Trinucleotide repeat expansions in neurological disease · Trinucleotide repeat expansions in...

Trinucleotide repeat

Stephen T.

expansions in neurological disease

Warren and David 1. Nelson

Emory University School of Medicine, Atlanta, USA and Baylor College of Medicine, Houston, USA

During the past year, new examples of human neurological disease

have been discovered that have an unprecedented type of mutation as their cause: the remarkable expansion of trinucleotide repeats. These

triplet repeats are normally polymorphic and exonic, though not always

coding. In disease states they become markedly unstable and may expand

moderately or by thousands of repeats in a single generation, influencing

gene expression, message stability or protein structure.

Current Opinion in Neurobiology 1993, 3:752-759

Introduction able within families where the gene becomes penetrant only when maternally transmitted and the chance of penetrance increases in successive generations. This peculiar inheritance pattern has been referred to as the Sherman paradox [4]. In 1991 a series of reports delineated the mutation and gene involved in FraX syndrome [5-81. The FMRl (Fragile X Mental Retardation-l) gene was discovered to contain a CGG-repeat within the first exon that was normally polymorphic, exhibiting between 6-52 copies with a mean of 29. Among affected individuals, the repeat length was dramatically increased well beyond 230 repeats, usually 600 or more. Concomitant with the large expansion, referred to as the full mutation, was abnormal methylation of restriction sites within a region rich in CG dinucleotides (a CpG-island) immediately upstream of the gene [9,10]. Pieretti et al. [ 111 demonstrated the absence of FMRl transcription from full mutation alleles. Non-penetrant carrier males and many such females had repeat lengths of intermediate size without abnormal methylation, called premutations. These sizes ranged from approximately 50 to 200 and, upon transmission to offspring, displayed remarkable instability, with offspring usually exhibiting allele sizes different from the transmitting parent and distinct from other siblings. The change tends to increase the repeat length and, in the maternal premutation size range, the repeat length is proportional with the risk of full expansion and therefore penetrance [121.

For decades, mutational mechanisms that lead to human genetic disease have followed rules and examples set forth in model systems such as Drosophila and yeast. In recent years, however, new mechanisms responsible for genetic disease have emerged where little or no prece- dent had been established in other genetically studied organisms. One such mechanism is trinucleotide repeat expansions [ l*,2,3*]. Since early 1991, when the mutation responsible for the fragile X syndrome was uncovered as an astounding expansion of an exonic CGG-repeat, four other human genetic diseases have been similarly demonstrated to be due to such mutations. In most cases, these mutations occurred in genetic diseases that display some degree of unusual inheritance patterns, such as incom- plete penetrance or genetic anticipation, where severity of symptoms increases and/or age-of-onset decreases in subsequent generations of a single kindred. These previ- ously poorly understood genetic phenomena can now be satisfactorily explained by the behavior of unstable and expanding trinucleotide repeats. However, much remains to be understood, in particular the mechanisms responsible for this unprecedented mutational change. Below are short descriptions of the genetic diseases currently recognized to exhibit these mutations and what is known about each trinucleotide repeat, its behavior and the gene it influences.

Fragile X syndrome

Fragile X (FraX) syndrome is an X-linked dominant disorder with reduced penetrance. The syndrome is the leading cause of inherited mental retardation in hum mans and is associated with a chromosomal fragile site at Xq27.3 (see Fig, 1). The reduced penetrance is vati-

Sutcliffe et al. [13**] studied chorionic villi and fetal tissue of a male fetus with a full mutation where the chorionic villi were unmethylated and the fetal tissue methylated. Expression of FMRl was demonstrated from the unmethylated sample, suggesting gene expression is repressed by the abnormal methylation. The abnormal methylation was shown to include not only the CpG- island but the entire repeat itself, in a pattern similar to that observed on the inactive X-chromosome [14*,15*].

752

Abbreviations

AR-androgen receptor; DM-mytonic dystrophy; FMRl-Fragile X Mental Retardation-l gene; FraX-Fragile X; HD-Huntington’s disease; Mt-PK-myotonic-protein kinase;

SBMA-spinal and bulbar muscular atrophy; SCAl-spinocerebellar ataxia type 1.

@ Current Biology Ltd ISSN 0959-4388

Trinucleotide repeat expansions in neurological disease Warren and Nelson 753

The notion that the absence of FMKl gene product is solely responsible for this phenotype is supported by patients with FraX phenotype (without the cytogenetic fragile site) that have deletions including FMRl [ 16*,17*] and one patient with a missense mutation in FMRl [ 18**].

The nature of the repeat expansion remains unclear. Richards et al. [ 19”] and Oudet et al [20**] demonstrated linkage disequilibrium between nearby polymorphic markers and the FraX chromosome suggesting a limited pool of founder chromosomes. Smits et al. [21*] presented evidence that the premutation may be quite long-lived in the population supporting a step-wise mutation rate from a normal allele ( < 35 CGGs) to normal alleles with larger repeats (3550 CGGs) to premutation and then full mutation [ 22*,23*]. The parent-of-origin dif- ferences in expansion to full mutations is perhaps being resolved. Willems et al. [24] showed that a mosaic male (with both full mutations and premutations) transmitted a premutation to his daughter who subsequently had an affected son. Reyniers et al [25**] showed only premutation sperm in affected males with full mutations. Tissue expression studies of FMRl showed normal expression in testes [ 26.1. Bachner et al. [ 27**] presented evidence to support the idea of FMRl expression being required for normal spermatogenesis, such that full mutation or mosaic males would produce premutation sperm due to selection for FMRl expression. Under this hypothe- sis, affected males cannot transmit full mutations, fitting the observations of Sherman et al. [4].

