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

The diagnosis of SCA2 ...22 Reference...23 CHAPTER 2 Simplified Methylation-specific PCR Detection of Fragile X Syndrome Expansion Mutations in Males and Females...32 2.1.. ABBREVIATION

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

TABLE OF CONTENT

ACKNOWLEDGEMENTS i

TABLE OF CONTENT ii

LIST OF FIGURES AND TABLES iv

ABBREVIATION vi

SUMMARY vii

CHAPTER 1 GENERAL INTRODUCTION 1

1.1 Trinucleotide repeat diseases 2

1.1.1 Overview of trinucleotide repeat diseases 2

1.1.2 Classification of trinucleotide repeat diseases 3

1.1.3 Shared defining features of trinucleotide repeat diseases 3

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 8

1.2.1 Overview and clinical features of fragile X syndrome 8

1.2.2 The cause of fragile X syndrome 8

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 2 (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 2 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 3 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

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 4 Spinocerebellar Ataxia Type 2 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

LIST OF FIGURES AND TABLES

Table 1-1 Summary of the repeat expansion disorders .4

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 non-methylated and non-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 1 .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 1 and flanking intronic region in 4 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 7

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

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

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 1 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 1 and flanking intronic

sequence 114

Figure 4-4 Polymorphisms found in other SCA2 exons and flanking intronic sequences

(partial) 115

ABBREVIATION

3' UTR 3' untranslated region

5' UTR 5' untranslated region

FMR1 fragile X mental retardation-1

FMRP fragile X mental retardation protein

FRAXA fragile site, X chromosome, A site

HUMARA Human androgen-receptor gene

SCA spinocerebellar ataxia

SNP single nucleotide polymorphism

STR short tandem repeat

TDI-FP Template-directed Dye-terminator Incorporation with

Fluorescence Polarization detection

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 3 affected members who had typical phenotypic and MRI features of spinocerebellar ataxia type 2 (SCA2) was studied

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

CHAPTER 1 GENERAL INTRODUCTION

1.1 Trinucleotide repeat diseases

1.1.1 Overview of trinucleotide repeat diseases

Trinucleotide, or triplet repeats consisting of 3 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, 6 and 7, and fragile X syndrome (the most common

form of inherited mental retardation) (Margolis et al 1999)

1.1.2 Classification of trinucleotide repeat diseases

Trinucleotide repeat diseases can be classified into two sub-categories based on the relative location of the trinucleotide repeat to a gene (Table 1-1) The first sub-category, having its repeats in non-coding sequences [in the 5’ untranslated region (5'UTR), the 3’ untranslated region (3'UTR) or the intronic region] is typically characterized by large and variable repeat expansions And the larger mutations often are transmitted from a small pool of clinically silent intermediate size expansions, termed premutations The second sub-category, characterized by exonic CAG repeats that code for polyglutamine tracts, is much smaller in size and variation This latter class is also referred as “polyglutamine

diseases” (Bowater and Wells 2001; Cummings and Zoghbi 2000; Margolis et al 1999)

1.1.3 Shared defining features of trinucleotide repeat diseases

Trinucleotide repeat diseases share several defining features First of all, most of the trinucleotide repeat diseases display the clinical features of anticipation, defined as a more severe form and/or earlier age of onset of disease with successive family generations Second, an earlier age of onset and increasing severity of phenotype are generally correlated with larger repeat length Third, the mutant repeats show both somatic and germline instability Fourth, mutant repeats frequently expand rather than contract in successive transmissions Fifth, the parental origin of the disease allele can often affect anticipation, with paternal transmissions carrying a greater risk of expansion for many of CAG repeat diseases and maternal transmission for fragile X syndrome, Friedreich ataxia, and myotonic dystrophy (Cummings and Zoghbi 2000; Gusella and MacDonald 1996; La Spada 1997) Finally, most of the trinucleotide repeats are GC rich, which makes the molecular diagnosis by PCR very difficult, especially to detect expanded alleles

