Molecular genetic studies of fragile x syndrome and spinocerebellar ataxia type 2

ACKNOWLEDGEMENTS I would like to express my sincere gratitude to all those who gave me the possibility to complete this thesis The first person I would like to thank is my supervisor, Associate Professor Samuel Chong, for his broad and profound knowledge, for his detailed suggestions and patient instructions, and for his warm smile and strong support He could not even realize how much I have learnt from him I would like to express my appreciation to all SSC lab mates, both present and past: to Arnold, who always offered his hand to me from my first day till now; to Felicia, who remembered my birthday every year; to Zeng Sheng, who trouble shot my Southern blot; to Liang Dong, who picked me up at the Airport on my first day; to Ya yun, who handled the purchase order; to Hai Bo, who extracted good quality DNA; to Wang Wen, who accompanied me in the small room after office hour; to Ben Jin, who cheered me up during boring experiments; to Xiao Tao, who brought back delicious food; to Jane, Chin Yan, Siew Hong and Hai Dong, who brought happiness to the lab Away from the lab, I want to thank Dr Caroline Lee and Tang Kun from Department of Biochemistry (NUS) for their interesting and valuable suggestions in the fragile X haplotype analysis I would like to thank Lily from Molecular Diagnostic Center (NUH) for her StB12.3 probe I would also express thanks to Celeste and Hong Keat for their help in the haplotype genotyping experiments Last but certainly not least, I want to devote this thesis to my parents, for their love and encouragement throughout my life i TABLE OF CONTENT ACKNOWLEDGEMENTS i TABLE OF CONTENT ii LIST OF FIGURES AND TABLES iv ABBREVIATION vi SUMMARY vii CHAPTER GENERAL INTRODUCTION .1 1.1 Trinucleotide repeat diseases .2 1.1.1 Overview of trinucleotide repeat diseases 1.1.2 Classification of trinucleotide repeat diseases 1.1.3 Shared defining features of trinucleotide repeat diseases 1.1.4 Molecular mechanisms of trinucleotide repeat diseases .6 1.1.5 Trinucleotide repeat diseases I have studied .6 1.2 Fragile X syndrome 1.2.1 Overview and clinical features of fragile X syndrome .8 1.2.2 The cause of fragile X syndrome 1.2.3 The prevalence of fragile X syndrome 12 1.2.4 The diagnosis of fragile X syndrome 13 1.2.5 FMR1 haplotypes in fragile X syndrome 16 1.3 Spinocerebellar ataxia type (SCA2) 18 1.3.1 Overview of SCA2 18 1.3.2 Clinical features of SCA2 20 1.3.3 The cause of SCA2 20 1.3.4 The prevalence of SCA2 22 1.3.5 The diagnosis of SCA2 .22 Reference .23 CHAPTER Simplified Methylation-specific PCR Detection of Fragile X Syndrome Expansion Mutations in Males and Females .32 2.1 Introduction 33 2.2 Materials and Methods .34 2.2.1 DNA samples 34 2.2.2 DNA extraction from cells (lymphoblast) .35 2.2.3 Sodium Bisulfite treatment .36 2.2.4 Methylation-specific PCR (ms-PCR) 36 2.2.5 Southern Blot 41 2.2.6 HUMARA Assay 45 2.3 Results 46 2.4 Discussion 59 Reference .64 CHAPTER HAPLOTYPE AND AGG INTERSPERSION ANALYSIS OF THE FMR1 CGG REPEAT IN THE SINGAPORE POPULATION 66 3.1 Introduction 67 3.2 Materials and Methods .70 3.2.1 DNA samples 70 ii 3.2.2 STR Genotyping 70 3.2.3 CGG repeat array sequencing 71 3.2.4 SNP Genotyping 72 3.2.5 FRAXAC1, FRAXAC2 and DXS548 sequencing 73 3.2.6 Statistical methods 74 3.3 Results 75 3.3.1 General diversity in the Singapore population 75 3.3.2 CGG structure in the Singapore population 76 3.3.3 FMR1 haplotypes in the unaffected and fragile X population 76 3.3.4 Association of (CGG)n locus and FMR1 flanking markers 81 3.3.5 Distribution of interspersion patterns among FRAXA haplotypes 85 3.3.6 Origin of instability 89 3.4 Discussion 91 3.4.1 “Odd-numbered” DXS548 allele 91 3.4.2 Relationship between ATL1 and IVS10 SNPs and 5’ AGG interruption position 91 3.4.3 Susceptible repeat structure .92 3.4.4 Difference among populations 95 Reference .95 CHAPTER Spinocerebellar Ataxia Type with Focal Epilepsy – an Unusual Association 100 4.1 Introduction 101 4.2 Materials and Methods 101 4.2.1 DNA samples 101 4.2.2 Oligonucleotide design 102 4.2.4 Purification of PCR product 107 4.2.5 Cycle sequencing 108 4.2.6 Ethanol precipitation of sequencing products 109 4.2.7 Gel electrophoresis 109 4.3 Results 110 4.4 Discussion 112 Reference .119 Appendix: Solutions 121 iii LIST OF FIGURES AND TABLES Table 1-1 Summary of the repeat expansion disorders Table 1-2 Fragment sizes (in kb) using Southern blot for molecular diagnosis of fragile X syndrome 14 Table 2-1 Primers used in specific