Spinal-bulbar muscular atrophy

Spinal and bulbar muscular atrophy (SBMA), also known as Kennedy’s disease, is a rare X-linked recessive with occasional manifesting females. Clinically, SBMA is characterized by a slowly progressive spinal and bulbdr muscular atrophy affecting the anterior horn cells, resulting in weakness and cramping of the proximal limb muscles (spinal) and of th e aci an c ewing muscles, including f ‘al d h the tongue (bulbar) with fasciculations. Gynecomastia is also frequently seen, as is testicular atrophy and azosper- mia, at the later stages of the disease. Age of onset is usually in the third to fifth decade, although by history, many patients describe mild symptoms much earlier.

Fischbeck et al. [ 281 mapped SBMA to Xql l-12 (see Fig. 1), which overlapped the position of the androgen receptor (AR). As patients exhibit some features of androgen insensitivity and the occasional finding of diminished androgen binding to SBMA fibroblasts, Ia Spada et al. [29] studied the AR gene in SBMA and correlated the length of an exonic CAG-repeat with the disease.

The CAG-repeat of the AR is normally polymorphic [30] and encodes a long tract of glutamine residues near the amino-terminus of the protein. Among normal individuals, there is an average of 21 repeats with a range of 12 to 30 [30,31*]. Among patients with SBMA, the repeats range from 40 to 62 [ 29,32,33**], The SBMA repeat allele is relatively unstable when transmitted, varying by 2-3

repeats [31*,33**]. This instability was observed by ~a Spada et al. [YY] to be more common upon paternal transmission rather than maternal (57% compared to 13%). Igarashi et al. [34] and La Spada et al. [33**] demonstrated a strong correlation between the length of the CAG-repeat and the age of onset of muscle weakness: those with the smallest abnormal length (43 repeats) had onset in the sixth decade, whereas those with the largest expansion (51 repeats) had onset between 22 and 31 years of age. Apart from the onset of muscle weakness, no correlation with other clinical parameters was observed.

SBMA is clearly a gain-of-function, as can be exquisitely demonstrated by considering testicular feminization, which is due to the absence of the AR. Testicular feminization individuals, some with deletions of the entire AR gene, exhibit abnormal fetal sexual differentiation leading to an external female phenotype, despite an XY kary- otype, with no evidence of muscle weakness. SBMA patients have normal fetal differentiation with comparatively mild signs of androgen insensitivity, therefore suggesting that the AR with the expanded glutamine tract is at least partially functional, As ARs are normally concentrated in spinal and bulbar motor neurons [35], the expanded glutamine repeat may allow a more promiscuous interaction with gene promoters, resulting in degeneration of these cells in SBMA

Myotonic dystrophy

Myotonic dystrophy (DM) is an autosomal dominant multi-systemic disorder characterized by dystrophic muscular weakness with myotonia and muscle wasting. Pa- tients often exhibit additional signs of cataracts, cardiac conduction defects and, in males, premature balding and testicular atrophy. This clinical picture is highly variable and the mildest form (cataracts with little or no muscle in- volvement) tends to become progressively worse in subsequent generations, sometimes leading to a congenital form of the disease, which is associated with muscular hypoplasia, mental retardation and neonatal mortality. In addition, the age-of-onset decrease in subsequent generations, resulting in a classic example of genetic anticipation, was until recently without molecular explanation.

In early 1992 several groups published the identification of the mutant gene involved in DM [36,37,38**40**]. The DM mutation was localized to chromosome 19q13.3 (see Fig. 1) and identified as a CTG-repeat. The CTG- repeat was found to reside within the 3’ untranslated region of a gene, designated myotonin kinase, because the predicted amino acid sequence shares homologies with other serine-threonine protein kinases. The repeat is normally polymorphic with a mean size of 5 triplets. Affected individuals display repeat lengths over a con- siderable range from approximately 50 triplets to over 2000. Within this range, the trinucleotide repeat is unstable in both meiosis and mitosis. The DM mutation is in strong linkage disequilibrium with a nearby polymorphism [37,38**,41], leading Imbert et al. [42**] to

754 Disease, transplantation and regeneration

16 Y HD 15.3 15.2 25 15.1 24

13.1 13.2 13.3 21.1 21.2 21.3

22 23

24

25

26

27

33.3

32

33

34 u

351 I

23

22.3 SCAl 22.2 22.1

15 16.1

1%

13.3 n

13.2

131

1: 1:

13.2

13.3 DM 134

19

22.3

22.2 22.1 R

21.3 21.2 21.1 11.4 11.3 6 11.23

11:;: 11.1

ii.1 12.2 SBMA 13

21.3 22.1 22.2 22.3 23

24

25

26

27 FRAXA 26

X Repeat sequences in human disorders due to trinucleotide repeat expansion

Disease Gene Repeat Location Normal lengtfi Disease length’“’

HD Huntingtin CAG Chromosome 4 11-36 42-l 00 poly-gin? 1 St axon

translated?