Table 1-1 Summary of the repeat expansion disorders (Bowater and Wells 2001; Cummings and Zoghbi 2000; Margolis et al 1999)

Amino Acid encoded normal expanded Inheritance

Mutation type

Parental gender bias Type 1 disorders:

LOF Fragile site

Non-Deletion Fragile site

Maternal

Friedreich's

ataxia

X25 9q13-21.1 Frataxin GAA intron 1 6-34 80 (PM)

112-1700 (FM) Autosomal recessive† LOF (partial) Maternal Myotonic

dystrophy

recessive†

Unknown Maternal SCA8 SCA8 13q21 None CTG 3' of RNA 16-37 107-127 Autosomal

recessive LOF? Maternal SCA12 SCA12 5q31-33 PP2A-PR55β CAG 5'-UTR 7-28 66-78 Autosomal

HD 4p16.3 Huntingtin CAG Glutamine 6-35 36-121 Autosomal

DRPLA DRPLA 12p13.31 Atrophin-a CAG Glutamine 6-35 49-88 Autosomal

SCA1 SCA1 6p23 Ataxin-1 CAG Glutamine 6-38 39-83 Autosomal

SCA2 SCA2 12q24.1 Ataxin-2 CAG Glutamine 14-31 32-77 Autosomal

SCA3 SCA3 14q32.1 Ataxin-3 CAG Glutamine 12-39 56-86 Autosomal

Table 1-1 (continued)

Amino Acid encoded normal expanded Inheritance

Mutation type

Parental gender bias

SCA6 SCA6 19p13 α1A-voltage

dependent calcium channel subunit

CAG Glutamine 4-19 20-30 Autosomal

SCA7 SCA7 13p12-13 Ataxin-7 CAG Glutamine 7-35 38-200 Autosomal

PM: premutation, FM: full mutation

† :Anticipation

LOF: loss of function, GOF: gain of function

ND: not determined

1.1.4 Molecular mechanisms of trinucleotide repeat diseases

As for the molecular mechanism of these diseases, replication of the DNA molecule is a prime candidate for the process that generates repeat tract instability A central aspect of the role of replication in generating repeat tract instability is that slippage

of the complementary DNA strands of the repeat may occur during movement of the polymerase (Paulson and Fischbeck 1996; Pearson and Sinden 1998b; Sinden 1999) Expansions of repeat tracts will occur if an unusual DNA structure, such as a hairpin, occurs within the newly synthesized DNA (Fig 1-1)

Despite the similarities mentioned above, the trinucleotide repeat diseases vary in many aspects, and it is clear that the particular trinucleotide repeat sequence and its location with respect to a gene are important defining factors in dictating the unique mechanism of pathogenesis for each disease The pathogenic mechanism will also vary from disease to disease, depending on the consequences of the lost function of the respective proteins or, in some cases, acquired function of a toxic transcript (Table 1-1) (Cummings and Zoghbi 2000)

1.1.5 Trinucleotide repeat diseases I have studied

Among all the trinucleotide repeat diseases, I focused mainly on fragile X syndrome, a type 1 disorder, with a cryptic repeat and relatively high frequency among the general population, to develop new diagnostic methods and identify potential cis-acting factors involved in triplet repeat expansion In addition to fragile X syndrome, I also performed molecular analysis on a family segregating with SCA2, a type 2 disorder

Figure 1-1 Molecular mechanism of expansions and deletions of DNA triplet repeats (adapted from Bowater and Wells 2001)

Deletions occur when hairpins form on the template strand and expansions occur when hairpins form on the nascent strand Original DNA is shown in line and newly synthesized DNA is shown in dash Thicker lines or dashes indicate regions of repetitive DNA, and arrows show the direction of DNA synthesis

