20.\o. ldent¡f¡cat¡on of Novel Genes for X-linked Mental Retardation Adelaide A thesis submitted for the degree of Dootor of Philosophy to the University of by Marie Mangelsdorf BSc (Hons) School ofMedicine Department of Paediatrics, Women's and Children's Hospital May 2003 Corrections The following references should be referred to in the text as: Page 2,line 2: (Birch et al., 1970) Page 2,line 2: (Moser et al., 1983) Page 3, line 15: (Martin and Bell, 1943) Page 3, line 4 and line 9: (Stevenson et a1.,2000) Page 77,line 5: (Monaco et a|.,1986) And in the reference list as: Birch H. G., Richardson S. ,{., Baird D., Horobin, G. and Ilsley, R. (1970) Mental Subnormality in the Community: A Clinical and Epidemiological Study. Williams and Wilkins, Baltimore. Martin J. P. and Bell J. (1943). A pedigree of mental defect showing sex-linkage . J. Neurol. Psychiatry 6: 154. Monaco 4.P., Nerve R.L., Colletti-Feener C., Bertelson C.J., Kurnit D.M. and Kunkel L.M. (1986) Isolation of candidate cDNAs for portions of the Duchenne muscular dystrophy gene. Nature 3232 646-650. Moser H.W., Ramey C.T. and Leonard C.O. (1933) In Principles and Practice of Medical Genetics (Emery A.E.H. and Rimoin D.L., Eds). Churchill Livingstone, Edinburgh UK Penrose L. (1938) A clinical and genetic study of 1280 cases of mental defect. (The Colchester survey). Medical Research Council, London, UK. Stevenson R.E., Schwartz C.E. and Schroer R.J. (2000) X-linked Mental Retardation. Oxford University Press. Page 7 line l: 'better known an better studied' should read 'better known and better studied'. Page 1 3 line 8 : 'in vitro' should be ' in vitro' . Page 16 line 2: '("g.)' should read'(eg. William's syndrome) Page 22line 20: causing should not be italicised. Page24line 2: should read 'The human genome sequence is expected to be fully completed soon, with the maps of individual chromosomes being published by the end of 2003'. Page34,line 4: 'was' should read'were'. Page 58, line 6: (company) should read (Multi cel (one L), Thermo Trace Ltd). Page 77 legend Figure 3.8: + and - refer to RT-PCR with and without the addition of reverse transcriptase respectively. Page 84 line 15: 'these results' should read 'the spanning signals for these clones' Page 100 line l6: The oRF of PRRGI was screened by direct sequencing. Page 139 line 17: 'Panay and island of the Phillipines' should read 'Panay an island of the Phillipines'. Page 153 line 5: 'lysine at position (K56 paired{ype) at position 50' should read 'lysine (Kso paired-type) at position 50'. Page 160 line 1 :'evalutaion' should be'evaluation,. I i;:#f '#:,;'#å'f;,i':í;i'wr:i!,ihü:!'# life üself." FRANCIS CRrCK (1988) CONTENTS ii Statement and Declaration 111 Contents iv List of Abbreviation vi List of Gene Names and Abbreviations v111 Summary xii Acknowledgements 1 CHAPTER 1 Introduction 29 CHAPTER 2 Materials and Methods X cltomosome 66 CHAPTER 3 Molecular characterisation of ,.**g"*"nts involving and Xq28 breakpoint in three patients with mental retardation gene screening in a family 172 CHAPTER 4 Fine mapping and candidate ;lth ;;_;¡oiaro*i, X_linked menral retardation mapping to either Xq13.1 otXq23 in the homeobox gene ARX n t4l CHAPTER 5 Identification of mutations puti"tttt with syndromic and non-syndromic XLMR 182 CHAPTER 6 Conclusions 186 References Appendices IV LIST OF ABBREVIATIONS A,C,G,T nucleotides : adenine, cyto sine, guanine, thymine BAC bacterial artificial chromosome BLAST basic local alignment search tool bp base pairs cDNA complementary DNA cM centimorgans CNS central nervous sYstem cos cosmid dNTP deoxynucleo side triPho sPhate ddNTP dideoxynucleo side triPho sPhate DNA deoxyribonucleic acid dup duplicaion EST expressed sequence tag FISH fluorescence in situ hybridisation FRAXA rare, folic acid type, fragite site, fra(X)(q2l.3) FRAXE rare, folic acid type, fragite site, fra(X)(q28) gDNA genomic DNA GDP guanine diphosPhate GLA y-carboxy glutamic acid GTP guano sine tripho sPhate IL-1 interleukin I Ins insertion inv inversion IQ intelligence quotient kb kilo base pairs LCR locus control region lod log ofthe odds LR-PCR long range PCR LRR leucine-rich rePeat tvfb megabase MR mental retardation mRNA Mru( MRXS NCBI NS)(LMR non-syndromic X-linked mental retardation ODzoo optical density at a wavelength of 260 nm oDeoo optical density at a wavelength of 600 nm OMIM online Mendelian Inheritance in Man OP.F open reading frame PAC Pl artificial chromsome PCR polymerase chain reaction rec recombinant RNA ribonucleic acid RT-PCR reverse transcribe PCR v SLRP small leucine rich protein SNP single nucleotide po lymorPhism SSCA single strand conformation analysis STS sequence tagged site UTR untranslated region vlv volume per volume w/v weight per volume WCH Women's and Children's HosPital )il,MR X-linked mental retardation YAC yeast artificial chromo some vl LIST OF GENE AI\D PROTEIN NAME ABBREVIATIONS ABCDl ATP-binding cassette, sub-family D (ALD)' member 1 AGTR2 angiotensin II recePtor , tYPe 2 (GEF) 6 ARHGEF6 RíclCdc42 guaninã nucleotide exchange factor ARX ari stal e s s related homeobox ATP2B3 plasma membrane, calcium transporting, ATPase 3 ATRX ãtpt a thalassemia/mental retardation syndrome BGN biglycan CBP CREB binding Protein Cdc42 cell division cycle 42 (GTP binding protein) CNG2 CYFIPI DNCH] 1 DUSP9 dualsp 9 ELKl ELKI, gene familY rePair ERCC6 excisio menting rodent deficiency, complementation group 6 ESD esterase D F2 coagulation factor II (thrombin) accelerator) F7 coaiulation factor VlÌ 1."