SCAl ??? CAG Chromosome 6 ??!

25-36 43-81

DM DM kinase CTC Chromosome 19 5-30 so->2000 (CAG) last exon

untranslated

SBMA AR CAG Chromosome X 12-30 40-62 poly.gin 1 St exon

translated

FRAXA FMRl CGG Chromosome X h-52 230.>2000 1 st exon untranslated

‘“‘Number of trinucleotide repeats

propose that DM mutations are derived from normal alleles of 19-30 repeats, which themselves are derived from the 5 repeat alleles in a multi-step mechanisms similar to that proposed for FraX syndrome.

The length of the abnormal DM repeat has been correlated with disease severity and age-of-onset [43,44]. Harley et al. [45*] has shown that patients with repeats toward the lower abnormal range (- 5Ck-100 triplets)

Fig. 1. Summary of human disorders due to trinucleotide repeat expansions. The chromosomal locations of the loci are indicated on the ideograms adjacent to the disease notation. The repeat is ori- ented relative to the sense strand, when known. FRAXA-FraX locus.

have the mildest disease, presenting with cataracts and minimal or no muscle weakness, whereas classic DM and congenital DM patients have progressively larger expansions. Abeliovich et al. [46], Mulley et al. [47*] and Lavedan et al. [48] have demonstrated that when an expanded DM paternal allele is transmitted, the repeat length often contracts, whereas similar sized maternal alleles continue to demonstrate continued expansion. Therefore, the earlier observation that congenital DM


are born to affected mothers rather than to affected fathers may be explained by this paternal size reduction, as congenital DM is always associated with very large CTG-repeat expansions [49*]. O’Hoy et al. [ 50**] showed that some apparent reductions in repeat length are due to gene conversion events where the expanded DM repeat is converted to the size of the repeat on the normal allele.

The mechanism(s) by which the DM repeat expansion leads to disease is largely unknown. Fu et al. [51.*] and Sabourin et al. [52**] have shown, however, that mRNA levels of the myotonin kinase gene are abnormal in DM tissue. Although it remains unclear what the precise change is in the normal allele, as well as the DM allele, changes in message half-life could alter protein levels [5I**] resulting in abnormal kinase activity.

Huntington’s disease

Huntington’s disease (HD) is an autosomal dominant disorder and has a frequency of approximately 1 per 10 000 individuals. It is a neurodegenerative disease characterized by choreiform movement and progressive intellec- tual deterioration associated with atrophy of the cau- date and putamen. Age-of-onset is usually in middle age (35-50 years), though juvenile cases, which are typically more severe, are occasionally seen and are usually pater- nally inherited [ 531.

In one of the first successful attempts at linkage analysis between a restriction fragment length polymorphism and an unmapped human disease, Gusella et al. [ 541 assigned HD to chromosome 4. Intense efforts over the following decade refined the position to 4~16.3 (see Fig. 1) and generated a high-resolution physical map of the area likely to include the HD gene. MacDonald et al. [55] identified the presence of strong linkage disequilibrium between the HD locus and several polymorphic markers suggesting a region of 500kilobases containing the HD mutation, which is found on a common haplotype in approximately one-third of HD chromosomes,

The Huntington’s Disease Collaborative Research Group [56**] recently reported the identification of the HD gene by exon-trapping from a cosmid contig of this region. The HD transcript, referred to as IT15, identifies a 10.4kilobase message predicting a 348kDa protein. Near the 5’ end of the message, a repeat of 21 CAG trinucleotides was observed predicting a polyglutamine stretch in the protein. This trinucleotide repeat was found to be normally polymorphic with allele length ranging from 11 to 36 CAG-repeats with 98% of normals having alleles at or below 24 triplets. Among patients with HD, this repeat expands to sizes between 42 and approximately 100 repeats. When the HD alleles are transmitted, they exhibit instability usually resulting in larger alleles, though occasional reductions are observed. The length of the repeat appears to be correlated with the severity of the disease such that juvenile cases fall within the high end of the abnormal allele lengths, Two new mutations

were reported where the abnormal alleles were inherited from a parent with an allele at the high end of the normal range (e.g. a 36 to 44 repeat transition from parent to affected offspring).

The change in the CAG trinucleotide repeat would predict a variable number of glutamines within the HD protein (termed huntingtin). Since normal message levels were observed in cell lines derived from patients, including a homozygous affected, the increase of glutamines beyond 44 residues likely result in a gain-of-function.

Spinocerebellar ataxia type 1

Spinocerebellar ataxia type 1 (SCAl) is a progressive neurodegenerative disease with variable presentation that is inherited as an autosomal dominant. The disorder is characterized by ataxia, ophthalmoparesis and motor weakness believed to be due to the selective neuronal loss in the cerebellum, inferior olive, and spinocerebellar tracts. The age of onset is typically in the third or fourth decade of life, although juvenile cases have been reported with onset as early as age 4. Indeed, these juvenile onset individuals appear in later generations, thus exhibiting anticipation [ 57,581.