1.2 Fragile X syndrome

1.2.1 Overview and clinical features of fragile X syndrome

In 1943, Martin and Bell described the first extended kindred with mental retardation segregating in an X-linked manner However, it was not until the early 1970s that X-linked inheritance of mental retardation was considered a contributing factor to the excess of males in retarded populations (Lehrke 1974; Martin and Bell 1943; Turner 1983)

Somatic features generally appear in male childhood and include increased head circumference, coarsening of facial features, hypotonia, and prominence of the ears, forehead and jaw (Hagerman and Synhorst 1984; Hagerman et al 1984; Loehr et al 1986; Opitz et al 1984) Macroorchidism is uncommon prior to puberty but is present in nearly

90 percent of postpubertal males (Sutherland et al 1985a)

Mental retardation is common but varies in severity in males with the fragile X syndrome Over 90 percent of male patients have IQ scores in the range of 20 to 60, with

a mean between 30 and 45 (Sutherland et al 1985b)

Facial features similar to those seen in affected males may be present in retarded female heterozygotes too Approximately 50 percent of females carrying the full mutation show mental impairment, with intellectual deficits ranging from learning disability with normal formal IQ testing to severe retardation (Brainard et al 1991; Fryns 1986)

1.2.2 The cause of fragile X syndrome

In 1969, Lubs first described a marker X chromosome in mentally retarded males (Lubs 1969) The marker consisted of a constriction or gap at the distal end of the long

arm of the X chromosome Around 1976-1977, the secondary constriction (widely known

as fragile X site) was shown to be localized to the interface between Xq27 and q28 (Giraud et al 1976; Harvey et al 1977; Sutherland 1977), and was later specified to Xq27.3 (Harrison et al 1983)

In 1991, a number of research groups isolated large segments of human DNA that spanned the fragile site (Heitz et al 1991; Hirst et al 1991; Kremer et al 1991b) Southern blot analysis revealed that in normal individuals the fragile site could be localized to a very small region of <200bp that contained a CGG trinucleotide repeat ranging from 5 to 55 repeats (Fu et al 1991; Kremer et al 1991a; Oberle et al 1991; Yu et

al 1991)

These different triplet lengths in normal individuals indicate that it is a genetic polymorphism, and that it is inherited stably in a Mendelian manner However, in individuals affected with the fragile X syndrome, the length of the repeat is increased and the stability of the amplified fragment is abnormal Transmitting males and many unaffected female carriers have CGG repeats in the premutation range, from 55 to 200 repeats Though premutations are not related to clinical disease, they are of major importance genetically because they are unstable when inherited, especially through maternal transmission; the carrier mother is at a high risk to transmit a larger repeat to her offspring Once the expansion ranges between >200 repeats and several thousand repeats,

it is accompanied by clinical and cytogenetic manifestations of the fragile X syndrome (Heitz et al 1992; Smits et al 1992) Once the CGG repeat expands beyond 200 repeats, the repeats become mitotically unstable (Devys et al 1992; Mornet et al 1993)

Fragile X syndrome is caused by the mutation of the FMR1 gene (Pieretti et al 1991; Verkerk et al 1991) The FMR1 gene consists of 17 exons spanning 38 kb and

encodes a 4.4 kb transcript (Fig 1-2) (Eichler et al 1993) The unstable CGG repeat is located in the 5’UTR of the gene (Ashley et al 1993a) The repeat is highly polymorphic

in the general population with a range of 5 to 55 and a mode of 30 (Fu et al 1991; Snow et

al 1993)

95% of patients with fragile X syndrome are caused by massive CGG repeat expansion When the CGG repeats exceed 200, the repeats and the upstream CpG island

are methylated and the expression of FMR1 is silenced (Hornstra et al 1993; Pieretti et al

1991; Sutcliffe et al 1992; Warren and Sherman 2001)