amplification of sodium bisulfite treated nonmethylated and methylated FMR1 alleles 39 Table 2-2 Assay optimization on female and male lymphoblastoid cell lines carrying normal, premutation, and/or full mutation FMR1 (CGG)n alleles 52 Table 2-3 Assay validation on peripheral blood DNAs of normal, premutation, and/or full mutation females and males 55 Table 2-4 FMR1 ms-PCR result interpretation chart 63 Table 3-1 Diversity of FMR1 haplotypes and CGG repeat arrays in the Singapore population (Chinese, Malays and Indians) compared with other world populations 77 Table 3-2 CGG Repeat Lengths And Patterns In The Three Ethnic Groups 78 Table 3-3 FMR1 Haplotypes In The Three Ethnic Groups .79 Table 3-4 LD and association analysis of the most common haplotype alleles of the FRAXA CGG locus and the flanking markers 82 Table 3-5 LD and association between 5' CGG substructures and ATL1 and IVS10 SNP loci .85 Table 3-6 FMR1 CGG patterns present on various FMR1 haplotypes in the Singapore population 86 Table 3-7 Expected heterozygosity of FMR1 haplotypes and CGG patterns based on position of AGG loss (A) or CGG repeat length (B) .90 Table 4-1 Primers used in specific amplification and sequencing of SCA2 exon 105 Table 4-2 Primers used in specific amplification and sequencing of SCA2 exons 2-25 106 Table 4-3 Sequencing result of SCA2 promoter region, exon and flanking intronic region in family members 111 Table 4-4 SCA2 exon1 coding nucleotide changes and effect on amino Acid codon 112 Table 4-5 SCA2 sequencing result of exons 2-25 and flanking intronic region 113 Figure 1-1 Molecular mechanism of expansions and deletions of DNA triplet repeats Figure 1-2 Schematic representation of the genomic structure of FMR1 11 Figure 1-3 Schematic diagram of restriction map around FMR1 CGG locus 15 Figure 1-4 Schematic representation of the genomic structure of SCA2 21 Figure 2-1 Methylation-specific PCR analysis at the FMR1 (CGG)n locus 37 Figure 2-2 Schematic representation of triple ms-PCR agarose gel results expected from females and males with various FMR1 CGG genotypes .42 Figure 2-3 Schematic representation of Southern blot results from EcoRI and Bsp68I (NruI) digested females and males with various FMR1 CGG genotypes 44 iv Figure 2-4 FMR1 ms-PCR results of sodium bisulfite treated genomic DNA from female and male lymphoblastoid cell lines carrying normal, premutation, and/or full mutation FMR1 (CGG)n alleles 47 Figure 2-5 Southern blot result from EcoRI and Bsp68I (NruI) digested female and male lymphoblastoid cell line DNA carrying normal, premutation, and/or full mutation FMR1 (CGG)n alleles 49 Figure 2-6 HUMARA X-inactivation patterns tracings .51 Figure 2-7 FMR1 ms-PCR results of sodium bisulfite treated female and male PBL genomic DNA carrying normal, premutation, and/or full mutation FMR1 (CGG)n alleles …………………………………………………………………………………….54 Figure 3-1 STR and SNP markers around the Fragile X locus on Xq27.3 (not to scale) Filled boxes represent FMR1 exons 69 Figure 4-1 Pedigree of the family with SCA2, using standard nomenclature .103 Figure 4-2 Schematic representation of the genomic structure of exon of the SCA2 gene and specific primers used to amplify and sequence this region .104 Figure 4-3 Polymorphisms found in SCA2 promoter region, exon and flanking intronic sequence 114 Figure 4-4 Polymorphisms found in other SCA2 exons and flanking intronic sequences (partial) 115 v ABBREVIATION 3' UTR 5' UTR bp CH CI EH FM FMR1 FMRP FRAXA HUMARA IN LD ML ms-PCR NL PCR PFM PM RNP SCA SNP STR TDI-FP 3' untranslated region 5' untranslated region base pair Chinese Confidence Interval expected heterozygosity full mutation fragile X mental retardation-1 fragile X mental retardation protein fragile site, X chromosome, A site Human androgen-receptor gene Indian linkage disequilibrium Malay methylation-specific PCR normal polymerase chain reaction pre/full mutation premutation ribonucleoprotein spinocerebellar ataxia single nucleotide polymorphism short tandem repeat Template-directed Dye-terminator Incorporation with Fluorescence Polarization detection vi SUMMARY The unique phenotypic and genotypic characteristics of the trinucleotide repeat expansion disorders present a new perspective from which to view human disease The discovery of each new trinucleotide repeat disorder brings tremendous clinical benefits, offering better classification of the diseases and facilitating early diagnosis and genetic counseling The combined usage of basic biochemistry and genetics and molecular and cellular biology has