Linkage analysis has placed SCAl in the region 6p22-~23 [59,6O] (see Fig. 1). Using a YACcontig spanning 1.2 megabases inclusive of the candidate region, Orr and colleagues [ 61**] identified cosmid subclones containing a common CAG-repeat. The repeat was normally polymorphic with alleles ranging from 25 to 36 triplets (99% of normal alleles less than 34 repeats). In patients with SCAl, an abnormal allele in excess of 43 repeats was observed in addition to a normal allele. The largest abnormal allele length observed consisted of 81 repeats and those patients with juvenile onset SCAl had repeat lengths in this range (59 to 81 repeats). Instability was observed in transmission of the abnormal allele with a preponderance of male transmissions of the juvenile cases. A remarkable correlation was demonstrated between the length of the abnormal CAG-repeat and the age-of-onset, providing compelling evidence for this repeat expansion to be the mutation site in SCAl [61**].

Although the data thus far has analyzed genomic DNA, the sequence surrounding the CAG-repeat is an open reading frame and is detectable by reverse transcrip- tase polymerase chain reaction, both suggestive of an exon. The CAG-repeat in one direction would predict a polyglutamine tract which would abnormally expand in length, similar to SBMA and HD.

Conclusions

Within the past two years, five human diseases have been shown to be the result of the novel mutation mechanisms of trinucleotide repeat expansion. All five disorders are neurological and share other similarities. Although the


mechanism(s) of the repeat expansion remain poorly understood, it is clear from these five examples that even if the mechanism of repeat expansion is similar, the consequence of that expansion may be unique. In FraX syndrome expansion leads to transcriptional silenc- ing, whereas in DM similar expansions appear to result in changes in message stability. In SBMA, HD and SCAl, the expansion changes the length of glutamine tracts perhaps separating critical domains, leading to a gain- of-function mutation. Neufeld et al. [62] has shown that in Drosophila proteins with similar amino acid repeats (opa repeats) the length of the repeats are evolutionarily conserved supporting the notion of glutamine repeats serving as spacers between domains. Unlike the repeats in these human disorders, the opa repeats of Drosophila are cryptic, that is, not pure repeats of identical trinucleotides. Gostout et al [63*] has found evidence suggesting that pure repeats are evolutionarily derived from cryptic repeats. Therefore, the homologous genes for the live human disorders may not contain pure triplet repeats, and thus may account for the current limitation of trinucleotide repeat expansion mutations to humans.

It is likely that additional examples of trinucleotide repeat mutations will be found in humans. Riggins et al. [64-l demonstrated the occurrence of triplet repeats in numerous human genes: seemingly more frequent in genes expressed in the brain. Schalling et al. [65*] recently described an approach to search for expanded triplet repeats, documenting a locus on chromosome 18, in an apparently normal family, which contains an expanded and unstable CTG-repeat. As the live disorders thus far known to be due to expansion mutations result in vati- able expressivity, different ages-of-onset, and/or incom- plete penetrance, disorders that are known not to follow strict patterns of Mendelian inheritance, such as psychi- atric disorders, may be particularly good candidates for further study.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as: . of special interest . . of outstanding interest

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256~784789. This paper reviews FraX syndrome, DM and SBMA from a number of perspectives, including the clinical/diagnostic areas as well as the molecular findings. Hypotheses to account for the high mutation frequencies of the triplet repeat alleles as well as the pathophysiology are proposed.

2. RICHARDS RI, SUTHERLAND GR: Dynamic Mutations, a New Class of Mutations Causing Human Disease. Cell 1992, 70:70%712.

3. MANDEL JL: Questions of Expansion. Nature Genet 1993, 48-9.

kis is a recent review of the molecular findings in FraX syndrome, SBMA, DM and HD. It provides a valuable comparison of the four triplet repeat diseases.

4. SHERMAN SL, JACOBS PA, MORTON NE, FROSTER-ISKENIUS U,

HOWARD-PEEBLFZ PN, NIEISEN KB, PARTINGTON MW,

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RICHARDS S, VERKEFX AJMH, HOIDEN JJA, FENWICK RG, WARREN

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D, WARREN ST: DNA Methylation Represses FMRl Transcrip- tion in Fragile X Syndrome. Hum Mol Genet 1992, 1:397PioO.

This paper describes identification of unmethylated chorionic tillus cells in a male fetus with a full FraX mutation. The finding of FMRl mRNA in the cells of the chorion in spite of the presence of a full mutation argues quite strongly for methylation as the mediator of FMRl down-regulation, Fetal tissue was found to be nearly completely methyl lated and fo express little FMRl mRNA.

14. HANSEN RS, GARTER SM, Sco-rr CR, CHEN SH, IAIR~ CD: . Methylation Analysis of CGG Sites in the CpG Island of

the Human FMRl Gene. Hum Mol Genet 1992, 1:571-578. A description of methylation of the region including the CGG-repeats at the FraX site in normal and FraX individuals using methylation-sensitive restriction enzymes. This is the first demonstration of methylation of the CGG-repeats, and a more complete analysis of CpG methylation in the region. The authors suggest abnormal X-inactivation plays a role in FraX syndrome, due to similarities in the methylation patterns of inactive and FraX chromosomes.

15. HORNSTRA IK, NELSON DL, WARREN ST, YANG TP: High Resolu- . tion Methylation Analysis of the FMRI Gene Trinucleotide

Repeat Region in Fragile X Syndrome. Hum Mel Genet 1993, in press.

