d. Genet., Vol. 75, Number 2, August 1996, pp. 193-217. ~) Indian Academy of Sciences

REVIEW ARTICLE

Dynamic in human : a review of trinucleotide repeat diseases

JOHN W. LONGSHORE and JACK TARLETON Greenwood Genetic Center, 1 Gregor Mendel Circle, Greenwood, SC 29646, USA

MS received 8 April 1996

Abstract. Dynamic mutations in human genes result from unstable trinucleotidc repeats embedded within the transcribed region. The changeable nature of these mutations from generation to generation is in contrast to the static inheritance of other single- mutational events, e.g. point mutations, deletions, insertions and inversions, typically associated with patterns, lntergenerational instability of dynamic mutations within families has provided an explanation for the genetic , leading to increasing severity or earlier age of onset in successive generations, associated with certain inherited disorders. While models for genomic instability presume that trinucleotide repeat expansion results fl'om disruption of the DNA replication process, experimental evidence has not yet been obtained in support of this contention. Nevertheless, examples of unstable trinucleotide repeats continue to increase, although not all are associated with a specificphenotype. Five disorders resulting from small-scale expansions of CAG repeats within the protein- are known: spinobulbar muscular atrophy, Huntington's disease, type 1, dentatorubral- pallidoluysian atrophy (DRPLA) and Machado-Joseph disease. A sixth disorder, Haw River syndrome, is allelic to DRPLA. Five folate-sensitive chromosomal fi'agile sites characterized to date, viz. FRAXA, FRAXE, FRAXF, FRAIlB and FRA16& all have large-scale CGG repeat expansion. Two disorders, and FRAXE mental retardation, result from instability of CGG repeats in the 5' untranslated region of FMRI and FMR2 genes respectively. FRA11B lies close to chromosome 1lq endpoints in many Jacobsen syndrome patients and may be related to the deletion event producing partial for 1lq. Expansion of FRAXF and FRA16A has not been assodated with a phenotype. results from a large-scale CTG expansion in the 3' untranslated region of the myotonin protein kinase gene while Friedreieh's ataxia has recently been found to have a large-scale GAA repeat in the first of X25. This article reviews the characteristics of trinucleotide repeat disorders and summarizes current understanding of the molecular pathophysiology.

Keywords. Dynamic mutations; trinucleotide repeats; microsatellite instability.

1. Introduction

During the last five years an extraordinary type of mutational event, dynamic resulting from expansion of trinucleotide repeats embedded in certain genes, has been shown to be involved in a growing number of human neurological disorders. is yet another addition to the list of non-Mendelian genetic events that began when McClintock's discovery of transposable elements suggested that are not static. The disorders associated with unstable trinucleotide repeats account worldwide for millions of individuals with physical and mental impairments, and have tremendous social and psychological costs to the affected individuals~ their families, their communi- ties, and governments. The biological basis for the connection between trinucleotide repeat genes and neurological disorders is unknown; so is the molecular basis for instability of only

193 194 John W. Longshore and Jack Tarleton trinucleotide repeats (as opposed to mononucleotide, dinucleotide or tetranucleotide repeats) in human disease. The one common feature of all the trinucleotide repeats that cause human disorders is the presence of the repeats within the transcribed region of genes. The molecular characterization of dynamic mutations has provided an explanation for the high variability and unusual patterns of inheritance found in some human disorders. This new understanding naturally leads to the hope that complex familial inheritance patterns, such as those found with bipolar disorder and manic-depressive psychosis, will eventually be unravelled. In addition, new information on the products of genes containing unstable trinudeotide repeats is likely to provide fresh insights about cellular physiology. In this review we describe the properties of trinucleotide repeats and summarize current understanding of the molecular pathophysiology of the known trinucleotide repeat diseases. Most of the information presented is recent, and more complete for some disorders than for others. For example, substantial information has accumulated regarding fragile X syndrorne but very little is known about Machado-Joseph disease. However, the intriguing nature of trinucleotide repeat mutations and the impact of the disorders on human health are sure to lead to many more investigations and eventually to better understanding of the disease genes and their relation to cellular processes.

2. Types of trinucleotide repeat diseases

Unstable DNA sequences composed of repeated trinucleotides were first discovered in 1991 in two X-linked human genes, AR (androgen receptor) (LaSpada et at. 1991) and

Table 1. Characterized trinueleotide repeats.

Year gene Disorder (involved gene) Product characterized

Disorders with CAG repeats in the protein-coding region Spinobulbar muscular atrophy (AR) Xq 13 Androgen receptor 1991 Huntington disease (ITIS) 4p16 Huntingtin 1993 Spinocerebellar ataxia (SCAt) 6p22 Ataxin I993 Dentatorubral-pallidoluysian atrophy (B37) 12p12 Atrophin 1994 Haw River syndrome (B37) Machado-Joseph disease (MJD) 14q23.3 Not named 1994 Disorders associated with fragile sites Fragile X mental retardation (FMR1) FRAXA FMRP 1991 Xq27.3 Fragile X mental retardation (FMR2) FRAXE Not characterized 1993 Xq28 1lq - Jacobsen syndrome (CBL2) FRAllB CBL2 protein 1995 (FRAllB) 11 @3.3 CTG repeat in 3' untranslated region Myotonic dystrophy (MTPK) 19q13 Myotonin protein 1992 kinase GAA repeat within an intron Friedreich's ataxia (X25) 9q13 Frataxin 1996 Characterized sites containing (CGG)~ but producing no known phenotype when expanded FRAXF - Xq28 FRA16A- 16p13.1 Trinucleotide repeat diseases 195

FMR1 (fragile X mental retardation) (Kremer et al. 1991; Oberle et at. 1991; Verkerk er al. 1991;Yu et al. 1991). Instability of the trinucleotide repeat in these genes leads to spinobulbar muscular atrophy (SBMA) and the fragile X syndrome respectively, the first disorders in any known to result from heritable unstable DNA repeat sequences. Since the discovery of these two disorders, other trinucleotide repeat disorders have been discovered (table 1). In retrospect, AR and FMR1 contain the first examples of two subgroups of trinucleotide repeats: (i) small-scale expansions in CAG repeats (encoding polyglutamine segments) in protein-coding regions (figure 1) and (ii) large-scale expansions in CGG repeats in the untranslated portion of gene transcripts that span all folate-sensitive chromosomal fragile sites ctaaracterized to date (figure 2b). (The folate-sensitive fragile sites are chromosomal constrictions occasionally expressed when cells are cultured in a folate-depleted medium.) In the fragile site-associated group, permutations of the CGG repeat may vary with strand or reading frame (figure 3). The recent characterization of the gene involved in Friedreich's ataxia, which contains the first example of an unstable GAA repeat, documents that other triplet types may also cause instability (Campuzano et al. 1996). The largest group of diseases associated with dynamic mutations involve CAG expansions and disruption of normal neurological processes. In addition to AR, genes containing CAG embedded in the protein-coding region include I T-15 (Huntington's

(a) Ib) : Ce) " I -] n t ! u l

i

Figure 1. Polymerase chain reaction detection of CAG repeats. Primers comprising se- quences that flank the CAG repeat region were used to generate PCR products labelled with 32p. Separation was by electrophoresis on polyacrylamide sequencing gels. Pathological alleles are toward the upper end of the gels (upper bracket) and normal alleles are below (lower bracket). (a) Abnormal HD alleles with 39 to 45 repeats (6 normal controls are included). Normal alleles shown here have 15-25 repeats. (b) DRPLA patient with 66 repeats. Normal alleles shown here have 8-16 repeats. (c) Machado-Joseph disease patient with 77 repeats. Normal alleles shown here have 14-30 repeats. (Autoradiographs provided by Dr Nick Potter, University of Tennessee, Knoxville) 196 John W. Longshore and Jack Tarleton

(a) (b)

0

Ot

- tlbO= -o ,tt:w o mw o

1 12 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 1 2 3 4 5 6 7 8 910

Figure 2. Southern blot detection of fragite X syndrome and myotonic dystrophy patients. (a) CTG expansions in myotonic dystrophy patients. In lanes 1-5, DNA was digested with EcoRI. In lanes 6-10, the same patient samples were digested with BamHI. Lanes 2 and 7 contain DNA from a normal control. The two different enzymes are used to detect the range of expanded alleles. For example, the sample in lanes 3 and 8 can be more precisely sized in the BamHI digest. The arrows indicate normal bands in both digests. The EcoRI digest detects a of 8"5-kb and 9-5-kb bands as seen in lane 2. (b) Premutations and full mutations in fragile X patients as assayed by the double restriction enzyme digestion method described in Rousseau et al. 199l. The enzymes used were EcoRi and EagI. EagI is methyla- tion-sensitive and is used to detect both the normal methylation associated with X inactivation and the aberrant methylation associated with expanded CGG repeats. The arrows indicate the 2.8-kb DNA fragment detected in normal males (lanes 10-17, 20 and 22) and the additional 5.2-kb fragment due to X inactivation detected in normal females (lanes 1, 5 and 7-9). Males with fully expanded and hypermethylated alleles are shown in lanes 3 and 18. A female with a normal allele and a fully expanded, hypermethylated allele is shown in lane 4. A premutation male (lane 19) and premutation females (lanes 2 and 6) are shown. The male sample in lane 21 has a fnlly expanded allele but evidence of incomplete methylation in some cells. The CGG Repeat Family

CGG GGC GCG 5' 3' G' CGG'C' GGCiG )GCG/G 3'CtGCCIG, CCG,CtCGC,CS' CCG GCC CGC

Figure 3. Pemautationsin frame and DNA strand~in the CGG family oftrinucleotide repeats. CGG, GGC and GCG are possibilities on the top strand while CGC, GCC and CCG are possible reading frames on the bottom strand.

disease, HD), SCAI (spinocerebellar ataxial), B37 (dentatorubral-pallidoluysian atro- phy, DRPLA), and MJD (Machado-Joseph disease) (table 1). Haw River syndrome (HRS) is caused by a CAG expansion in B37 identical to that found in DRPLA. HRS TrinucIeotide repeat diseases 197

Table 2. Normal, transitional and pathological ranges for the trinucleotide repeat disorders.