produced remarkable insights into these unusual mutations during the past decade Fragile X syndrome is the most common inherited mental retardation disorder, and is caused by instability and hyperexpansion of a polymorphic CGG trinucleotide repeat in the 5’ untranslated region of the FMR1 gene More than a decade after its gene identification, molecular diagnosis of this disorder, especially in females, continues to rely on Southern blot analysis In this study, a rapid and reliable methylation-specific PCR system for detecting normal, premutation, and full-mutation FMR1 alleles in both males and females was developed To fully understand the CGG repeat dynamics and to potentially apply the findings to risk ascertainment, CGG repeat structures were examined and haplotype studies were carried out in a large Singaporean population, including three ethnic groups: Chinese, Malay and Indian These comprehensive data and specific findings from this population suggested many potential mutation pathways In addition to fragile X syndrome, a family with affected members who had typical phenotypic and MRI features of spinocerebellar ataxia type (SCA2) was studied Two affected members had focal epilepsy, which has not been associated with SCA2 vii previously Trinucleotide expansions in the pathological range were found in the SCA2 gene, confirming SCA2 Sequencing of the expanded SCA2 gene did not reveal any new mutations that could account for epilepsy It was hypothesized that the new feature of focal epilepsy was due to co-existence of an epilepsy susceptibility gene with the expanded SCA2 gene viii CHAPTER GENERAL INTRODUCTION 1.1 Trinucleotide repeat diseases 1.1.1 Overview of trinucleotide repeat diseases Trinucleotide, or triplet repeats consisting of nucleotides consecutively repeated within a region of DNA were once thought to be commonplace iterations in the genome All possible combinations of nucleotides are known to exist as trinucleotide repeats, though some (e.g., CGG and CAG) are more common than others (Beckman and Weber 1992; Stallings 1994) In 1991, trinucleotide repeats were found to undergo a new type of genetic mutation, known as a dynamic or expansion mutation In this kind of mutation, the number of triplets in a repeat region increases Expanded repeats tend to be unstable: an expanded repeat passed from one generation to the next will usually vary in length, typically becoming longer On the other hand, the repeats of normal length will rarely change in length (Pearson and Sinden 1998a) Over the past decade, dynamic mutations responsible for more than 20 serious human genetic diseases have been traced to the genetic variation in the lengths of specific trinucleotide repeats in the genome Many of the diseases associated with this form of mutation affect the neurological or neuromuscular systems and include myotonic dystrophy (the most common form of muscular dystrophy), Huntington's disease, spinocerebellar ataxia types 1, 2, 3, and 7, and fragile X syndrome (the most common form of inherited mental retardation) (Margolis et al 1999) 70% ethanol, vacuum dried, then resuspended in 50 µl of deionized water for subsequent sequencing 4.2.5 Cycle sequencing 4.2.5.1 Exon fragments For fragments ARb and EC, each 20 µl sequencing reaction contained µl of BigDye™ version2 sequencing mix, àl of 2.5ìdilution buffer, M of betaine, and 0.2 mM of sequencing primer For fragment FB, the amount of BigDye™ sequencing mix used was µl, without any additional 2.5×dilution buffer added The volume of PCR product template to be added per sequencing reaction depended on the concentration and the length of the fragment (10 ng for fragments between 200bp to 500bp; 20 ng for fragments between 500bp to 1kb; and 40 ng for fragments between 1kb to 2kb) For fragment ARb sequenced with primer 1-Rb, fragment EC sequenced with both primers 1-C and 1-E, and fragment FB sequenced with primer 1-F, the sequencing reaction conditions were 25 cycles of 96 °C for 30 s, 50 °C for 15 s and 60 °C for For fragment FB sequenced with primer 1-B and fragment FD sequenced with primer 1-D, sequencing conditions were an initial denaturation at 98 °C for followed by 25 cycles of 98 °C for min, 55 °C for 15 s and 60 °C for 4.2.5.2 Exons 2-25 fragments Each 20 µl sequencing reaction contained µl of BigDye™, àl of 2.5ìdilution buffer and 0.2 mM of each primer For exons 2-7, 9, 11-18 and 21-25, cycle sequencing conditions were 25 cycles of 96 °C for 30 s, 50 °C for 15 s and 60 °C for For 108 exons 8, 10 and 19-20, cycle sequencing