The definitive description of methylation of the CGG repeats and other CpGs at the FraX site using genomic sequence analysis to assess each nucleotide in the region.

16. WOHRLE D, KOTZOT D, HIR~T MC, MANCA A, KORN B,

. SCHMIDT A, BARBI G, Roar HD, POUSTKA A, DAVIES KE,

STEINBACH P: A Microdeletion of Less than 250kb, Includ- ing the Proximal Part of the FMRI Gene and the FragiIe X Site, in a Male with the Clinical Phenotype of Fragile X Syndrome. Am J Hum Genet 1992, 51:29+306.


These authors provide evidence for FMRl’s role in FraX syndrome by identification of a patient with FraX features exhibiting a deletion en- compassing the gene. Other closely linked genes cannot be ruled out as playing a role in the disorder from these data.

FraX in great numbers. This view is entirely compatible with the mode1 of Morton and MacPherson [22-j.

17. GEDEON AK, BAKER E, ROBINSON H, PARTINGTON IMW, GROSS . B, MANCA 4 KORN B, POLJSTKA A, YU S, SUTHERIAND GR,

MULLEY JC: Fragile X Syndrome Without CCG AmplIfica- tion has an FMRl Deletion. Nature Genet 1992, 1:341&344.

24. WILLEMS PJ, VAN ROY B, DE BOUILI! K, Vr13 L, REYNIERX E, BECK 0, DUMON JE, VERKERK AJMH, OOSTRA BA: Segregation of the Fragile X Mutation from an Affected Male to His Normal Daughter. Hum Mol Genet 1991, 1:511-515.

This paper also describes a deletion of FM/?1 in a patient with FraX features. Here, the deletion is extensive, with loss of the entire FMRl gene and over 2.5 million flanking base pairs of DNA, suggesting that no other essential genes are in this region.

25. REYNIER.5 E, VITS L, DE BOULLE K, VAN ROY B, VAN VEIZEN D, . . DE GRAFF E, VERKERK AJMH, JORENS HZJ, DARSY JK, OOSTRA B,

WILI~MS PJ: The Full Mutation in the FMRI Gene of Male Fragile X Patients is Absent in their Sperm. Nature Genet 1993, 4:14>146.

18. DE BOIJ~LE K, VERKERK AJMH, REYNIERS E, VITS L, HENDRICKX . . J, VAN ROY B, VAN DEN Bos F, DE GRAFF E, OOSTRA BA,

WILLEMS PJ: A Point Mutation in the FMRl Gene Associ- ated with Fragile X Mental Retardation. Nature Genet 1993, 3:31-35.

The finding of a retarded male with features of FraX syndrome exhibiting a new missense mutation in the FMRl gene offers definitive evidence of the role of FMRl in the etiology of FraX syndrome. The finding of a more severe phenotype in this patient suggests that this aberrant protein is more deleterious than the absence of the FMRl gene product. Whether this mutation is dominant cannot be determined as it is absent in the patient’s mother.

This surprising finding begins to unravel the peculiar maternal-only expansion of premutation-sized repeats to the full mutations in FraX families. The finding that males with full mutations in somatic cells (e.g. blood) demonstrate solely premutation-bearing sperm suggests two possibilities, either expansion is specific to somatic cells and the germ-line is exempt, or FMRl protein is required in sperm development and selection for premutation-carrying sperm producing cells is operating in the germ line. The observation of transiently high levels of FMRl mRNA in developing spermatagonia (see [ 27**] ) suggests the latter.

19. RICHARDS RI, HOLMAN K, FRJEND K, KREMER E, HUN D, . . STAPLES A, BROWN WI’, G~~NEWARDENA P, TARLETON J,

SCHWARTZ C, SUTHERLAND GR: Evidence of Founder Chromo- some in Fragile X Syndrome. Nature Genet 1992, 1:257-260.

26. HINDS HL, ASHLEY CT, NELSON DL, WARREN ST, HOUSMAN DE, . !%XALLtNC M: Tissue Specify Expression of FMRl Provides

Evidence for a Functional Role in Fragile X Syndrome. Nature Genet 1993, 3:3643.

A survey of FMRl mRNA expression in the developing and adult mouse. The finding of higher levels of expression in brain and testes suggests that FMRl is involved in the phenotype of FraX syndrome.

This paper reports identification of common haplotypes on FraX chromosomes. This was a surprising finding, since X~linked genetically ‘lethal’ disorders typically involve new mutations, and the frequency of FraX syndrome in the population has been taken to indicate a very high new mutation frequency ( - 50% of carrier mothers would have new mutations). Whether this indicates a common chromosome susceptible to the mutation or long-term inheritance of ‘predisposed chromosomes is unclear.

27. BACHNER D, STEINBACH P, WOHIUE D, JUST W, VOGEL M, . . HAMEISTER H: Enhanced Fmr-1 Expression in Testis. Nature

Genet 1993, 4:ll>ll6. This correspondence regarding FMRl mRNA expression levels in murine testes suggests that developing spermatagonia produce high levels of FMRl transiently prior to production of sperm. This observation may help to explain the lack of transmission of full mutations from males.

