the other hand, the RET gene plays a critical role in kidney development and it has been 25 found involved in isolated congenital anomalies of the kidneyl I • Considering that, like HSCR, vesico-ureteral reflux is a genetically heterogeneous trait which may occur as a result of the complex inheritance, we suggest that contemporary defects in the two pathways (decreased RET expression and TIAF1 haploinsufficiency) may be critical for the onset of vesico-ureteral reflux in our proband and that these different pathways may be involved in other cases of either syndromic or isolated vesico-ureteral reflux or renal malformations. Further screening of the TIAF1 gene in other HSCR patientswith association of vesico-uretral reflux or other renal malformations may be helpful to better understand the molecular basis of the disease.

Acknowledgement The financial support of Italian Telethon (Grant GGP04257 to IC) is gratefully acknowledged.

97 Chapter ------

7 References

The Me tabolic & Mo lecular Bases of lnherited Disease. (1) Chakravarti A, Lyonnet S: Hirschsprung Disease; in: Scriver CR, Beaudet AL, Valle D, Sly WS, Childs B, Kinzler KW, Vogelstein B (eds). New York: International Edition, McGraw-Hill, 2001, 8{1V): 6231-55. (2) Amiel J, Lyonnet S. Hirschsprung disease, associated syndromes, and genetics: a review. J Med Genet 2001 Nov;38(11):729-39. (3) Mowat DR, Wilson MJ, Goossens M. Mowat-Wilson syndrome. J Med Genet 2003 May;40(5):305-10. (4) Brooks AS, Bertoli-Avella AM, Burzynski GM, Breedveld GJ, Osinga J, Boven LG, Hurst JA, Mancini GM, Lequin MH, de Coo RF, Matera I, de Graaff E, Meijers C, Willems PJ, Tibboel D, Oostra BA, Hofstra RM. Homozygous nonsense mutations in KIAA1279 are associated with malformations of the central and enteric nervous systems. Am J Hum Genet 2005 Jul;77(1):120-6. (5) Weller S, Gartner J. Genetic and clinical aspects of X-linked hydrocephalus (Ll disease): Mutations in the LlCAM gene. Hum Mutat 2001;18(1):1-12. (6) Moore SW. The contribution of associated congenital anomalies in understanding Hirschsprung's disease. Pediatr Surg Int 2006 Apr;22(4):305-15. (7) Hofstra RM, ElfferichP, Osinga J, Verlind E, Fransen E, Lopez Pison J, Die-Smulders CE, Stolte-Dijkstra I, Buys CH. Hirschsprung disease and LlCAM: is the disturbed sex ratio caused by LlCAM mutations? J Med Genet 2002 Mar;39(3):E11. (8) Parisi MA, Kapur RP, Neilson I, Hofstra RM, Holloway LW, Michaelis RC, Leppig KA. Hydrocephalus and intestinal aganglionosis: is LlCAM a modifier gene in Hirschsprung disease? Am J Med Genet 2002 Feb 15;108(1):51-6. (9) Okamoto N, Del Maestro R, Valero R, Monros E, Pao P, Kanemura Y, Yamasaki M. Hydrocephalus and Hirschsprung's disease with a mutation of LlCAM. J Hum Genet 2004;49(6):334-7. (10) Bocciardi R, Giorda R, Marigo V, Zordan P, Montanaro D, Gimelli S, Seri M, Lerone M, Ravazzolo R, Gimelli G. Molecular characterization of a t(2;6) balanced translocation that is associated with a complex phenotype and leads to truncation of the TCBAl gene. Hum Mutat 2005 Nov;26(5):426-36. (11) Lantieri F, Griseri P, Puppo F, Campus R, Martucciello G, Ravazzolo R, Devoto M, Ceccherini I. Haplotypes of the human RET prate-oncogene associated with Hirschsprung disease in the Italian population derive from a single ancestral combinationof alleles. Ann Hum Genet 2006 Jan;70(Pt 1):12-26. (12) Lantieri F, Griseri P, Ceccherini I. Molecular mechanisms of RET-induced Hirschsprung pathogenesis. Ann Med 2006;38(1):11-9. (13) Emison ES, Mccallion AS, Kashuk CS, Bush RT, Grice E, Lin S, Portnoy ME, Cutler DJ, Green ED, Chakravarti A. A common sex-dependent mutation in a RET enhancer underlies Hirschsprung disease risk. Nature 2005 Apr 14;434(7035):857-63. (14) Yang CH, Szeliga J, Jordan J, Faske S, Sever-Chroneos Z, Dorsett B, Christian RE, Settlage RE, Shabanowitz J, Hunt DF, Whitsett JA, Chroneos ZC. Identification of the surfactant protein A receptor 210 as the unconventionalmyosin 18A. J Biol Chem 2005 Oct 14;280(41):34447-57. (15) Chang NS, MattisonJ, Cao H, Pratt N, Zhao Y, Lee C. Cloning and characterization of a novel transforming growth factor-betal-induced TIAFl protein that inhibits tumor necrosis factor cytotoxicity. Biochem Biophys Res Commun 1998 Dec 30;253(3):743-9. (16) Khera S, Chang NS. TIAFl participates in the transforming growth factor betal--mediated growth regulation.Ann NYAcad Sci 2003 May;995:11-21. (17) Schultz L, Khera S, Sieve D, Heath J, Chang NS. TIAFl and p53 functionally interact in mediating apoptosis and silencing of TIAFl abolishes nuclear translocation of serine 15-phosphorylated p53. DNA Cell Biol 2004 Jan;23(1):67-74. (18) Griseri P, Sachetti T, Puppo F, Lantieri F, Ravazzolo R, Devoto M, Ceccherini I. A common haplotype at the 5' end of the RET proto-oncogene, overrepresented in Hirschsprung patients, is associated with reduced gene expression. Hum Mutat 2005 Feb;25(2):189-95. (19) Gabriel SB, Salomon R, Pelet A, Angrist M, Amiel J, Fornage M, Attie-Bitach T, Olson JM, Hofstra R, Buys C, Steffann J, Munnich A, Lyon net S, Chakravarti A. Segregation at three loci explains familial and population risk in Hirschsprung disease. Nat Genet 2002 May;31(1):89-93. (20) Heanue TA, Pachnis V. Expression profiling the developing mammalian enteric nervous system identifies

98 HSCR disease in a patientwith an LlCAM mutation

marker and candidate Hirschsprung disease genes. Proc Natl Acad Sci U SA2006 May 2;103(18):6919- 24. (21) lkawa H, Kawano H, Takeda Y, Masuyama H, Watanabe K, Endo M, Yokoyama J, Kitajima M, Uyemura K, Kawamura K. Impaired expression of neural cell adhesion molecule Ll in the extrinsic nerve fibers in Hirschsprung's disease. J Pediatr Surg 1997 Apr;32(4):542-5. (22) Anderson RB, Turner KN, Nikonenko AG, Hemperly J, Schachner M, Young HM. The cell adhesion molecule 11 is required for chain migration of neural crest cells in the developing mouse gut. Gastroenterology 2006 Apr;130(4):1221-32. (23) de Pontual L, Pelet A, Clement-Ziza M, Trochet D, Antonarakis SE, Attie-Bitach T, Beales PL, Blouin JL, Dastot-Le Moal F, Dollfus H, Goossens M, Katsanis N, Touraine R, Feingold J, Munnich A, Lyonnet S, Amie! J. Epistatic interactions with a common hypomorphic RET allele in syndromic Hirschsprung disease. Hum Mutat 2007 Aug;28(8}:790-6. (24) Yim HE, Bae IS, Yoo KH, Hong VS, Lee JW. Genetic control of VEGF and TGF-betal gene polymorphisms in childhood urinary tract infection and vesicoureteral reflux.Pediatr Res 2007 Aug;62(2):183-7. (25) Jain S, Encinas M, Johnson EM, Jr., Milbrandt J. Critical and distinct roles for key RET tyrosine docking sites in renal development. Genes Dev 2006 Feb 1;20(3):321-33.

99

Chapter 8

X-ch romosome-specific array-CGH analysis indicates new genes, other than the L1CAM gene, involved in Ll syndrome

Yvonne J. Vos, Krista K. van Dijk-Bos, Christine S. van der Werf, Harke Alkema, Robert M.W. Hofstra and Klaas Kok

Department of Genetics, University Medical Centre Groningen, University of Groningen, the Netherlands

Submitted Chapter s Abstract

Ll syndrome is an X-linked recessive disorder caused by mutations in the L1CAM gene. Because only 20% of the patients clinically suspected of having Ll syndrome carry a mutation in the L1CAM gene, we hypothesize that other genes must be involved. We searched for a second gene on the X-chromosome by screening for deletions in 60 patients using a high density X-chromosomal array. Analysis of the results revealed eleven candidate disease-causing deletions in twelve patients, involving eleven candidate genes: DGAT2L6, POU3F4, GD/1, ASMTL, ATP6AP1, M/01, FRMPD4, MAP3K15, TGIF2LX, DDX53, and TMEM27. Sequence analyses of five of these genes in 95 Ll syndrome patients showed only one gene, GD/1, to be involved: in addition to the deletion found we detected one mutation, c.153-3C>T (probably pathogenic). From the low deletion and mutation frequency fo und, we conclude that at least the five selected genes do not appear to be major players in Ll syndrome, but our findings do not exclude a role for these in the disease development. Not finding a frequently involved second L1 syndrome gene might be caused by the selection of the candidate genes (maybe, one or more of the remaining six genes do harbor more mutations) or by the initial screening method searching for micro deletions (>2kb). By using this method, genes that only have point mutations or small deletionsco uld have been missed.

102 X-chromosome-specific array-CGH analysis

Introduction

Ll syndrome is an X-linked recessive neurological disorder, caused by mutations in the LlCAM gene 11· 21, a gene located on the X-chromosome at Xq28. The phenotypic spectrum associated with mutations in LlCAM includes four different neurological disorders: X-linked Hydrocephalus, also referred to as Hydrocephalus due to stenosis of the Aqueduct of Sylvius (HSAS, MIM #307000), MASA syndrome standing for Mental Retardation (MR), Aphasia, Shuffling gait and Adducted thumbs (MIM #303350)131, X-linked complicated hereditary Spastic Paraplegia type 1 (SPGl, MIM # 303350) and X-linked complicated Corpus Callosum Agenesis (X-linked ACC, MIM # 304100)14·51• The seven most important characteristics of these four disorders are hydrocephalus, aqueduct stenosis, mental retardation, adducted thumbs, agenesis/dysgenesis of the corpus callosum, aphasia and spastic paraplegia. We conducted an LlCAM mutation analysis study in our lab with 367 patients, most of whom were suspected to have L1 syndrome. We were able to detect a mutation in almost 20% of these patients. This rate increased to 85% for patients showing at least three of the seven major Ll syndrome characteristics mentioned above and with at least one affected relative16I. These results indicate that the chance of detecting an LlCAM mutation increases with the number of characteristics supporting the Ll syndrome diagnosis. However, this also raises the question of whether there are other, not yet identified, genes involved in Ll syndrome and whether they might explain part, if not all, of the remaining 80% of the patients who do not have a mutation in the LlCAM gene. To exclude potentially overlooked mutations in the non­ coding regions of the LlCAM gene, we analyzed the promoter region, the Neural Restrictive Silencer Element (NRSE) and the Homeodomain and Paired Domain Binding site (HPD), but found no mutations[61. To date, the possibility of heterogeneity in Ll syndrome has not been studied in detail, partly because the results from a linkage study in a number of families with congenital hydrocephalus and MASA syndrome[7I revealed a clear 1.5 Mb region at Xq28[8I that harbors the LlCAM gene. Shortly afterwards, the LlCAM gene was shown to be responsible for the disorder 11, 9•111. The search for new genes involved in the Ll syndrome has so far been limited to animal models. Recently it was reported112I that, in mice, apart from a mutation in the LlCAM gene, a so-called Llcam hydrocephalus modifier 1 locus [Llhydrol] had been found that might have an effect on the severity of the hydrocephalus. This modifier locus is located on chromosome 5 and harbors a few candidate genes that could be an Ll modifier. The human homologues have not yet been associated with hydrocephalus1121. It is known from animal models that abnormal brain development, caused by abnormal cellular signaling and functioning during early development, can lead to congenital hydrocephalus. In animals (rat, mouse and zebrafish), at least 43 loci and 14 genes have been linked to hereditary hydrocephalus to date 112·131, coding for cytokines and growth factors, in addition to molecules active in the cellular signal pathways during early brain development, and those involved in ciliary function. Thus, different groups of genes may be involved in congenital hydrocephalus in animals. We cannot exclude the possibility that more than one gene might be involved in the development of the Ll syndrome or congenital hydrocephalus in humans. To test this hypothesis we have focused on the X chromosome, since 95% of all Ll syndrome patients (with and without an LlCAM mutation) are males and familial cases mostly show an X-linked recessive inheritance.

103 Chapter 8 We searched for small deletions using a new, high density, X-chromosomal oligonucleotide array, with one oligonucleotideevery 1.9 Kb on average. Our analysis was performed mainly on male patients, but also included a few females diagnosed with ll syndrome. We included them because a few female Ll syndrome patients have been shown to have a mutation in the LlCAM gene 114• 151 (Chapter 2, appendix). The best candidate genes detected with this method were then analyzed for possible point mutations in a cohort of 95 patients suspected of having Ll syndrome.

Materials and methods

Patients used for array CGH About 370 patients had been referred to our laboratory for diagnostic LlCAMtesting. We selected 60 patients without a mutation in their LlCAM gene; these included 8 patients with a strong suspicion of L1 syndrome (they had at least three characteristics of Ll syndrome and one or more affected family members161, and 52 patients belonging to a less stringent group (table 1). The study included 49 males and 11 females; 7 unaffected females who were related to male index patientsfor whom we had no DNA (mother n=5 or sister n=2) and 4 females with characteristics of ll syndrome.

Ta ble 1. Patients lA. Number of affectedfamily members and number of clinical characteristicsin eight patientsbelonging to the group highly suspected of having Ll syndrome

No. of affectedfamily members No. of features* No. of patients in this group 2 3 2 2 4 2 2 5 2 3 4 1 6 3 1 1B. Number of affected family members and number of clinical characteristics in the 52 patients belonging to the less stringent group

No. of affected family members No. of features* No. of patients in this group Index patient: male 1 2 14 1 3 6 1 4 5 1 5 5 1 6 1 2 1 7 2 2 5 3 1 2 5 1 2 6 1 1 Index patient: female 1 4 3 2 (including one male} 1 1

* Features: hydrocephalus (HC), aqueduct stenosis (AqS), adducted thumbs (AT}, a/dysgenesis corpus callosum (ACC/DCC), mental retardation (MR), aphasia (Aph), spastic paraplegia (SP), hypotonia (HP)

104 X-chromosome-specificarr ay-CGH analysis Patients used for sequence analysis of candidate genes From the approximately 370 patients, we selected a group of 95 patients to sequence the candidate genes; they did not have a mutation in their LlCAM gene, but were suspected of having L1 syndrome. From these 95 patients 27 were also included in the array experiments.

X- chromosome-specific array-based comparativegenomic hybridization (array CGH) An 8-plex 60K array was custom-designed using eArray. The array (10_023317, Agilent Technologies Inc., Santa Clara, CA, USA) contains 48,325 probes evenly distributed over the X chromosome, with an average distance of 1.9 Kb between the probes. All the probes were selected from the groups present in the catalogue for lM and 400K arrays {10_021529 and 10_021850, respectively, Agilent). In addition, the array contains 10,178 probes from chromosome 22. Mixes of either 40 healthy male or female DNA samples were used as a reference. 500 ng DNA samples were labeled using the genomic DNA enzymatic labeling kit (Agilent). After hybridization and washing (procedures carried out according to the manufacturer's protocols), the slides were scanned at 2 �tm resolution (microarray scanner, Agilent), and the resulting Tiffimages were processed with Feature Extraction (FE) software V.9.1 (Agilent). The output tilesfrom FE were imported into DNA analytics V.4.081 (Agilent). Next 2IogR data from all the arrays were exported from DNA analytics into Excel for further analysis. Chromosome-X oligo's deleted in males and homozygously deleted in females were identifiedin Excel by a 2IogR value < -1.5. A sensitivityand specificityof at least 95% is reached with two consecutive probes.

Sequence analysis of candidate genes Mutation analysis of all the coding exons and flanking intronic sequences of the genes DDX53, POU3F4, DGAT2L6, GD/1 and TGIF2LX genes was carried out using flanking intronic primers (primer sequences available upon request). PCR was performed in a total volume of 15 µI containing 10 µI AmpliTag Gold ®Fast PCR Master Mix (Applied Biosystems), 1.5 pmol/µI of each primer (Eurogentec, Serian, Belgium) and 2 µI genomic DNA. The samples were PCR amplified on a Perkin-Elmer (ABI) Geneamp 9700 using the following program: an initial denaturation at 94°C for 1 minute, followed by 5 cycles of denaturation at 94°C for 5 seconds, annealing starting at 65 °C for 30 seconds with a stepdown of 1 °C every cycle, and elongation at 72°C for 1 minute, followed by 20 cycles of denaturation at 94°C for 5 seconds, annealing at 60°C for 30 seconds, and elongation at 72°C for 1 minute, followed by 15 cycles of denaturation at 94°C for 5 seconds, annealing at 55°C for 30 seconds, and elongation at 72°C for 1 minute with a final step at 72°C for 5 minutes, after which the samples were cooled down to 20°C. 5 µI of the PCR products were loaded with 5 µI loading buffer and run on a 2% agarose gel with a FastRuler Low Range DNA Ladder (Fermentas) for comparison. The remaining PCR products were purified with ExoSAP-IT (Amersham Pharmacia Biotech, Biscataway, NY) and subjected to direct sequencing on an ABI 3730 automated sequencer, using the specific primers.