In normal individuals, the FMR1 gene is expressed and its translation product is

called fragile X mental retardation protein (FMRP), and its extensive alternate splicing of exons produces a number of protein isoforms (Ashley et al 1993; Eichler et al 1993) In

contrast, expression of FMR1 is greatly reduced or absent in affected individuals (Pieretti

et al 1991) FMRP has functional domains in common with proteins known to form large

ribonucleoprotein (RNP) complexes in vivo Three RNA binding domains are present in

FMRP: two KH domains (KH1, KH2) that show homology to hnRNP K, and an RGG box which is similar to hnRNP U (Ashley et al 1993b; Kiledjian and Dreyfuss 1992; Siomi et

al 1993a; Siomi et al 1993b) Therefore, FMRP was confirmed to indeed be an RNA binding protein (Brown et al 1998)

Figure 1-2 Schematic representation of the genomic structure of FMR1 (drawn to scale) The 17 exons of the gene span

approximately 38 kb of genomic DNA The solid boxes indicate exons: black color indicates translated region and grey color indicates untranslated region The open boxes indicate introns Tel.: Telomere; Cen.: Centromere

If the CGG repeat is between 55 and 200, the allele is called as premutation Though premutation individuals are clinically unaffected, the premutation alleles are unstable and tend to expand when transmitted A premutation can undergo a small expansion to another, usually larger allele in the premutation range, or it can undergo massive expansion to a full mutation This massive expansion to full mutation only occurs by maternal transmission Furthermore, the risk of expansion to the full mutation

is determined by the premutation’s size; the larger the premutation is, the more likely it will expand to a full mutation (Fu et al 1991; Heitz et al 1992) Some premutation individuals are not truly unaffected, but exhibit subtle fragile X-like features (Hull and Hagerman 1993; Loesch et al 1994; Riddle et al 1998) as well as premature ovarian failure in females and Parkinsonism in elderly males (Allingham-Hawkins et al 1999; Hagerman et al 2001; Uzielli et al 1999) These latter two features are remarkable in that they are unique to the premutation, because full mutation individuals are not affected

Since some groups have observed that premutation alleles express FMR1 mRNA at higher

levels and FMRP at lower levels than in normal controls, the unique features in

premutation individuals may be caused by the higher level of FMR1 mRNA (Kenneson et

al 2001; Tassone et al 2000)

1.2.3 The prevalence of fragile X syndrome

Fragile X syndrome is estimated to affect 1 in 4500 males and 1 in 9000 females The prevalence of the premutation allele is estimated to be 1 out of 1000 males and 1 out

of 400 females (Warren and Sherman 2001)

1.2.4 The diagnosis of fragile X syndrome

A diagnosis of fragile X syndrome is often suspected based on clinical phenotype and family history of X-linked mental retardation (Hagerman et al 1991) However, there still remains the problem in families with nonspecific X-linked mental retardation lacking associated phenotypic features that would help in distinguishing them from the fragile X syndrome Thus, diagnosis of the fragile X syndrome should not be made on phenotypic grounds alone, but must be confirmed with direct analysis of the CGG repeat expansion in

FMR1

In the early 1990s, laboratory diagnosis of fragile X syndrome was done by cytogenetic analysis using specialized growth medium (Dewald et al 1992; Jacky et al 1991) The key features of the protocol include the use of two or more fragile X induction systems, the analysis of at least 50 to 100 cells in males and 75 to 150 cells in females, and a standard constitutional chromosome analysis to rule out other cytogenetic etiologies for the mental retardation However, cytogenetic studies are generally insensitive for detection of premutation carriers Additionally, the presence of three other fragile sites in distal Xq (FRAXD, FRAXE and FRAXF), which cannot be distinguished from FRAXA

by cytogenetic studies, indicates that a positive cytogenetic finding may not be specific for fragile X syndrome and requires confirmation by direct molecular testing