20. OUDET C, MORNET E, SEFXE JI THOMAS F, LENTES-ZENGERLING . . S, KRETZ C, DELUCHAT C, TEJADA I, BouB J, BouB A, MANDEL JL:

Linkage Disequilibrium between the Fragile X Mutation and Two Closely Linked CA Repeats Suggests that Frag- ile X Chromosomes are Derived from a Small Number of Founder Chromosomes. Am J Hum Genet 1993, 52:297-304.

28. FISCHBECK KH, IONA~ESCU V, RITIER AW, IONASESCU R, DAVIES K, BALL S, BOSCH P, BLJRNS T, HAUSMANOWA-PETRUSEWICZ I, BORKOWSKA J, ET AL: Localization of the Gene for X-Lied Spinal Muscular Atrophy. Neurology 1986, 36:1595-1598.

Extending the observation of common haplotypes in FraX chromosomes, this paper suggests as few as six founder chromosomes making up the majority of present-day FraX mutations.

29. L4 SPADA AR, WILsON EM, I.U&~HN DB, HARDING & FISCHBECK KH: Androgen Receptor Gene Mutations In X- Linked Spinal and Bulbar Muscular Atrophy. Nature 1991, 352177-79.

21. SMITX APT, DREESEN JCFM, POST JG, SMEETS DFCM, DE DIE- . SMUIDERS C, SPAANS-VAN DER BIJI. T, GOVAERTS LCP, WARREN ST,

ROSTRA BA, VAN Oos’r BA: The Fragile X Syndrome: No Evidence for any Recent Mutations. J Med &net 1993, 30:9496.

30. EDWARDS A, HAMMON HA, JIN L, CASKF~ CT, CHAKRAB OR’I?’ R: Genetic Variation at Five Trimeric and Tetrameric Tandem Repeat Loci in Four Human Population Groups. Genomics 1992, 12:241-255.

Identification of a series of affected FraX males with common ancestry dating to the mid-1700s sugRest that initial mutations in FraX can persist for many generations prior to phenotypic effect. This, combined with the observation of no new mutations in a large number of families, suggest that predisposition to FraX is not deleterious and begIns to explain the obsetvation of common haplotypes in FraX chromosomes.

31. BIANCAIANA V, SERVILLE F, POMMIER J, JUUEN J, HANAUER A, . MANDEL JL: Moderate Instabiity of the Trinucleotide Repeat

in Spin0 Bulbar Muscular Atrophy. Hum Mol Genet 1992, 1:255-258.

22. MORTON NE, MACPHERSON JN: Population Genetics of the . Fragile X Syndrome. Multiallelic Model for the FMRl Locus.

Proc Nat1 Acud Sci USA 1992, 89~42154217.

This paper describes small changes in the number of repeats upon transmission of mutant alleles in SBMA in a four generation pedigree. Of 17 transmissions, 7 increased in size, 9 remained the same and 1 decreased. The mutant alleles varied in length from 46 to 53 repeats.

A mathematical treatment of the origin of FraX chromosomes, suggesting that several transitions in size and/or stability are required (each with different rates) for eventual FraX development. This model would explain founder chromosomes as well as the high frequency of the sym drome without high mutation frequency.

32. YAhL4MOTO Y, KAWAI H, NAKAHARA K, 0s~ M, NAKAT~UJI Y, KISHIMOTO T, SAKODA S: A Novel Primer Extension Method to Detect the Number of CAG Repeats In the Androgen Receptor Gene in Families with X-Linked Spinal and Bul- bar Muscular Atrophy. Biccbem Biophys Res Commun 19992, 182:507-513.

23. CHAKRAvARn A: Fragile X Founder Effect? Nature Genet 1992, 33. IA SPADA AR, ROLING DB, HARDING AE, WARNER CL, SPIEGEL R, . 1:237-238. . . HAUSMANOWA-PETRUSEWZ I, YEE WC, FISCHBECK KH: Meiotic This news and views regarding [ 19**] suggests that tluctuation anal@s Stability and Genotype-Phenotype Correlation of the Trin- is the appropriate view for the common haplotypes seen in modem- ucleotide Repeat in X-Linked Spinal and BuIbar Muscular day FraX chromosomes. In this view, the most common haplotypes Atrophy. Nature Genet 1992, 2:301-304. obsetved are those on which the initial mutation arose at the appropri- This extensive analysis of instability in SBMA observed 45 transmissions ate number of generations ( -30) in the past for them to be causing of mutant alleles, with 12 changes in length, of which 2 were clear de-


creases. Correlation between repeat length and age of onset of a variety that congenital DM caSes have the longest repeat length and that their of symptoms was noted. mothers have larger mean repeat sizes.

34. IGAFMHI S, TANNO Y, ONODERA 0, Y -1 M, SAT-0 S, ISHIKAWA A, MNATANI N, NAGASHIMA M, ISHIKAWA Y, SAHAsHI K, ETA: Strong Correlation Between the Number of CAG Re- peats in Androgen Receptor Genes and the Clinical Onset of Features of Spinal and Bulbar Muscular Atrophy. Neuro logy 1992, 42:230&2302.

46. AE%EL~OV~CH D, LEER I, PASHUT-IAVON I, SCHMUEU E, RAAS- ROTHSCHILO A, FRYDMAN M: Negative Expansion of the Myotonic Dystrophy Unstable Sequence. Am J Hum Genet 1993, 52:1175-1181.