Constructs for Ex Vivo Splicing Assay Wild type and mutant GD/1 exon 3 and exon 9, including approximately 80 bp of 5' and 3'flanking intronic sequences, were amplified from control and patient genomic DNA, using the primers GCCGCC-EcoRl,5'-CACAGGGTGGAAGTAGCTC-3', and GGGCGC-BamHl,5'­ CAACATCAGCACGGCTCTC-3' for the intron 2 mutation and the primers GCCGCC-EcoRl,5' -

105 Chapter 8 GTAGCCACCACAACTTGGAG-3' and GGGCGC-BamHI, 5'CCTCACAATGGGCCATGCC-3' for the intron 8 mutation. The PCR was carried out using Phusion High-Fidelity DNA polymerase (NEB) in the following steps: denaturation at 98°C for 30 seconds, followed by 35 cycles of denaturation at 98°C for 10 seconds, annealing at 63°C for 30 seconds, and elongation at 72°C for 30 seconds, and a final step at 72°C for 10 minutes. The products were analysed on, and extracted from, an agarose gel (Qiagen kit). To obtain 3'A-overhangs, 7 µI of purified PCR product, 1 µI Taq polymerase, 1 µI l0xRB and 1 µI dNTPs were incubated at 72°C for 15 minutes and put on ice for 2 minutes. 4 µI of this product was added to 1 µI pCR2.1 TOPO plasmid (lnvitrogen) and 1 µI salt and incubated for 5 minutes at room temperature. The pCR2.1 TOPO constructs were then transformed to DHSa competent cells, according to the manufacturer's protocol: "Transforming One Shot Machl-TlCompetent Cells". 40 µI of X-gal (20 mg/ml) was spread over ampicillin- containing agar plates. Then the transformed DH5a cells were plated and incubated for 16 hours at 37°C. White colonies were picked and grown in Low Broth medium for 16 hours at 37° C and 225 rpm. The DNA constructs isolated from these cells using the Machery-Nagel kit were checked by BamHI and EcoRI restriction enzyme analysis and direct sequencing. Then the correct plasmids and the exon trapping vector pSPL3 (lnvitrogen) were digested, loaded on and extracted from an agarose gel (Qiagen). 100 ng pSPL3 and 50 ng insert were put together with 3 µI l0x ligation buffer (Biolabs) and 1 µI T4 Ligase (Biolabs) in a total volume of 30 µI. Ligation was performed overnight at 16° C and 2 µI of the product was transformed to DH5a competent cells as described. The finalDNA constructs were checked by restriction enzyme analysis and direct sequencing.

Transfection of HEK 293 cells and RT-PCR Human embryonic kidney (HEK) 293 cells were plated in 6-well plates containing 6 x 105 cells/well and cultured in DEMEM supplemented with glutamine, 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (penicillin 10,000 U/ml, streptomycin 10,000 µg/ml) and ° incubated at 37 C in air with 5% CO2 • After 24 hours the cells were transfected with 1 µg plasmid DNA using polyethylenimine according the manufacturer's instructions (Polyscience INC). The plasmid containing the wild-type GOil sequences and the pSPL3 vector, as such, were used as controls. After48 hours the cells were lysed and RNA was isolated according the manufacturer's instructions (Qiagen). 4.5 µg total RNA was used as a template to synthesize cDNA using the cDNA primers pd(N)6 (GE Healthcare). Then PCR was performed using the primers (SD6) 5' -CTGAGTCACCTGGACAACC-3' and (SA2) 5' -ATCTCAGTGGTATTTGTGAGC-3' and the following amplification program: 5 minutes at 94°C, followed by 35 cycles of 1 minute at 94 °C, 1 minute at 60°C and 5 minutes 72°C, and a finalelo ngation time of 10 minutes at 72°C. The PCR products were analyzed by gel electrophoresis and direct sequencing.

Non random X-inactivation A possible pathogenic mutation in the GOil gen was detected in a female patient, so we were interested whether this patient showed non random X-inactivation. HPAll-digestion of the DNA sample was performed overnight at 37°C in a total volume of 20 µI, containing 2 µg DNA, 2 µI Hpall (l0U/µI, New England Biolabs), 2 µI l0x restriction buffer. The next day a PCR was performed in a total volume of 20 µI, containing 2 µI l0xPCR buffer (Amersham Biosciences), 0.1 µI rTaq polymerase (5 U/µI, Amersham Biosciences), 0.5 µI of 20 pmol of Forward Androgen Receptor (AR) primer (5' -GCTGTGAAGGTTGCTGTTCCTCAT) and 0.5 µI of

106 X-chromosome-specific array-CGH analysis 20 pmol of Reverse AR primer (5'-TCCAGAATCTGTTCCAGAGCGTGC) (Eurogentec) 2 µI 4x2mM dNTP's (Amersham Pharmacia Biotech), 3 µI digested DNA (or a non-digested sample) and MilliQ water. The samples were PCR amplified on a Perkin-Elmer (ABI) Geneamp 9700 using the following protocol: an initial denaturation at 95°C for 5 minute, followed by 32 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 1 minute, elongation at 72°C for 1 minute with a final step at 72°C for 5 minutes, after which the samples are cooled down to 20°C. 1 µI of each PCR-product is used in a formamide dilution and analyzed on an ABI 3130x/ (Applied Biosystems).

Results

X-chromosome-specijic array-based comparativegenomic hybridization (array CGH) Deletions encompassing at least two consecutive oligonucleotides After screening the DNA of 49 male patients suspected of having Ll syndrome by array-CGH and analyzing the data obtained, we detected 74 deletions that encompassed at least two consecutive oligonucleotides. Several of these were either known polymorphisms (CNVs listed in the Database of Genomic Variants at http://projects.tcag.ca/variation) or deletionsnot including all or part of a known gene (CCDS or RefSeq track, UCSC genome browser at http://genome.ucsc. edu/, build 36.1). These deletions were excluded from further analysis. In this way we ended up with 8 candidate disease-associated deletions. We also identified two single-copy deletions in pseudo-autosomal regions, which included the TG/21X/Y gene and part of the ASMTL gene, respectively. We decided to include these two deletions in our further analysis, making a total of 10 candidate disease-associated deletions (table 2). In the group of 11 females, we identified one deletion, resulting in the loss of one copy of part of the ASMTLgene. This deletion was also found in one of the male patients (patient 4). In the deletions we identified 11 genes that could be involved in the Ll syndrome (table 2) in 12 different patients. The candidate genes were divided over four groups (table 2): Group A contains the most likely candidate genes based on the known characteristics of the gene product and/ or their expression in brain/neuronal tissue; Group B contains candidate genes for which it is unknown whether they are expressed in brain/neuronal tissue; Group C comprises candidate genes selected because the gene is expressed in neuronal tissue and the deletions, comprising several probes in an intron, may extend into an exon of the gene (because the flanking probe is positioned in, or downstream of, the adjacent exon); and Group D contains those candidate genes selected using the same criteria as for Group C but which are not extending into an exon. We selected only those deletions which comprised more than one probe, but if several patients showed a deletion in the same region of a gene, we deviated from this criterion (patients 121, 44), giving 11 candidate disease-associated deletions (table 2). In three DNA samples, we detected more than one deletion (patients 4, 6 and 44). Two candidate genes were found in patients belonging to the highly suspected group; these were DGAT2L6 (patient 207) and M/D1 (patient 121). Deletions in the ASMTL gene, the M/Dlgene, and the MAP3K15 gene were detected in several patients. Deletionsencompassing only one oligonucleotide In additionto the 74 deletions that encompassed at least two consecutiveoligonucleotides, we identified 319 deletions in 46/49 male patients that comprised only one oligonucleotide (162

107 I-' 9 �� . �- - 'i'Jr �-_:_ .. ,�-� ----. ,------I ------·- Kb � Table 2. Summary deletions found by Agilent X-chromosomal array .., II I ___ _J Patient Sex Phenotype # Affected Gene Start - end # Deleted Remarks relatives Probes

Group A* Diacylglycerol Opacyltransferase 2-like prom, 207 M HC, AqS, AT 2 DGAT2L6 69310516 -69315731 3 5.2 protein 6. Expressed in brain, among other so exon 1 tissues. Neural transcriptionfactor. Expressed during 397 M MR, AT 1 POU3F4 823477751 -82669656 321.9 whole gene embryonic development in brain. Disease gene: X-linked deafness. Expressed primarily in neural and sensory prom, 4 M HC, MR, Aph, HP 1 GDll 153316250 - 153320060 2 3.8 tissue. Mutations in this gene have been exon 1 linked to non-specific MR.

Group B* 1 Also called CAGE gene (Cancer associated 31 M HC, AT DDX53 22867019 - 23018419 53 151.4 whole gene gene), expressed in a variety of cancers and 44 in testis. Exclusively expressed in kidneys, not in adult M HC, MR, Aph, DCC 1 TMEM27 15574355 - 15634089 31 59.7 exons 1-3 brain. Is detectable at day 13 of gestationin mice mRNA (Zhang etal. 2001}. 71 Transcription factor. Testis-specific M HC 3 TGIF2LX 88943182 - 89400685 69 457.5 whole gene expression. mRNA from brain tissuehas been reported twice

Group C* Acetylserotonin methyltransferase-like Normal, carrier intronl- 289 F 2 ASMTL 1528326 - 1530474 4 2.1 protein. Expressed in brain, among other Son: HC exonl? tissues. intronl- 4 M HC, MR, Aph, HP 1 ASMTL 1528326 - 1530474 4 2.1 exonl? Vacuolar AT Pase. Expressed in many tissues, 3'UTR-last 4 M HC, MR, Aph, HP 1 AT P6AP1 153316250 - 153320060 2 3.8 including foetal brain. Many functions such exons? as fibroblastgrowth factor receptor binding. Sex Phenotype # Affected Gene Start - end # Kb Deleted Remarks relatives 11 II 'Probes Patient ------lf --,;------i1 Group D* Mid line 1, member of the tripartite M motiffamily. Mutationsin this gene have 367 HC 2 MIDl 10654513 - 10663078 3 8.6 intronl been associated with Opitz syndrome. I� I Expressed in brain, among other tissues. 44 M I 121 HC, MR, SP 2 MIDl 10486705 - 10492151 1 5.4 intron2 M HC, MR, Aph, DCC 1 MIDl 10486705 -10492151 1 5.4 intron2 M FERM and PDZ domain containing 4. It's in 6 HC, ACC, AT 1 FRMPD4 12343603 - 12349210 2 5.6 intronl vivo function is yet unknown. Expressed in 2 brain, among other tissues. 44 Member of the mitogen-activated M protein-kinase kinase kinases. In contrast HC, MR, Aph, DCC 1 MAP3K15 19375526 - 19381726 6.2 intronS to some members, the function of M MAP3K15 is still unknown. Expressed in nervous system, among others. 6 HC, ACC, AT 1 MAP3K15 idem 2 6.2 intron 5 M 392 HC, MR, ACC, SP, AT 1 MAP3K15 idem 2 6.2 intron 5 M 3 79 HC, DCC 1 MAP3Kl5 idem 2 6.2 intron 5 0 0 3 Legend. The deletions groups are based on several criteria: Group A t

- Deletions comprising several probes covering all or part of a coding area of a gene. The gene is expressed in neurons and/or in the brain. r, Group B Cl - Deletions comprising several probes covering all or part of a coding area of a gene. The gene is probably not expressed in neurons and/or in the brain. "f Group C - Deletions comprising several probes in an intron or the promoter area of a gene. Probably, the deletion also comprises coding regions of the gene. The gene is

expressed in neurons and/or in the brain. Cl I-' Group D: � - Deletions comprising several probes in an intron of a gene or deletions comprising only one probe in an intron of a gene but the deletion has been found in more than .;;· one patient. The gene is expressed in neurons and/or in the brain. Chapter 8

Table 3. Summary of one probe deletions found by Agilent X-chromosomal array j j Patient Sex Phenotype # Affected Gene Start - end bp relatives 53 M HC, AqS, MR 1 WWC3 10059340 - 10063447 4107 138 M HC, MR, Aph, DCC, SP 1 MAP3K15 19383842 - 19386854 3012 110 M HC, AqS, ACC, AT 2 SH3KBP1 19634524 - 19637815 3291 121 M HC, MR, SP 2 FAM47C 36939186 - 36947481 8295 Normal, carrier 391 F 2 WNK3 54267613 - 54274012 6399 Son: HC 41 M HC, AT 1 LOC554203 73081201 - 73086192 4991 178 M HC, MR, AT 1 BCORLl 128943192 - 128946728 3536 109 M HC, MR, Aph, ACC 1 F8 153781200 -153783605 2405 mapped within a transcribed sequence, representing 65 different loci). Since the likelihood of fa lse-positives is rather high in this group of 319 one probes deletions, we applied the fo llowing four, rather stringent, selectioncr iteria: (1) only results with a high quality score were included; (2) we excluded deleti ons that were frequently lost homozygously or heterozygously in a control group of 17 healthy females; (3) we excluded deletionstha t were known CNVs or that mapped within segmenta l duplications. This reduced the group from 319 to 97 deletions; and (4) we further refined this group by selectingonly those deletions that partly overlapped with an exon of a gene (UCSC genome browser, CCDS or RefSeq track). For this, the size of the deletion was calculated as the segment between the two fl anking (non-deleted) oligos. Our fi nal list contained only 8 possible candidate deletions associated with Ll syndrome for 8 individual ca ndidate genes in 7 patients and in 1 mother of a deceased patient (table 3). The oligo of one of the deletionswas fo und in an exon (WNK3 gene) and another deleted oligo was nearby an exon - the oligo comprised the conserved splice site of the intron - (WWC3 gene). In one patient, a deletion was found in the MAP3K15 gene, giving a total of 5 patients with a deletion in this gene (tables 2 and 3), and thus making it a very promising candidate gene for Ll syndrome. Two other candidate genes were fo und in patients belonging to the highly suspected group, i.e. SH3KBP1 (patient 110), and FAM47C (patient 121). In the latter, a deletion in the M/01 gene was also found (table 2).

Sequence analysis of candidate genes In order to determine whether these genes play a more general role in patients suspected of having Ll syndrome, we sequenced the coding and fl anking intronic regions of five candidate genes from Groups A and B, i.e. DGAT2L6, POU3F4, and GD/1 (Group A), and DDX53 and TGIF2LX (Group B) in a group of 95 patients suspected of having Ll syndrome. In the genes from Group A no mutationswere detected in the DGAT2L6 gene or the POU3F4 gene. In the GD/1 gene two variants were detected: c.154-3C>T on one allele in intron 2, in a young girl with Ll syndrome and c.992-lOC>T in intron 8 in an affected boy. These variants are no known SNPs according to the NCBI SNP database and were not detected in 100 healthy male controls. In Group B we detected one variant in the DDX53 gene, c.185T>C, implying an amino acid substitution from valine to alanine, p.Val62Ala. However, as this variant is mentioned in the NCBI SNP database, it is considered to be a polymorphism.

110 X-chromosome-specificM WT Mutarray -CGHM pSPL3 analysis Bl

A � B Exon + mutation\ _.0, .,.' ..I...

SD SA SD SA Wild type ® © pSPL3 Mutant

I: Mutant II: Wild type

cDNA ���

PCR

Figure 1. A. Principle of exon trapping. SA = splice acceptor site, SD = splice donor site. B. cDNA from wild type and mutated intron2/exon3. M = marker DNA, WT = wild-type intron2/exon 3, Mut = c.154-3C>T mutation in intron 2, pSPL3 = empty vector, Bl = blank.

In the TGIF2LX gene, we found the variant, c.421T>C, at the protein level resulting in the replacement of a serine by a praline (p.Ser141Pro), a missense mutation of unknown pathogenicity. This variant is no known SNP according the NCBI SNP database. Prediction programs do not indicate a high likelihood of pathogenicity for proline141 due to the moderate physicochemical differencebetween serine and praline (Grantham score: 74) and the moderate conservation of serine 141 and its flanking amino acids.

Ex Vivo Splicing assay intronic GD/1 mutation Because RNA from the girl carrying the c.154-3C>T variant in the GOil gene was not available, we investigated the possible influence of this variant on RNA splicing using an exon trapping system (figure lA). We cloned exon 3 of the GOil gene, including its flanking intron regions, both with and without the mutation in pSPL3. RNA isolated from transfected HEK93 cells was converted, using reverse transcriptase, to cDNA. After PCR of this cDNA with pSPL3 specific primers, we got two fragments in both cases (figure lB). Based on the sizes of these fragments we concluded that one fragment lacked exon 3 and the other one contained exon 3 (figure lB), which was then confirmed by sequence analysis of the fragments eluted from the agarose gel. So, from figure lB, which shows a clear difference in intensity of the bands, it may be concluded that the wild-type RNA represents mainly correctly-spliced RNA, while in case of the c.154-3C>T variant, mainly incorrectly-spliced RNA, skipping exon 3, is being formed. Based on these findings which were reproduced in independent experiments, we concluded that the patient with this c.154-3C> T variant in intron 2 of the GOil gene was mainly producing the shortened mRNA and this might well be the cause of her clinical phenotype. The c.992-l0C>T variant did not influence the proper RNA splicing using the ex vivo RNA splicing assay and is considered non-disease-causing.