The fundamental molecular assay for the fragile X syndrome is to detect the length

of CGG repeat in the FMR1 gene (Malmgren et al 1992; Oostra et al 1993; Pergolizzi et

al 1992; Rousseau et al 1991; Rousseau et al 1992; Snow et al 1992; Sutherland et al 1991; van Oost et al 1992; Verkerk et al 1991)

In Southern blots, the CGG repeat is located on a ~1 kb Pst I fragment, a 5.2 kb

EcoR I fragment, and a 12 kb Bgl II fragment in normal individuals (Table 2 and Fig

1-3) (Maddalena et al 2001) The choice of enzymes to use relies heavily on whether testing is being done for premutation or full mutation detection For general screening,

EcoRI generates an average size fragment and is particularly useful for double digestion

with a methylation sensitive enzyme, such as EagI The full mutation sometimes will

result in an indistinct smear ranging upwards from 5.7 kb because of somatic instability in full mutation patients, and the difference between a large normal allele and a small premutation allele is indistinguishable on a Southern because of the almost same migration rate of the two digested fragments on a gel

Fortunately, PCR is technically feasible and reliable in this particular range Furthermore, to save labor and cost, PCR has obvious advantages over Southern blot to

detect the size of the CGG repeat in the FMR1 gene Unfortunately, it is very difficult for

Table 1-2 Fragment sizes (in kb) using Southern blot for molecular diagnosis of

Probe A: pfxa3 (Yu et al 1991) or Ox0.55 (Nakahori et al 1991)

Probe B: StB12.3 (Rousseau et al 1991) or E5.1 (Verkerk et al 1991) or Ox1.9(Nakahori et

al 1991)

n.a.: not applicable

*EagI can be replaced by other methylation sensitive enzymes within the range, such as PauI,

NruI, etc The changes of fragment size will be less than 100 bp

Figure 1-3 Schematic diagram of restriction map around FMR1 CGG locus Asterisk (*) is used to indicate methylation sensitive

enzyme Two probes, A and B are used for Southern blot analysis (referred to Table 1-2)

PCR to detect the larger repeat, especially when there is a smaller repeat to compete against Therefore, the general method used currently in molecular diagnosis for fragile X syndrome is a combination of Southern blot and PCR

Besides these methods, RT-PCR and immunohistochemical analyses are available

to confirm a loss of FMR1 expression by direct analysis of FMR1 RNA or FMRP (Oostra

and Willemsen 2001; Pai et al 1994) However, only male individuals can be reliably evaluated

Recent strategies based on methylation-specific PCR (ms-PCR) (Panagopoulos et

al 1999; Weinhausel and Haas 2001) have unsatisfactorily addressed the major difficulty

of detecting very large premutation and full mutation alleles, especially in females The

refractiveness of the FMR1 (CGG)n allele to PCR amplification is directly proportional to the size of the CGG repeat The various combinations of normal, premutation, and full mutation alleles together with the possibility of skewed X-chromosome inactivation and size mosaicism still pose a diagnostic challenge that has so far been impossible to be overcome with one simple diagnostic procedure Therefore, I worked on this field and finally developed a comprehensive ms-PCR system to detect the full spectrum of (CGG)n

alleles (normal, premutation and full mutation) in both males and females

1.2.5 FMR1 haplotypes in fragile X syndrome

In 1991, Richards et al found clear evidence of linkage disequilibrium between fragile X syndrome and 2 polymorphic microsatellite (dinucleotide repeat) markers that

flank the FMR1 CGG repeat, FRAXAC1 and FRAXAC2 No recombination was

observed between these markers either in normal pedigrees or affected fragile X pedigrees

And the haplotype evidence suggested that there was a founder effect in the fragile X mutation

In 1992, Riggins et al found another highly polymorphic dinucleotide repeat

marker which is approximately 150 kb proximal to the FMR1 CGG repeat, DXS548 This

marker was also very useful in studying the origin of the fragile X mutation, since it was tightly linked to the fragile X syndrome locus without recombination