reaction conditions were 25 cycles of 96 °C for 30 s, 55 °C for 15 s and 60 °C for 4.2.6 Ethanol precipitation of sequencing products Two microliters of M sodium acetate (pH 4.6) and 50 µl of 95% ethanol were added to each 20 µl sequencing product The mixtures were kept in the dark at -20 °C for 25 to aid in precipitation of the sequencing products Mixtures were then centrifuged for 25 at 16,000 ìg Each pellet was rinsed with 250 àl of 70% ethanol, vacuum dried for min, then kept at -20 °C until further use 4.2.7 Gel electrophoresis Ethanol-precipitated pellets were re-suspended in µl loading dye which consisted of a 5:1 mixture of deionized formamide to 50 mM blue dextran in 25 mM EDTA Resuspended sequencing products were denatured at 95 °C for min, and 1.5 µl was loaded into the wells of a 4% polyacrylamide sequencing gel Each 50 ml of gel mix contained ml of 40% (19:1 ratio of acrylamide to bis) polyacrylamide stock, ml of 10×TBE, and 18 g of urea topped up with dH2O, and deionized in the presence of 0.5 g of Amberlite™ resin (ICN) After removal of the resin, 250 µl of 10% ammonium persulfate and 35 µl of TEMED were added to the gel solution to induce polymerization after casting Electrophoresis was carried out for h using the run module: Seq Run 36E-1200 Sequencing gel running and data processing were extracted using ABI PRISMTM 377 DNA Sequencer and accompanying Macintosh based data collection software (version 2.0, Perkin Elmer, USA) 109 4.3 Results The sequencing results are listed in Tables 4-3, 4-4 and 4-5, and exact positions of the identified polymorphisms are highlighted in Figures 4-3 and 4-4 A total of 11 single nucleotide differences were identified among the four family members studied, of which involved amino acid changes Both amino acid changes were in exon CD07 was heterozygous for a C to G change in codon 29 (Ser 29 Trp), while both CD03 and CD09 were heterozygous for a C to G change in codon 106 (Leu 106 Val) CD03 and CD09 were also heterozygous for a C to T change in codon 129, but this change was silent Additionally, a c/t polymorphism was observed in the promoter; both CD02 and CD07 were homozygous t/t, while CD03 was homozygous c/c and CD09 was heterozygous c/t A c/t polymorphism was also identified in the 5’UTR; both CD02 and CD07 were homozygous c/c while both CD03 and CD09 were heterozygous c/t An a/g polymorphism was identified in the 3’UTR; both CD02 and CD07 were heterozygous a/g while both CD03 and CD09 were g/ g The remaining nucleotide polymorphisms were all intronic CD09 was not sequenced for exons 2-24, due to insufficient DNA Even so, it was clear that none of the differences were observed only in the affected or the unaffected individuals Since both affected and unaffected samples shared the same genotype in those different nucleotide sites, these polymorphisms are unlikely to have a causal effect Therefore, a second SCA2 mutation separate from the CAG repeat expansion could not be identified to account for the additional feature of focal epilepsy in the index patient (CD02) and his similarly-affected son (CD09) 110 Table 4-3 Sequencing result of SCA2 promoter region, exon and flanking intronic region in family members (nucleotide numberings are based on GenBank sequence #AC004085) Amplicon Sequencing primer: CD02 CD03 CD07 CD09 nucleotides screened (proband) (unaffected wife) (unaffected sister) (affected son) FB 1-F:64910→64736 64781:t/t 64781:c/c 64781:t/t 64781:t/c (promoter) FD 1-F:64735→64574 (5’UTR) 64705:c/c 64705:t/c 64705:c/c 64705:t/c 1-D:64381→64573 64486:C/C* 64486:C/C* 64486:C/G* 64486:C/G* 1-B:64291→64380 √ √ –– √ EC 1-E: 64290→(CAG)n 1-C: 63972→(CAG)n ARb 64256:exp: C * nml: C 64185:exp: C* nml: C 64256:C/G* 64256:C/C* 64185:T/C* 64185:C/C* exp:(CAG)n=39; CAA=0 nml:(CAG)n=22; CAA=1 √ exp:(CAG)n=39; CAA=0 nml:(CAG)n=22; CAA=1 (CAG)n=22; CAA=2 (CAG)n=22; CAA=1 √ (CAG)n=22; CAA=2 √ (CAG)n=22; CAA=1 √ √ √ √ 1-Rb: 63847→63971 √ 1-Rb: 63691→63846 (intron) √ √ : no polymorphism in the fragment –– : not sequenced due to insufficient DNA exp: expanded allele; nml: normal allele * : refer to Table 4-4 64256:exp:C* nml:G 64185:exp:C* nml:T exp:(CAG)n=52; CAA=0 nml:(CAG)n=22; CAA=2 √ exp:(CAG)n=52; CAA=0 nml:(CAG)n=22; CAA=2 √ √ 111 Table 4-4 SCA2 exon1 coding nucleotide changes and effect on amino Acid codon (nucleotide numbering based on GenBank sequence #AC004085) nucleotide change 64486: C→G 64256: C→G 64185: C→T Codon change TCG→TGG CTC→GTC CGC→CGT Amino Acid change Ser 29 Trp Leu 106 Val Arg 129 Arg 4.4 Discussion The SCAs are