111 Chapter 8 Non random X-inactivation In order to confirm the relevance of the c.154-3C>T mutation in the GOil gene in a female patient suspected of having L1 syndrome, we investigated DNA from her blood for non random X-inactivation. We could not detect non random X-inactivation.

Discussion

Because almost 20% of the patients suspected of having Ll syndrome carry a mutation in the LlCAM gener61 we started to search for one or more potentially disease associated gene(s) on the X chromosome. We choose for the X chromosome since 95% of all Ll syndrome patients reffered to us are male and because familial cases mostly show X-linked recessive inheritance. In our screening of 60 patients using a high density X-chromosomal array, we identified eleven candidate deletions containing eleven candidate genes (table 2). These deletionsencompassed at least two consecutive oligonucleotides to prevent false positive results (or only one probe, when the deletionwas found in more than one patient).In our laboratory the Agilent system is validated for diagnostic purposes. A sensitivity and specificity of at least 95% is reached with two consecutiveprobes. We classified the eleven genes into four groups and hypothesized that genes involved in Ll syndrome should be expressed in the brain or neurons in an early stage of fetal development. As shown in table 2, at least 8/11 genes are being expressed in the brain or in neurons indeed (OGAT2L6, POU3F4, GOil, ASMTL, ATP6AP1, M/01, FRMPO4, and MAP3K15} (Groups A, C and D) and at least 3/8 were expressed in an early stage of development (POU3F4, GOil, and ATP6AP1). The other three candidate genes were less easily to categorize. Nonetheless we selected OOX53 and TM EM27 (Group B), as they harbored distinct, non-CNV deletions in patients who certainly had Ll syndrome and TGIF2LX, because brain cDNA clones have been described (BI830352 and BC029920). The candidate genes are found in patients belonging to the stringent group as well as in patients from the less stringent groups. From the literature it is known that three out of these eleven candidate genes are disease­ causing when mutated, i.e. the POU3F4 gener161, M/01 gener171 and GOil genel181. The POU3F4 gene Mutations in the POU3F4 gene cause X-linked non-syndromic deafness1161• In one family patients have been described with X-linked non-syndromic deafness and MR1191. Their disease­ causing mutation turned out to be a deletion of at least 200 kb, lying between 300 and 700 kb upstream of the POU3F4 gene. The deafness in this family might be explained by the deletion as it likely affects a regulatory region controlling POU3F4 expression. As the POU3F4 gene could not directly explain the MR in the family, it was hypothesized that within this 200 kb­ sized DNA sequence a gene should reside that could explain the MR, although a causative gene was not identified1191 . As our deletion overlapped with the deletion found in this family, the MR in our patients might be explained by the same genetic aberration. There is some doubt as to whether this is a gene since analysis of the region revealed no genes. We reduced the region to approx 300 kb upstream of the POU3F4 gene, as a result of the upstream border of the deletion in our patient. Hildebrand's1191 and our data can also be seen as an indication

112 X-chromosome-specificarr ay-CGH analysis that the POU3F4 gene itself might be involved in mental retardation, which was reason for us to sequence the POU3F4 gene in 95 patients suspected of having Ll syndrome. However, we found no mutations. Thus, the POU3F4 gene does not seem to be part of the cause of Ll syndrome or of an atypical appearance thereof, except for very seldom cases, as might be the case with our patientwith the deletion. The M/01 gene The M/01 gene is involved in Opitz syndrome, as demonstrated by approximately 80 mutations reported for this gene1171. These mutations are scattered over the entire gene and consist of missense, nonsense and splice site mutations, small insertions and deletions, and deletions of either single exons or the entire M/01 coding region. They did not report intronic deletions in patients having Opitz syndrome, as we detect in our patients (table 2). This can explain why the three patients in our study do not have Opitz syndrome. Some minor characteristics of this syndrome show overlap with Ll syndrome, notably MR, developmental delay and agenesis of the corpus callosum1171. 98% of the patients carrying a mutation in M/01 suffer from hypertelorism1171, but this is not seen in patients suspected of having Ll syndrome, so it seems unlikely that mutations in the M/01 gene would be involved in causing Ll syndrome or an atypical appearance thereof. This was the reason we did not sequence the cohort of 95 patients suspected of having Ll syndrome for mutations in this gene. However, since 3/60 patients did show a deletion in this gene (twice in intron 2 and once in intron 1), some involvement in Ll syndrome as a result of these deletions cannot be excluded. The GD/1 gene In contrast, the GD/1 gene, identified as being one of the non-syndromic MR genes11s1, was thought to be an attractive candidate second Ll syndrome gene. It codes for a protein, involved in the neuronal differentiationduring brain development, neurite extension and neurotransmitter release1201. So far, only three mutationsin the GD/1 gene have been detected1181; two mutations in large families from linkage data mapping to Xq28 by D'Adamo et al.1201, and a third in a study of 164 patients suffering from non-syndromic MR1211. We sequenced this gene in our group of 95 patients suspected of having Ll syndrome and we detected two differentmutations in two different L1 syndrome patients: c.153-3C>T in intron 2 and c.992-lOC>T in intron 8. Both mutations are no known SNPs according the NCBI SNP database and could not be detected in 100 male healthy controls. Although, the c.992-lOC>T variant is considered non-disease­ causing, since it did not influence the proper RNA splicing. Ex vivo RNA splicing analysis of the mutation in intron 2 showed two distinct splice forms (figure18), one with exon 3 present and one with exon 3 lacking. The splice form without exon 3 proved to be the most pronounced band. In contrast the splice form with exon 3 proved the main product for the wild type construct. As we did see a quantitativedifference between the transcripts of the wild type and mutant constructs we concluded that the c.153-3C>T mutation might be considered pathogenic. This mutation was found in a newborn girl suffering from hydrocephalus, aqueduct stenosis, agenesis of the corpus callosum and adducted thumbs. As this heterozygous mutation was found in a female patient, non-random X-inactivation in the nerves and brain would be needed for this mutation to be causative, but this can obviously not be confirmed experimentally. In blood we did not detect non-random X-inactivation. It is interesting to note that in one of the two families described by D'Adamo, carrier females were also affected1221. We conclude that the GD/1 gene could well be involved in generating non­ UCAM mediated Ll syndrome.

113 Chapter 8 For the other 8 candidate genes no disease associated mutations are described yet. DGAT2L6 gene The DGAT2L6 gene encodes a diacylglycerol O-acyltransferase 2-like protein 6 and is expressed in many different types of tissues, including the brain1231 • Studies with this gene, cloned in yeast revealed its role in triacylglycerol synthesis1231 • The DGAT2L6 protein shows significant homology with a large number of acyl-CoA diacyl-glyceroltransferases. If DGAT2L6 has a comparable function (acyltransferase), it could be a candidate (early stage membrane synthesis?). However, detailed DNA analysis of 95 patients suspected of having Ll syndrome did not reveal any mutations in this gene. Besides the possible involvement of the mutated gene in the disease of the patient of this study (patient 207), we do not foresee an important role for the DGAT2L6 gene in typical or atypical Ll syndrome development. The DDX53 gene The DDX53 gene, which codes for a protein with possibly helicase activity and plays a role in cell proliferation1241, was the only gene in the 150 kb deletion seen in one of our Ll syndrome patients and we therefore considered it to be a potential candidate gene. However, the analysis of the DDX53 gene in the 95 patientsdid not reveal any mutations. Besides its possible role in the patient with the deletion, the DDX53 gene does not appear to play a key role in typical or atypical Ll syndrome development. The TGIF2LX gene The TGIF2LX gene codes for one of the so-called TG-interacting factors (TGIFs) belonging to the family of TA LE-homeodomain proteins. This family includes TGIF, TGIF2, and TGIF2LX/Y, which play an important role in the developmental stage of various tissues. TGIF and TGIF2 are transcriptional repressors1251 • Mutations in hTGIF, a close family member of TGIF2LX, have been associated with holoprosencephaly, a birth defect involving malformation of the anterior ventral midline, including forebrain and facial structures1261 • The function of TGIF2LX/Y is unknown. In a Drosophila model system it was shown, that human TGIF2LX inhibits cell division and cell growth, in fat body cells, epithelial cells and neuronal cells, amongst others. It is unknown whether these findings are relevant in humans1251, but we thought it sufficient reason to sequence this gene in our group of 95 patients. We found one variant in this gene, c.421T>C (p.Ser141Pro), but this variant is probably non-pathogenic. Thus, the relevance of TGIF2LX as a second gene related to the Ll syndrome is judged to be limited. The testisspecific expression of the gene is in line with this conclusion. The MAP3K15 gene This gene is a member of the apoptosis signal-regulating kinase (ASK) family and, more specifically, of the mitogen-activated protein kinase kinase kinases (MAP3K) subfamily. These MAP3Ks phosphorylate and activate MAPK kinases, which in turn phosphorylate and activate MAPKs during signaling cascades, which are important in mediating cellular responses to extracellular signals. Although the function of MAP3K5 (ASKl) and MAP3K6 (ASK2) is known, it is unknown for the MAP3K15 gene. However, if we assume a role in signal transduction for MAP3K15 as well, this makes it an attractive candidate for involvement in Ll syndrome development, since Li-mediated neurite outgrowth involves intracellular signaling1271• A role for the MAPK cascade in the Ll-guided activation has been described1281• We identified five deletions in five different patients suspected of having Ll syndrome: four patients had a deletion in intron 6 of the MAP3K15 gene, and one patient had a deletion in intron 5 (one probe). Intrans 5 and 6 are part of the non-coding segment of the MAP3K15 gene (the first ten

114 ------

X-chromosome-specific array-CGH analysis exons of the gene are non-coding, according to RefSeq track UCSC genome browser). Thus, although it is clear that these mutations do not cause an altered gene product as such, they might disturb the proper processing of the primary transcript and might therefore be relevant to Ll syndrome development. It is noteworthy that neither MAP3K15 deletions were present in our control group, nor are they described as known CNVs. However, in a study concerning X-linked mental retardation (X LMR) ([29], Tarpey et al. found two truncating mutations in the coding sequences of the MAP3K15 gene in male controls (p.Arg611X (2/272) and p.Arg597X (1/243)), making the MAP3K15 gene an unlikely candidate in Ll syndrome development. Although these data do not implicate MAP3K15, the identifieddeletions might still be involved by influencing the expression of other gene(s) on the X-chromosome and thereby contributing to Ll syndrome development. TM EM27. FRMPD4. ASMTL. and ATP6AP1 The other four genes TM EM27, FRMPD4, ASMTL, and ATP6AP1, were not analyzed further, since we judged them to be less likely candidate genes due to their (tissue) specific function or because the deletion may not comprise coding regions of the gene (table 2). We also did not examine candidate genes detected by only one probe any further. It is obvious that these deletions will need independent confirmation by an independent approach, prior to a final decision about their relevance.

Based on the low deletion- and mutation frequency found in our group of respectively 60 and 95 patients, we conclude that at least the five genes we selected for mutation analysis do not appear to be major players in the development of Ll syndrome. However, our findings cannot exclude a certain role for these genes in the disease phenotype, because in all cases we have found a deletion of (or part of) these genes in at least one patient and six genes have not been screened for mutations yet. The high frequency of deletions found in the MAP3K15 gene (in 5 out of 60 patients) is striking, and it was not detected in controls. However, the gene as such is not a likely candidate due to the finding of truncating mutations in male controls by Tarpey et al. l291• For one candidate gene, GD/1, however, the potential involvement in the typical or atypical Ll syndrome is more likely, since we could relate Ll syndrome phenotypes with GD/1 gene abnormalities in 2 patients. In one patient we found a deletion in the gene and in a second patient we found a splice site mutation (c.153-3C>T in intron 2) that likely is pathogenic. This makes the GD/1 gene the strongest candidate gene that emerged from this study. We also conclude that a putative, second Ll syndrome gene on chromosome X is apparently not characterized by deletions (>2 kb) in a considerable proportion of the patients. Novel techniques that allow the detection of smaller aberrations, including X-chromosome-specific exome resequencing approaches, are thus needed to further elucidate the role of these and other genes in Ll syndrome development.

Acknowledgements We thank Connie van Ravenswaaij for discussion and Jackie Senior for editing the manuscript.

115 Chapter B References

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(21) Bienvenu T, des Portes V, Saint MartinA, McDonell N, Billuart P, Carrie A, Vinet MC, Couvert P, Toniolo D, Ropers HH, Moraine C, van Bokhoven H, Fryns JP, Kahn A, Beldjord C, Chelly J. Non-specific X-linked semidominant mental retardation by mutations in a Rab GDP-dissociation inhibitor. Hum Mol Genet 1998 Aug;7(8):1311-5. (22) des Portes V, Billuart P, Carrie A, Bachner L, Bienvenu T, Vinet MC, Beldjord C, Ponsot G, Kahn A, Boue J, Chelly J. A gene for dominant nonspecificX-linked mental retardation is located in Xq28. Am J Hum Genet 1997 Apr;60(4):903-9. (23) Turkish AR, Henneberry AL, Cromley D, Padamsee M, Oelkers P, Bazzi H, Christiano AM, Billheimer JT, Sturley SL. Identificationof two novel human acyl-CoA wax alcohol acyltransferases: members of the diacylglycerol acyltransferase 2 (DGAT2) gene superfamily. J Biol Chem 2005 Apr 15;280(15):14755- 64. (24) Cho B, Lim Y, Lee DY, Park SY, Lee H, Kim WH, Yang H, Bang VJ, Jeoung DI. Identification and characterization of a novel cancer/testis antigen gene CAGE. Biochem Biophys Res Commun 2002 Apr 5;292(3):715-26. (25) Wang Y, Wang L, Wang Z. Transgenic analyses of TGIF family proteins in Drosophila imply their role in cell growth. J Genet Genomics 2008 Aug;35(8):457-65. (26) Gripp KW, WottonD, Edwards MC, Roessler E, Ades L, Meinecke P, Richieri-Costa A, Zackai EH, Massague J, Muenke M, Elledge SJ. Mutations in TGIF cause holoprosencephaly and link NODAL signalling to human neural axis determination. Nat Genet 2000 Jun;25(2):205-8. (27) Kenwrick S, Watkins A, De Angelis E. Neural cell recognitionmolecule Ll: relating biological complexity to human disease mutations. Hum Mol Genet 2000 Apr 12;9(6):879-86. (28) Schaefer AW, Kamiguchi H, Wong EV, Beach CM, Landreth G, Lemmon V. Activationof the MAPK signal cascade by the neural cell adhesion molecule Ll requires Ll internalization. J Biol Chem 1999 Dec 31;274(53):37965-73. (29) Tarpey PS, Smith R, Pleasance E, Whibley A, Edkins S, Hardy C, O'Meara S, Latimer C, Dicks E, Menzies A, Stephens P, Blow M, Greenman C, Xue Y, Tyler-Smith C, Thompson D, Gray K, Andrews J, Barthorpe S, Buck G, Cole J, Dunmore R, Jones D, Maddison M, Mironenko T, Turner R, Turrell K, Varian J, West S, Widaa S, Wray P, Teague J, Butler A, Jenkinson A, Jia M, Richardson D, Shepherd R, Wooster R, Tejada Ml, Martinez F, Carvill G, Goliath R, de Brouwer AP, van Bokhoven H, Van Esch H, Chelly J, Raynaud M, Ropers HH, Abidi FE, Srivastava AK, Cox J, Luo Y, Mallya U, Moon J, Parnau J, Mohammed S, Tolmie JL, Shoubridge C, Corbett M, Gardner A, Haan E, Rujirabanjerd S, Shaw M, Vandeleur L, Fullston T, Easton DF, Boyle J, Partington M, HackettA, Field M, Skinner C, Stevenson RE, Bobrow M, Turner G, Schwartz CE, Gecz J, Raymond FL, Futreal PA, Stratton MR. A systematic, large-scale resequencing screen of X-chromosome coding exons in mental retardation. Nat Genet 2009 May;41(5):535-43.

117

Chapter 9

Discussion and future perspectives Chapter 9 This thesis has addressed different aspects of genetic analysis in patients clinically suspected of having L1 syndrome and it set out to resolve various questions: Who should be analyzed for mutations in the LlCAM gene? Is there a correlationbetween the disease phenotype and the mutations identified in LlCAM? How should missense mutations be interpreted in terms of their potential disease-causing effects and what about variants influencing the RNA splicing? Are genes other than the LlCAM gene involved in Ll syndrome or an atypical form of it? In this chapter, I discuss these issues and the results of my research.

PART I Mutation analysis of the L1CAM gene in patients suspected of having Ll syndrome

We performed mutation analysis of the LlCAM gene in a large group of patients (n=367) suspected of having Ll syndrome and referred to our laboratory for diagnostic LlCAM testing. To date, LlCAM is the only gene that has been clearly shown to be involved in L1 syndrome11-51• In this group of patients, only �20% harbored a mutation in LlCAM, implying that 80% did not have a mutation in this gene; this raised several questions. Had we overlooked any mutations? Were the patients referred to us truly suspected of having Ll syndrome or was the test requested to exclude this possibility? Are there more genes involved in Ll syndrome or an atypical form of it?