In 1995, Eichler et al did a population survey of the FMR1 CGG repeat structure

and suggested that the biased loss of the most 3’ AGG interruption likely plays an

important role in predisposing FMR1 (CGG)n alleles to expansion mutations More recently, Crawford et al (2000) discovered that the lack of a 5’ AGG interruption may also be a factor involved in CGG repeat instability, especially in the African-American population, and may be an alternative pathway to that proposed from Caucasian association studies Furthermore, another recent study showed that loss of an AGG interruption was a late event in the generation of a fragile X chromosome (Dombrowski et

al 2002)

In 1998, Gunter et al found a single nucleotide polymorphism (SNP) in intron 1 of

the FMR1 gene: ATL1 The two alleles of ATL1 (A/G) revealed a highly significant

linkage disequilibrium with fragile X chromosomes and with the 5’ end of the CGG repeat itself, specifically the position of the first AGG interruption

Another nucleotide change in intron 10 of FMR1: IVS10+14C-T, which was

identified by Wang et al (1997) as a mutation, was later discovered to be a SNP (Vincent

and Gurling 1998) According to a study by Xu et al (1999), linkage disequilibrium was not found with the IVS10 and the fragile X chromosome

In addition to these microsatellites and SNPs, some researchers have also been

interested in FRAXE/FMR2, located on Xq28 600kb distal to FRAXA/FMR1 Previous studies on the association of expanded CGG repeat alleles at FMR1 and the number of GCC repeats at FMR2 in Caucasians have yielded contradictory results Murray et al

(1997) have reported that large expansions at FRAXA are significantly associated with

small alleles at FRAXE, whereas Brown et al (1997) identified expanded alleles at FMR2

in persons with expansions at the FMR1 locus

The above conclusions were mainly drawn from Caucasian populations It has been suggested that investigations of different ethnic groups could provide important fundamental information such as evaluation of the culture influence on patient outcome and dynamics of CGG repeat expansion (McCabe et al 1999) Some previous studies even suggested that Southeast Asian populations may have a distinctive genetic history worthy of independent study (Faradz et al 2001) Therefore, I carried out a haplotype study on three Singaporean ethnic groups, the Chinese, Malay and Indian This

comprehensive study offers detailed new information on Southeast Asian FMR1

family, extending through 5 generations, indicated autosomal dominant inheritance In

1992, Lazzarini et al encountered a large, previously unreported branch of the “W” family that shared a common ancestor 8 generations removed from the patients reported by Boller and Segarra They suggested that the disorder was a form of spinocerebellar ataxia (SCA) and named it SCA2

In 1993, Gispert et al found that in a large Cuban kindred which failed to show linkage to markers at the SCA1 locus on chromosome 6, the disease could be mapped to 12q23-24.1 by linkage analysis Pulst et al (1993) also identified a pedigree with linkage

to 12q, and established closer flanking markers for SCA2 than had been achieved in the Cuban pedigree They also suggested that an expanded triplet repeat underlies SCA2 as it does in SCA1

In 1996, Pulst et al identified the SCA2 gene using a combination of positional

cloning and candidate gene approaches Meanwhile, Sanpei et al (1996) isolated a series

of cDNA clones using primer sequences that flanked a CAG repeat in patients with SCA2

At the same time, Imbert et al (1996) screened cDNA expression libraries using an antibody specific for polyglutamine repeats and identified 6 novel genes containing (CAG)n stretches One of the genes was mutated in patients with spinocerebellar ataxia

linked to chromosome 12q They also reported that normal alleles of the SCA2 gene

contained 1 to 3 CAA interruptions within the CAG repeats while patient alleles contained pure (CAG)n stretches Furthermore, the latter two groups also reported that there was a strong inverse correlation between the size of the repeat and the age of onset of disease