Disease/gene Repeat Location Normal range Transitional" Full mutation b

AR CAG Coding region 17-26 40-52 HD (IT-I5) CAG Coding region 9-34 38-121 SCA1 CAG Coding region 25-36 43-81 DRPLA (B37) CAG Coding region 8-25 49-75 MaD CAG Coding region 13-36 68-79 MTPK CTG 3' Untranslated 5-37 42-150 > 150 FRDA (X25) GAA Intron 1 7-22 Unknown 200-900 FMRI CGG 5' Untranslated 6-54 55-230 > 230 FMR2 CGG Unknown 6--35 Up to ~ 200 > 200 CBL2 CGG 5' Untranslated 8-14 Up to ~ 100 > 100

"Premutation in CGG repeats/mild symptoms in CAG repeats bPathological alleles. has a phenotype that is similar, but not identical, to DRPLA and is caused by expansions with copy numbers overlapping with the range found in some DRPLA patients (Burke et al. 1994). All the CAG expansion disorders have five basic characteristics: neurological function is affected by the mutation, the expanded repeat encodes a polyglutamine motif, the repeats are both transcribed and translated, the repeat numbers are highly polymorphic among normal individuals, and distinct repeat numbers are associated with normal and disease-associated alleles (table 2). Mutations of this type have been referred to as 'gain of function' or 'change of function' as they impart a new or altered function to the encoded protein. This type of dominantly inherited alteration differs from both 'loss of function' mutations commonly seen with recessive disorders, where both copies of a gene must be altered for onset of disease, and dominant negative mutations, in which a mutant gene product interferes with the normal functioning product. Five rare chromosomal fragile sites have been characterized to date at the DNA level and each is composed of extended stretches of (CGG),, (table 1). Genes that span fragile sites may be disrupted by both repeat expansion and aberrant overmethylation of CpG dinucleotides contained in the promoter and transcribed regions--as demonstrated to occur in FMR1 (spanning FRAXA), FMR2 (spanning FRAXE) and CBL2 (spanning FRA11B). (Fragile site terminology used: FRAgile site; chromosome X, 11, or 16; site A, B, E or F). Two other fragile sites produced by expansion of(CGG),, and hypermethyla- tion have been characterized but appear not to be associated with a phenotype when expanded (FRAXF and FRA16A). Whether transcription occurs at or near these fragile sites has not been established conclusively, but the absence of any phenotype in individuals who have expansion of the repeats suggests that no gene is present. Deletions of the terminal region of chromosome 1 lq often occur near the fragile site FRAllB, resulting in individuals with Jacobsen syndrome, a contiguous gene syn- drome caused by partial aneuploidy of chromosome 1 lq (Jones et al. 1994). Recent work has shown that the protooncogene CBL2 bridges FRAllB (Jones et al. 1995). DNA sequencing of CBL2 revealed a (CGG), region contained within the 5' untran- slated portion of the gene. Myotonic dystrophy (DM) and Friedreich's ataxia (FRDA) are the 0nly examples to date of other unstable triplet types. The myotonin protein kinase gene MTPK, 198 John W. Longshore and Jack Tarleton involved in the causation of DM, contains a CTG repeat in the 3' untranslated portion (CAG on the DNA template strand). Recent evidence suggests that expansion of the CTG repeat in MTPK may disrupt nearby genes as well as the protein kinase gene (Boucher et al. 1995). While expanded CGG repeats in fragile sites become methylated, the CTG repeat is not a target for cellular methyltransferases. Fragile sites cannot be induced in the chromosome 19 region containing the MTPK gene although many of these patients have large-scale expansions similar in copy number to those associated with fragile sites (Wenger et" aI. 1996). Such findings suggest that hypermethylationmay be a requirement for fragile-site expression. X25, recently implicated in FRDA, contains a GAA repeat in the first intron (Campuzano et al. 1996). Expansion of the GAA repeat in X25 may disrupt stability of the primary mRNA. Usually both of a patient's alleles contain expanded GAA repeats since this disorder is an autosomal recessive, but some FRDA patients have a on one allele and GAA expansion on the other.

3. Instability of trinueleotide repeats

Dinucleotide, trinucleotide and tetranucleotide repeats belong to the group of repeti- tive termed microsatellites, which are spaced nearly at random throughout the . In the trinucleotide repeat disorders, these naturally polymorphic microsatel- lites may increase in copy number upon transmission to offspring fl'om a carrier parent with repeat copy number beyond a certain threshold specific for each disease (table 2). Increase of copy number, or less frequently repeat contraction, results in dynamic mutations with non-Mendelian characteristics, in contrast to stable molecular alter- ations (e.g. point mutations, deletions, duplications and insertions) which may occur spontaneously but are then typically inherited in Mendelian fashion. Each trinucleotide repeat disorder has a normal range of repeat copy number within which the polymorphic alleles are stable and inherited in Mendelian fashion. Abnor- mal, pathological alleles are those found in individuals manifesting symptoms of the particular disorder. Transitional alleles (termed 'premutations' in FMRI and FMR2) are those present in unaffected individuals which may be transmitted to offspring with either minor instability, perhaps changing by a few repeats, or major instability leading to great increases in repeat copy number. Generally, higher the repeat copy number in transitional alleles greater the propensity for further expansion. The inconsistent stability of the transitional alleles leads to the non-Mendelian inheritance patterns found in the disorders. For example, FMRI premutations may segregate within a family for many generations before expansion into a full mutation results in an affected individual (Tarleton et aI. 1992). Each disorder typically has a 'grey zone' composed of high normal (i.e. stable) alleles that overlap slightly in repeat copy number with low abnormal alleles. Thus instability upon transmission of grey-zone alleles may stem from factors beyond total repeat copy number. For example, the CGG repeat found in FMRI is interrupted by occasional AGG repeats believed to confer stability on the repetitive segment (Eichler et aI. 1994). Grey-zone alleles of the CAG repeat- containing genes may result in minor disease symptoms that perhaps may go unno- ticed. Interestingly, few spontaneous mutations occur in any of the trinucleotide repeat disorders, i.e. expanded alleles from which disease is produced derive rarely from Trinucleotide repeat diseases 199 parental alleles containing repeats in the normal range for each disorder. The lack of new mutations leads to a dilemma in respect of the fragile X syndrome, where males with expanded mutations rarely reproduce. How are expanded alleles, lost h'om the gene pool through lack of reproduction, replaced? The low reproductive fitness of fragile X males is in stark contrast to the relatively high frequency of the disorder. Several models have suggested that new mutations occur over long periods involving many generations as normal alleles change to potentially unstable alleles capable of expansion and loss of (Morton and MacPherson 1992; Ashley and Sherman 1995). Goldberg et al. (1993) have found evidence to support of intermediate alleles in HD (30-38 repeats) into pathological range during a single transmission from parent to child. In HD the intermediate alleles are meiotically unstable when they are transmitted through sperm. While several mechanisms have been proposed to account for repeat instability in the trinucleotide repeat diseases, experimental data to confirm these mechanisms have not been extensively compiled to date. The paucity of data is in great part due to the only recent discovery of trinucleotide repeats. Hypotheses to explain repeat instability have focussed on DNA replication errors related to lagging-strand slippage or intra- strand secondary structures of DNA segments containing repeats, or both (Eichler et al. 1994; Fry and Loeb 1994; Richards and Sutherland 1994; Gacy et al. 1995; Kang e~ al. 1995; Usdin and Woodford 1995). As previously rnentioned, interspersed AGG triplets found in FMRI (and CAT triplets in SCAI) are found within otherwise 'pure' stretches of trinucleotide repeats. These appear to ~anchor' the repeat and increase stability (Orr et al. 1993; Eichler et al. 1994; Zhong et al. 1995). AGG repeats in trMR1 and CAT repeats in SCA1 are referred to as cryptic repeats. The protective effect of cryptic repeats may result from a decrease in the thermodynamic energy of potential intrastrand secondary DNA structures (Gacy et al. 1995). Point mutations in the cryptic repeats, changing AGG to CGG in FMR1 for example, may be an event leading to instability. Pure repeats beyond about 34-38 may be prone to small expansion which becomes larger by passage through cell division events--either meiotic or mitotic (Eichler et al. 1994; Kunst and Warren 1994). Both meiotic and mitotic instability of expanded trinucleotide repeats have been described in DM (Jansen et aI. 1994) and fragile X syndrome (Wohrle et al. 1993). In both DM and fragile X syndrome the repeat expansions are similar in tissues derived from the same embryonic cell lineage. It appears that both disorders manifest repeat instability post-conceptionally during early embryonic mitotic events. In contrast to these disorders, HD displays only gametic instability and no evidence of somatic instability (MacDonald et al. 1993). In addition, germline instability has been demon- strated for SBMA and SCA1 (Nelson 1993). These data suggest that several mechan- isms may be responsible for trinucleotide repeat instability in the . That errors in replication may lead to microsatellite instability is further suggested by generalized genomic microsatellite instability found in some . Although there is no known direct connection between general genome instability and the gene-specific instability found in trinucleotide repeat diseases, defects in DNA replica- tion may be a common thread between the two. Microsatellite instability in tumour DNA versus DNA isolated from normal surrounding tissues may reflect defects in the human homologues of the bacterial DNA repair genes (rout genes in bacteria and MSH genes in humans) (Kolodner 1995). These genes encode components of a complex involved in DNA mismatch repair, the loss or inefficient function of which may lead to 200 John W. Longshore and Jack Tarleton strand slippage at microsatellites. Inability to repair DNA leads to tumour progression as unrepaired mutations accumulate in the tumour cells.

4. Non-Mendelian inheritance

Unstable trinucleotide repeat sequences have several interesting properties. A hall- mark of the trinucleotide repeat diseases, with the exception of Friedreich's ataxia and SBMA, is genetic anticipation, the increasing severity of a disorder as it is inherited through successive generations. The validity of genetic anticipation was debated among human geneticists for decades before the discovery of these genes. Molecular characterization of the trinucleotide repeat disorders provided an explanation for genetic anticipation: increases in repeat number through subsequent generations is accompanied by increasing disease severity, decreasing age of onset, or increasing penetrance. As a result, anticipation in the fragile X syndrome, the so-called '', is now understood to reflect the increasing propensity for repeat expansion as it is transmitted through generations in a family (Fu et al. 1991). Sherman et al. (1984) had observed that the risk for males of having fragile X syndrome in a family where the disorder was segregating depended upon their position in the pedigree. Nonpenetrant (transmitting) males were more likely to be found in earlier generations than in the most contemporary generation, reflecting the increasing likelihood of transition from premutation to full mutation. During the 1940s Julia Bell first observed anticipation, which she referred to as 'antedating', in families with myotonic dystrophy (Bell 1947). However, notable geneticists defined anticipation as resulting from ascertainment bias (Penrose 1948). The explanation for the generational variability found in myotonic dystrophy families was said to lie in the method by which these families came to medical attention. Congenitally affected children came to immediate attention because of the severe nature of their symptoms, while ascertainment in other family members with less severe symptoms came to light only after the initial diagnosis. In retrospect, the low parent-child correlation of symptoms actually resulted from true anticipation related to molecular events and not to ascertainment bias. Thus anticipation has been redefined by the characterization of the trinucleotide repeat diseases. Today, human geneticists actively search for any hint of anticipation when perform- ing pedigree analysis since its presence for a disorder is considered good evidence of trinucleotide repeat involvement. In addition to fragile X syndrome and lnyotonic dystrophy, Huntington's disease demonstrates anticipation in some families; so do SCA1, DRPLA and Machado-Joseph disease. Some spinocerebellar ataxia families with an abnormality unrelated to SCA1 (or to other loci with somewhat overlapping phenotype, the DRPLA or MJD loci) also appear to demonstrate anticipation, although this putative SCA2 locus has not yet been isolated. Anticipation appears in some families with schizophrenia, and at least one preliminary study of schizophrenic patients has found indications of repeat expansion using the RED (repeat expansion detection) technique (O'Donovan ee al. 1995). Differences in disease severity may also depend on which parent the abnormal allele was inherited from, the so-called 'parent-of-origin' effect. Parent-of-origin effects suggest the possibility that genetic imprinting may play a role in some trinucleotide repeat disorders. In particular, Huntington's disease may have an earlier onset if the Trinucleotide repeat diseases 201 abnormal allele is inherited from the father (Snell et al. 1993; Trottier et al. 1994). The same phenomenon has been observed in families with SCA1 (Chung et aL 1993). The severe congenital form of myotonic dystrophy occurs almost exclusively through inheritance of the abnormal allele from the maternal line. In the fragile X syndrome instability of the CGG repeat embedded in FMR1 leads to large repeat expansions only through the maternal line. Minor fluctuations involving a few repeats in FMRI premutations (alleles that have from ~ 55 to 200 CGG repeats) may occur but typically premutation alleles passed from father to daughter are remarkably stable. FMRI premutation alleles may remain stable or expand from the premutation range to a full mutation (hypermethylated alleles containing many hundreds or thousands of repeats) when passed from the mother to a son or daughter (Kremer et al. 1991; Oberle et al. 1991; Verkerk et al. 1991). Neither the mechanisms for the parent-of-origin effects nor the physical basis for imprinting are understood.