trinucleotide repeat expansion disorders, with the exception of SCA10 where a pentanucleotide repeat is involved (Grewal et al 2002) Antenatal diagnosis is possible The family studied here showed phenotypic features consistent with SCA2 (Geschwind et al 1997) They had ataxia, markedly slowed eye saccades, and peripheral neuropathy The SCAs also exhibit genetic anticipation, where the disease begins earlier with each succeeding generation Anticipation is well correlated to increasing size of trinucleotide expansions with each succeeding generation; all the SCAs, except SCA5, show more dramatic anticipation with paternal transmission (Evidente et al 2000) The family in this study demonstrates this feature clearly; paternal transmission from the index patient to his son resulted in a dramatic increase in repeat size from 39 to 52, resulting in the son becoming symptomatic before even his father or uncle who had a 37 repeat allele Maternal transmission from the index patient's mother did not show such marked anticipation The expanded SCA genes produce ataxins with abnormally long glutamine stretches, which then aggregate in the neurons of affected patients to form neuronal intranuclear inclusions (NII) The probability of NII formation increases with the number of repeats (Klockgether et al 2000) This could account for the phenomenon of 112 Table 4-5 SCA2 sequencing result of exons 2-25 and flanking intronic region (nucleotide numbering based on GenBank AC002395) exon Nucleotide Coverage 16830→17037* 16979→17015# 18657→18834* 18681→18740# 19871→20200* 19921→19992# 20431→20870* 20468→20618# 47291→47726* 47582→47706# 51781→52034* 51925→52016# 52600→53030* 52822→53019# 54351→54704* 54471→54649# 56461→56940* 56535→56744# 59322→59710* 59351→59541# 62151→62640* 62316→62513# 62711→63240* 62900→63007# 63008→63393* 63279→63349# 84001→84422* 84118→84422# 86074→86372* 86074→86137# 87021→87280* 87033→87185# 87281→87657* 87567→87627# 102141→102486* 102156→102340# 102601→103080* 102658→102790# 108069→108310* 108183→108236# 115471→115754* 115539→115684# 116521→116869* 116642→116869# 118941→119312* 119053→119221# 119946→120447* 120058→120447# 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 CD02 Patients' sample CD03 CD07 Region containing polymorphism CD09 √ √ √ –– 18657: 13t/14t √ 18657: 13t/14t √ 18657: 12t/13t √ –– 20723: t/t 20723: g/g 20723: t/t –– √ √ √ –– √ √ √ –– √ √ √ –– √ √ √ –– √ √ √ –– √ √ √ –– √ √ √ –– √ √ √ –– √ √ √ –– √ √ √ –– √ √ √ –– √ √ √ –– 87285: t/t 87285: c/t 87285: t/t –– √ √ √ –– √ √ √ –– √ √ √ –– 115499: c/c 116630: a/a √ 115499: c/g 116630: g/g √ 115499: c/c 116630: a/a √ –– intron 21 –– intron 22 120105: a/g 120105: g/g 120105: a/g 120105: g/g intron –– intron intron 17 –– 3' UTR * : sequences including flanking intronic sequences # : sequence of the exon only √ : no polymorphism in the fragment ––: not sequenced due to insufficient DNA 113 63601 tcttgaccgc 63661 cagaggaccc 63721 cccgcgctgc 63781 cggcgcgggc 63841 acccacCTGC 63901 GCCGTGGCCG 63961 GCTAGAAGGC 64021 TGCTGCTGCT 64081 GGCTTCAGCG 64141 GGAGCCGGGC 64201 GAGGCGCGGG 64261 ACGAAGGGGC 64321 CCGCCGCCGT 64381 GAGGGAGGGG 64441 GCGCCACCGC 64501 CACCTGGCTG 64561 CCGCTGAGCG 64621 agggactctt 64681 ggccgggagg 64741 gagggccagg 64801 ggagggcgtg 64861 tttcttctcc 64921 acctgatgca 64981 gctacgtgta cgggggaggg ggcggggatg ctgcgggagg ctggacaggc ctgacaatcc cggctgcgcc caccggccga gcctcggggc tcaggcccga gcgagtctcc cggcccccag cccaccccgg gtagcccggc gggtcacggg gcggggacgg cgcgggggag gggacgccgg gcccggagcg gagggggctg gggtgccgac CCAGGCCGGG CCTCCCGCCG CCGGAGGTCG CCGCGACCAC CGAGGAGGGA AGGACGAGGA GACCGAGGAC GAGGACGGCG AAGGCGCGGC GGCGGGCGAC CGCTGCCGCC GGGCTTGCGG ACATTGGCAG CCGCGGGCGG CGG[CTGCTGC GCTGCTGTTG CTGCTGCTGC TGCTGCTGCT GCTGCTGCTG CTGCTGCTG]G ACATGGTGAG GGGCCCATAC ACCGGCTCGC ACGCCGGGCG GGGACAGCCG GCGCCAAGGA GACGCCGGAA CGCGGCGGGG ACGCGCGGGC GCCGAGCGGG TTGGCGCGGC CGGAGGGGCG CCCGGGCTGG CGAGGGGGAG AAGGAGGACG GGGGAGGCCC GCCGAGACCA AGGAGCCGCC GGGAGCCGGG CCGAAACGCG TGCCGTTGCT ACCAAAACAG TCTGAGGCGG AGGGAGGCGA GCTCTGCCGG GGCCGGGGCC GGGCGGAGGA AGGGCGGCGG AGGGATACGG TCCCGGGGCC CGCCCCGCCC GCTCCGCCGC GCCGGCCGCT GGAGCGAGCG CCACCCGGGC CGGCGAA*CGG CGAGACTCGG TGGCCACCGC GGGACTCCGA GGAGCTGCGG CATcggaggg cgggcgcgcc gaggcgccgg gtgggagcgg aggtgcggat taccggaagt cggaggggtc agacggaagc agaacgtgag gtggccccgg gacacgtgag gagcgggcgg cgcgctgggt tgctttctcg ggggtcgggt cggcggtccc tacccggatc cgccttcctc aaggcgggtc tgcccttcgg tcggtctcag cctctggggc ctgcctggtg tggaggacag gagagaagca tactgtattc cttcagcatc ttcacttctg cggggaccta gtttgccccc acccagaaaa tggactgact gcgagtgcct tggggcggag