Had we overlooked any mutations? The mutation analysis we performed was based on a prescreening method called denaturing gradient gel electrophoresis (DGGE), followed by direct sequencing of any fragments showing an aberrant DGGE pattern. We might have overlooked some mutations using this technique. However, the DGGE technique had been validated by testing 175 BRCAl and BRCA2 gene mutations with a sensitivity of 100%l61• Such validation for the LlCAM DGGE system was not possible due to the limited number of mutations available when we started this work. Nevertheless, all the known LlCAM mutationsdetected by other techniques could be detected using our DGGE system. A full sequence analysis of the gene in 15 patients not showing a mutationby DGGE but who were clinically very likely to have a mutation in the LlCAM gene did not produce any missed mutations, corroborating that we had not overlooked many mutations by using DGGE. Since we analyzed only the coding sequences and the exon-intron boundaries, we were not able to exclude mutations residing in regions regulating gene expression. Searching 100 patients for mutations in the LlCAM promoter and two other regulatory regions, namely the neural restrictive silencer element (NRSE) and the homeodomain and paired domain-binding (HPD) site, did not reveal any additional mutations(Chapter 2). The probability of mutationsin these regions being responsible for the Ll syndrome in our group of 367 patientswas therefore judged to be very low. Thus, the low frequency of mutation detection could not be explained by mutations overlooked in these regulatory sequences. To check whether we had overlooked the possibility of large duplications in the LlCAM gene

120 Discussion and future perspectives (this analysis is no standard procedure in our diagnostic DNA screening) as a cause of Ll syndrome, we analyzed a subgroup of 98 patients for this type of genetic aberration. We found a duplicationin only one of these patients and concluded that this type of mutationcould also not explain the low frequency of detection(Chapter 2). Sometimes we analyzed DNA from the mother instead of from the index patient. We cannot exclude missing a mutationin 1 or 2 index patientsby doing this (see Germ line mosaicism), but again, this does not explain why so many patientsdid not have an L1CAM mutation. One region we did not screen for possible mutationswas the 3' part of the gene. Apart from the AAUAAA element, which signals 3' cleavage for most mRNAs, it is known that microRNAs (miRNAs) regulate gene expression post-transcriptionally by pairing with the 3' UTR of target mRNAs171• Some genetic variants in this region, which alter miRNA recognition sites, may play a pathogenic role in human diseases181 as demonstrated for two point mutationsin the 3'UTR of the REEP1 gene, which is associated with spastic paraplegia type 3119• 101. Thus, we cannot fully exclude possible mutations being located in this region of the L1CAM gene. We will look for this type of mutation in the near future, but at the moment we assume the likelihood of findinganything will be rather low. In conclusion we are confidentthat it is very unlikely that we have overlooked many mutations in the L1CAM gene.

Does the mutation detection rate depend on the pre-selection of patients? We concluded from a questionnairesent to clinicians that approximately 20% of the patients who were analyzed were referred to us in order to exclude a diagnosis of Ll syndrome (Chapter 2). These patients most probably do not have an L1CAM mutation. If we exclude such patients from our analyses, the mutationfrequency becomes 25% instead of 20%. The Ll syndrome diagnosis is based on specific clinical characteristics. The seven most important are hydrocephalus, aqueduct stenosis, mental retardation, adducted thumbs, agenesis/dysgenesis of the corpus callosum, aphasia and spastic paraplegia. We showed that the chance of detecting a mutation is positively correlated with the number of clinical characteristics (Chapter 2). Besides these clinical characteristics, a positivefamily history also proved to be a powerful criterion. A combination of these two characteristics - that is, patients with a positive family history for Ll syndrome and with three or more clinical characteristics - resulted in a higher detectionrate of 85% in a well documented subgroup of 106 patients (OR 10.4, 95%CI 3.6 to 30.1) (Chapter 2). Thus, the mutation detection rate does depend on the pre-selection of cases.

This finding is in line with the results of two other studies, although it is more pronounced. Finckh et a/Y11 reported a rather low mutation frequency (30%) in an unselected group of L1 syndrome patients. Much higher frequencies of 46% and 74% were reported in pre-selected groups with a positivefamily history for L1 syndrome by Kanemura et a/.1121 and Finckh et a/.f11l, respectively. This supports our own finding that pre-selection of patients raises the mutation frequencies. Our mutation detection rate increased even more, from 20% (unselected group) to 85% when we selected for patients more stringently, that is with three or more clinical characteristics in addition to a positive family history. Indirectly this also corroborates our conclusion that we had not overlooked many mutations.

121 �------

Chapter0,9 9

Q) 0,8 ro 0,7 C 0 ·.p 0,6 ,. Q) / .•

Q) 0,5

C 0,4 0 _._ 2or more ·.p ro 0,3 affected fa mily members 0,2 -- •·- 1 affected fa mily 0,1 _,_.------member 0 ..

Number of clinical characteristics

Mutation detection rate versus number of age-independent clinical characteristics and number of affected fa mily members. Figure 1. A mutation determination model Using these data - the mutation analysis results, the clinical data and the fa mily anamneses - we were able to develop a powerful tool for clinicians and genetic counselors (Chapter 2) to determine the chance of fi nding a mutation in a target patient before DNA diagnostic testing, see figure 1. Since diagnostic DNA analysis is time consuming and costly, it is important to be well informed prior to requesting a DNA test. Figure 1 shows the mutation detection rate in relation to the number of age-independent clinical characteristics and the number of affected fa mily members. These interrelationships were determined from data from 338 patients suspected of having Ll syndrome (338 = 367 minus 29 patients who were only analyzed to exclude Ll syndrome). Despite the relevance of this tool, it is important to realize that mutations might also be detected in patients with only one or two clinical characteristics and with a negative family 11 1 14 anamnesis, as seen in our data and from the literature1 • 3• 1.

Genotype-phenotype correlations In trying to identify genotype-phenotype corre lations in a group of patients (n=33) (Chapter 2), we demonstrated that children with a truncating mutation were more likely to die before the age of 3 years (52%) than children with a missense mutation (8%}. These results are statistically signifi cant and relevant for geneticcou nseling related to prenatal DNA diagnostics. They correspond with previous reports by Fransen et af. 1151 and Ya masaki et al_l161• Fransen et al. reported early death percentages of 100% (truncating)ver sus 58% (missense) before the age of 2 and Ya masaki et al. reported 50% (truncating) versus 32% (missense) before the age of 1. In our group of patients we did not detect a correlation between the severity of hydrocephalus and the location of the missense mutation in either the key residues or the surface residues of the Ll protein, in contrast to what Michaelis et al.f171 and Kamiguchi et alY81 detected. Key residues are amino acid residues crucial for the structural build up of the protein domains and

122 Discussion and future perspectives a surface residue occurs at the surface site of a protein and is important for intermolecular interactions. We determined that severe hydrocephalus is due to key residue changes in 57% of the patients and due to surface residue changes in 50% of the patients, which are much closer rates than the 67% and 42%, respectively, reported by Michaelis et a/.l171, or the 78% and 28%, respectively, determined by Kamiguchi et a/Y81• This discrepancy may be explained by differences in the sizes of the target groups (Michaelis' group was three times larger than ours). Another explanation for the genotype-phenotype differences might lie in the relevance of genetic modifiers. The contribution of modifiers to the severity of X-linked hydrocephalus in 4 mouse models has recently been describedr 1 • Likewise the differences in the occurrence and

19 20 severity of hydrocephalus, which has been documented among related malesr , 1 carrying the 15 same mutationr 1, might also be explained by the action of different modifiers.

GermlineLlCAM mo saicism Most mutations found in the gene are private mutations, occurring only in a single family. In this thesis (Chapter 2) we have described 58 private mutationsout of the 68 mutations identifiedand shown that 7% ofLlC allAM mutations detected in our index patientsoccurred de nova, 2 which is in the same range as that published by others, e.g. 4%r111 and ll%r 11• For one of the patients with a de nova mutation, we detected a germline mosaicism in the mother, a 11 21 22 phenomena that has already been described for L1 syndrome r , • 1 • Although the frequency of germline mosaicism in the mothers of index patients is not precisely known, we estimate it to be 5-10% based on our own screening and data from the literature, assuming all de nova mutations are germline mosaicisms. This relatively high percentage is important, because it is the frequency of germline mosaicism in the mother that determines the risk of having an affectedchild in a next pregnancy (if no mutation is detected in DNA isolated from the mother's blood) and therefore warrants prenatal DNA diagnostics. In addition, based on the frequency of de nova mutations/germline mosaicism to be 7% in our group of 367 patients, we determined that we may have overlooked 1 or 2 index patients whose mothers (n=25) were analyzed due to lack of DNA from the index patient and found negative for a mutation, thus leading us to assume that the index patient did not have a mutation, whereas in fact it had. The occurrence of de nova mutations in mothers of index patients or a germline mosaicism 23 24 in a grandparent of the index patient has also been describedr , 1 and was determined to be about 10% in our study. This and the 7% de nova mutations in the index patients means that at least one-fifth (20%) of the mutations causing the Ll syndrome in our group has arisen recently. This was also reported by Finckh et a1.r111, who showed that eight mutations out of 46 (17%) emerged recently: originating maternally (n = 2), in a grandfather (n = 4), in a grandmother (n = 1) and in a great-grandfather (n = 1).

Mutations disturbing proper RNA splicing lntronic but also silent and missense mutations might disturb proper RNA splicing and thereby explain the phenotype. In order to study this possibility we selected four variants (2 missense, 1 silent and 1 intronic mutation) for which it was unclear whether they were disease causing, to check for potentialinvolvement in pre-mRNA splicing. Via analysis of the RNA obtained from the patient's blood we were able to demonstrate that the

123 Chapter 9 SpliceSiteFinder-like [0-100] 74.6 MaxEntScan [0-12] 8.3 NNSPLICE [0-1] Wild type, no predicted splice site 0.9 GeneSplicer [0-15] 5.4 1 c.645 exon intron Wild- se uence ACGCCCACITCCCAGGCACCAGGACCATCATICAGAAGGAACCCATIGACCTCCGGGT CAAGGCCA t tc S'splice site

SpliceSiteFinder-like [0-100] "- 71.2 74·6 MaxEntScan [0-12] � 7.9 Mutant, strong predicted splice site 8.3 NNSPLICE [0-1] 0.9 0.9 GeneSplicer [0-15] 9.7 4.61

I Mutated sequence ACGCCCACITCCCA'jffACCAGGACCATCATTCAGAAGGAACCCA TTGACCTCCGGGTCAAGGCCAgtgagtd 5'splice site

Figure 2. RNA splicing prediction by four different methods: Splice Site Finder-like, Max Ent Scan, NNSPLICE and Gene Splicer, comparing the wild-type sequence with the mutant sequence (c.645C>T). The strength of the S'splice site is expressed on different scales, 0-100, 0-12, 0-1 and 0-15, respectively. The new splice donor site is much stronger according to GeneSplicer, 9.7 instead of 4.6. silent mutation, c.645C>T in exon 6 of the LlCAM gene was indeed pathogenic, because this mutation generates a new splice donor site (Chapter 2). Another silent mutation detected by Du et a/Y51 in exon 8 of the LlCAM gene, c.924C>T, was also shown to be a splice site mutation. Since RNA research is lengthy and rather costly, it is not part of the standard diagnostic tool kit for demonstratingthe pathogenicity of a mutation. However, due to our findings and those of Du et a/Y51, the possibility that a (silent) mutationmight disturb proper RNA splicing and be pathogenic should be noted. Splice site prediction programs can be a great help in indicating the pathogenicity, as shown in figure 2. The c.645C>T mutation is predicted by four different methods to create a splice donor site not occurring in the wild-type gene which is much stronger than the splice site at the beginning of intron 6 according to one of the methods (figure 2). Irrespective of the usefulness of the predictions, final proof should always come from RNA analysis (Chapter 2), not in the least because it might lead to RNA splice site mutations not predicted by these models. Since it is not always possible to isolate and analyze RNA from patients, a reliable alternativemight be an ex vivo RNA splicing assay, as described in Chapter 5. By applying this assay, we showed that the three other selected variants are now considered pathogenic due to disturbance of the correct RNA splicing: two mutations detected in an exon, c.1546G>T (p.Asp516Tyr) and c.1546G>A (p.Asp516Asn) and one detected in an intron, c.76+5G>A. Consequently, we recommend checking all mutations of unknown pathogenicity in both intrans and exons for their influence on RNA splicing. The number of mutations detected in our group of 367 patients and considered to be disease­ causing increased with this result from 61 (Chapter 2) to 64 (Chapter 5) and the number of likely disease-causing mutations decreased (from 7 to 4).

Interpretation of missense mutations In the 367 patients investigated, we found 73 patients with 68 different disease-causing or likely disease-causing mutations. Of these, 40 mutations were truncating and therefore considered to be disease-causing. The remaining 28 mutations were missense mutations

124 Discussion and future perspectives (n=23), in-frame deletions or duplications (n=3), and intronic variants outside the splice site consensus sequences (n=2), of which 24 were considered to be disease-causing and 4 as likely disease-causing. The missense mutations in the L1CAM gene are considered to be disease causing, for various reasons:

1. In Ll syndrome the number of de nova mutations detected in index patients is very high at "'7%.A de nova mutation found in the index patient is always considered to be pathogenic while a de nova mutationfound in the mother of an affectedchild is always seen as a strong indication. The number of de nova mutations in mothers or a germ line mosaicism in a grandparent of the index patientis ,.., 10%. In silica analysis of the protein plays an important role 2. The Ll protein is highly conserved. The replacement of a strongly conserved amino acid contributes positively to deciding that a missense mutation is considered to be pathogenic. 26 3. If a mutation influences the protein structure as described by Bateman et a/.! 1, notably 26 if it involves replacement of key residuesl 1, this is seen as a strong indication of pathogenicity because disturbance of the protein structure and therefore a change in the proper protein functionality are likely to occur. Further arguments are: 4. Co-segregation of the variant in the family is also seen as an indication for pathogenicity. In most cases, however, only a few family members are available so that this is not a very reliable tool, although excluding the mutationas pathogenic by finding it in unaffected males in the family does help. 5. Missense and silent mutationsshown to be responsible for incorrect RNA splicing are considered to be pathogenic. If they are only pinpointed by prediction programs, they are considered to be potentiallypathogenic. 6. Loss of function in a functional analysis. If a mutation affects the axon growth or influences the Ll protein dimer interaction or disturbs Ll protein interactionwith other relevant molecules needed for optimal functioning of the L1 protein, this also strongly indicates pathogenicity of the mutation.

Obviously, prior to finally labeling an L1CAM mutation as pathogenic, it is important to determine most of the above issues in order to make the conclusion as strong as possible.

Several studies concerning changes in protein functionality due to a mutation in the LlCAM 27 3 gene have been describedl - 11 • Figure 3 gives a schematic overview of the protein-protein 32 interactions and the pathways activated by the wild-type Ll proteinl 1 •

It was proven that some mutations influence the proper functioning of the L1 protein, since they disturb the interactionof the Ll protein with itself or with other proteins such as axonin-1 and Fll (both cell adhesion molecules) and neurocan, or they disturb the transfer of the 27 29 protein to the exterior of the celll , 1• Others have studied the effect that mutations may have on the growth and branching of axonsl30•311. Although these functional studies seem to be rather straightforward, they are not without interpretation problems and not indisputably predictive.

125 Chapter 9

Heterophilic interactions: Extracellular matrix components e.g. lam in in, neurocan, phosphocan, tenascin-R; lg superfamily CAMs e.g. DMl GRASP, F11/F3, Promote, Ll,Ll TAG-1/axonin-1 (ii \ ,;,,;,, \!) w """ '"''"" Required for I � neurite outgrowth ------Stimulationupon Ll:Ll binding

FGFR Plasma membrane

Tyrosine kinase domains

Signaling Phosphorylationby: cascade P90rsk, CKII " ERK2, CekS fl

Neurite outgrowth

Figure 3. The complexity of L1 protein interactions. The protein shows interactions with itself and with other proteins to initiate neurite outgrowth (adapted from Kenwrick et al.(32]). Sometimes normal protein interactions occur, irrespective of the pathogenicity of certain 29 mutations, as illustrated by mutation p.Lys655Glu[ 1, a pathogenic mutation segregating with the disease in a large X-linked pedigreer331_ From analyses by De Angelis et al. 1291 it may be concluded that this mutant protein still interacts normally with wild-type Ll protein and with axonin-1 and is correctly being transported to the exterior of the cell. However, because the overall interaction pattern of the Ll protein is quite complex[321 (figure 3), it cannot be ruled out that the pathogenicity is caused by disturbance of the interactionwith another protein, for instance in this example Fll or an unknown protein. Basically, these problems also apply to studies that look at the growth and branching of axons. Michelson et al.f3°1 seems to outline a nice method to confirm the pathogenicity of missense mutations. Four mutationsconsidered to be pathogenic were tested. All four showed a clearly reduced outgrowth of neurons, whereas studies by Cheng et alY11 indicate that most of the six considered pathogenic Ll mutations they tested did not affect neurite length significantly, but rather led to a decrease in branching. This clearly demonstrates the need for validated functional assays with known sensitivityand specificity, although these are extremely hard to determine. In order to do this one would need two sets of unclassified variants (UVs), one very likely to be pathogenic and the other very unlikely to be pathogenic, to be selected from all the UVs identified so far and based on the criteria mentioned. There is still a long way to go and at this point in time a functional assay with 100% reliability to confirm the pathogenicity of certain mutationsis not yet available.