5. Mechanisms of pathophysiology: loss of gene function versus change of function

The phenotype of fragile X syndrome and FRAXE mental retardation syndrome occurs as a result of loss of gene expression. Aberrant overmethylation of CpG dinucleotides within and near the FMR1 repeat segment typically accompanies repeat expansion with the result that transcription is inhibited or completely eliminated (Pieretti et al. 1991; Sutcliffe et al. 1992). Methylation of CpG dinucleotide may inhibit transcription factors from binding to their target in the FMRI regulatory regions (Richards et al. 1993). Instability in X25 producing FRDA also occurs through a loss of function since GAA expansion appears to lead to mRNA instability (Carnpuzano et al. 1996). In contrast, expanded CAG repeats in the protein-coding region appear to cause ,heir effect through a change of function, i.e. new biochemical properties are gained by the addition of glutamine residues in the gene product. A mutant product of the Huntin- gton's disease gene with (CAG),~ has been shown to exclusively bind a novel intracellular protein, HAP-1 (huntingtin-associated protein). The products of normal alleles do not bind HAP-1 (Li et aI. 1995). Burke et al. (1996) have recently obtained fascinating expmimental results--that several of the proteins containing expanded polyglutamine tracts (the products of the IT-15, B37 and SCAI genes) tightly bind glyceraldehyde-3- phosphate dehydrogenase (GAPDH), an enzyme that functions in the glycolytic path- way. Could aberrantly expanded polyglutamine tracts modulate the activity of this enzyme and thus have an effect on energy metabolism? The authors suggest that perhaps slow neurodegeneration processes occur over many years in the late-onset diseases associated with expanded glutamine repeats, and that these disorders may have a com- mon thread in inefficient energy metabolism. Perhaps this reduction in energy produc- tion efficiencyresults in long-term neuronal losses characteristic of degenerative diseases.

6. Trinueleotide repeat diseases and the associated genes

6.1 Spinobulbar muscular atrophy

SBMA, also known as Kennedy's disease, was the first syndrome found to be asso- ciated with CAG repeat expansion. This X-linked recessive condition is characterized 202 John W. Longshore and Jack Tarleton by slowly progressive muscle weakness and atrophy, and partial insensitivity to androgens. This androgen insensitivity may result in gynecomastia in males and testi- cular atrophy. Mental retardation results from the onset oflnotor neuron degradation. In 1991 the expansion of a CAG repeat in the androgen receptor gene, which mapped to Xql3, was implicated in the disease (LaSpada et al. 1991, 1992). Affected individuals had between 40 and 52 copies of the CAG repeat, whereas normal individuals had copies within a range of 17 to 26. The expanded repeat, located in the first exon of the gene, encodes a glutamine repeat, which results in production of defective receptors. The gene product, a DNA-binding protein, may also be involved in regulation of transcription. However, the expanded repeat is located in a portion of the androgen receptor protein not known to be involved in hormone or DNA binding. Thus the pathophysiology of SBMA may not be related to disruption of normal function but the acquisition of new properties. The degree of expansion is correlated with disease severity. However, anticipation has not been observed in this disorder (Ross et al. 1993).

6.2 Hun.tington's disease

HD is a progressive neuromuscular disorder characterized by movement disorder, cognitive loss and psychiatric manifestations. The phenotype is caused by atrophy of the putamen and caudate nuclei in the affected individual's brain. It is inherited as a true autosomal dominant and affects approximately one in 10,000 individuals. Huntington's disease was one of the first disorders for which linkage analysis was used. Gusella et aI. (1983) demonstrated an RFLP (restriction fragment length polymorphisln) linking the disease to chromosome 4. The D4S10 marker of Gusella et al.'s work mapped to chromosome 41916.3 in the Wolf-Hirschhorn syndrome region. After ten painstaking years of confusing linkage data, the search for the Huntington gene was concluded with the discovery of the IT-15 gene (Huntington's Disease Collaborative Research Group 1993). The 1T-15 gene spans 180 kb and has 67 exons (Ambrose et al. 1994). The gene is disrupted by expansion of a CAG repeat located in the 5' end of the coding region. Normal individuals have fi'om 9 to 34 repeats while HD patients have more than 37 repeats (Read 1993) (figure la). A CCG repeat distal to the CAG repeat region encodes a small proline repeat. This second repeat also is polymorphic and was a source of concern for early polymerase chain reaction-based diagnoses. Subsequently, additional PCR primers have been designed to exclude this potenti~ source of error (Reiss et al. 1993). The age of onset in HD patients typically occurs in the fourth or fifth decade of life. This is a distressing feature as most persons with an abnormal allele are likely to have passed a mutated copy of the gene to their offspring before the onset of symptoms. The repeat length has been found to be highly variable in transmission between generations. Patients with longer repeats have recently been shown to have earlier onset of the disease state (Trottier et aI. 1994). In maternal transmission the repeat generally increases slightly in length. However, paternal transmission may trigger dramatic expansion with a doubling in repeat number in approximately one-third of the cases (Duyao et al. 1993). The IT-15 gene has been shown to be widely expressed in the brain and associated tissues as an 1 l-kb transcript coding for a 348-kDa protein known as huntingtin. The major areas of huntingtin synthesis appear to be the hippocampus, cerebellum and frontal cortex (Strong er al. 1993). Trinucleotide repeat diseases 203

6.3 Spinocerebellar ataxia type 1

The gene responsible for SCA 1, an autosomal dominant progressive neurodegenerative disease with ataxia, vision problems and varying motor weakness, was mapped to 6p23 (Ranum et al. 1991; Zoghbi et aI. 1991). Anticipation occurs in SCA1 families, and before the isolation of the SCA1 gene an unstable-repeat type of mutation was postulated. Unaffected individuals have 36 or fewer repeats while patients have more than 43 copies of the transcribed repeat. Trinucleotide expansion length correlates well with disease severity although the role of the expansion in pathogenesis of the disease has not been determined (Orr et al. 1993). The SCAt gene encodes a protein product known as ataxin-1 (Servadio et al. 1995). The gene was found to encode a 10-kb mRNA, which spanned 450 kb of DNA sequence and comprised nine exons. The coding region, which contains the expandable (CAG), motif, fails within the last two exons (Banff et al. 1994). The ataxin-1 protein shows nuclear localization in examined cells. However, cytoplasmic localization of the protein also was observed in cerebellar Purkinje cells. The pattern of synthesis of ataxin-1 appears to be identical in SCA1 and normal individuals (Servadio et al. 1995).

6.4 Dematorubral-pallidoluysian atrophy and Haw River syndrome

The clinical presentation and features of DRPLA have often been misdiagnosed as Huntington's disease and overlap somewhat with those of SCA1 as well. DRPLA is characterized by cerebellar atrophy, dementia, ataxia and hyperkinetic involuntary move- ments (Koide et al. 1994; Nagafuchi et al. 1994). Before the discovery of the gene involved in DRPLA, the distinction between the two disorders was made during autopsy. The gene involved was mapped to chromosome 12p12-pter before it was isolated (Nagafuchi et al. 1994). An unstable repeat had been found to be associated in affected individuals with human brain genes containing polymorphic trinucleotide repeats map- ping to chromosome 12 (Li et al. 1993). Using this approach to identify candidate cDNAs, a gene, B37, was isolated and found to contain CAG expansion in DRPLA patients (Koide et al. 1994; Nagafuchi et al. 1994). Normal individuals possess 8 to 25 copies of the CAG repeat while DRPLA patients may have a copy number ranging from 49 to 75 repeats (figure lb) (Koide et al. 1994). An inverse correlation has existed between age of onset and number of CAG repeats (Nagafuchi et al. 1994). The transcript of the B37 gene has 4294 bases and encodes a protein of 1184 amino acids. Northern blot analysis detects a ~ 4.5-kb transcript in all body tissues tested (Nagafuchi et al. 1994). DRPLA is an autosomal dominant syndrome considered to be very rare in North American and European populations but found more commonly in the Japanese. Anticipation occurs in DRPLA families with the age of symptom onset ranging from the first to the eighth decade. Juvenile onset may occur after transmission from an affected father (Nagafuchi et al. 1994). The protein product has been postulated to play a role in protein-protein interac- tions (Yazawa et al. 1995). The DRPLA protein was found, with the use of polyclonal antibodies, to be approximately 190 kDa in normal tissue, but approximately 205 kDa in DRPLA-affected brain tissues as assessed by electrophoretic mobility assays. In addition, the protein was found specifically in the cytoplasm of neurons in both affected and normal brains. 204 John W. Longshore and Jack Tarleton

Haw River syndrome (HRS) is a disorder allelic to DRPLA (Burke et aL 1994). Clinical expression and pathology of Haw River syndrome is similar to those of DRPLA, as well as HD and SCA1. Myoclonic seizures, a consistent finding in DRPLA, are absent in HRS. However, extensive demyelination of the subcortical white matter, calcification of basal ganglia, and neuroaxonal loss are found in HRS but not in DRPLA (Burke et al. 1994). Affected members of a large African-American HRS kindred from North Carolina have CAG expansions in B37 whose repeat numbers overlap with the numbers found in individuals of other ethnic background affected with DRPLA. It has been postulated that the different clinical presentations between the two disorders are due to factors other than total CAG repeat length (Burke et aI. 1994). Interestingly, the strong negative correlation between repeat length and age of onset in Japanese DRPLA families is not seen in the HRS family. For example, an affected father in the HRS family with 60 repeats had age of onset of 45 while his daughter with 61 repeats had disease onset at 18 (Burke et al. 1994). The role of genetic background in disease presentation has not yet been explored in either DRPLA or HRS. Further study of factors beyond repeat expansion should reveal fascinating information about the pathophysiology of both disorders.

6.5 Machado-Joseph disease

MJD is a dominantly inherited disorder also known as spinocerebellar ataxia type 3 (SCA3). The disorder is characterized by cerebellar ataxia, peripheral nerve palsy, pyramidal and extrapyramidal signs, external ophthalmoplegia, facial and lingual fasciculation, and bulging eyes (Kawaguchi et al. 1994). The gene involved maps to chromosome 14q32.1 (Sasaki et al. 1995). In late 1994 a Japanese group reported isolation of a new gene that contained a CAG repeat in persons affected with MJD. Normal controls possessed between 13 and 36 copies of the expandable repeat, whereas MJD patients had 68 to 79 repeats (Kawaguchi et al. 1994) (figure le), An inverse correlation between expansion size and age of onset was noted. The predicted protein sequence from the MJD gene showed no homology to any known sequences. However, it was speculated that the MJD protein may be an intracellular protein because it lacked a signal sequence or a transmembrane domain (Kawaguchi et al. 1994).