1.3.2 Clinical features of SCA2

SCA2 manifests itself with progressive gait and limb ataxia, dysarthria, slow saccadic eye movements with ophthalmoparesis, extrapyramidal signs, areflexia, and cognitive dysfunctions, which largely overlap with clinical manifestations of other types

of the autosomal dominant cerebellar ataxias (Imbert et al 1996; Pulst et al 1996; Sanpei

et al 1996) Epilepsy is not a feature of the SCAs, with the exception of SCA10 (Grewal

et al 2002)

1.3.3 The cause of SCA2

The SCA2 mutation is an expansion of an unstable CAG repeat located in exon 1

of the SCA2 gene (Fig 1-4) on chromosome 12q24.1 (Imbert et al 1996; Pulst et al 1996;

Sanpei et al 1996) This CAG repeat encodes a polyglutamine tract (Trottier et al 1995)

The SCA2 gene is encompassed of 25 exons spanning 130 kb of genomic DNA with a 4.7

kb transcript The protein is called ataxin-2 which encodes a 1,313 amino acid polypeptide (Sahba et al 1998; Sanpei et al 1996)

Typically, in asymptomatic individuals there are between 14 and 31 copies of

CAG in the SCA2 allele In a person with the disease, however, the allele has anywhere

between 36 and 77 copies Each CAG trinucleotide codes for a single glutamine, so these CAG repeats result in a long string of glutamines (also known as a polyglutamine) within the peptide Individuals with between 31 and 36 copies of CAG may or may not develop the symptoms of the disease (individual results vary) (Imbert et al 1996; Pulst et al 1996; Sanpei et al 1996)

1 2 3 4 5 6 7 8 9 10 11 121314 15 161718 19 20 21 22 23 24 25

(CAG)n

1 2 3 4 5 6 7 8 9 10 11 121314 15 161718 19 20 21 22 23 24 25

Figure 1-4 Schematic representation of the genomic structure of SCA2 (drawn to scale) The 25 exons of the gene span

approximately 130 kb of genomic DNA The solid boxes indicate exons, and open boxes indicate introns Tel.: Telomere; Cen.: Centromere Details of exon1 are described in Figure 4-2; detailed exon1 and exon25 sequences are listed in Figure 4-3 and 4-4, respectively

1.3.4 The prevalence of SCA2

The exact number of people affected by SCA2 is unknown, but Pulst et al (1996) estimates that the number could be as high as one or two in every 100,000 people The

frequency of SCA2 mutations in patients with hereditary spinocerebellar ataxia differs

between populations Normally, SCA2 accounts for 6-15% of patients with autosomal dominant cerebellar ataxia, intermediate between SCA1 and SCA3 in relative percentages (Geschwind et al 1997)

1.3.5 The diagnosis of SCA2

The diagnosis of SCA2 rests upon the use of DNA-based testing to detect an

abnormal CAG trinucleotide repeat expansion in the SCA2 gene Most affected individuals

have alleles with 36-64 CAG trinucleotide repeats Rare cases of extreme CAG repeat expansion (>200) have been reported Routine molecular analysis of SCA2 by PCR and denaturing PAGE detects approximately 100% of cases except for very large expansions

(Mao et al 2002)

SCA2 is inherited in an autosomal dominant manner It is necessary to confirm the

clinical diagnosis in an affected person by using DNA-based testing of the SCA2 gene to

assess the size of the CAG trinucleotide repeat as part of the genetic counseling and testing of asymptomatic family members at risk Offspring of an affected individual have

a 50% chance of inheriting the gene mutation

A family with SCA2 was studied Though PCR had confirmed SCA2 in the patients, it was difficult to explain the additional feature of epilepsy in this family

Therefore I carried out PCR and sequencing of 4 family members to determine if a second

mutation within the SCA2 gene could account for the epilepsy

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CHAPTER 2 SIMPLIFIED METHYLATION-SPECIFIC PCR DETECTION OF FRAGILE X SYNDROME EXPANSION MUTATIONS IN MALES AND

FEMALES