35. SAR M, STUMPF WE: Androgen Concentration in Motor Neu- rons of Cranial Nerves and Spinal Cord. Science 1977, 197:77-80.

36. A.SL&NIDIS C, JANSEN G, AMEMNA C, SHUTLER G, MAHADEVAN M, TSILFIDIS C, CHEN C, ALLEMAN J, WORMSKAMP NGM, VOOIJS M, ET AL.: Cloning of the Essential Myotonic Dystrophy Re- gion and Mapping of the Putative Defect. Nature 1992, 355:54%551.

47. MULLEY JC, STAPLES A, DONNELLY A, GEDEON AK, HECKT BK, . NICHOLSON GA, HAAN EA, SUTHERLAND GR: Explanation for

Exclusive Maternal Inheritance for Congenital Form of Myotonic Dystrophy. Lancet 1993, 34 1~236237.

A brief letter describing data showing that paternal DM alleles with long repeats are less likely to expand, and even contract, when transmitted as compared to similar sized maternal alleles. The authors suggest that the exclusive maternal origin of congenital DM is due to this phenomenon.

37. HARLEY HG, BROOK JD, RUNDLE SA, CROW S, REARDON W, BUCKLER AJ, HARPER PS, HOUSMAN DE, SHAW DJ: Expansion of an Unstable DNA Region and Phenotypic Variation in Myotonic Dystrophy. Nature 1992, 355:545-546.

48. IAXDAN C, HOFFMAWRALWAWI H, RABEs JP, ROUME J, JUNIEN C: Different Sex-Dependent Constraints in CTG Length Vari- ation as Explanation for Congenital Myotonic Dystrophy. Lancet 1993, 341:237.

38. WEVAN M, TSILFIDIS C, SABOURIN L, SHUTIER G, AMEMNA . . C, JANSEN G, NEVILLE C, NARANG M, BARCELO J, O’Hou K, ~7

AL: Myotonic Dystrophy Mutation: An Unstable CTG Repeat in the 3’ Untranslated Region of the Gene. Science 1992, 255:1253-1255.

See [4000].

49. TSILFIDIS C, MACKENZIE AE, METTLER G, BARCEL.~ J, . KORNEL~JK RAG: Correlation Between CG Repeat Length and

Frequency of Severe Congenital Dystrophy. Nature Genet 1992, 1:192-195.

A determination of the DM repeat length in 272 patients that demonstrates a direct correlation between repeat length and disease severity.

39. FU YH, Pzzu’n A, FENWOCK RG, KING J, RAJNARAYAN S, DUNNE . . PW, DUBEL J, NASSER GA, A.SHUAWA T, DE JONG P, ~7’ AL: An

Unstable Triplet Repeat in a Gene Related to Myotonic Muscular Dystrophy. Science 1992, 255:125&1258.

see [40**1.

40. BRCOK JD, MCCURRACH ME, HARLEY HG, BUCKLER AJ, . . CHURCH D, AEXIRATANI H, HUNTER K, STANTON VP, THIRION JP,

HUDSON T, ET a: Molecular Basis of Myotonic Dystrophy: Expansion of a Trinucleotide (CTG) Repeat at the 3’ End of a Transcript Encoding a Protein Kinase Family Member. Cell 1992, 68:79%808.

50. O’Hou KL, TSILFIDIS C, MAHADEVAN MS, NE~ILLE CE, BARCEL~) J, . . HUNTER AGW, KORNELUK RG: Reduction in Size of the

Myotonic Dystrophy Trinucleotide Repeat Mutation Dur- ing Transmission. Science 1993, 259:80+812.

Describes three cases of reduction of the repeat upon transmission. One case wa.s most likely due to a gene conversion event that changed the expanded repeat of the DM allele to the repeat length of the normal allele in normal offspring who inherited the DM haplotype from an affected father.

The papers by Mahadevan et al. [3Boo], Fu et a(. [39**], and Brook ef al. [40**] all describe the simultaneous discovery of the mutation responsible for DM as an expanding CTG-repeat in a gene with homology with serine-threonine protein kinase.

51. Fu YH, FRIEIXVIAN DL, FUcbmx S, PEARIMAN JA, GIBBS . . R& PI~~IJTI A, A.sHI~_AWA T, PERRYW MB, SCARLA1’0

G, FENWOCK RG, C~SKEY CT: Decreased Expression of Myotonin-Protein Kinase Messenger RNA and Protein in Adult Form of Myotonic Dystrophy. Science 1993, 260:235-238.

41. YAMAGATA H, MIKI T, OGIHARA T, NAKAGAWA M, HIGUCHI I, 0s~ M, SHELBOURNE P, DAVIES J, JOHNSON K: Expansion of Unstable DNA Region in Japanese Myotonic Dystrophy Patients. Lancet 1992, 339~692.

The paper shows evidence for reduced levels of myotonic~protein kinase (Mt.PK) mRNA as well as protein in muscle from adult DM patients. Also described are antibodies against Mt~PK protein.

42. . .

IMBERT G, KRETz C, JOHNSON K, MANDEL JL: Origin of the Expansion Mutation in Myotonic Dystrophy. Nature Genet 1993, 4~72-76.