126 Discussion and future perspectives llCAM database Since technologies for detecting mutationsar e becoming more and more advanced, the number of mutationside ntifiedis growing enormously, also implying an increase in the number of UVs identified. As discussed, additional research and tool development is needed to cope with this. One such tool is our L1CAM mutation database (described in Chapter 4), which is freely accessible and contains not only a list of mutations but also other relevant information. This extra information might outline clinical characteristics of patients, co-segregation in the fa mily, and results from functional assays, RNA splicing experiments, in silico analysis and the like. Our L1CAMmu tation database not only contains mutations detected in our own lab, but also published mutations including any relevant information given and, if necessary, supplemented by us with in silica data. As a result this database is a good tool for researchers and counselors, providing access to all the data in a fast and simple way and enabling them to draw conclusions on the pathogenicity and severity of L1CAM mutationsbased on multiplefactors.

Other Ll syndrome genes Although the frequency of mutation detection in specific well-defined patient groups is high {85%), a large group of patients clearly suspected of having Ll syndrome still remains unexplained. It is therefore temptingto assume that the L1CAM gene is not the only gene involved in Ll syndrome development. One of the aims of this thesis was to identify potentially new genes, as described in Chapter 8, and this work will be further discussed in Part Ill of this chapter.

Future perspectives Although we have been able to improve the diagnostic tool box for patient care, we need to upgrade it continuously, for instance by: ;,.- Sequencing the 3' end of the L1CAM gene This region of the L1CAM gene will be sequenced to look for possible mutations which alter miRNA recognitionsi tes, consequently making them potentially disease­ causing. };;> Functional assays A good functionalassay is still lacking to determine whether a missense mutation is pathogenic. This needs to be a fast and reliable assay for standard applicationin DNA diagnostics which we feel may be best based on an assay to measure the outgrowth and debranching of neurons[3o1. };;> Incorrect pre-mRNA splicing lntronic mutationsar e found regularly, but we do not know if they are pathogenic. After the introductionof next generation sequencing technology, it is expected that this number will increase quite dramatically and there will be a need for a reliable ex vivo RNA splicing assay. This should also be less dependent on the availability of patientRN A. The first experiments we performed look very promising (described in Chapter S) and further research will lead to a standard application in DNA diagnostics. Furthermore, with this assay, all mutations in exons of unknown pathogenicity will be checked for their influence on RNA splicing.

127 Chapter 9 PART II Syndromic patients including characteristics of the Ll syndrome

Patients with several congenital abnormalities in which the Ll syndrome characteristics are part of the phenotype have been described in literature134·411• In most of these patients a mutation in the LlCAM gene was found, sometimes in addition to another chromosomal aberration136•371• We investigated two such patients, one with Nephrogenic Diabetes lnsipidus (NDI) and Ll syndrome (Chapter 6) and one with Hirschsprung's disease (HSCR) and Ll syndrome (Chapter 7). The reasons to study these patients were to determine whether the clinical picture caused by mutations in the LlCAM gene is broader than thought or that these clinical presentations are not (solely) based on a mutationin the LlCAM gene, but due to an aberrationin a different gene or a large deletion including the LlCAM gene. If we could detect new genes by such an approach it would also be interesting to check whether mutationsin these genes could explain the clinical features of some of the L1 syndrome patients who do not have a mutation in the LlCAM gene.

Can Nephrogenic Diabetes lnsipidus {NOi) be explained by a mutation in the l1CAM gene alone? Two patients have been described suffering from both Ll syndrome and NDI: one is described in Chapter 6 and one was previously reported1361• NDI is characterized by the excessive loss of dilute urine due to the inability of the renal collecting tubules to respond to vasopressin and it is caused by mutationsin the AVPR2 gene142A31_ Since the LlCAM and AVPR2 genes are located close to each other on the long arm of the X chromosome, we analyzed our patient for the presence of a deletion on the X chromosome including both genes as an explanationfor the dual phenotype. We found a deletion of 61.577 kb encompassing the entire LlCAM and AVPR2 genes, thereby confirming the linked clinical characteristic in our patient. The second patient was described as having virtually the same phenotype1361 and showed a deletion of 32.7 kb from LlCAM intron 1 to AVPR2 exon 2, thus confirming the results for our patient. Finding this deletion as an explanationfor the linked phenotype led us to conclude that the NDI phenotypic features are caused by the loss of the AVPR2 gene rather than the loss of the LlCAM gene.

Hirschsprung's disease {HSCR) and l1 syndrome At least 13 cases concerning patients with joint HSCR and Ll syndrome characteristics have been reported in literaturel22, 36•38·41A4-461· In 11 of these patients, a mutation in the LlCAM gene could be detected122, 36•38·41A4A5l_ No pathogenic mutation in the RET gene, which is associated with HSCR disease, was detected in these patients as far as it was tested for. In our study (Chapter 7) we carried out the molecular characterization of a ten-year old boy affected with HSCR disease, X-linked hydrocephalus and vesico-ureteral reflux. A de nova reciprocal balanced translocation, t(3;17)(p12.l;q11.21), was found by karyotyping. Mutation analysis of the LlCAM gene and the RET gene showed a frame shift mutation in the

128 Discussion and future perspectives LlCAM gene, c.2265delC, leading to a premature stop codon in the LlCAM gene, whereas no pathogenic mutation could be detected the RET gene. Although we cannot exclude an involvement of the translocation being involved in the phenotype of our patient, our data in combination with the data presented in the literature showed that a large proportion of patients with Ll syndrome in combination with HSCR do in fact have an L1CAM mutation. The frequency is far higher than one would expect based on random matingl45l, suggesting that there might be a link between LlCAM mutations and HSCR. The low incidence of HCSR disease in patients with LlCAM mutations (about 3%} and the absence of L1CAM mutations in males affectedwith isolated HSCRf45l suggest that LlCAM is not a causative gene of HSCR, but that it likely acts as a modifier gene in the development of HSCRl3s,4s,45l_ Additional factors might promote the occurrence of HSCR, these include the RET predisposing haplotype, which is a specific combination of genetic variants located between the promoter and exon 2 of the genel471 • Our patientproved heterozygous for this particular haplotype. This hypothesis requires further work to prove it valid. In conclusion the intestinalaganglionosis of HSCR might be due in part to LlCAM mutations.

Future perspectives We have described the possible modifier role that the L1CAM gene might have in HSCR. It would be interesting to prove the gene plays this role by finding evidence for the hypothesis that the RET predisposing haplotype contributes to the intestinal aganglionosis under the influence of an L1CAM mutation. To prove this we need to demonstrate that the presence of the RET haplotype in patients with an LlCAM mutation is significantly higher in those with HSCR disease than those without HSCR disease, but as the number of patients available is very small, this will be hard to prove.

PART Ill Do other Ll syndrome genes exist?

Only -20% of all the patients referred to our laboratory for LlCAM testing turned out to harbor a mutation in the LlCAM gene, implying that 80% of them did not have a mutation in this gene. If we correct for the number of patientsthat were analyzed to simply exclude the Ll syndrome, the number suspected of having Ll syndrome but with no mutation in the LlCAM gene drops to 75%. This is still very high. Even when patientsare very stringently pre-selected using the clinical criteria mentioned, we still do not find mutations in 15-20% of our patients. These findings led to the hypothesis that other genes might be involved in L1 syndrome or an atypical form of it. In order to test this hypothesis we attempted to find such genes by selecting patients without an LlCAM mutation and analyzing them for large deletions using a high-density, X-chromosome-specific array-CGH (Chapter 8).

X-chromosome-specific array-CGH analysis An X-chromosome-specific array-CGH analysis was used to detect deletions that could include genes potentially involved in Ll syndrome. We focused on the X-chromosome since 95% of all referred patients suspected of having L1 syndrome are males and familial cases mostly show

129 Chapter 9 an X-linked recessive inheritance. In order to increase the chance of finding a new gene, we designed a very dense array. All the deletions found could in principle be associated with the Ll syndrome, unless they concerned a polymorphism (Chromosomal Neutral Variant (CNV)). Deleted genes were classified as candidate Ll syndrome genes based on different criteria, for instance their gene product or their expression in tissuesof interest, such as the brain or neurons, or their expression in an early stage of fetal development, or because of the features of the deletion itself, namely its size and/or localization (within coding or non-coding regions of the gene). By screening 60 patients, selected because they were clinically suspected of having Ll syndrome, we identified 11 candidate deletions containing 11 candidate genes (Chapter 8). At least eight out of eleven genes are expressed in the brain or in neurons (DGAT2L6, POU3F4, GD/1, ASMTL, ATP6AP1, M/01, FRMPD4 and MAP3K15}, and at least three out of eight are expressed in an early stage of development (POU3F4, GD/1 and ATP6AP1}. The other three candidate genes are TGIF2LX, DDX53 and TMEM27. As our analysis only detected deletions and no point mutations, which are probably far more common, we selected five genes as being the best candidates (based on different criteria, see table 2 Chapter 8) for further sequence analysis (POU3F4, DGAT2L6, GD/1, DDX53 and TGIF2LX}. The analysis was performed in 95 patients suspected of having Ll syndrome without an LlCAM mutation. In three out of the 95 patients we detected a variant: two intronic variants in GD/1, c.153-3C>T in intron 2 and c.992-lOC>T in intron 8, and one missense mutation in TGIF2LX, c.421T>C (p.Ser141Pro). The c.153-3C>T variant is most likely to be disease-causing, while the other two are probably not disease-causing (Chapter 8). We therefore concluded that in these 95 patients the evidence for a second gene involved in Ll syndrome is still rather weak, with perhaps the exception of the GD/1 gene which seemed to be the most likely candidate. The remaining six candidate genes were not further analyzed by sequencing the DNA of the 95 patients, because they were estimated to be less likely candidate genes. However, at first glance the M/01 gene seemed to be of interest, because we detected a deletion in three different patients (table 2, Chapter 8), but since the deletions comprised only intrans and no coding regions, this gene was also labeled as a less probable candidate for Ll syndrome. Basically the same also applied to the MAP3K15 gene. These results show that we still lack hard evidence for any gene other than the LlCAM gene to explain Ll syndrome in many patients. However, the possible involvement of the candidate genes we identified cannot be excluded in patients suspected of having Ll syndrome, because of the presence of deletions in or from these genes and for one gene an additional point mutationin this gene.

Future perspectives The existence of a second gene explaining Ll syndrome in patients suspected of having Ll syndrome has not yet been clearly demonstrated. We might have overlooked putative new L1 syndrome genes due to the design of our study: we only looked for reasonably large deletions and so we would have missed genes that only have point mutations or small deletions. Most probably a more advanced technology will be needed to detect other genes involved in L1 syndrome. We therefore plan to perform X-chromosome-specific exome resequencing of patients clinically suspected of having Ll syndrome and not carrying an LlCAM mutation. This

130 Discussion and future perspectives will allow us to draw better conclusions as to the involvement of other genes in Ll syndrome in the near future. Finally, we should point out that perhaps it is not a matter of one or two unknown genes but rather a whole set of genes each of them explaining the disorder in a few patients suspected of having Ll syndrome and with no mutationin the LlCAM gene.

131 Chapter 9 References

(1) Jouet M, Feldman E, Yates J, Donnai D, Paterson J, Siggers D, Kenwrick S. Refining the genetic location of the gene for X linked hydrocephalus within Xq28. J Med Genet 1993 Mar;30(3):214-7. (2) Jouet M, Rosenthal A, MacFarlane J, Kenwrick S, Donnai D. A missense mutation confirms the L1 defect in X-linked hydrocephalus (HSAS). Nat Genet 1993 Aug;4(4):331. (3) Coucke P, Vits L, Van Camp G, Serville F, Lyonnet S, Kenwrick S, Rosenthal A, Wehnert M, Munnich A, Willems PJ. Identification of a 5' splice site mutation in intron 4 of the LlCAM gene in an X-linked hydrocephalus family. Hum Mol Genet 1994 Apr;3(4):671-3. (4) Tapanes-Castillo A, Weaver EJ, Smith RP, Kamei Y, Caspary T, Hamilton-Nelson KL, Slifer SH, Martin ER, Bixby JL, Lemmon VP. A modifierlocus on chromosome 5 contributes to Ll cell adhesion molecule X-linked hydrocephalus in mice. Neurogenetics 2010 Feb;ll(l):53-71. (5) ZhangJ, Williams MA, RigamontiD. Genetics of human hydrocephalus. J Neurol 2006 Oct;253(10):1255- 66. (6) van der Hout AH, van den Ouweland AM, van der Luijt RB, Gille HJ, Bodmer D, Bruggenwirth H, Mulder IM, van der Vlies P, Elfferich P, Huisman MT, Ten Berge AM, Kromosoeto J, Jansen RP, van Zan PH, Vriesman T, Arts N, Lange MB, Oosterwijk JC, Meijers-Heijboer H, Ausems MG, Hoogerbrugge N, Verhoef S, Halley DJ, Vos YJ, Hogervorst F, Ligtenberg M, Hofstra RM. A DGGE system for comprehensive mutation screening of BRCAl and BRCA2: application in a Dutch cancer clinic setting. Hum Mutat 2006 J ul;27(7) :654-66. (7) Diena AF, PattonJG. Genomic organization of microRNAs. J Cell Physiol 2010 Mar;222(3):540-5. (8) Meola N, Gennarino VA, Banfi S. microRNAs and genetic diseases. Pathogenetics2009;2(1):7. (9) Beetz C, Schule R, Deconinck T, Tran-Viet KN, Zhu H, Kremer BP, Frints SG, Zelst-Stams WA, Byrne P, Otto S, Nygren AO, Baets J, Smets K, Ceulemans B, Dan B, Nagan N, Kassubek J, Klimpe S, Klopstock T, Stolze H, Smeets HJ, Schrander-Stumpel CT, Hutchinson M, van de Warren burg BP, Braastad C, Deufel T, Pericak-Vance M, Schols L, de Jonghe P, Zuchner S. REE Pl mutation spectrum and genotype/phenotype correlation in hereditary spasticparaplegia type 31. Brain 2008 Apr;l31(Pt 4):1078-86. (10) Zuchner S, Wang G, Tran-Viet KN, Nance MA, Gaskell PC, Vance JM, Ashley-Koch AE, Pericak-Vance MA. Mutations in the novel mitochondrial protein REEPl cause hereditary spastic paraplegia type 31. Am J Hum Genet 2006 Aug;79(2):365-9. (11) Finckh U, Schroder J, Ressler B, Veske A, Gal A. Spectrum and detection rate of LlCAM mutations in isolated and familial cases with clinically suspected Ll-disease. Am J Med Genet 2000 May 1;92(1):40- 6. Nov;lOS(S (12) Kanemura Y, Okamoto N, Sakamoto H, Shofuda T, Kamiguchi H, Yamasaki M. Molecular mechanisms and neuroimaging criteria for severe Ll syndrome with X-linked hydrocephalus. J Neurosurg 2006 Suppl):403-12. (13) Graf WD, Born DE, Shaw DW, Thomas JR, Holloway LW, Michaelis RC. Diffusion-weighted magnetic resonance imaging in boys with neural cell adhesion molecule Ll mutations and congenital hydrocephalus. Ann Neurol 2000 Jan;47(1):113-7. (14) Weller S, Gartner J. Genetic and clinical aspects of X-linked hydrocephalus (Ll disease): Mutations in the LlCAM gene. Hum Mutat 2001;18(1):1-12. (15) Fransen E, Van Camp G, D'Hooge R, Vits L, Willems PJ. Genotype-phenotype correlationin Ll associated diseases. J Med Genet 1998 May;35(5):399-404. (16) Yamasaki M, Thompson P, Lemmon V. CRASH syndrome: mutations in LlCAM correlate with severity of the disease. Neuropediatrics 1997 Jun;28(3):175-8. Nov;35( (17) Michaelis RC, Du YZ, Schwartz CE. The site of a missense mutation in the extracellular lg or FN domains of LlCAM influencesinfant mortality and the severity of X linked hydrocephalus. J Med Genet 1998 11) :901-4. (18) Kamiguchi H, Hlavin ML, Yamasaki M, Lemmon V. Adhesion molecules and inherited diseases of the human nervous system. Annu Rev Neurosci 1998;21:97-125. (19) Schrander-Stumpel C, Howeler C, Jones M, Sommer A, Stevens C, Tinschert S, Israel J, Fryns JP. Spectrum of X-linked hydrocephalus (HSAS), MASA syndrome, and complicated spastic paraplegia (SPGl): Clinical review with six additional families. Am J Med Genet 1995 May 22;57(1):107-16.