aatgtgtctt aagcacctag aaccctgcca gattcagggg acacagcatc ctgccgtttt *: a G is missing in the GenBank sequence Figure 4-3 Polymorphisms found in SCA2 promoter region, exon and flanking intronic sequence (GenBank #AC004085) Sequence shown is of the antisense strand CAG repeats are in brackets Exon sequences are indicated in bold with 5’ untranslated region indicated by small letters and translated sequences indicated in capital letters The ATG start codon is italicized and underlined Highlighted letters indicate positioned polymorphisms 114 18541 caagcactaa gtataatgca aatatcccca aatccgaaaa aaatccgcag tctaaaatac 18601 ttctggtccc aagcatttta gatgaggaag attcagtttg tactaatttc taatagtttt 18661 tttttttttt aatattccag ATTTCTTTTG ATGGAATCTA TGCAAATATG AGGATGGTTC ex3 18721 ATATACTTAC ATCAGTTGTT gtaagttatt agattattgg ggataaactg ccttgggggt 18781 agaataaagt aattccatga agttaaaatg tggataaatg attgtcaaag taacattgct 20401 ctaaagaatt cctaacaaat tttattttgt aaaggtttgg agtacttact tgtgtttttc 20461 attttagTGT GATTTGGTAC TTGATGCCGC ACATGAGAAA AGTACAGAAT CCAGTTCGGG ex5 20521 GCCGAAACGT GAAGAAATAA TGGAGAGTAT TTTGTTCAAA TGTTCAGACT TTGTTGTGGT 20581 ACAGTTTAAA GATATGGACT CCAGTTATGC AAAAAGAGgt gggttttgat ttcctaaata 20641 tgcctcatgg tttattagat ttattcaagc aaagattttc acagtgatct tacaaacttt 20701 ttttaaagaa atatctgggc tgggtatggc ggctcattcc tgtaatctta gcacttaggg 20761 aggctgaggc gggtggatca cctgaggtca ggagttcgag accagcctgg ccaacatggc 87001 tgagataatt gaatttattg ttgtttttgt agCCAAAGCC TTCTACTACC CCAACTTCAC ex17 87061 CTCGGCCTCA AGCACAACCT AGCCCATCTA TGGTGGGTCA TCAACAGCCA ACTCCAGTTT 87121 ATACTCAGCC TGTTTGTTTT GCACCAAATA TGATGTATCC AGTCCCAGTG AGCCCAGGCG 87181 TGCAAgtaag tcatagaatt tgatgttcac ttagcctccc caattgtttg tatctgacac 87241 caagcactct ttaggttttc agtgacttga gggtgtgatg gttatgcata tgcatttgaa 87301 acagacaggc atgcagagat tcagtgtgtt gttaagtatg aggacctaaa tctgagaatg 115441 attgtaacca tcaagaaagt tcagttgatg aagtgtagag gagcgatgga ggttgtcaga 115501 catcggttgt gtacatgctc ctttttcttt cactttagTT TCCACGGGCT CCCTTGCTCA ex22 115561 GCAGTATGCG CACCCTAACG CTACCCTGCA CCCACATACT CCACACCCTC AGCCTTCAGC 115621 TACCCCCACT GGACAGCAGC AAAGCCAACA TGGTGGAAGT CATCCTGCAC CCAGTCCTGT 115681 TCAGgtaagg gcaactcaga ggtctgcatg gagtggcttc tttatcctag tatctgagtg 115741 ctttcttcag gtgccaggta tcgcatcgtc agaacacatg gcatgtccac cctcgtgaag 116521 cctgtacttt ccagtgaccc tcatcatagg cccaagtgtg caaagcttag ctttgtgggt 116581 atcccttggc tgcttttcat taaagaagtt ttcctctcaa ttctttcctg tcgctttgca 116641 gCACCATCAG CACCAGGCCG CCCAGGCTCT CCATCTGGCC AGTCCACAGC AGCAGTCAGC ex23 116701 CATTTACCAC GCGGGGCTTG CGCCAACTCC ACCCTCCATG ACACCTGCCT CCAACACGCA 116761 GTCGCCACAG AATAGTTTCC CAGCAGCACA ACAGACTGTC TTTACGATCC ATCCTTCTCA 116821 CGTTCAGCCG GCGTATACCA ACCCACCCCA CATGGCCCAC GTACCTCAGg taataccagc 119941 attttgagtt ttgttcagct agcacgagga tagtttacaa tcatgtgctg cagagacact 120001 aggctgatgt gtggtgttgc cagttttctg tttcaatgtt cgcttttctt tttacagTAC ex25 120061 AAGCCCACCA CCAACAGCAG TTGTAAggct gccctggagg aaccgaaagg ccaaattccc 120121 tcctcccttc tactgcttct accaactgga agcacagaaa actagaattt catttatttt 120181 gtttttaaaa tatatatgtt gatttcttgt aacatccaat aggaatgcta acagttcact 120241 tgcagtggaa gatacttgga ccgagtagag gcatttagga acttgggggc tattccataa 120301 ttccatatgc tgtttcagag tcccgcaggt accccagctc tgcttgccga aactggaagt 120361 tatttatttt ttaataaccc ttgaaagtca tgaacacatc agctagcaaa agaagtaaca 120421 agagtgattc ttgctgctat tactgctaaa aaaaaaaaaa aaaaaaaatc aagacttgga Figure 4-4 Polymorphisms found in other SCA2 exons and flanking intronic sequences (partial) (GenBank #AC002395) Bold letters define exon sequences Small letters in exon 25 indicate 3’ untranslated region, while capital letters indicated translated sequences The TAA stop codon is italicized and underlined, while the highlighted letters shown the positions of the polymorphisms 115 anticipation The exact pathophysiological mechanism by which NIIs cause cellular dysfunction is uncertain, but binding and inactivation of transcription factors by the abnormal proteins has been postulated (Klockgether et al 2000) Ataxia and anticipation are core features of the SCAs Epilepsy, however is not a feature of the SCA family, except in SCA10 and a related disorder, dentatorubralpallidoluysian atrophy (DRPLA) SCA10, described predominantly in patients of Mexican descent (Matsuura et al 2002), is distinct from the other SCAs phenotypically because of epilepsy It is also unusual in that an ATTCT pentanucleotide repeat expansion, and not a trinucleotide repeat, is the genetic culprit (Grewal et al 2002) The studied family with ataxia and epilepsy in this study