6.6 Myotonic dystrophy

Myotonic dystrophy (DM), inherited as an autosomal dominant, is the most common adult-onset muscular dystrophy with a general prevalence of about one in 8000. Higher or lower incidence is found in different ethnic groups or geographic settings (Harper i989). Myotonia, a delay in muscle relaxation after contraction, is a characteristic feature but the phenotype impacts a variety of systems. Wasting of facial muscles results in a characteristic appearance of ptosis and expressionless appearance. Other muscle groups such as the sternomastoids and distal limb muscles are usually affected as well. Smooth and cardiac muscle involvement is a common finding as the disorder prog- resses into adult life. Sudden death is not uncommon, typically resulting from degener- ation of cardiac conduction tissue. Premature balding and ocular abnormalities such as cataract and retinal degeneration may occur. In the congenital form of DM, mental Trinucleotide repeat diseases 205 retardation and generalized hypotonia occur. Until isolation of the mutations respon- sible for DM, the aetiology of the disorder and the reason for variable age of onset were not understood. A gene involved in DM was isolated by several groups simultaneously (Aslanidis et al. 1992; Brook et al. 1992; Buxton et al. 1992; Fu et al. 1992; Harley et al. 1992; Mahadevan et al. 1992, 1993). The mRNA, composed of 15 exons spanning 13 kb of genomic DNA, is 2.5 kb long and the predicted product has 624 amino acids (Shaw et al. 1993). The amino terminal region is highly similar to that of cAMP-dependent serine-threonine protein kinases (Fu et al. 1992). Thus the gene has been referred to as the myotonin protein kinase gene (MTPK). Alternative splicing in brain and heart was demonstrated by Jansen et al. (1992). Unlike the situation with trinucIeotide repeat disorders associated with CAG in protein-coding regions or CGG at fragile sites, the MTPK gene contains a CTG repeat in the last exon, 3' of the termination codon. Variability in DM severity is correlated with repeat copy number to a large degree but not absolutely. The normal range of CTG repeats observed is 5-37 (Brunner et al. 1992), and 42 to 150 repeats are found in mildly affected individuals, perhaps having only cataracts or muscle weakness. Patients with 150 to 800 repeats usually have adult onset of symptoms. Severe congenital DM occurs when an infant has 500 to 2000 repeats (Harley et al. 1993) (figure 2a). However, exceptional cases where repeat number does not correlate well with the congenital form are not uncommon, leading to suggestions of additional factors beyond repeat size to explain severity (Barcelo et at. 1994; Novelli et al. 1995). When various tissues were examined from an individual with a large repeat copy number, considerable variation of CTG copy number was found in different tissues (Levadan el al. 1993). This result suggests an explanation for why the repeat number in lymphocytes does not correlate absolutely with disease severity: variable DM symptoms may reflect tissue-to-tissue variation of repeat number. It is unclear how repeat expansion in the 3' untranslated region could lead to the clinical features of myotonic dystrophy. The dominant inheritance pattern suggests a gain or change of function, but the repeat does not lie within the protein-coding region as would be expected for these functional effects. Wang et al. (1995) found evidence in muscle tissue that transcripts containing an expanded MTPK allele may alter accumulation of poly (A)-messenger RN A. If it is confirmed, this is the first report of a dominant negative effect at the mRNA level and may help to explain the dominant inheritance pattern found in myotonic dystrophy. The possibility also exists that the repeat expansion affects multiple genes in close proximity or that expansion disrupts chromatin structure so that other transcription units are affected. In fact, the region of chromosome 19 within which the MTPK gene resides is gene rich. Boucher et al. (1995) identified a nearby candidate gene for involvement in myotonic dystrophy, the DM locus-associated homeodomain gene. The protein product of this gene is expressed in tissues involved in producing the phenotype: skeletal muscle, heart and brain.

6.7 Fragile X syndrome

The fragile X syndrome is the most common inherited form of mental retardation and is second only to Down's syndrome in causation of mental impairment. The of fragile X syndrome is unusual: the mode of inheritance is X-linked dominant, with reduced penetrance in females. Most males who inherit the full mutation have 206 John W. Longshore and Jack Tarleton moderate to severe mental impairment and have a more or less definitive phenotype (Tarleton and Saul 1993). The predominant features in prepubertal males are behav. ioral--developmental delay and abnormal temperament. The physical features, which are more obvious after puberty, involve the craniofacies (long face, prominent forehead, large ears and prominent jaw) and genitalia (macroorchidism). Females who have inherited a full mutation (hypermethylated alleles expanded beyond ,-, 230 repeats) are typically less severely affected than males. About one-half of the females who inherit the full mutation are mentally retarded (Rousseau et al. 1991). Presumably, skewed X inactivation within the tissues responsible for the phenotype may result in either severely affected females or females who have little or no impairment. Females who inherit the premutation (ranging from ,,~ 55 to 200 repeats) fi'om their ~transmitting male' fathers are unaffected since little or no change in repeat copy number occurs. Interestingly, anecdotal reports of early menopause (at < 35 years of age) have surfaced in premutation females (Schwartz et aI. 1994). While surveys have documented many instances of early menopause, no systematic studies have yet been done to investigate the physiological basis for the phenomenon. The association between fragile X syndrome and marker X chromosomes containing a fragile site in Xq27.3, FRAXA (Lubs 1969; Sutherland 1977), leads to the presump- tion of a gene or genes near FRAXA involved in the syndrome. In 1991 FMR1 was isolated using yeast artificial chromosomes spanning FRAXA (Kremer et al. 1991; Oberle et al. 1991; Verkerk et al. 1991; Yu et al. 1991). DNA sequencing of FMRI revealed a trinucleotide repeat, composed primarily of CGG, very close to the 5' end of the gene. Instability of the repeat is the major mutational mechanism that disrupts FMR1 and leads to FRAXA expression (Verkerk et al. 1991) (figure 2b). The gene occupies 38 kb of genomic DNA and has 17 exons (Eichler et al. 1993) represented in a messenger RNA of ~-, 4kb (Verkerk et al. 1991). The trinucleotide repeat segment is within the untranslated portion of exon 1, ending 69 base pairs upstream of the translational start (Ashley et al. 1993). Alternative splicing of FMRI occurs toward the 3' end of the mRNA (Ashley et al. 1993; Eichler et al. 1993; Verkerk et al. 1993). The FMRI protein product, FMRP, is widely expressed in a variety of tissues but is most abundant in neurons (Devys et al. 1993). The protein contains two KH binding domains, which are found in other proteins with RNA-binding properties, and appears to function as a cytoplasmic RNA-binding protein that interacts with a subset of brain mRNAs (Ashley e~ al. i993; Devys et al. 1993; Gibson et al. 1993; Verheij et al. 1993; Siomi et al. 1994). FMRP is associated with the 60S ribosome subunit and may be a nonintegral ribosomal protein (Khandjian et al. 1996). It has been proposed that fragile X mental retardation results from defects in the translation machinery due to lack of FMRP (Khandjian et al. 1996). Expansion of the trinucleotide repeat in exon 1 accounts for > 99 % of the mutations. Expansion of the repeat copy number beyond about 230, accompanied by abnormal overmethylation of the deoxycytosine residues in the FMR1 promoter, inhibits or reduces FMR1 transcription, resulting in loss of the protein product (Pieretti et al. 1991; Sutcliffe et al. 1992). It is the loss of FMRP that produces the fragile X syndrome. Normal amounts of FMRP are usually found in premutation carriers, but recent data indicate that large premutations ( > 180 CGG repeats) may have an effect on transla- tion efficiency, producing some reduction of FMRP (Feng et al. 1995). Rare patients with deletions of all or part of FMR1 (Gedeon et al. 1992; Wohrle et al. 1992; Tarleton ?u repeat diseases 207 et al. 1993) or point mutations in FMR1 (De Boulle et al. 1993; Lugenbeel et al. 1995) have been reported but probably account for much less than 1% of patients with fragile X menta! retardation. The deletion cases confirm that the syndrome is due to lack of FMR1 expression. A patient with a point mutation in one of the two KH domains found in FMRP and normal mRNA levels reportedly has a more severe phenotype than that associated with CGG repeat expansion (De Boulle et al. 1993). Thus, while not rigidly proven, mutations in functional protein domains may lead on rare occasions to gain-of-function alterations. Stable (i.e. normal) FMR1 alleles in the population have been observed to range from 6 to ,-0 54 repeats and have a trimodal distribution of trinucleotide repeats with the major peak around 30 repeats and minor peaks around 20 and 40 repeats (Fu er al. 1991; Snow et al. 1993). Alleles with > 55 repeats are potentially unstable. However, rare instances of instability have been observed in alleles with repeat copy number in the 40s (Reiss et al. 1994). Thus the inconsistency of stability in alleles falling in a so-called 'grey zone' of from ,-, 46 to --~ 54 repeats has led to investigations of factors involved in stability of FMR1 alleles. A major finding regarding the DNA sequence of FMR1 alleles is that the CGG repeats, which comprise most of the trinucleotide repeat region, are interrupted by AGG at about repeats 10 and 20 (and occasionally repeat 30) (Eichler et al. 1994). These AGG repeats appear to 'anchor' the segment against expansion by disruption of DNA secondary structures which may form during DNA replication. Long sequences, rnore than about 34-38 repeats, of uninterrupted CGG repeats beyond the last AGG repeat ('pure repeats') appear to increase the risk of instability for maternal alleles upon transmission to offspring (Eichler et al. 1994; Kunst and Warren 1994). Alleles in the premutation range contain long stretches of pure repeats and clearly are at risk of expansion to full mutations (Warren and Nelson 1994). On the basis of detection using methylation-sensitive restriction enzymes and Southern blot analysis (Rousseau et al. 1991), the vast majority of patients with > 230 repeats will have aberrant methylation of deoxycytosine residues in FMR1. However, not infrequently a patient will be found to have some cells in which methylation of FMR1 has occurred and some cells in which there is no methylation of FMR1 (McConkie-Rosell et al. 1993). Less frequently, a patient will be identified who appears to have no cells in which the abnormal methylation events have occurred even when > 230 CGG repeats are found. The former have been referred to as 'methylation mosaics' and their Southern blot pattern represents the presence of cellular mosaicism involving FMR1 methylation events. The latter have usually been described as 'unmethylated full mutations'. A second and fairly common type of mosaicism for molecular events in FMR1 describes patients who have some cells with methylated full mutations and some cells with premutations. These individuals have been termed simply 'mosaics'. Collectively, all the mosaicism types comprise perhaps 15% of FMRI mutations (Nelson 1993). Recent experimental evidence suggests that FMRP interacts with the products of two newly discovered autosomal genes, FXR1 and FXR2 (Siomi et aI. 1995; Zhang et al. 1995). Both have similarity to FMRP and to each other, and the presumed RNA-binding domains, KH1 and KH2, are conserved in them. This finding provides the first clues towards understanding phenocopies of fragile X syndrome, patients who appear to have the syndrome but who do not have any detectable alteration in FMR1. 208 John W. Longshore and Jack Tarlewn