*tn prothrombin conversion haemophila F9 coaiulation factor IX (plasma thromboplastic component, FACL4 long-chain 4 FATE transcriPt FGDl faciogenital dysplasia (Aarskog-Scott syndrome) fragile site FMRl fragile mentai rêtardation 1 gene, associated with the FRAXA with the fragile site FMR2 fragile mental retardation 2 gene, associated FRAXE FMRP GABRA3 GABRE GABRQ GAP GTP-ase activating Protein GAS6 growth arrest-sPecific 6 GDIl GDP dissociation inhibitor 1 GEF guanine nucleotide exchange factor GPR5O G protein-couPled recePtor 50 GRIA3 glutamate receptor, ionotrophic, AMPA 3 ILlRAPLI interleukin 1 receptor accessory protein-like I KLFS kruppel-like factor 8 MECP2 -"ttwt CpG binding protein 2 (Rett syndrome) genase-like NSDHL NnOipl ãependent steroid dehydro NXF5 nuclear RNA exPort factor 5 OPFINl oligophrenin 1 PAK3 p21 (CDKNlA)-activated kinase 3 PAX6 paired box gene 6 (aniridia, keratitis) vll PIN4/bPARI4 - protein ) NlMA-interacting PRRG] proline PoþePtide ! PRRG2 proline PoþePtide 2 PRSSl2 serine protease 1 2 (neurotr¡psin) RACl ras-relãted C3 botulinum toxin substrate 1 (rho family, small GTP binding protein) RLGP Ras-like GTPase KP56KA3 ribosomal protein 56 kinase, 90kDa, poþeptide 3 RP56KA6 ribosomal protein 56 kinase, 90kDa, poþeptide 6 SHH sonic hedgehog homolog (Drosophila) SLC6A8 solute .utri.t ømity 6 (creatine neurotransmitter transporter), TM4SF2 transmembrane 4 superfamily member 2 TMG3 transmembrane gaÍrma-carboxyglutamic acid gene 3, TMG4 transmembrâne gammâ-carboxyglutamic acid gene4 TREX2 three prime repair exonuclease 2 VCX-A variable charge protein on X with eight repeats ZNF185 zinc frnger gene 185 (LIM domain) ZNF26] zinc finger gene26l ZNF275 zinc finger gene275 ZXDA/7XDB duplicated zinc finger, X-linked, A and B vlll SUMMARY Mental retardation (Nß) is estimated to affect 7-2Vo of the populatior¡ and is due to both environment and improperly functioning genes. A higher incidence of MR in males suggests a significant proportion of MR with a genetic cause is due to mutations in genes on the X- chromosome. The aim of this thesis is to identifr novel genes involved in X-linked mental retardation Qil-NR). Understanding the genetic causes of MR will result in better diagnosis, and as a result, will lead to improved patient management and counselling of family members. It will also contribute to the understanding of the genes and mechanisms required for normal cognitive function. The first part of the project involved molecular characterisation of the breakpoints of three X chromosome rearrangements in three unrelated patients with lvÍR, to identifu candidate genes for familial )0MR. For Patient I an inversion breakpoint was found to lie within the 3' untranslated region (3'UTR) of the biglycan gene (BGÀl), such tløt BGN in this patient obtained a new 3'UTR and polyadenylation signal from Xql3.1. The open reading frame of BGN remained intact, and apparently normal levels of mRNA transcribed. 3'UTRs have been shown to contain elements, important for mRNA localization, stability and translation efftciency. Therefore, in this patient disruption of elements in the 3'UTR may affect the levels of BGN protein produced and thereby cause MR. Characterisation of this inversion has identified BGI/ as a candidate gene for )0MR. For Patient 2, who also has an inversion of the X chromosome, all BAC clones derived from Xq2S that were used as probes for FISH gave spanning signal. This suggested that this patient lx inversion. had a previously undetected duplication of Xq28 as well as the initially detected Thus the MR This duplication covers at least 2.7 \{Ibof Xq.28, an extremely gene rich region. Xq28' This in this patient is likely to be the result of functional disomy of many genes from duplication work suggests that some familial cases of )(LMR may be due to submicroscopic methods of of many genes, a mechanism that would not be detected by current PCR based gene screeilng. For patient 3 a translocation of the X chromosome associated with MR and cardiomyopathy inactivated was characterised. In this patient, the normal X chromosome is preferentially responsible for the suggesting that disruption of a gene on the X chromosome may be not phenotype. The break at Xq28 was located within a 75 kb BAC clone.