however had SCA2, not SCA10 DRPLA is another trinucleotide repeat neurodegenerative disorder (Evidente et al 2000), that was considered as a differential diagnosis in this family It also presents with ataxia and epilepsy However, choreoathetosis, dementia and myoclonus are also features of DRPLA that were absent in the family Genetic analysis of the DRPLA locus at chromosome 12p in the family excluded DRPLA Complete sequencing of the SCA2 gene did not yield any additional consistent differences between affected and unaffected members that could explain the epilepsy It is unlikely that the expanded SCA2 gene alone could account for the epilepsy, as no previously described families with SCA2 have had epilepsy, even at advanced stages of the disease Several mutations causing focal epilepsy have been described in the past decade; all except one are ion channel disorders (Kalachikov et al 2002; Winawer 2002) Could one of these known mutations causing epilepsy be linked with the SCA2 gene? This is felt 116 unlikely; all existing identified genes causing non-progressive epilepsy syndromes are located on other chromosomes and none are on 12q24 The combination of epilepsy and ataxia could also suggest a channelopathy and mouse models have been described (Fletcher and Frankel 1999) Rare families and individuals with mutations in the potassium (Zuberi et al 1999) and calcium channels (Jouvenceau et al 2001) causing epilepsy and ataxia have also been described However, neither these two mutations, nor any of the other described channelopathies causing neurological dysfunction in humans (Kullmann 2002) are close to the SCA2 gene Therefore a linked channelopathy is less likely in the family It was postulated, therefore, that there may be a separate unlinked susceptibility gene for focal epilepsy in this family The susceptibility gene by itself is insufficient to manifest epilepsy and may be a mild non-manifesting mutation in one of the existing ion channels However, co-inheritance with the expanded SCA2 gene causes a complex interaction which triggers epilepsy Both genes need to be present to manifest the epilepsy Such an oligogenic (as opposed to monogenic) model of epilepsy has recently been proposed as a potential genetic model for generalised epilepsy (Durner et al 2001) Under this hypothesis, the focal epilepsy in the affected son of the index is ascribed to coinheritance of both the SCA2 gene and the epilepsy-susceptibility gene from his affected father Likewise, the absence of epilepsy in the SCA2-affected brother of the index is attributed to absence of the epilepsy-susceptibility gene in him The hypothesis of an epilepsy-susceptibility gene requiring the presence of the expanded SCA2 gene for manifestation is also consistent with the absence of epileptic findings in the other family members without SCA2 117 SCA10 is the only other SCA with epilepsy In one study of two large families with SCA10, half of the affected patients overall had epilepsy (Grewal et al 2002) However, the inter-family frequency of epilepsy in affected individuals was significantly different (80% vs 25%, p=0.01) between these two families No patients without SCA10 had epilepsy There was no correlation between the pentanucleotide repeat size and epilepsy, and the mechanism by which the SCA10 mutation leads to epilepsy has yet to be elucidated The authors concluded that other genetic influences in different families with SCA10 may modify the phenotypic expression of the SCA10 mutation This lends support to the hypothesis that a co-inherited epilepsy susceptibility gene, together with an expanded SCA2 gene, can account for the finding of focal epilepsy in only some affected patients with SCA2 in the family Both genes must be present to cause epilepsy This susceptibility gene could be a mild non-manifesting mutation in one of the existing ion channels known to cause focal epilepsy, or it could be present in a asyet undescribed location Assuming an oligogenic model, locating and identifying this epilepsy susceptibility gene in the family by linkage analysis will be a daunting challenge Given the small size of the SCA2 family and the small number of affected individuals, as well as the lack of other families with similar findings, it would also be very difficult to validate the hypothesis using existing disease-association strategies In conclusion, I describe here a family with confirmed SCA2 with the previously undescribed phenotypic feature of focal epilepsy in affected members, a feature which had previously been