132 Discussion and future perspectives

(20) Willems PJ, Brouwer OF, Dijkstra I, Wilmink J. X-linked hydrocephalus. Am J Med Genet 1987 Aug;27(4):921-8. (21) Saugier-Veber P, Martin C, Le Meur N, Lyonnet S, Munnich A, David A, Henocq A, Heron D, Jonveaux P, Odent S, Manouvrier S, Monda A, Morichon N, Philip N, Satge D, Tosi M, Frebourg T. Identification of novel LlCAM mutations using fluorescence-assisted mismatch analysis. Hum Mutat 1998;12(4):259- 66. (22) Vits L, Chitayat D, Van Camp G, Holden JJ, Fransen E, Willems PJ. Evidence for somatic and germline mosaicism in CRASH syndrome. Hum Mutat 1998;Suppl 1:5284-5287. (23) Du JS, Bason L, Woffendin H, Zackai E, Kenwrick S. Somatic and germ line mosaicism and mutation origin for a mutation in the Ll gene in a family with X-linked hydrocephalus. Am J Med Genet 1998 Jan 13;75(2):200-2. (24) Jouet M, Strain L, Bonthron D, Kenwrick S. Discordant segregation of Xq28 markers and a mutation in the Ll gene in a family with X linked hydrocephalus. J Med Genet 1996 Mar;33(3):248-50. (25) Du YZ, Dickerson C, Aylsworth AS, Schwartz CE. A silent mutation, C924T (G308G), in the LlCAM gene results in X linked hydrocephalus (HSAS). J Med Genet 1998 Jun;35(6):456-62. (26) Bateman A, Jouet M, MacFarlane J, Du JS, Kenwrick S, Chothia C. Outline structure of the human Ll cell adhesion molecule and the sites where mutations cause neurological disorders. EMBO J 1996 Nov 15;15(22):6050-9. (27) De Angelis E, MacFarlane J, Du JS, Yeo G, Hicks R, Rathjen FG, Kenwrick S, Brummendorf T. Pathological missense mutations of neural cell adhesion molecule Ll affect homophilic and heterophilic binding activities. EMBO J 1999 Sep 1;18(17):4744-53. (28) Needham LK, Thelen K, Maness PF. Cytoplasmic domain mutationsof the Ll cell adhesion molecule reduce Ll-ankyrin interactions. J Neurosci 2001 Mar 1;21(5):1490-500. (29) De Angelis E, Watkins A, Schafer M, Brummendorf T, Kenwrick S. Disease-associated mutations in Ll CAM interfere with ligand interactions and cell-surface expression. Hum Mol Genet 2002 Jan 1;11(1):1- 12. (30) Michelson P, Hartwig C, Schachner M, Gal A, Veske A, Finckh U. Missense mutations in the extracellular domain of the human neural cell adhesion molecule Ll reduce neurite outgrowth of murine cerebellar neurons. Hum Mutat 2002 Dec;20(6):481-2. (31) Cheng L, Lemmon V. Pathological missense mutations of neural cell adhesion molecule Ll affect neurite outgrowth and branching on an Ll substrate. Mol Cell Neurosci 2004 Dec;27(4):522-30. (32) Kenwrick 5, Watkins A, De Angelis E. Neural cell recognition molecule Ll: relating biological complexity to human disease mutations. Hum Mol Genet 2000 Apr 12;9(6):879-86. (33) Vits L, Van Camp G, Coucke P, Fransen E, De Bou lie K, Reyniers E, Korn B, Poustka A, Wilson G, Schrander­ Stumpel C, . MASA syndrome is due to mutations in the neural cell adhesion gene LlCAM. Nat Genet 1994 Ju1;7(3):408-13. (34) Bott L, Boute 0, Mention K, Vinchon M, Boman F, Gottrand F. Congenital idiopathic intestinal pseudo­ obstruction and hydrocephalus with stenosis of the aqueduct of sylvius. Am J Med Genet A 2004 Sep 15;130(1):84-7. (35) Liebau MC, Gal A, Superti-Furga A, Omran H, Pohl M. LlCAM mutation in a boy with hydrocephalus and duplex kidneys. Pediatr Nephrol 2007 Jul;22(7):1058-61. (36) Tegay DH, Lane AH, Roohi J, Hatchwell E. Contiguous gene deletion involving LlCAM and AVPR2 causes X-linked hydrocephalus with nephrogenic diabetes insipidus. Am J Med Genet A 2007 Mar 15;143(6):594-8. (37) Hiraki Y, Okamoto N, Ida T, Nakata Y, Kamada M, Kanemura Y, Yamasaki M, Fujita H, Nishimura G, Kato M, Harada N, Matsumoto N. Two new cases of pure lq terminal deletion presenting with brain malformations. Am J Med Genet A 2008 May 15;146A(10):1241-7. (38) Okamoto N, Del Maestro R, Valero R, Monros E, Poo P, Kanemura Y, Yamasaki M. Hydrocephalus and Hirschsprung's disease with a mutation of LlCAM. J Hum Genet 2004;49(6):334-7. (39) Basel-Vanagaite L, Straussberg R, Friez MJ, lnbar D, Korenreich L, Shohat M, Schwartz CE. Expanding the phenotypic spectrum of LlCAM-associated disease. Clin Genet 2006 May;69(5):414-9. (40) Nakakimura 5, Sasaki F, Okada T, Arisue A, Cho K, Yoshino M, Kanemura Y, Yamasaki M, Todo S. Hirschsprung's disease, acrocallosal syndrome, and congenital hydrocephalus: report of 2 patients and literature review. J Pediatr Surg 2008 May;43(5):El3-E17.

133 Chapter 9

(41) Jackson SR, Guner YS, Woo R, Randolph LM, Ford H, Shin CE. LlCAM mutation in association with X-linked hydrocephalus and Hirschsprung's disease. Pediatr Surg Int 2009 Jul 30. (42) Rosenthal W, Seibold A, Antaramian A, Lonergan M, Arthus MF, Hendy GN, Birnbaumer M, Bichet DG. Molecular identificationof the gene responsible for congenital nephrogenic diabetes insipid us. Nature 1992 Sep 17;359(6392):233-5. (43) Frattini A, Zucchi I, Villa A, Patrosso C, Repetto M, Susani L, Strina D, Redolfi E, Vezzoni P, Romano G. Type 2 vasopressin receptor gene, the gene responsible nephrogenic diabetes insipidus, maps to Xq28 close to the LICAM gene. Biochem Biophys Res Commun 1993 Jun 30;193(3):864-71. (44) Okamoto N, Wada Y, Goto M. Hydrocephalus and Hirschsprung's disease in a patient with a mutation of LlCAM. J Med Genet 1997 Aug;34{8):670-1. (45) Parisi MA, Kapur RP, Neilson I, Hofstra RM, Holloway LW, Michaelis RC, Leppig KA. Hydrocephalus and intestinalaga nglionosis: is LlCAM a modifier gene in Hirschsprung disease? Am J Med Genet 2002 Feb 15;108(1):51-6. (46) Hofstra RM, Elfferich P, Osinga J, Verlind E, Fransen E, Lopez Pison J, Die-Smulders CE, Stolte-Dijkstra I, Buys CH. Hirschsprung disease and LlCAM: is the disturbed sex ratiocaused by LlCAM mutations? J Med Genet 2002 Mar;39(3):E11. (47) Burzynski GM, Nolte IM, Branda A, Bos KK, Osinga J, Plaza Menacho I, Twigt B, Maas S, Brooks AS, Verheij JB, Buys CH, Hofstra RM. Identifyingcandidate Hirschsprung disease-associated RET variants. Am J Hum Genet 2005 May;76(5):850-8.

134

Chapter 10

Summary Chapter 10 Ll syndrome is an X-linked recessive disorder with a prevalence of 1:30,000 newborn males. It comprises four neurological syndromes, all associated with mutations in the L1CAM gene. The most severe of the four different forms is X-linked hydrocephalus, also referred to as hydrocephalus due to stenosis of the aqueduct of Sylvius (HSAS). Other clinical characteristics are adducted thumbs, mental retardation, and lower limb spasticity. In most fa milies, the affectedboys die before, during, or soon afterbir th. A less severe form of the disease has been published under the acronym MASA syndrome. This syndrome consists of light to moderate mental retardation, .§.phasia, �huffiing gait and .§.dducted thumbs. X-linked complicated hereditary spastic paraplegia type 1 and X-linked complicated corpus callosum agenesis (X-linked ACC) are also parts of the Ll syndrome, but are seen much less frequently. The complicated forms may be combined with mild to moderate mental retardation.Overa ll, the seven most important characteristics of Ll syndrome are hydrocephalus, aqueduct stenosis, mental retardation, adducted thumbs, agenesis/dysgenesis of the corpus callosum, aphasia (delayed speech development) and spastic paraplegia. The L1CAM gene, located on the X-chromosome at Xq28, codes for the neural cell adhesion molecule Ll (Ll protein) and is primarily expressed in the nervous system. The gene consists of 29 exons and encodes a protein of 1,257 amino acids. The first exon of 125 bp is non-coding. The Ll protein is a transmembrane glycoprotein of approximately 200 kDA belonging to the immunoglobulin superfamily cell adhesion molecules and contains thirteen distinct domains, i.e. six immunoglobulin- (lg) and fivefi bronecti n (Fn) Ill-like domains at the extracellular surface, one single-pass transmembrane domain, and one short cytoplasmic domain. The Ll protein is involved in neuronal cell migration, neurite outgrowth and axon bundling (fasciculation), myelination, and cell-cell adhesion on differentiated neurons.

The aims of this thesis (Chapter 1) were: to develop a tool to predict the chance of detecting a mutation in the L1CAM gene in patients suspected of having Ll syndrome (Chapter 2); to look for genotype-phenotype correlations (Chapter 2); to set up an easily accessible L1CAM mutation database and include all disease-causing and non-disease-causing mutations, all unclassified variants (UVs) and all relevant patientda ta (Chapter 4); to prove the pathogenicity of some UVs caused by incorrect RNA splicing (Chapter S); to look for possible other clinical features caused by an L1CAM mutation (Chapters 6 and 7); and to identify new genes involved in L1 syndrome (Chapter 8). In Chapter 2 the DNA of 367 referred patients suspected of having Ll syndrome was analyzed for mutations in the coding sequences and the exon-intron boundaries of the L1CAM gene. A subgroup of 100 patients was also investigated for mutations in the promoter and two regulatory sequences of the L1CAM gene, e.g. the neural restrictive silencer element (NRSE) and the homeodomain and paired domain binding site (HPD), and for large duplications to determine whether there were additional mutations in these regulatory regions or any duplications. As a result in 73 patients (20%) 68 diffe rent mutations were detected: 61 are considered to be disease-causing and 7 to be unclassified variants (UVs). In one patient a duplication from exons 2-10 was identified. No mutations were detected in the promoter, the HPD or the NRSE sites of the 100 patients analyzed. Based on these results it was concluded, that broadening the DNA screening procedure to include regulatory sequences analysis is not worthwhile, although screening for duplications might be beneficial. Furthermore, clinical data on 106 patients, 31 of whom carried a mutation, were collected

138 Summary and used for statistical analysis. In patients with three or more clinical characteristics of Ll syndrome, the mutation detection rate was 66% compared to 16% in patients with one or two characteristics (odds ratio (OR) 10.1, 95% confidence interval (Cl} 3.8 to 27.1). The detection rate was 18% in families with one affected male and 51% in families with more than one affected family member. A combinationof these two characteristics, i.e. patients having a positive family history for Ll syndrome and having three or more clinical characteristics, results in a detection rate of 85% (OR 10.4, 95% Cl 3.6 to 30.1). Using the data of the whole group a diagram could be drawn up, showing the mutation detection rate related to the number of clinical characteristics and the number of affected family members. By using this diagram, clinicians can now accurately predict the chance of finding a mutation in a patient, thus fulfilling one of the aims of the thesis. The analysis also confirmed a previously reported genotype-phenotype correlation: children harboring a truncating mutation died more frequently (52%) before the age of 3, than children with a missense mutation (8%} (Fisher exact p=0.02). In contrast to the findings of other groups, we did not find a difference in the severity of hydrocephalus in patients with a missense mutation in key residues versus surface residues. Finally, the occurrence of (maternal) germ line mosaicism for an L1CAM mutation was proven in this study and three females with Ll syndrome carrying an L1CAM mutationwere reported. The latterfinding, the detection of L1CAM mutations in women with the Ll syndrome, is an important observation. To date, it was believed that mutations in the L1CAM gene did not really cause Ll syndrome in women, since only a very mild phenotype was observed in approximately 5% of the mutation carriers. Our results show that also severe phenotypes of the Ll syndrome do occur in women, which has very important consequences for counseling, notably for prenatal diagnostics. Chapter3 describes a novel mutationin the L1CAM gene in a male with X-linked hydrocephalus who had multiplesmall gyri, hypoplasia of the white matter, agenesis of the corpus callosum, and lack of cleavage of the thalami. Three brothers with congenital hydrocephalus died in the neonatal period. Mutation analysis of the L1CAM gene revealed a missense mutation, p.Ala415Pro in the lg 4 domain of the Ll protein. Since alanine 415 is considered a key residue crucial for protein folding, it is likely that the X-linked hydrocephalus and cerebral dysgenesis are a result of the abnormal structure and function of the mutant Ll protein. In Chapter 4 the construction of the L1CAM mutation database is described. About 240 different L1CAM mutations have been reported in the literature, disease-causing and non­ disease-causing mutations as well as unclassified variants. Since the amount of information is growing rapidly and it is important for geneticists and clinicians to have easy access to this information, the existing database was renewed and updated. New information recorded in the database comprises: (1) clinical data (including age at early death), (2) family anamnesis, (3) the reported clinical classification,(4) elements which co-determine the possible pathogenicity of a variant, e.g. co-segregationof the variant in the family, de nova appearance in the patient or de nova appearance in the mother of the patient (5) in silica data concerning the variant, (6) effectof a variant in a functionalassay, and (7) effectof a variant on the pre-mRNA splicing. As a result, the database now offers much more complete information, allowing conclusions to be drawn on the pathogenicity and severity of L1CAM mutations based on multiple factors. The database will be updated monthly with newly published data. The L1CAM Mutation Database is at: www.llcammutationdatabase.info.

139 Chapter 10 In Chapter 5 the results are described from using an ex vivo RNA splicing assay as a tool to further substantiatethe assumed pathogenicty of certain LlCAM variants. Analyzing the seven unclassified variants (UVs) detected in the study described in chapter 2 with in silico prediction programs, it was concluded that at least 3/7 UVs might be involved in aberrant splicing of the pre-mRNA. In order to prove this effect an ex vivo RNA splicing assay was used and all three UVs (c.76+5G>A, c.1546G>A (Asp516Asn) and c.1546G>T (p.Asp516Tyr)) were fo und to be pathogenic. The reliability of the ex vivo RNA splicing assay was confirmed with the LlCAM c.645C>T mutation, a mutation previously proven to be pathogenic due to incorrect splicing (Chapter 2). In conclusion, 64 (instead of 61) of the 68 mutations described in chapter 2 are now considered as pathogenic. Chapter 6 deals with the geneticanal ysis of a boy with Ll syndrome and nephrogenic diabetis insipidus (NDI). We wondered whether this phenotype could be explained by mutationsin the LlCAM gene alone or by a large deletioninclud ing the LlCAM and the AVPR2 genes. The deletion hypothesis was based on the fact that NDI is caused by mutationsin the AVPR2 gene, which is located close to the L1CAM gene on the X chromosome. A deletionof 61,577 bp was detected, encompassing the entire LlCAM and AVPR2 genes. In conclusion, the NDI phenotypic features are caused by the loss of the AVPR2 gene rather than the loss of the LlCAM gene. In Chapter 7 the molecular characterizationof a patient affectedwith X-linked hydrocephalus and Hirschsprung's (HSCR) disease is outlined. Karyotype analysis showed a de nova reciprocal balanced translocation t(3;17)(p12;q21). Mutation analysis of the LlCAM gene and the RET gene, which is associated with HSCR disease, showed a frame shift mutation in the LlCAM gene, c.2265delC, leading to a premature stop codon in the LlCAM gene, whereas in the RET gene no pathogenic mutation could be detected. Characterization of the chromosome 17 breakpoint showed interruption of the MYOlBA and TIAFl genes. Besides the LlCAM mutation, a possible involvement of the MYO18A and TIAFl genes on the phenotype cannot be excluded. To date, most patients reported with co-occurrence of HSCR and Ll syndrome were proven to have mutations in the LlCAM gene, which was proposed as a candidate HSCR gene. But the low incidence of HCSR disease in patients with LlCAM mutations(a bout 3%) and the absence of LlCAM mutations in males affected with isolated HSCR suggest that LlCAM is not a causative gene for HSCR, but that it likely acts as a modifier gene for the development of HSCR. Because only 20% of the patients clinically suspected of having L1 syndrome carry a mutation in the LlCAM gene, it was hypothesized that other genes might be involved in Ll syndrome. In Chapter 8 we searched for such genes on the X-chromosome by screening for deletions in 60 patients suspected of having Ll syndrome using a high density X-chromosomal array, assuming that the deletionwo uld harbor a new Ll syndrome gene. Analysis of the results revealed eleven candidate disease-causing deletions in twelve patients, involving eleven candidate genes: OGAT2L6, POU3F4, GOil, ASMTL, ATP6AP1, M/Dl, FRMPO4, MAP3K15, TGIF2LX, ODX53, and TMEM27. To substantiatetheir involvement in Ll syndrome it was decided to sequence the five most promising candidate genes, OGAT2L6, POU3F4, GOil, TGIF2LX and OOX53 in 95 L1 syndrome patients. This analysis showed only one gene, GOil, to be further involved, as in addition to the deletion found a variant was also detected, c.153-3C>T, in one of the 95 patients.Thi s variant is most probably disease causing because it

140 Summary influenced correct RNA splicing in an ex vivo RNA splicing assay. We concluded that these five genes do not appear to be major players in Ll syndrome, but a role could not be excluded for these genes in the disease development. Furthermore, as only five of the eleven genes were sequenced, it is still possible, that one or more of the remaining six genes could be more frequently involved in patients suspected of having L1 syndrome. Since we searched for new genes by looking for deletions (> 2kb), we would have missed any genes that only have point mutations or small deletions. To study this X-chromosome-specific exome resequencing will be carried out for a selected group of patients suspected of having Ll syndrome by using next generation sequencing technique. We concluded that several more genes, in addition to the LlCAM gene, might be involved in Ll syndrome: so far the GD/1 gene emerges as the best gene, but further research will be necessary. In Chapter 9the results described in this thesis are discussed and the future plans are presented. The most important plans for our future research are to: (1) set up a good functional assay and (2) search for new genes that can explain the L1 syndrome in patients suspected of having Ll syndrome but who do not carry an LlCAM mutation.