6.8 FRAXE mental retardation

Reports of individuals with mental retardation and the presence of a fragile site in the Xq28 region, but normal FMRt, led to the discovery ofFRAXE (Sutherland and Baker 1992). FRAXE cannot be reproducibly distinguished by cytogenetic analysis froin either of the nearby folate-sensitive fragile sites, FRAXA or FRAXF, and molecular diagnosis is used to confirm involvement of FRAXE. The presence of apparently normal males with cytogenetic FRAXE expression has complicated understanding of the phenotype so that few blanket statements regarding the disorder can be made with certainty (Knight et al. 1994; Mulley et al. 1995). In one study expansion in FRAXE was associated with mild mental retardation and severe language delay in affected males, but no consistent physical findings (Hamel et aI. 1994). Females with FRAXE are typically normal, suggesting that the disorder is caused by a hemizygous loss of gene function in males and is X-linked recessive. The FRAXE-associated mental retardation appears to be relatively rare since few families with the expansion have been found or because the phenotype is so mild that patients do not easily come to medical attention (Allingham-Hawkins and Ray 1995}. The FRAXE site was characterized by Knight et at. (1993) and shown to be due to expansion of a GCC repeat. The presence of a CpG island near the GCC repeat suggested a gene organization similar to FMRI, and methylation of the CpG island was found to occur when the triplet repeat expanded beyond ~ 200. Screening of cDNA libraries, however, failed to detect a transcription unit and the association of FRAXE in some individuals with mild mental retardation was called into question. More recent work has identified candidate transcripts (Chakravarti et al. 1996; Gu et al. 1996) for the FRAXE-associated gene, which has been referred to as FMR2 (Gu et al. 1996). However, the complete characterization of these transcripts has not yet been performed and the gene expression has not yet been fully analysed. Chakravarti et al. (1996) identified a cDNA (OX19) that contains 1495 nucleotides and an open reading frame encoding 436 amino acids. The protein encoded by this transcript is predicted to have homology with the human AF-4 gene, a putative transcription factor. When the OX19 cDNA was used to screen mRNA from specific adult brain tissue, a 9-5-kb transcript was detected in most tissues but the most intense signals came from the hippocampus and amygdala. The larger transcript appears to be the same as that isolated by Gu et al. (1996). This latter cDNA is 9.5 kb with an open reading frame of 3828 bp encoding a predicted protein product of 1276 amino acids. The predicted product has similarity to MLLT2, a protein synthesized from a gene on chromosome 4 occasionally involved in translocations found in acute lymphocytic leukaemia. Although not formally proven, the possibility exists that the OX19 tran- script is an alternative splice product of the 9"5-kb transcript. Gedeon et al. (1995) reported that a DNA probe (DXS296) located ~ 175 kb distal to FRAXE identifies a 9"5-kb transcript in mRNA isolated from placenta and brain, and a 2'5-kb transcript in other tissues. This 9.5-kb transcript also is likely to be the same as that found by Gu et at. (1996) but has not been characterized yet. Subclones of OX19 cDNA cross-hybridized with the DXS296 probe and also detected a 9.5-kb transcript in placenta, adult brain, adult lung, foetal brain and foetal kidney, but no 2.5-kb transcript. These results provide further suggestions that all the transcripts arise from FMR2. The lack of detection of the 2.5-kb transcript found by Gedeon et al. suggests that FMR2 may have a complex splicing pattern. Trinucleotide repeat diseases 209

The original difficulty in finding a transcript originating from the FRAXE region was complicated by the presence of an extremely large intron (> 170kb) (Chakravarti et al. 1996). The 5' end of the mature transcript identified by Chakravarti et al. (1996) begins 331 bp distal to the FRAXE repeat and contains only 135 transcribed nucleotides before the beginning of the large intron. The clones from the region immediately surrounding FRAXE initially used to screen cDNA libraries did not contain the transcribed nucleotides. The finding of the FRAXE-associated transcripts is only the initial step in under- standing the phenotype associated with FMR2 disruption. Clarification of the molecu- lar genetics should lead to better comprehension of the pathophysiology but little headway has yet been made. The disorder is striking because of the absence of physical findings and the mildness of the associated mental retardation. Perhaps the most intriguing component of the phenotype is the pronounced language delay found in affected males. Gedeon et al. (1995) speculated that perhaps this gene is a 'language gene' and that characterization of the gene product will be useful in understanding language delays in children and general speech pathology.

6.9 Friedreich's ataxia

To the great surprise of geneticists, X25, the gene associated with the autosomal recessive disorder Friedreich's ataxia, contains a GAA repeat (Campuzano et al. 1996). The repeat occurs not in the protein-coding region or the flanking transcribed region, but in the first intron. Expansion of the repeat within a primary mRNA appears to interfere with nuclear RNA processing, resulting in the absence of mature message. Thus the repeat instability leads to loss of gene function. However, these findings are so recent that only the initial speculations concerning mutation effect and protein fnnction are included here. Campuzano et al. (1996) isolated the gene after a protracted search of candidate genes in the chromosome 9q13 region. Preliminary analysis of the gene indicates that there are at least seven exons with a main opening reading frame (ORF) within the first five exons. Alternative splicing involving the fifth, sixth and seventh exons appears to occur, giving rise to a secondary ORF. The main ORF is predicted to encode a 210-amino-acid product, called frataxin, while the secondary ORF is predicted to encode a shorter 171-amino-acid isoform of frataxin. Campuzano et al. (1996) found that individuals affected with Friedreich's ataxia have expansion ranging fl'om 200 to 900 repeats. The patients had either two alleles with the expanded GAA or one expanded allele and one allele containing a point mutation. The discovery of an expanded GAA repeat was quite unexpected. Friedreich's ataxia is an autosomal recessive, does not appear to demonstrate genetic anticipation, and provides the first example of an intronic unstable repeat. Owing to thermodynamic considerations, GAA repeats were not considered a likely candidate for trinucleotide repeat instability. All other unstable repeats have been variations on (CGG)~ or (CAG)n. Up to the finding of instability in a GAA repeat all the previously characterized repeats seemed to be related through a C-Pu-G motif (where Pu represents either of the purines, A or G).

6.10 CBL2 and Jacobsen syndrome

Jacobsen syndrome is the result of partial aneuploidy of 1 lq. The typical features are psychomotor retardation, trigonocephaly, particular facial features, cardiac defects 210 John W. Longshore and Jack Tarleton and thrombocytopaenia. The majority, but not all, of Jacobsen syndrome patients have deletion breakpoints near FRA11B at [email protected] (Penny et al. 1995), suggesting that the fragile site may play a role in causing llq- deletions. Jones et at. (1994) established that the CGG repeat at FRAllB is near the 5' end of CBL2, a protooncogene responsible for prolonged survival of lymphocytes and protection from programmed cell death. Jones et at. (1995) further established the localization of FRA11B within the 5' untranslated region of CBL2. No studies have been performed to date following observation of expression of CBL2 in individuals with expression of FRA 11B and little is known of the phenotype, if any, in such individuals. The phenotype found in Jacobsen syndrome is presumed to be related to haploinsufficiency for genes on chromosome 1 lq and not necessarily related to involvement of CBL2 in the deletion events. Interestingly, CBL2 may be the only trinucleotide repeat-containing gene discovered to date that does not have a known neurological connection. However, the gene has not been well chara- cterized and a connection may yet be found. CBL2 is normally expressed in a variety of haemopoietic cell lines and may be involved in translocations observed in some malignancies. If CBL2 is similar to other genes that span fragile sites, expansion of the CGG repeat might be expected to result in loss of gene expression. Since the CBL2 protein is involved in protection from programmed cell death, it may be that the loss of expression leads to shorter lymphocyte lifespans. Such an effect may be lethal. In contrast, overexpression of CBL2 has been observed in patients with systemic lupus erythematosus, where the role of the CBL2 product may be related to aberrantly prolonged survival of autoimmune memory cells (Graninger 1992).

6.11 FRAXF and FRAI6A

FRAXF was described in 1993 and cloned in 1994 (Hirst et at. 1993; Parrish et al. 1994; Ritchie et al. 1994). FRAXF is located distal to FRAXA and FRAXE, and is composed of a complex (GCCGTC),~(GCC), array. In contrast, FRA16A is composed of a CGG repeat which may be interrupted by CTG (Nancarrow et al. 1994, 1995). Both fragile sites are located near CpG islands but no transcription units appear to be derived from the region surrounding either site. Expansion of the respective repeats does not lead to any recognizable phenotype. Methylation of the region occurs when the repeat is expanded. Both repeats are polymorphic, with surveys of normal chromosomes finding from 6 to 29 triplets in FRAXF and 9-30 repeats in FRA16A.

To date, only the CAG, CGG and GAA repeat motifs have been implicated in causation of genetic disease. However, it seems likely that other trinucleotide repeats may be involved in dynamic mutations. By use of repeat expansion detection technique, three additional trinucleotide groups have been shown to be potentially unstable (Lindbald et al. 1994). These include the ATG, CCT and CCA motifs. Expansions in the GAA repeat group, recentty implicated in Friedreich's ataxia, were also noted (Lindblad e~ al. 1994). Hybridization selection has been used to isolate many polymor- phic triplet repeats (Armour et aI. 1994), and genes containing AAT and CCA repeat polymorphisms have been reported (Margolis et al. 1993). Trinucleotide repeat diseases 211

7. Conclusion

With the progression of the human genome project, additional examples of disease- associated trinucleotide repeats are certain to be found and longstanding puzzles concerning complex disorders may be solved as a result. Any neurological diseases that show anticipation or non-Mendelian inheritance patterns are candidates for trinuc- leotide repeat expansion. Often mentioned as possible candidate disorders are domi- nant hereditary ataxias, bipolar disorder, schizophrenia, autism, and some cases of familial breast cancer (Mandel 1993). Additionally, at least five dominant neuro- degenerative disorders have been shown to manifest anticipation. These include SCA2, SCA4, SCA5, SCA7 and familial spastic paraplegia (FSPI) (Gispert er al. 1995; Junck and Fink 1996). As recent experience with Friedreich's ataxia has demonstrated, similar examples of autosomal recessive diseases may exist as many autosomal recessive disorders remain uncharacterized. These are indeed exciting times in human molecular genetics as exploration of the effects of dynamic mutations continues.