exclusive to SCA10 among all the SCAs It is believed that this new feature reflects the influence of a co-existing epilepsy susceptibility gene on the expanded SCA2 gene Future clinicians, when confronted with a patient with a spinocerebellar ataxia and focal epilepsy, should consider both SCA10 and SCA2 as possible diagnoses 118 Reference Bennett RL, Steinhaus KA, Uhrich SB, O'Sullivan CK, Resta RG, Lochner-Doyle D, Markel DS, Vincent V, Hamanishi J 1995 Recommendations for standardized human pedigree nomenclature Pedigree Standardization Task Force of the National Society of Genetic Counselors Am J Hum Genet 56: 745-52 Durner M, Keddache MA, Tomasini L, Shinnar S, Resor SR, Cohen J, Harden C, Moshe SL, Rosenbaum D, Kang H, Ballaban-Gil K, Hertz S, Labar DR, Luciano D, Wallace S, Yohai D, Klotz I, Dicker E, Greenberg DA 2001 Genome scan of idiopathic generalized epilepsy: evidence for major susceptibility gene and modifying genes influencing the seizure type Ann Neurol 49: 328-35 Evidente VG, Gwinn-Hardy KA, Caviness JN, Gilman S 2000 Hereditary ataxias Fletcher CF, Frankel WN 1999 Ataxic mouse mutants and molecular mechanisms of absence epilepsy Hum Mol Genet 8: 1907-12 Geschwind DH, Perlman S, Figueroa CP, Treiman LJ, Pulst SM 1997 The prevalence and wide clinical spectrum of the spinocerebellar ataxia type trinucleotide repeat in patients with autosomal dominant cerebellar ataxia Am J Hum Genet 60: 84250 Grewal RP, Achari M, Matsuura T, Durazo A, Tayag E, Zu L, Pulst SM, Ashizawa T 2002 Clinical features and ATTCT repeat expansion in spinocerebellar ataxia type 10 Arch Neurol 59: 1285-90 Jouvenceau A, Eunson LH, Spauschus A, Ramesh V, Zuberi SM, Kullmann DM, Hanna MG 2001 Human epilepsy associated with dysfunction of the brain P/Q-type calcium channel Lancet 358: 801-7 Kalachikov S, Evgrafov O, Ross B, Winawer M, Barker-Cummings C, Martinelli Boneschi F, Choi C, Morozov P, Das K, Teplitskaya E, Yu A, Cayanis E, Penchaszadeh G, Kottmann AH, Pedley TA, Hauser WA, Ottman R, Gilliam TC 2002 Mutations in LGI1 cause autosomal-dominant partial epilepsy with auditory features Nat Genet 30: 335-41 Klockgether T, Wullner U, Spauschus A, Evert B 2000 The molecular biology of the autosomal-dominant cerebellar ataxias Mov Disord 15: 604-12 Kullmann DM 2002 The neuronal channelopathies Brain 125: 1177-95 Matsuura T, Ranum LP, Volpini V, Pandolfo M, Sasaki H, Tashiro K, Watase K, Zoghbi HY, Ashizawa T 2002 Spinocerebellar ataxia type 10 is rare in populations other than Mexicans Neurology 58: 983-4 119 Subramony SH, Filla A 2001 Autosomal dominant spinocerebellar ataxias ad infinitum? Neurology 56: 287-9 Winawer MR 2002 Epilepsy genetics Neurologist 8: 133-51 Zuberi SM, Eunson LH, Spauschus A, De Silva R, Tolmie J, Wood NW, McWilliam RC, Stephenson JP, Kullmann DM, Hanna MG 1999 A novel mutation in the human voltage-gated potassium channel gene (Kv1.1) associates with episodic ataxia type and sometimes with partial epilepsy Brain 122: 817-25 120 APPENDIX: SOLUTIONS Growth medium (for cell culture) 15% inactivated fetal bovine serum, 1% L-Glutamine, 1% HEPES, top up with RPMI 1640 Freeze medium (for cell culture) 90% inactivated fetal bovine serum 10% DMSO TKM buffer (for DNA extraction) Tris-HCl, pH7.6 10mM KCl 10mM MgCl2 10mM EDTA, pH8.0, 2mM TKM buffer (for DNA extraction) Tris-HCl, pH7.6 10mM KCl 10mM MgCl2 10mM EDTA, pH8.0, 2mM NaCl 0.4M Rehydrating Solution (Tris-EDTA) Tris-HCl, pH7.6 10mM EDTA, pH8.0, 1mM (pH=8.0) TAE (50×) Tris base Glacial acetic acid EDTA, pH8.0 (pH=7.8) 2M 1M 50mM Denaturation solution (for Southern blot) NaOH 0.5M NaCl 1.5M Neutralization solution (for Southern blot) Tris, pH7.4 1M NaCl 1.5M 121 SSC (20×) NaCl Na3Citrate⋅2H2O (pH=7.0) 3M 300mM Blocking buffer A (for Southern blot) 0.2% Blocking Reagent 1× PBS 0.1% Tweenđ 20 Wash Buffer A (for Southern blot) 1ìPBS 0.1% Tween® 20 Assay Buffer (for Southern blot) 0.1M Diethanolamine (DEA) (Intergen) 1mM MgCl2 pH 10.0 2.5× Dilution Buffer (for sequencing) Tris-HCl 200mM MgCl2 5mM 122 ... features of fragile X syndrome .8 1 .2. 2 The cause of fragile X syndrome 1 .2. 3 The prevalence of fragile X syndrome 12 1 .2. 4 The diagnosis of fragile X syndrome 13 1 .2. 5... out of 1000 males and out of 400 females (Warren and Sherman 20 01) 12 1 .2. 4 The diagnosis of fragile X syndrome A diagnosis of fragile X syndrome is often suspected based on clinical phenotype and. .. haplotypes in fragile X syndrome 16 1.3 Spinocerebellar ataxia type (SCA2) 18 1.3.1 Overview of SCA2 18 1.3 .2 Clinical features of SCA2 20 1.3.3 The cause of