141

Nederlandse samenvatting Nederlandse samenvatting

Het Ll syndroom is een X chromosoom gebonden recessieve aandoening, die bij een op de 30.000 pasgeboren jongetjes voorkomt. Vier verschillende neurologische aandoeningen, die alle vier veroorzaakt warden door mutaties in het L1CAM gen, behoren tot het syndroom. De meest ernstige vorm van de vier aandoeningen staat bekend ender de naam X-gebonden hydrocefalie (waterhoofd), oak wel bekend als hydrocefalie veroorzaakt door een vernauwing van het aqueduct van Sylvius (HSAS: b.vdrocefalie veroorzaakt door �tenose van het--9.queductvan ,S_ylvius).Andere klinische kenmerken van ditsyndroom zijn duimen gefixeerd in de adductiestand (zie figuur 1 pagina 12), mentale retardatie (verstandelijke beperking) en spasticiteit van de benen (loopstoornis). In de meeste families overlijden de aangedane jongetjes voor, tijdens of vlak na de geboorte. Een veel minder ernstige uiting van de aandoening staat bekend onder het acroniem MASA syndroom, dat gekenmerkt wordt door een lichte of matige vorm van mentale retardatie, afasie (een spraakstoornis, meestal vertraagde spraakontwikkeling), een loopstoornis en eveneens geadduceerde duimen. X-gebonden gecompliceerde erfelijke spastischeparaplegie type 1 (spastischeverlamming aan beide zijden van het lichaam) (X-linked SPGl) en de X-gebonden gecompliceerde vorm van de agenesie van de corpus callosum (het gedeeltelijk of geheel ontbreken van de hersenbalk) (X-linked ACC) behoren ook tot het Ll syndroom, maar komen zeer zelden voor. Deze laatste twee vormen kunnen gepaard gaan met een lichte mate van mentale retardatie. Samengevat zijn de zeven belangrijkste kenmerken van het Ll syndroom; een hydrocefalie, een aqueductstenose, mentale retardatie, duimen in de adductiestand, agenesie of dysgenesie van de corpus callosum, afasie en spastische paraplegie. Zoals gezegd warden deze syndromen veroorzaakt door mutaties in het L1CAM gen, dat gelegen is op het X-chromosoom op positie Xq28. Het gen codeert voor het neurale celadhesie molecuul Ll (Ll eiwit) en komt voornamelijk tot expressie in het zenuwstelsel. Het bestaat uit 29 exonen en codeert voor een eiwit van 1257 aminozuren. Het eerste exon van 125 bp is niet coderend. Het Ll eiwit is een transmembraan glycoprotein van ongeveer 200 kD behorende tot de immunoglobuline superfamilie van celadhesie moleculen en bevat dertien verschillende domeinen, te weten zes immunoglobuline- en vijf fibronectine 111-achtige extracellulaire domeinen, een transmembraan domein en een kart cytoplasmisch domein. Het L1 eiwit is betrokken bij neuronale eel migratie, neuriet groei en axon bundeling, myeline vorming en eel-eel adhesie van gedifferentieerdeneuronen.

De doelstellingen van het promotieonderzoek (Hoofdstuk 1) zoals beschreven in dit proefschrift waren: een methode ontwikkelen die het mogelijk maakt om een uitspraak te doen over de kans om bij een patientverdacht van het L1 syndroom een mutatiein het LlCAM gen te vinden; het vaststellen van mogelijke genotype-fenotype correlaties (Hoofdstuk 2}; het opzetten van een goede LlCAM mutatie databank met alle beschreven en door ons gevonden pathogene en niet-pathogene mutaties, met alle niet geclassificeerde varianten (UV's) en met relevante patientgegevens (Hoofdstuk 4); te bewijzen dat sommige niet geclassificeerde varianten pathogeen zijn, omdat ze een foutievepre-mRNA splicing veroorzaken (Hoofdstuk 5); te zoeken naar mogelijke andere klinische kenmerken veroorzaakt door L1CAM mutaties (Hoofdstuk 6 en 7); en het identificeren van nieuwe genen, die als ze ontbreken of zijn gemuteerd het L1 syndroom zouden kunnen veroorzaken (Hoofdstuk 8). In Hoofdstuk 2 is het DNA van 367 L1 syndroom verdachte patientengeanalyseerd op mutaties in de coderende exonen en de exon-intron overgangen van het LlCAM gen. Een subgroep

144 Nederlandsesamenvatting van 100 patienten werd ook onderzocht op mutaties in de promotor en twee regulatoire gebieden van het L1CAM gen, te weten het "Neural Restrictive Silencer Element" (NRSE) en het "Homeodomain en Paired Domain" bindingsgebied (HPD). Ook is in een subgroep onderzoek gedaan naar grate duplicaties. Als resultaat werden in 73 patienten (20%) 68 verschillende mutaties gevonden, waarvan 61 door ons werden geclassificeerd als pathogene mutatiesen 7 als UV's. In een patient werd een duplicatievan exon 2 tot en met exon 10 gevonden. Er werden geen mutaties aangetoond in de promotor, de NRSE of het HPD gebied. Op basis hiervan is geconcludeerd dat uitbreiding van het DNA mutatieonderzoek met regulatoire gebieden, niet lonend is, terwijl onderzoek naar duplicatieswel iets oplevert. Van 106 patienten,waarvan 31 met een mutatie, werden de klinische gegevens verzameld en deze werden gebruikt voor een statistische analyse. Hieruit bleek dat in patientenmet drie of meer van de zeven hierboven beschreven klinische Ll kenmerken de mutatie detectiegraad 66% is en slechts 16% in patienten met een of twee Ll karakteristieken (odds ratio (OR) 10,1; 95% betrouwbaarheids interval (Cl) 3,8 tot 27,1). Verder bleek de detectiegraad 18% in families met een aangedaan persoon en 51% in families met meer dan een aangedaan familielid. Een combinatie van deze twee, d.w.z. patienten met een positievefamilie geschiedenis voor het Ll syndroom en met drie of meer klinische Ll kenmerken, leidt tot een detectiegraadvan 85% (OR 10,4; 95% Cl 3,6 tot 30,1). Door gebruik te maken van de gegevens van de gehele groep patienten,kon er een grafiekwarden gemaakt, waaruit de mutation detectiegraad kan warden afgelezen als functie van het aantal klinische Ll kenmerken van een patient en het aantal aangedane familieleden. Met deze grafiek, een goede tool voor artsen, kan vrij nauwkeurig warden voorspeld hoe groat de kans is om in een patientmet het Ll syndroom een mutatie te vinden. De analyse bevestigde ook een eerder gepubliceerde genotype-fenotype correlatie: kinderen met een truncerende mutatie stierven vaker (52%) voor hun derde levensjaar dan kinderen met een missense mutatie (8%) (Fisher exact p=0.02). In tegenstelling tot de bevindingen van anderen, vonden wij geen verschil in de ernst van de hydrocefalie bij patienten met een missense mutatie in 'key' aminozuren versus aminozuren aan de buitenkant van het eiwit. Tot slot is in een familie bewezen dat er sprake was van een maternale kiemcel moza'iek en werd bij drie vrouwen met het Ll syndroom een mutatie in het LlCAM gen aangetoond. De laatste bevinding, het vinden van l1CAM mutaties in vrouwen met het Ll syndroom, is een belangrijke observatie. Tot nu toe was de gedachte dat mutaties in het l1CAM gen eigenlijk geen volledig L1 syndroom gaven bij vrouwen. Wat wel werd gezien was een zeer mild fenotype bij ongeveer 5% van de mutatiedraagsters.Onze resultaten laten zien dat ook ernstigevormen van het Ll syndroom bij vrouwen kunnen voorkomen. Deze bevinding heeft implicaties voor de counseling, zeker in geval van prenatale diagnostiek. Hoofdstuk3 beschrijfteen nieuwe mutatie in het LlCAM gen van een jongetje met X-gebonden hydrocefalie, met meerdere kleine gyri (hersenwindingen), onderontwikkeling van de wittestof in de hersenen, afwezigheid van de hersenbalk en niet gescheiden thalami. Drie broertjes met een aangeboren hydrocefalie stierven neonataal. Mutatie analyse van het L1CAM gen toonde een missense mutatieaan, p.Ala415Pro, in het lg 4 domein van het Ll eiwit. Aangezien alanine 415 beschouwd wordt als een heel belangrijk (key) aminozuur, dat cruciaal zou zijn voor de correcte eiwit vouwing, is het zeer waarschijnlijk dat de hydrocefalie en de hersenafwijkingen in dit patientjehet gevolg zijn van een abnormale structuur en functievan het Ll eiwit. In Hoofdstuk 4 wordt de opbouw van de L1CAM mutatie databank beschreven. Tot op

145 Nederlandse samenvatting heden zij n er in de literatuur ongeveer 240 verschillende LlCAM mutaties gerapporteerd, waaronder ziekteverwekkende, niet-ziekteverwekkende en UV's. Aangezien de hoeveelheid informatie sterk groeit, is het van belang dat artsen en genetici snel en gemakkelijk toegang hebben tot deze informatie. De bestaande, verouderde, databank werd daarom vernieuwd en uitgebreid. De databank bevat nu, naast een ge-update lijst van mutaties, oak informatie zoals: (1) klinische gegevens (incl. leeftijd bij vroegtijdig overlijden) (2) familie anamnese, (3) de gerapporteerde klinische classificatie van de mutaties, (4) parameters die van belang zijn voor het vaststellen van de mogelijke pathogeniteit van een mutatiezoals bijvoorbeeld cosegregatie van de mutatie in de familie, het de nova voorkomen van de mutatie bij de patient of bij de moeder van de patient (5) in silica gegevens betreffende de mutatie, (6) gegevens van het gedrag van een variant in een functionele test en (7) het effect van de mutatie op de mRNA splicing. Het resultaat is een databank met veel meer informatie dan voorheen, waardoor conclusies betreffende de pathogeniteit en de ernst van de mutaties in het L1CAM gen op basis van meer factoren gedaan kan warden. De database zal iedere maand warden ge-update met de nieuw gepubliceerde en door ans gegenereerde gegevens en is te vinden op: www.llcammutationdatabase.info. In Hoofdstuk5 warden de resultaten van een ex vivo RNA splicing onderzoek beschreven. Een analyse van 7 UV's uit het onderzoek beschreven in Hoofdstuk 2, met verschillende RNA splicing predictieprogramma's, leidde tot de conclusie dat tenminste 3 van de 7 aanleiding zouden kunnen geven tot afwijkende mRNA splicing. Om dit te bewijzen werd gebruik gemaakt van een ex vivo RNA splicing test. Alie drie UV's (c.76+5G>A, c.1546G>A (Asp516Asn) en c.1546G>T (p.Asp516Tyr)) bleken pathogeen te zijn, vanwege incorrecte splicing. De betrouwbaarheid van de ex vivo RNA splicing test kon warden aangetoond met de LlCAM c.645C>T mutatie, waarvan eerder met een andere methode werd vastgesteld (Hoofdstuk 2) dat deze pathogeen was vanwege incorrecte RNA splicing. Als gevolg warden thans 64 (ipv 61) van de 68 mutaties, beschreven in hoofdstuk 2 als pathogeen beschouwd. Hoofdstuk 6 handelt over het genetisch onderzoek bij een jongetje met Ll syndroom en tevens nefrogene diabetes insipidus (NOi). De vraag hierbij was of het fenotype verklaard kon warden door mutaties in het LlCAM gen alleen, of door een grate deletiedie zowel het L1CAM gen als het AVPR2 gen bevat. De deletie hypothese was gebaseerd op het feit dat NOi veroorzaakt wordt door mutatiesin het AVPR2 gen, dat vlak bij het LlCAM gen op het X chromosoom ligt. Er werd een deletie aangetoond van 61.577 bp, die het volledige LlCAM en AVPR2 gen bevat. Het NDI fenotype wordt dus veroorzaakt door verlies van het AVPR2 gen en niet door een mutatie in het L1CAM gen. In Hoofdstuk 7 wordt de moleculaire karakterisatie van een patient met een X-gebonden hydrocefalie en de ziekte van Hirschsprung (HSCR) beschreven. Karyotypering liet een de novo reciproke gebalanceerde translocatie t(3;17)(p12;q21) zien. Mutatie analyse van het L1CAM gen en het RETgen, dat betrokken is bij de ziekte van HSCR, toonde een frameshift mutatie aan in het L1CAM gen, c.2265de1C, die leidt tot een vroegtijdig stop in het gen, terwijl in het RET gen geen pathogene mutatie kon warden gedetecteerd. Karakterisatie van het chromosoom 17 breukpunt liet een interruptie van de MY018A en TIAF1 genen zien, met als gevolg dat naast de mogelijke betrokkenheid van de LlCAM mutatie, oak de afwijkingen in het MY018A en T/AF1 gen van invloed zouden kunnen zijn op het fenotype. Tot op heden blijken de meeste patienten met zowel HSCR als Ll syndroom mutaties te hebben in het L1CAM gen, waardoor

146 Nederlandse samenvatting het L1CAM gen als een kandidaat gen voor de ziekte van HSCR werd gezien. Echter het feit dat slechts 3% van de patienten, die een mutatie in het L1CAMgen hebben oak de ziekte van HSCR hebben en de afwezigheid van L1CAM mutaties in jongens met alleen de ziekte van HSCR, suggereert dat het L1CAM geen oorzakelijk gen is voor HSRC, maar eerder een rol speelt als modifier in de ontwikkeling van deze ziekte.

Aangezien slechts 20% van de Ll syndroom patienteneen mutatie in het L1CAM gen hebben, kan warden verondersteld dat oak andere genen betrokken zouden kunnen zijn bij het Ll syndroom. In Hoofdstuk 8 beschrijven we het onderzoek naar dergelijke genen door te zoeken naar deleties in 60 Ll syndroom patienten m.b.v. een high density X-chromosomale array, aannemende dat deze deleties dergelijke nieuwe Ll syndroom genen zouden kunnen bevatten. Analyse van de resultaten leverde 11 kandidaat ziekte veroorzakende deleties op, in 12 patienten, die leidde tot 11 kandidaat genen t.w: DGAT2L6, POU3F4, GD/1, ASMTL, ATP6AP1, M/01, FRMPD4, MAP3K15, TGIF2LX, DDX53 en TM EM27. Om hun betrokkenheid bij het L1 syndroom te kunnen onderbouwen, werd besloten om van 95 L1 syndroom patienten de vijf meest belovende kandidaat genen; DGAT2L6, POU3F4, GD/1, TGIF2LX en DDX53 te sequencen. Uit dit onderzoek bleek a Ileen extra betrokkenheid voor het GD/1 gen, omdat in een patient een mutatie werd gevonden, c.153-3C>T. Dit is zeer waarschijnlijk een ziekte verwekkend mutatie, omdat de mutatie incorrecte RNA splicing veroorzaakt in een ex vivo RNA splicing test. De conclusie is, dat deze vijf genen geen belangrijke rol spelen bij de ontwikkeling van het Ll syndroom, maar een zekere rol m.n. voor het GD/1 gen, kan niet warden uitgesloten. Het is ook mogelijk dat een (of meer) van de zes niet geanalyseerde genen meer betrokken is bij de tot standkoming van het Ll syndroom. Omdat we gescreend hebben voor deleties (>2 kb) is het niet uit te sluiten dat we genen gemist hebben die alleen maar puntmutaties of heel kleine deleties hebben. Om mogelijk nieuwe genen op te sporen, zullen wij met behulp van de next generation sequencing techniek een X-chromosoom-specifieke exoom resequencing gaan uitvoeren bij een geselecteerde groep L1 syndroom patienten. Samenvattend concluderen wij dat er naast het L1CAM gen andere genen betrokken zouden kunnen zijn bij de ontwikkeling van het Ll syndroom, waarbij het GD/1 gen de beste kandidaat lijkt tot nu toe, maar verder onderzoek is nodig.

In Hoofdstuk 9 worden de verkregen resultaten bediscussieerd en de toekomst plannen gepresenteerd, waarvan de belangrijkste zijn: (1) het opzettenvan een betrouwbare functionele test en (2) het onderzoek naar nieuwe genen, waarmee het Ll syndroom bij patienten, die geen mutatie in het L1CAM gen hebben, kan warden verklaard.

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Dankwoord/ acknowledgements Dankwoord/acknowledgements En dan is het proefschrift opeens af ! Na de ups en downs in het onderzoek en vervolgens de inspanning om het op papier te krijgen, is dat een goed gevoel. Ondanks het feit dat mijn naam als enige op de omslag staat, is het zonneklaar dat het proefschrift niet tot stand zou zijn gekomen zonder de hulp van velen.