References

Allingham-Flawkins D. J. and Ray P. N. 1995 FRAXE expansion is not a common etiological factor among developmentally delayed males. Am. J. Hum. Genet. 57:72-76 Ambrose C. M., Duyao M. P., Barnes G., Bates G. P., Lin C. S., Srinidhi J., Baxendale S., Flummerich H., Lehrach H., Altherr M., Wasmuth J., Buckler A., Church D., Housman D. E., Berks M., Mickel G., Durbin R., Dodge A., Read A., Gusella J. F. and MacDonald M. 1994 Structure and expression of the Huntington's disease gene: evidence against simple inactivation due to an expanded CAG repeat. Somat. Cell Mol. Genet. 20:27-38 Armour J. A. L., Neumann R., Gobert S. and Jeffreys A. J. 1994 Isolation of human simple repeat loci by hybridization selection. Hum. Mol. Genet. 3:599-605 Aslanidis C., Jansen G., Amemiya C., Shutler G., Mahadevan M., Tsilfidis C., Chen C., Alleman J., Wormskamp N. G. M., Vooijs M., Buxton J., Johnson K., Smeets H. J. M., Lennon G. G., Carrano A. V., Komeluk R. G., Wieringa B. and de Jong P. 1992 of the essential myotonic dystrophy region and mapping of the putative defect. Nature 355:548-551 Ashley A. E. and Sherman S. L. 1995 Population dynamics of a meiotic/mitotic expansion model for the fragile X syndrome. Am. J. Hum. Genet. 57:1414-1425 Ashley C. T., Sutcliffe J. S., Kunst C. B., Leiner H. A., Eichler E. E., Nelson D. L. and Warren S. T. 1993 Human and murine FMR-I: alternative splicing and translational initiation downstream of the CGG-repeat. Nat. Genet. 4:244-251 Banff S., Servadio A., Chung M., Kwiatkowski T. J., McCall A. E., Duviek L. A., Shen Y., Roth E. J., Orr H. T. and Zoghbi H. Y. 1994 Identification and characterization of the gene causing type 1 spinocerebellar ataxia. Nat. Genet. 7:513-520 Barcelo J. M., Pluscauskas M., MacKenzie A. E., Tsilfidis C., Narang M. and Komeluk R. G. 1994 Additive influence of maternal and offspring DM-kinase gene CTG repeat lengths in the genesis of congenital myotonic dystrophy [-letter]. Am. J. Hum. Genet. 54:1124-1125 Bell J. 1947 Dystrophia myotonica and allied disease. In Treasury of human inheritance, 4th edn (ed.) L. S. Penrose (Cambridge: Cambridge University Press) pp. 343-410 Boucher C. A., King S. K., Carey N., Krahe R., Winchester C. L., Rahman S., Creavin T., Meghji P., Bailey M. E. S., Chartier F. L., Brown S. D., Siciliano M. J. and Johnson K. J. 1995 A novel homeodomain-encoding gene is associated with a large CpG island interrupted by the myotonic dystrophy unstable (CTG),, repeat. Hum. Mol. Genet. 4:1919-1925 Brook J. D., McCurrach M. E., Harley H. G., Buckler A. J., Church D., Aburatani H., Hunter K., Stanton V. P., Thirion L-P., Hudson %, Sohn R., Zemelman B., Snell R. G., Rnndle S. A., Crow S., Davies J., Shelborne P., Buxton J., Jones C., Juvonen V., Johnson K., Harper P. S., Shaw D. and Housman D. E. 1992 Molecular basis of myotonie dystrophy: expansion of a trinucleotide (CTG) repeat at the 3' end of a transcript encoding a protein kinase family member. Cell 68:799-808 212 John W. Longshore and Jack Tarleton

Brunner H. G., Nillesen W.0 van Oost B. A., Jansen G., Wieringa B., Ropers H. H. and Smeets H. J. 1992 Presymptomatic diagnosis of myetonic dystrophy. J. Med. Genet. 29:780-784 Burke J. R., Wingfield M. S., Lewis K. E., Roses A. D., Lee J. E., Hulette C., Perieak-Vance M. A. and Vance J. M. 1994 The Haw River syndrome: dentatorubropaUidoluysian atrophy (DRPLA) in an African- American family. Nat. Genet. 7:521-524 Burke J. R., Enghild J. J., Martin M. M., Jou Y.-S., Myers R M., Roses A. D., Vance J. M. and Strittmatter W. J. 1996 Huntingtin and DRPLA proteins seleeti,~ely interact with the enzyme GADPH. Nat. Med. 2:347-350 Buxton J., Shelbourne P., Davie J., Jones C., Van Tongeren T., Aslanidis C., de Jong P., Jansen G., Anvret M., Riley B., Williamson R. and Johnson K. 1992 Detection of an unstable fragment of DNA specific te individuals with myotonie dystrophy. Nature 355:547-548 Campuzano V., Montermini L., Molto M. D., Pianese L., Cossee M., Cavalcanti F., Monros E., Rodius F., Duclos F., Monticelli A., Zara F., Canizares J., Koutnikova H., Bidichandani S. I., Gellera C., Brice A., Trouillas P., De Michele G., Filla A., De Frutos R., Palau F., Patel P. I., Di Donato S., Mandel J.-L, Cocozza S., Koenig M. and Pandolfo M. 1996 Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion. Science 271: 1423-1427 Chakravarti L., Knight S. J. L., Flannery A. V. and Davies K. E. 1996 A candidate gene for mild mental handicap at the FRAXE fragile site. Hum. Mol. Genet. 5:275-282 Chung M. Y., Ranum L. P., Duvick L. A., Servadio A., Zoghbi H. Y. and Orr H. T. 1993 Evidence for a mechanism predisposing to intergenerational CAG repeat instability in spinocerebellar ataxia type I. Nag. Genet. 5:254-258 De Baulle K., Verkerk A. J. M. H., Reyniers E., Vits L., Hendriekx J., Van Roy B., Van Den Bos F., de Graaff E., Oostra B. A. and Willems P. J. 1993 A point mutation in the FMR-1 gene associated with fragile X mental retardation. Nat. Genes. 3:31-35 Devys D., Lutz Y, Rouyer N., Bellocq J.-P., and Mandel J.-L. 1993 The FMR-1 protein is cytoplasmic, most abundant in neurons and appears normal in carriers of a fragile X premutation. Nat. Genet. 4:335-3413 Duyao M., Ambrose C., Myers R., Novelletto A., Persiehetti F., Fontali M., Folstein S., Ross C., Franz M., Abbott M., Gray J., Cenneally P, Young A., Penney J., Hollingsworth Z., Shoulson I., Lazzirini A., Falek A., Koroshetz W., Sax D., Bird E., Voasattel J., Bonilla E., Alvir J., Conde J. B., Cha J.-H., Dure L., Gomez G., Ramos MI, Sanehez-Ramos J., Snodgrass S., de Young M., Wexler N., Moscowitz C., Penchaszadeh G., MaeFarlane H., Anderson M., Jenkins B., Srinidhi J., Barnes G., Gusella J. F. and MacDonald M. 1993 Trinucleotide repeat length instability and age of onset in Huntington's disease. Nat. Genet. 4:387-392 Eiehler E. E., Richards S., Gibbs R. A. and Nelson D. L. 1993 Fine structure of the human FMR1 gene. Hum. Mol. Genet. 2:1147-1153 Eichler E. E., Holden J. J. A., Popovich B. W., Reiss A. L., Snow K., Thibodeau S. N., Richards C. S., Ward P. A. and Nelson D. L. 1994 Length of uninterrupted CGG repeats determines instability in the FM R 1 gene. Nat. Genet. 8:88-94 Feng Y., Zhang F., Lokey L. K., Chastain J. L., Lakkis L., Eberhart D. and Warren S. T. 1995 Translational suppression by trinucleotide repeat expansion at FMR1. Science 268:731-734 Fry M. and Loeb L. A. 1994 The fragile X syndrome d(CGG), nucleotide repeats form a stable tetrahelical structure. Proc. Natl. Acad. Sci. USA 91:4950-4954 Fu Y.-H., Kuhl D. P. A., Pizzuti A., Pieretti M., Sutcliffe J. S., Richards S., Verkerk A. J. M. H., Holden J. J. A., Fenwick R. G., Warren S. T., Oostra B. A., Nelson D. L. and Caskey C. T. 1991 Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox. Cell 67: 1047-1058 Fu Y.-H., Pizzuti A., Fenwiek R. G., King J., Rajnarayan S., Dunne P. W., Dubel J., Nasser G. A.0 Ashizawa T., de Jong P., Wieringa B., Korneluk R., Perryman M. B., Epstein H. F. and Caskey C. T. 1992 An unstable triplet repeat in a gene related to myotonic muscular dystrophy. Scfence 255:1256-1258 Gaey A. M., Goellner G., Juranic N., Macura S. and McMurray C. T. 1995 Trinucleotide repeat structures that expand in human disease form hairpin structures in vitro. Cell 81:533-540 Gedeon A. K., Baker E., Robinson H., Partington M. W., Gross B., Manta A., Korn B., Poustka A., Yu 8., Sutherland G. R. and Mulley J. C. 1992 Fragile X syndrome without CCG amplification has an FMItl deletion. Nat. Genet. 1:341-344 Gedeon A. K., Keinanen M., Ades L. C., Kaafiainen H., Gecz J., Baker E., Sutherland G. R. and Mulley J. C. 1995 Overlapping submicroscopic deletions in Xq28 in two unrelated boys with developmental dis- orders: identification of a gene near FRAXE. Am. J. Hum. Genet. 56:907-914 Gibson T. J., Rice p, M., Thompson J. D. and Heringa J. 1993 KH domains within the FMR1 sequence suggest that fragile X syndrome stems from a defect in RNA metabolism, Trends Biochem. Sci. 18:331-333 Trinucleotide repeat diseases 213

Gispert S., Santos N., Damen R., Volt T., Schulz J., Kloekgether T., Orozco G., Kreuz F., Weissenbach J. and Auburger G. 1995 Autosomal dominant familial spastic paraplegia: reduction of the FSP1 candidate region on chromosome 14q to 7 cM and locus heterogeneity. Am. J. Hum. Genet. 56:183-187 Goldberg Y, P., Kremer B., Andrew S. E., Theilmann J., Graham R. K., Squitieri F., Telenius H., Adam S., Sajoo A., Starr E., Heiberg A., WolffG. and Hoyden M. R. 1993 Molecular analysis of new mutations for Huntington's disease: intermediate alleles and sex of origin effects. Nat. Genet. 5:174-179 Graninger W. B. 1992 Transcriptional overexpression of the proto-oncogene bcl-2 in patients with systemic lupus erythematosus. Wien. Kiln. Wochenschr. 104:205-207 Gu Y., Shen Y., Gibbs R. A. and Nelson D. L. 1996 Identification of a novel gene (FMR2) associated with the FRAXE CCG repeat and CpG island. Nat. Genet. 13:109-113 Gusella J. F., Wexler N. S., Conneally P. M., Naylor S. L, Anderson M. A., Tanzi R. E., Watkins P. C., Ottina K., Wallace M. R., Sakaguci A. Y., Young A. B., Shoulson I., Bonilla E. and Martin J. B. 1983 A polymorphie marker genetically linked to Huntington's disease. Nature 306:234 Hamel B. C. J., Smits A. P. T., de Graaff E., Smeets D. F. C. M., Schoute F., Eussen B. H. J., Knight S. J. L., Davies K. E., Assman-Hulsmans C. F. C. H. and Oostra B. A. 1994 Segregation of FRAXE in a large family: clinical, psychometric, cytogenetic, and molecular data. Am. J. 1-Ium. Genet. 55:923-931 Harley H. G., Brook J. D., Rundle S. A., Crow S., Reardon W., Buckler A. J., Harper P. S., Housman D. E. and Shaw D. 1992 Expansion of an unstable DNA region and phenotypic variation in myotonic dystrophy. Nature 355:545-546 Harley H. G., Rundle S. A., MacMillan J. C., Myring J., Brook J. D., Crow S., Reardon W., Fenton I., Shaw D. J. and Harper P. S. 1993 Size of the unstable CTG repeat sequence in relation to phenotype and parental transmission in myotonic dystrophy. Am. J. Hum. Genet. 52:1164-1174 Harper P. S. 1989 Myotonie dystrophy, 2nd edn (Philadelphia: W. B. Sounders) Hirst M. C., Barnicoat A., Flynn G., Wang Q., Daker M., Buckle V. J., Davies K. E. and Bobrow M. 1993 The identification of a third fragile site, FRAXF, in Xq27-q28 distal to both FRAXA and FRAXE. Hum. Mol. Genet. 2:197-200 Huntington's Disease Collaborative Research Group 1993 A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. Cell 72:371-383 Jansen G., Mahadevan M., Amemiya C., Wormskamp N., Segers B., Hendriks W., O'Hoy K., Baird S., Sabourin L., Lennon G., Jap P. L., Iles D., Coerwinkel M., HoNer M., Carrano A. V., de Jong P., Korneluk R. G. and Wieringa B. 1992 Characterization of the myotonie dystrophy region predicts multiple protein isoform-eneoding mRNAs. Nat. Genet. 1:261-266 Jansen G., Willems P., Coerwinkel M., Nillesen W., Smeets H., Vits L., Howeler C., Brunner H. and Wieringa B. 1994 Gonosomal mosaicism in myotonie dystrophy patients: involvement of mitotic events in (CTG), repeat variation and selection against extreme expansion in sperm. Am. J. Hum. Genet. 54:575-585 Jones C., Slijepcevic P., Marsh S., Baker E., Langdon W. Y., Richards R. 1. and Tunnacliffe A. 1994 Physical linkage of the fragile site FRAllB and a Jacobsen syndrome chromosome deletion breakpoint in 11q23.3. Hum. Mol. Genet. 3:2123-2130 Jones C., Penny L., Mattina T., Yu S., Baker E., Voullaire L., Langdon W. Y., Sutherland G. R., Richards R. I. and Tunnacliffe A. 1995 Association of a chromosome deletion syndrome with a fragile site within the proto-oncogene CBL2. Nature 376:145-149 Junck L. and Fink J. K. 1996 Machado-Joseph disease and SCA3: the meets the phenotypes. Neurology 46:4-8 Kang S., Ohshima K., Shimizu M., Amirhaeri S. and Wells R. D. 1995 Pausing of DNA synthesis in vitro at specific loci in CTG and CGG triplet repeats from human hereditary disease genes. J. Biol. Chem. 270: 27014-27021 Kawaguchi Y., Okamoto T., Taniwaki M., Aizawa M., Inoue M., Katayama S., Kawakami H., N akamura S., Nishimura M., Akiguchi I., Kimura J., Narumiya S. and Kakizuka A. 1994 CAG expansions in a novel gene for Machado-Joseph disease at chromosome 14q32.1. Nat. Genet. 8:221-228 Khandjian E. W., Corbin F., Woerly S. and Rousseau F. 1996 The fi'agile X mental retardation protein is associated with ribosomes. Nat. Genet. 12:91-93 Knight S. J. L., Flannery A. V., Hirst M. J., Campbell L., Christodoulou Z., Phelps S. R., Pointon J., Middleton-Price H. R., Barnicoat A., Pembrey M. E., Holland J., Oostra B. A., Bobrow M. and Davies K. E. t993 Trinucleotide repeat amplification and hypermethylation of a CpG island in FRAXE mental retardation. Cell 74:127-134 Knight S. J. L., VoelckeI M. A., Hirst M, C., Flannery A. V., Moncla A. and Davies K. E. 1994 Triple repeat expansion at the FRAXE locus and X-linked mild mental handicap. Am..I. Hum. Genet. 55:81-86 214 John W. Longshore and Jack Tarleton

Koide R., Ikeuchi T., Onodera O., Tanaka H., Igarashi S., Endo K., Takahashi H., Kondo R., Ishikawa A., Hayashi T., Saito M., Tomoda A., Miike T., Naito H., lkuta F. and Tsuji S. 1994 Unstable expansion of CAG repeat in hereditary dentatorubral-pallidoluysian atrophy (DRPLA). Nat. Goner. 6:9-13 Kolodner R. D. 1995 Mismatch repair: mechanisms and relationship to cancer susceptibility. Trends Biochem. Sci. 20:397-401 Kremer E. J., Pritchard M., Lynch M., Yu S., Holman K., Baker E. Warren S. T., Schlessinger D, Sutherland G. R. and Richards R. I. 1991 Mapping of DNA instability at the fragile X to a trinucleotide repeat sequence p(CCG),. Science 252:1711-1714 Kunst C. B. and Warren S. T. 1994 Cryptic and polar variation of the fragile X repeat could result in predisposing normal alleles. Cell 77:853-861 LaSpada A. R., Wilson E. M., Lubahn D. B., Harding A. E. and Fischbeck K. H. 1991 Androgen receptor gene mutations in X-linked spinal and bulbar muscular atrophy. Nature 352:77-79 LaSpada A. R., Roling D. B., Harding A. E., Warner C. L., Spiegel R., Hausmanowa-Petrusewicz I., Yee W. C. and Fischbeck K. H. 1992 Meiotic stability and genotype-phenotype correlation of the trinueleetide repeat in X-linked spinal and bulbar muscular atrophy. Nat. Goner. 2:301-304 Levadan C., Hofmann-Radvanyi H., Shelbourne P., Robes J. P., Duros C., Savoy D., Dehaupas I., Luce S., Johnson K. and Junien C. 1993 Myotonic dystrophy: size and sex-dependent dynamics of CTG meiotic instability and somatic mosaicism. Am. d. Hum. Genet. 52:875-883 Li S.-H., McInnis M. G., Margolis R. L., Antonarakis S. E. and Ross C. A. 1993 Novel triplet repeat containing genes in human brain: cloning, expression, and length polymorphisms. 16:572-579 Li X.-J., Li S.-H., Sharp A. H., Nucifora F. C. Jr., Schilling G., Lanahan A., Worley P., Snyder S. H. and Ross C.A. 1995 A huntingtin-associated protein enriched in brain with implications for pathology. Nature 378: 398-402 Lindblad K., Zander C., Schalling M. and Hudson T. 1994 Growing triplet repeats. Nat. Genet. 7:I24 Labs H. A. 1969 A marker X chromosome. Am. J. Hum. Genet. 21:231-244 Lugenbeel K. A., Peier A. M., Carson N. L., Chudley A. E. and Nelson D. L. 1995 Intragenic loss of function mutations demonstrate the primary role of FMR1 in fragile X syndrome. Nat. Goner. 10:483-485 MeConkie-Rosell A., Lachiewicz A. M., Spiridigliozzi G. A., Tarleton J., Schoenwald S., Phelan M. C., Goonewardena P., Ding X. and Brown W. T. 1993 Evidence that methylation of the FMR-1 locus is responsible for variable phenotypic expression of the fragile X syndrome. Am. J. Hum. Goner. 53:800-809 MacDonald M. E., Barnes G., Srinidhi J., Duyao M. P., Ambrose C. M., Myers R. H., Gray J., Conneally P. M., Young A., Penney J., Shoulson I., Hollingsworth Z., Koroshetz W., Bird E., Vonsattel J., Bonilla E., Moscowitz C., Penchaszadeh G., Brzustowicz L., Alvir J., Condo J. B., Cha J.-H., Dure L., Gomez G., Ramos-Arroyo M., Sanchez-Ramos J., Snodgrass S., de Young M., Wexler N., MacFarlane H., Anderson M. A., Jenkins B. and Gusella J. F. 1993 Gametic but not somatic instability of CAG repeat length in Huntington's disease. J. Med. Genet. 30:982-986 Mahadevan M., Tsilfidis C., Sabourin L., Shutler G., Amemiya C., Jansen G., Neville C., Narang M., Barcelo J., O'Hoy K., Leblond S., Earle-Macdonald J., de Jong P., Wieringa B. and Korneluk R. G. 1992 Myotonic dystrophy mutation: an unstable CTG repeat in the 3' untranslated region of the gone. Science 255:1253-1255 Mahadevan M. S., Amemiya C., Jansen G., Sabourin L., Baird S., Neville C. E., Wormskamp N., Segers B., Batzer M., Lamerdin J., de Jong P., Wieringa B. and Korneluk R. G. 1993 Structure and genomic sequence of the myotonic dystrophy (DM kinase) gone. Hum. Mol. Genet. 2:299-304 MandeI J.-L. 1993 Questions of expansion. Nat. Goner. 4:8-9 Margolis R. L., Li S.-H., McInnis M. G. and Ross C. A. 1993 Identification of novel cDNA clones containing AAT or CCA trinucleotide repeats [abstract]. Am. J. Hum. Genet. 53: suppl., abstract 708 Morton N. E. and Macpherson J. N. 1992 of the fragile X syndrome: multiallelic model for the FMR1 locus. Prec. Natl. Acad. Sci. USA 89:4215-4217 Mulley J. C., Yu S., Loesch D. Z., Hay D. A., Donnelly A., Gedeon A. K., Carbonell P., Lopez I., Glover G., Gabarron I., Yu P. W. L., Baker E., Haan E. A., Hockey A., Knight S. J. L., Davies K. E., Richards R. I. and Sntherland G. R. 1995 FRAXE and mental retardation. J. Med. Genet. 32:162-169 Nagafuchi S., Yanagisawa H., Sate K., Shirayama T., Ohsaki E., Bundo M., Takeda T., Tadokoro K., Kondo I., Murayama N., Tanaka Y., Kikushima H., Umino K., Kurosawa H., Furukawa T., Nihei K., Inoue T., Sane A., Komure O., Takahashi M., Yoshizawa T., Kanazawa I. and Yamada M. 1994 Dentatorubral and pallidoluysian atrophy: expansion of an unstable CAG trinucleotide on chromosome 12p. Nat. Genet. 6:14-18 Trinucleotide repeat diseases 215