Als eerste wil ik mijn promotor, professor dr. R.M.W. Hofstra, noemen. Beste Robert, bedankt voor het vertrouwen dat je in mij had. De start was wat traag, omdat de diagnostiek voortdurend aan mij trok. Keer op keer maakten we een nieuw tijdpad en als ik een beetje bedenkelijk keek of het wel haalbaar was, kwam jij altijd met de opwekkende woorden: "gaat lukken, het meeste is al af". Je optimisme hielp mij om er weer tegenaan te gaan. Het laatste anderhalf jaar hebben we er heel hard aangetrokken en ik ben je heel dankbaar voor je hulp op de momenten dat ik dacht vast te lopen en voor de snelheid waarmee je mijn stukken van waardevol commentaar voorzag. Ook ben ik veel dank verschuldigd aan professor dr. C.H.C.M. Buys, omdat hij mij in 2000 de kans gaf om na een carriere in de industriele biotechnologie een nieuwe uitdaging aan te gaan in de humane genetica. Beste Charles, bedankt voor deze mogelijkheid en het geloof dat je in mij had dat ik die overstap zou kunnen ma ken. lk dank je ook voor de grate vrijheid, die jij mij gaf in mijn werk als hoofd van de DNA diagnostiek.

De beoordelingscommissie, professor dr. C.T.R.M. Schrander-Stumpel, professor dr. O.F. Brouwer en professor dr. H. van Bokhoven, wil ik hartelijk danken voor het positiefbeoordelen van het proefschrift. Een apart bedankje voor professor Schrander-Stumpel is hier zeker op zijn plaats. Beste Connie, bedankt voor de samenwerking op het gebied van het Ll syndroom. Onze gezamenlijke publicaties over het Ll syndroom kwamen tot stand doordat jij, als specialist op dit gebied (in 1995 promoveerde jij zelf op dit onderwerp), aan mij vroeg de moleculair genetischekant op mij te nemen.

Dit proefschrift zou niet tot stand zijn gekomen zonder de inspanningen van Krista van Dijk-Bos. Beste Krista, jou wil ik vooral bedanken voor de enorme hoeveelheid werk die je hebt verzet. Je hebt dit van het begin tot het eind met veel enthousiasme en inzet gedaan. Je had vaak aan een half woord genoeg om voortvarend aan de slag gaan. In het begin zat het onderzoek een beetje tegen, maar we bleven erin geloven en dat maakten de momenten dat je enthousiast met een goed resultaat de kamer kwam binnenstappen extra leuk. Wat mij betreft hebben we fijn en goed samengewerkt. Verder wil ik de analisten bedanken, die binnen de DNA diagnostiek de mutatie analyse van het LlCAM gen hebben uitgevoerd: Edwin Verlind, die de mutatie analyse heeft opgezet, Annet Vrieling, Gerwin Jansen, Bea Hiemstra en Annelies ten Berge. Jullie resultaten hebben mede tot een mooie publicatie geleid.

Ook een aantal stagiaires hebben een bijdrage geleverd aan het onderzoek. De LlCAM regulatioregebieden zijn door Alieke Souilljee gesequenced. Mireille Mulder heeft de MLPA analyse opgezet. Een moeilijk onderzoek, maar het heeft resultaat opgeleverd. Irene Molthof heeft een eerste aanzet gegeven tot het ontwikkelen van een functionele assay. Jammer genoeg zijn we hier niet zover meegekomen dat de resultaten konden warden opgenomen, maar het heeft wel een mooi plaatje opgeleverd voor de omslag van mijn proefschrift. Alie

150 Dankwoord/acknowledgements drie hartelijk dank. Martijn Bruining en Jenneke Stegeman, die beiden hun wetenschappelijke stage bij mij gedaan hebben, wil ik bedanken voor het opzetten van de patienten database en hun inspanning om een questionnairete maken, uit te zetten en te interpreteren. Het resultaat is terug te vinden in hoofdstuk 2. Met dank oak aan Irene Stolte-Dijkstra die als ervaren klinisch geneticus beide laatst genoemde stagiaires heeftbegeleid voor het klinische deel. Dank oak aan Hermien de Walle. Hermien bedankt voor het analyseren van de grote berg gegevens en voor het feit dat je gewoon weer aan de gang ging als ik met een nieuwe of aanvullende vraag kwam. Klaas Kok en Pieter van de Vlies wil ik bedanken voor het arraywerk en het meedenken met het onderzoek naar nieuwe genen. Klaas, bedankt oak voor de hulp bij het vastleggen van de resultaten in een artikel. Heel leuk vond ik het enthousiasme waarmee Christine van der Werf te werk ging bij het maken van constructen voor het RNA splicing onderzoek, onder tijdsdruk, en ondanks haar eigen drukke werkzaamheden. Samen met Krista is er een goed resultaat neergezet. Hartelijk dank.

Next, I would like to thank the many clinicians from all over the world for sending in patient material and for their trust in our diagnostic laboratory. Although I have never met most of you personally, over time I have developed a very friendly and fruitful relationship with many of you. I much appreciate all the clinicians who have spent some of their precious time, completing the questionnaire. Your efforts have proved to be very worthwhile. Last but not least, I would like to thank all my co-authors who were involved in the various scientific papers. I really enjoyed working with you.

De stafleden van de sectie DNA diagnostiek,Annemieke van der Hout, Renee Niessen, Henny Lemmink en Jan Jongbloed, verdienen een aparte vermelding. Zander hun extra inspanningen om een deel van mijn werk ten behoeve van de patientenzorg, vooral het laatste anderhalf jaar, over te nemen, was mijn promotie onderzoek waarschijnlijk niet tot een goed einde gekomen. Heel erg bedankt hiervoor. Uiteraard wil ik ook alle analisten van de sectie DNA diagnostiek bedanken voor de fijne samenwerking en de steun die ik heb gekregen tijdens het werk aan het boekje.

Jackie Senior wil ik heel hartelijk danken voor het corrigeren van het Engels. Het proefschrift is er absoluut beter van geworden. Margaret Burton wil ik bedanken voor het corrigeren van hoofdstuk 2.

Krista van Dijk en Arjan Bijlsma hebben beiden direct toegestemd toen ik hen vroeg mijn paranimf te worden. Krista, ik vind het een leuke afsluiting van onze samenwerking, want kart na het afronden van het Ll onderzoek ben je naar een ander team overgestapt en is onze samenwerking grotendeels beeindigd. Arjan, mijn neef, als student natuurkunde heb je inmiddels een aantal malen een promotie meegemaakt en vind je het heel leuk om eens "aan de andere kant" te staan en ik vind het leuk jou naast mij te hebben. Je nam, zoals altijd, ook deze taak heel serieus en je ging voortvarend aan de slag met je taak. Heel erg bedankt. Bob Vos, mijn andere neef, vond het een leuke uitdaging om voor mijn proefschrift het ontwerp

151 Dankwoord/acknowledgements voor de omslag te maken. lk was blij verrast toen ik het resultaat zag. Bob, heel erg bedankt, want door jouw creativiteit en artisticiteit, dekt de vlag heel fraai de lading.

Mijn familie, vrienden en kennissen wil ik bedanken voor het begrip dat ze vooral de laatste tijdgetoond hebben, voor het feit dat zo ik weinig tijdhad voor iedereen. De tijdwerd zelfs zo schaars dat ik een jaar lang mijn vrijwilligerswerk in Roden heb moeten stilleggen en deels aan anderen heb moeten overdragen. Fijn dat dit kon, bedankt, daar komt nu snel een eind aan. De banden warden weer aangetrokken en de activiteiten warden weer hervat.

En als laatste, maar een heel belangrijke factor in mijn leven, wil ik mijn ouders bedanken voor alles wat zij voor mij gedaan hebben en nog steeds doen. Lieve pa en ma, heel erg bedankt voor jullie onvoorwaardelijke steun, voor de kans die jullie mij gegeven hebben om te gaan studeren en voor alles wat jullie me hebben meegegeven. Van jongs af aan hebben jullie ons voorgehouden hoe belangrijk het is om een goede opleiding te volgen en onafhankelijk te zijn. U hebt helemaal gelijk gekregen en daar ben ik ben u heel dankbaar voor. lk ben een gelukkig mens en zonder jullie zou ik dit proefschriftnooit gerealiseerd hebben. Dikke zoen!

Yvonne

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List of publications List of publications (1) Sijmons RH, Vos VJ , Herkert JC, Bos KK, Holzik MF, Hoekstra-Weebers JE, Hofstra RM, Hoekstra HJ. Screening for germline DNDl mutations in testicular cancer patients. Fam Cancer 2010 Apr 22: [Epub ahead of print].

(2) Vos VJ , Hofstra RM. An updated and upgraded LlCAM mutation database. Hum Mutat 2010 Jan;31(1):E1102-E1109.

(3) Vos VJ, de Walle HE, Bos KK, Stegeman JA, Ten Berge AM, Bruining M, van Maarle MC, Elting MW, den Hollander NS, Hamel B, Fortuna AM, Sunde LE, Stolte-Dijkstra I, Schrander­ Stumpel CT, Hofstra RM. Genotype-phenotype correlations in Ll syndrome: a guide for genetic counselling and mutation analysis. J Med Genet 2010 Mar;4 7(3}:169-75.

(4) Griseri P, Vos V, Giorda R, Gimelli S, Beri S, Santamaria G, Mognato G, Hofstra R, Gimelli G, Ceccherini I. Complex pathogenesis of Hirschsprung's disease in a patient with hydrocephalus, vesico-ureteral refluxand a balanced translocation t(3;17)(p12;qll}. Eur J Hum Genet 2009 Apr;17(4):483-90.

(5) Wessels MW, van de Laar IM, Roos-Hesselink J, Strikwerda S, Majoor-Krakauer DF, de Vries BB, Kerstjens-Frederikse WS, Vos VJ, de Graaf BM, Bertoli-Avella AM, Willems PJ. Autosomal dominant inheritance of cardiac valves anomalies in two families: extended spectrum of left-ventricular outflow tract obstruction. Am J Med Genet A 2009 Feb;149A(2):216-25.

(6) Gomez LC, Marzese DM, Adi J, Bertani D, Ibarra J, Mol B, Vos VJ , De Marchi G, Roque M. MLPA mutation detection in Argentine HNPCC and FAP families. Fam Cancer 2009;8(1):67-73.

(7) Rump P, Dijkhuizen T,Sikkema-Raddatz B, Lemmink HH, Vos VJ , Verheij JB, van Ravenswaaij CM. Drayer's syndrome of mental retardation, microcephaly, short stature and absent phalanges is caused by a recurrent deletion of chromosome 15(q26.2-->qter). Clin Genet 2008 Nov;74 (5):455-62.

(8) Knops NB, Bos KK, Kerstjens M, van Dael K, Vos VJ . Nephrogenic diabetes insipidus in a patient with Ll syndrome: a new report of a contiguous gene deletion syndrome including LlCAM and AVPR2. Am J Med Genet A 2008 Jul 15;146A(14):1853-8.

(9) Marshall CR, Noor A, Vincent JB, Lionel AC, Feuk L, Skaug J, Shago M, Moessner R, Pinto D, Ren Y, Thiruvahindrapduram B, Fiebig A, Schreiber S, Friedman J, Ketelaars CE, Vos VJ , Ficicioglu C, Kirkpatrick S, Nicolson R, Sloman L, Summers A, Gibbons CA, Teebi A, Chitayat D, Weksberg R, Thompson A, Vardy C, Crosbie V, Luscombe S, Baatjes R, Zwaigenbaum L, Roberts W, Fernandez B, Szatmari P, Scherer SW. Structural variation of chromosomes in autism spectrum disorder. Am J Hum Genet 2008 Feb;82 (2):477-88.

(10) van der Hout AH, Oudesluijs GG, Venema A, Verheij JB, Mol BG, Rump P, Brunner HG, Vos VJ, van Essen AJ. Mutation screening of the Ectodysplasin-A receptor gene EDAR in hypohidrotic ectodermal dysplasia. Eur J Hum Genet 2008 Jun;16 (6):673-9.

156 List of publications (11) Stu Ip RP, Herkert JC, Karrenbeld A, Mol B, Vos VJ , Sijmons RH. Thyroid cancer in a patient with a germline MSH2 mutation. Case report and review of the Lynch syndrome expanding tumour spectrum. Hered Cancer Clin Pract 2008;6 (1):15-21.

(12) van Tintelen JP, Hofstra RM, Katerberg H, Rossenbacker T, Wiesfeld AC, du Marchie Sarvaas GJ, Wilde AA, van Langen IM, Nannenberg EA, van der Kooi AJ, Kraak M, van Gelder IC, van Veldhuisen DJ, Vos VJ , van den Berg MP. High yield of LMNA mutations in patients with dilated cardiomyopathy and/or conduction disease referred to cardiogenetics outpatient clinics. Am Heart J 2007 Dec;154 (6):1130-9.

(13) van Tintelen JP, Tio RA, Kerstjens-Frederikse WS, van Berlo JH, Boven LG, Suurmeijer AJ, White SJ, den Dunnen JT,te Meerman GJ, Vos VJ , van der Hout AH, Osinga J, van den Berg MP, van Veldhuisen DJ, Buys CH, Hofstra RM, Pinto YM. Severe myocardial fibrosis caused by a deletion of the 5' end of the lamin A/C gene. J Am Coll Cardiel 2007 Jun 26;49(25):2430-9.

(14) van der Hout AH, van den Ouweland AM, van der Luijt RB, Gille HJ, Bod mer D, Bruggenwirth H, Mulder IM, van der Vlies P, Elfferich P, Huisman MT, Ten Berge AM, Kromosoeto J, Jansen RP, van Zon PH, Vriesman T, Arts N, Lange MB, Oosterwijk JC, Meijers-Heijboer H, Ausems MG, Hoogerbrugge N, Verhoef S, Halley DJ, Vos VJ, Hogervorst F, Ligtenberg M, Hofstra RM. A DGGE system for comprehensive mutation screening of BRCAl and BRCA2: application in a Dutch cancer clinic setting. Hum Mutat 2006 Jul;2 7(7):654-66.

(15) Stulp RP, Vos VJ , Mol B, Karrenbeld A, de Raad M, van der Mijle HJ, Sijmons RH. First report of a de novo germline mutation in the MLHl gene. World J Gastroenterol 2006 Feb 7;12 (5):809-11.

(16) Verheij JB, Sival DA, van der Hoeven JH, Vos VJ , Meiners LC, Brouwer OF, van Essen AJ. Shah-Waardenburg syndrome and PCWH associated with SOXlO mutations: a case report and review of the literature. Eur J Paediatr Neural 2006 Jan;l0 (l):11-7.

(17) Rump P, Lemmink HH, Verschuuren-Bemelmans CC, Grootscholten PM, Fack JM, Hayflick SJ, Westaway SK, Vos VJ, van Essen AJ. A novel 3-bp deletion in the PANK2 gene of Dutch patients with pantothenate kinase-associated neurodegeneration: evidence for a founder effect. Neurogenetics 2005 Dec;6 (4):201-7.

(18) Schrander-Stumpel CT, Vos VJ . [From gene to disease; X-linked hydrocephalus and LiCAM]. Ned Tijdschr Geneeskd 2004 Jul 17;148(29):1441-3.

(19) Schrander-Stumpel CT, Vos VJ . Ll syndrome. In: Pagon RA, Bird TC, Dolan CR, Stephens K, editors. GeneReviews [Internet]. Seattle (WA}: University of Washington, Seattle; 2004.

(20) Rodriguez CG, Perez AA, Martinez F, Vos VJ , Verlind E, Gonzalez-Meneses LA, Gomez de TS, I, Schrander-Stumpel C. X-linked hydrocephalus: another two families with an Ll mutation. Genet Couns 2003;14 (1):57-65.

157 List of publications (21) Kariola R, Otway R, Lonnqvist KE, Raevaara TE, Macrae F, Vos VJ , Kohonen-Corish M, Hofstra RM, Nystrom-Lahti M. Two mismatch repair gene mutations found in a colon cancer patient--which one is pathogenic? Hum Genet 2003 Feb;112 (2):105-9.

(22) Sztriha L, Vos VJ , Verlind E, Johansen J, Berg B. X-linked hydrocephalus: a novel missense mutation in the LlCAM gene. Pediatr Neural 2002 Oct;2 7(4):293-6.

(23) Raevaara TE, Timoharju T, Lonnqvist KE, Kariola R, SteinhoffM, Hofstra RM, Mangold E, Vos VJ , Nystrom-Lahti M. Description and functionalanalysis of a novel in frame mutation linked to hereditary non-polyposis colorectal cancer. J Med Genet 2002 Oct;39(10):747- 50.

(24) Dorssers LCJ, Wagemakers G, Vos VJ , van Leen RW, Persoon MLN. Expression and purification of human interleukin-3 and muteins thereof. United States Patent 1990; US 005. 304.637

(25) de Heij HT, Lustig H, van Ee JH, Vos VJ,Groot GSP. Repeated sequences on mitochondrial DNA of Spirodela Oligorrhiza. Plant Mol Biol 1985; 4(4): 219-24

(26) Sanders JPM, van den Berg JA, Andreoli PM, Vos VJ,van Ee JH, Mulleners USM. Molecular cloning and expression in industrial microorganism species. European patent application 1984; EP O 134 048 Al

(27) de Kok A, Vos VJ , van Garderen C, de Jong T, van Opstal M, Frei RW, Geerdink RB, Brinkman UAT. Chromatographic determination of phenylurea herbicides and their corresponding aniline degradation products in environmental samples. J Chrom 1984; 288(1): 71-89

(28) van Ee JH, Vos VJ , Bohnert HJ, Planta RJ. Mapping of genes on the chloroplast DNA from the duckweed Spirodela Oligorrhiza. Plant Malec Biol 1982; 1(2): 117-31

(29) van Ee JH, Vos VJ , Planta RJ. Physical map of chloroplast DNA of Spirodela Oligorrhiza; analysis by restriction endonucleases Pst I, Xho I and Sac I. Gene 1980; 12 (3-4): 191- 200

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