Nancarrow J. K., Kremer E., Holman K., Eyre H., Doggett N. A., Le Paslier D., Callen D. F., Sutherland G. R. and Richards R. I. 1994 Implications of FRAI6A structure for the mechanism of chromosomal fragile site genesis. Science 264:1938-1941 Nancarrow J. K., Holman K., MangelsdorfM., Hori T., Denton M., Sutherland G. R. and Richards R. I. 1995 Molecular basis of p(CCG),, repeat instability at the FRA16A fragile site locus. Hum. Mol. Genet. 4: 367-372 Nelson D. L. 1993 Six human genetic disorders involving mutant trinueleotide repeats. In Genome rearrangement and stability (eds.) K. E. Davies and S. T. Warren [Plainview, New York: Cold Spring Harbor Laboratory Press) pp. 1-24 Novelli G., Gennarelli M., Menegazzo E., Angelini C. and Dallapieeola B. 1995 Discordant clinical outcomein myotonic dystrophy relatives showing (CTG),, greater than 700 repeats. Neuromusc. Disord. 5: 157-159 Oberle I., Rousseau F., Heitz D., Kretz C., Devys D., Hanauer A., Boue J., Bertheas M. F. and Mandel J.-L 1991 Instability of a 550-base-pair DNA segment and abnormal methylation in fragile X syndrome. Science 252:1097-1102 O'Donovan M. C., Guy C., Craddock N., Murphy K. C., Cardno A. G., Jones L. A., Owen M. J. and MeGuffin P. 1995 Expanded CAG repeats in schizophrenia and bipolar disorder. Nat. Genet. 10: 380-381 Orr H. T., Chung M., Banff S., Kwiatkowski T., Servadio A., Beaudet A. L., McCall A. E., Duvick L. A., Ranum L. P. W. and Zoghbi H. 1993 Expansion of an unstable trinucleotide CAG repeat in spinocerebel- far ataxia type 1. Nat. Genet. 4:221-226 Parrish J. E., Oostra B. A., Verkerk A. J. M. H., Richards C. S., Reynolds J., Spikes A. S., Shaffer L. G. and Nelson D. L. 1994 Isolation of a GCC repeat showing expansion in FRAXF, a fragile site distal to FRAXA and FRAXE. Nat. Genet. 8:229-235 Penny L. A., Dell'Aquila M., Jones M. C., Bergoffen J., Cunniff C., Fryns J. P., Grace E., Graham J. M., KousseffB., Mattina T., Syme J., Voullaire L., Zelante L., Zenger-Hain J.0 Jones O. and Evans G. A. 1995 Clinical and molecular characterization of patients with distal llq deletions. Am. J. Hum. Genet. 56: 676-683 Penrose L. S. 1948 The problems of anticipation in pedigrees of dystrophia myotonica. Ann. Ett~jen. 14: 125-132 Pieretti M., Zhang F., Fu Y.-H., Warren S. T., Oostra B. A., Caskey C. T. and Nelson D. L. 1991 Absence of expression of the FMR-1 gene in fragile X syndrome. Cell 66:817-822 Ranum L. P., Duvick L. A., Rich S. S., Schut L J., Litt M. and Orr H. T. 1991 Localization of the autosomal dominant HLA-linked spinocerebellar ataxia (SCA1) locus, in two kindreds, within an 8-cM subregion of chromosome 6p. Am. J. Hum. Genet. 49:31-41 Read A. P. 1993 Huntington's disease: testing the test. Nat. Genet. 4:329-330 Reiss A. L., Kazazian H. H., Krebs C. M., McAughan A., Boehm C. D., Abrams M. T. and Nelson D. L 1994 Frequency and stability of the fragile X premutation. Hum. MoI. Oenet. 3:393-398 Reiss O., Noerremoelle A., Soerensen S. A. and Epplen J. T. 1993 Improved PCR conditions for the stretch of (CAG),, repeats causing Huntington's disease. Hum. Mol. Genet. 2:637 Richards R. I. and Sutherland G. R. 1994 Simple repeat DNA is not replicated simply. Nat. Genet. 6:114-116 Richards R. I., Holman K, Yu S. and Sutherland G. R. 1993 Fragile X syndrome unstable element, p(CCG),, and other simple sequences are binding sites for specific nuclear proteins, Hum. Mot. Genet. 2:1429-1435 Ritchie R. J., Knight S. J. L., Hirst M. C., Grewal P. K., Bobrow M., Cross G. S. and Davies K. E. 1994 The cloning of FRAXF: trinucleotide repeat expansion and methylation at a third fl'agile site in distal Xqter. Hum. Mol. Genet. 3:2115-2121 Ross C. A., McInnis M. G., Margolis R. A. and Li S. 1993 Genes with triplet repeats: candidate mediators of neuropsychiatric disorders. Trends Neurosci. 16:254-260 Rousseau F., Heitz D., Biancalana V., Blumenfeld S., Kretz C., Boue J., Tommerup N., Van Der Hagen C., DeLozier-Blanchet C., Croquette M.-F., Gilgenkrantz S., Jalbert P., Voetckel M.-A., Oberle I. and Mandel J.-L. 1991 Direct diagnosis by DNA analysis of the fragile X syndrome of mental retardation. N. Engl. J. Med. 325:1673-1681 Sasaki H., Wakisaka A., Takada A., Yoshiki T., Ihara T., Suzuki Y., Hamada T., Iwabuchi K., Onari K., Tada J., Suzuki T. and Tashiro K. 1995 Mapping of the gene for Maehado-Joseph disease within a 3.6-cM interval flanked by D14S291/D148280 and D14S81 on the basis of studies of linkage and linkage disequilibrium in 24 Japanese families. Am. J. Hum. Genet. 56:231-242 216 John W. Longshore and Jack Tarleron

Schwartz C. E., Dean J., Howard-Peebles P. N., Bugge M., Mikkelsen M., Tommerup N., Hull C.,Hagerman R., Holden J. J. A. and Stevenson R. E. 1994 Obstetrical and gynecological complications in fragile X carriers: a multicenter study. Am. J. Med. Goner. 51:400-402 Servadio A., Koshy B., Armstrong D., Antalffy B., Orr H. T. and Zoghbi H. Y. 1995 Expression analysis of the ataxin-1 protein in tissues from normal and spinocerebellarataxia type 1 individuals. Nat. Genet, 10: 94-98 Shaw D. J., McCurrach M., Rund|e S. A., Harley H. G., Crow S. R., Sohn R., Thirion J. P., Hamshere M. G., Buckler A. J., Harper P. S., Housman D. E. and Brook J. D. 1993 Genomic organization and transcriptional units at the myotonie dystrophy locus. Genomics 18:673-679 Sherman S. L., Morton N. E., Jacobs P. A. and Turner G. 1984 The marker (X) syndrome: a eytogenetie and genetic analysis. Ann. Hum. Goner. 48:21-37 Siomi H., Choi M., Siomi M. C., Nussbaum R. L. and Dreyfuss G. 1994 Essential role for KH domains in RNA binding: impaired RNA binding by a mutation in the KH domain of FMR1 that causes fragile X syndrome. Cell 77:33-39 Siomi M. C., Siomi H., Sauer W., Srinivasan S, Nussbaum R. L. and Dreyfuss G. 1995 FXR1, an autosomal homolog of the fi'agile X mental retardation gone. EMBOJ. 14:2401-2408 Snell R. G., MacMillan J. C., Cheadle J. P., Fenton I., Lazarou L. P., Davies P., MacDonald M. E., Gusella J. F., Harper P. S. and Shaw D. J. 993 Relationship between trinucleotide repeat expansion and phenotypic variation in Huntington's disease. Nat. Genet. 4:393-397 Snow K., Doud L. K., Hagerman R., Pergolizzi R. G., Erster S. H. and Thibodeau S. N. 1993 Analysis of the CGG sequence at the FM R- 1 locus in fragile X families and in the general population. Am. J. Hum. Genet. 53:t217-1228 Strong T. V., Tagle D. A., Valdes J. M., Elmer L. W., Boehm K., Kaatz K. W., Swaroop M., Kaatz K. W., Collins F. S. and Albin R. L. 1993 Localization of Huntington's disease expression in rat and human brain. Nat. Genet. 5:259-265 Sutcliffe J. S., Nelson D. L., Zhang F., Pieretti M., Caskey C. T., Saxe D. and Warren S. T. 1992 DNA methylation represses FMR-1 transcription in fragile X syndrome. Hum. MoI. Goner. 1:397-400 Sutherland G. R. 1977 Fragile sites on human chromosomes: demonstration of their dependence on the type of tissue culture medium. Science 197:265-266 Sutherland G. R. and Baker E. 1992 Characterisation of a new rare fragile site easily confused with the fragile X. Hum. Mol. Goner. 1:111-113 Tarleton J. C. and Saul R. A. 1993 Molecular genetic advances in fragile X syndrome. J. Pediatr. 122:169-185 Tarleton J., Montjoy J., Dean J., Wong S., Schwartz C. and Saul R. 1992 Unification of"isolated" cases of the fragile X syndrome and molecular detection of the fragile X mutation. Proc. Greenwood Genetic Center t 1: 27-32 Tarleton J., Richie R., Schwartz C., Rao K., Aylsworth A. S. and Lachiewicz A. 1993 An extensive de novo deletion removing FMR1 in a patient with mental retardation and the fragile X syndrome phenotype. Hum. MoI. Genet. 2:1973-1974 Trottier Y., Biancalana V. and Mandel J.-L. 1994 Instability of CAG repeats in Huntington's disease: relation to parental transmission and age of onset. J. Med. Goner. 31:377-382 Usdin K. and Woodford K. J. 1995 CGG repeats associated with DNA instability and chromosome fragility form structures that block DNA synthesis in vitro. Nucl. Acids Res. 23:4202-4209 Verheij C., Bakker C. E., de GraaffE., Keulemans J., Willemsen R., Verkerk A. J., Gatjaard H., Rouser A. J,, Hoogeveen A. T. and Oostra B. A. 1993 Characterization and localization of the FMR-1 gene product associated with fragile X syndrome. Nature 363:722-724 Verkerk A. J. M.H., Pieretti M., Sutcliffe J. S., Fu Y.-H., Kuhl D. P. A., Pizzuti A., Reiner O., Richards S., Victoria M. F., Zhang F., Eussen B. E., van Ommen G.-J., Blonden L. A. J., Riggins G. J., Chastain J, L., Kunst C. B.0 Galjaard H., Caskey C. T., Nelson D. L., Oostra B. A. and Warren S. T. 1991 Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome. Ceil 65:905-914 Verkerk A. J. M. H., de Graaff E., De Boulle K., Eichler E. E., Konecki D. S., Reyniers E., Manca A., Poustka A., WiUems P. J., Nelson D. L. and Oostra B. A. 1993 Alternative splicing in the fragile X gone FMR1. Hum. Mol. Genet. 2:399-404 Wang J., Pegararo E., Menegazzo E., Gennarelli M., Hoop R. C., Angelini C. and Hoffman E. P. 1995 Myotonio dystrophy: evidence for a possible dominant-negative RNA mutation. Hum. Mot. Gener. 4:599-606 Warren S. T. and Nelson D. L. 1994 Advances in molecular analysis of fragile X syndrome. J. Am. Med. Assoc. 271:536-542 TrinucIeotide repeat diseases 217

Wenger S. L., Giangreco C. A., Tarleton J. and Wessell H. B. 1996 Lack of fragility at CTG repeats in congenital myotonic dystrophy. Am. J. Med. Genet. (in press) Wohrle D., Kotzot D., Hirst M. C., Manca A., Korn B., Schmidt A., Barbi G., Rott H.-D., Poustka A., Davies K. E. and Steinbach P. 1992 A microdeletion of less than 250kb, including the proximal part of the FMR-1 gene and the fragile-X site, in a male with the clinical phenotype of fragile X syndrome. Am. J. Hum. Genet. 51:299-306 Wohrle D., Hennig I., Vogel W. and Steinbach P. 1993 Mitotic stability of fragile X mutations in differentiated cells indicates early post-conceptional trinudeotide repeat expansion. Nat. Genet. 4: 140-142 Yazawa I., Nukina N., Hashida H., Goto J., Yamada M. and Kanazawa 1. 1995 Abnormal gene product identified in hereditary dentatorubral-paUidoluysian atrophy (DRPLA) brain. Nat. Genet. 10:99-103 Yu S., Pritehard M., Kremer E., Lynch M., Nancarrow J., Baker E., Holman K., Mulley J. C., Warren S. T., Schlessinger D., Sutherland G. R. and Richards R. I. 1991 Fragile X genotype characterized by an unstable region of DNA. Science 252:1179-1181 Zhang Y., O'Connor J. P., Siomi M. C., Srinivasan S., Dutra A., Nussbaum R. L. and Dreyfuss G. 1995 The fragile X mental retardation syndrome protein interacts with novel hemologs FXR1 and FXR2. EMBOJ. 14:5358-5366 Zhong N., Yang W., Dobkin C. and Brown W. T. 1995 Fragile X gene instability: anchoring AGGs and linked microsatellites. Am. J. Hum. Genet. 57:351-361 Zoghbi H. Y., Jodice C., Sandkuijl L. A., Kwiatkowski T. J. Jr., McCall A. E., Huntoon S. A., Lulli P., Spadaro M., Litt M., Cann H. M., Frontali M. and Terranto L. 1991 The gene for autosomal dominant spinocerebellar ataxia (SCA1) maps telomeric to the HLA complex and is closely linked to the D6S89 locus in three large kindreds. Am. J. Hum. Genet. 49:23--30