52. . .

SABOIWN LA, WEVAN MS, NARANG M, LEE DSC, SURH LC, KORNELUK RG: Effect of the Myotonic Dystrophy (DM) Mu- tation on mRNA Levels of the DM Gene. Nature Genel 1993, in press.

An insightful study of the linkage disequilibrium between the DM mutation and a nearby polymorphic marker. The authors interpret their data as an initial predisposing event of transition from a 5-repeat allele to alleles with 1930 repeats, which would constitute a resenroir for recurrent expansion mutations. This interpretation would suggest that alleles with 1 l-13 repeats rarely, if ever, undergo expansion mutations.

This study shows evidence for elevated levels of Mt-PK mFWA from the brain of an infant with congenital DM and suggests the expansion sta- bilizes the message resulting in increased levels of Mt-PK protein. Also described as an exonic polymorphism useful for distinguishing prod- ucts of each allele.

53. HARPER PS, MORRIS MJ, QUARREU. 0, SHAW DJ, ‘PnER A, YOUNGMAN S: Huntington’s Disease. Philadelphia: W.B. Saun- ders; 1991.

43. HARLF( HG, RLJNDLE SA, REARDON W, MYR~NG J, CROW S, BROOK JD, HARPER PS, SHAW DJ: Unstable DNA Sequence in Myotonic Dystrophy. Lancet 1992, 339:1125-l 128.

54.

44. KURDON W, HARLEY HG, BROOK JD, RUNDLE SA, CROW S, HARPER PS, SHAW DJ: Minimal Expression of Myotonic Dys- trophy: a Clinical and Molecular Analysis. J Med Genet 1992, 29:77&773.

GUSEIIA JF, WEXLER NS, CONNEALLY PM, NAYLOR SL, ANDERSON MA, TANZI RE, WATKINS PC, O’ITINA K, WALIACE MR, SAKAGUCHI AY, ET AL.: A polymorphic DNA Marker Ge- netically Linked to Huntington’s Disease. Nature 1983, 306:234-238.

55.

45. HARLEY HG, RUNDLE SA, MAcMll~rl JC, MYRING J, BROOK JD, . CROW S, REARDON W, FENTON I, SHAW DJ, HARPER PS: Size

of the Unstable CTG Repeat Sequence in Relation to Phe- notype and Parental Transmission in Myotonic Dystrophy. Am J Hum Genet 1992, 52:1164-1174.

MACDONALD ME, NOVELLETTO A, LIN C, TAGLE D, BARNES G, BATES G, TAYLOR S, ALurro B, ALTHERR M, MVERS R, ET AL.: The Huntington’s Disease Candidate Region Exhibits Many Different Haplotypes. Nature &net 1992, 1:9%103.

56. . .

A very complete analysis of 439 individuals of 101 kindreds, which demonstrates anticipation quite nicely as there is a correlation between repeat length and disease severity and age-of-onset. The data shows

MACDONALE ME, AMBROSE CM, DUYAO MP, MVERS RH, LIN C, SIUNIDHI L, BARNES G, TAYWR SA, JAMES M, GROOT N, ET AL. [THE HUNTING’I’ON’S DISEASE COUABORATNE RESEARCH GROUP]: A Novel Gene Containing a Trinucleotide Repeat that is Expanded and Unstable on Huntington’s Disease Chromo- somes. Ceil 1993, 72:971-983.


The long~standing search for this elusive gene is resolved in this paper, which identifies the HD gene and describes the mutation as a trinucleotide repeat expansion.

57.

58.

59.

60.

61. . .

ZOCHBI HY: Spinocerebellar Ataxia: Variable Age of Onset and Linkage to Human Leukocyte Antigen in a Large Kin- dred. Ann Neural 1988, 23:58&584.

HAINES JL, SCHUT LJ, WErr’&+MP LR Spinocerebellar Ataxis in Large Kindred: Age at Onset, Reproduction and Genetic Linkage Studies. Neurology 1984, 34:1542-1548.

RANIJM LPW: Localization of the Autosomal Dominant, HLA- Linked Spinocerebellar At&a (SCAl) Locus in Two Kin- dreds Within an 8cM Subregion of Chromosome 6p. Am J Hum Genet 1991, 49:31-41.

K~IATKOWSKI TJ: The Gene for Autosomal Dominant Spinocerebellar Ataxia (SCAl) Maps Centromeric to D6S89 and Shows no Recombination, in Nine Large Kindreds, with a Dinucleotide Repeat at the AM10 Locus. Am .J Hum Genet 1993, in press.

ORR HT, CH~NC M, BANFI S, KWAITKOWSKI TJ, SERVA~IO A, BEAUDET AL, MCCALL AE, DIJVCK IA, RANIJM LPW, ZOCHIU

HY: Expansion of an Unstable Trinucleotide (CAG) Re- peat in Spinocerebellar Ataxia Type 1. Nature Genet 1993, 4:221-226.

The latest example of a human neurological disease due to a trinucleotide repeat expansion.

62. NEUFELD SJ, SCHMID AT, YEDVOBNICK B: Homopolymer Length Variation in the Drosophila Gene Mastermind. J Mel Et101 1993, in press.

63. GOSTOLIT H, LKI Q, SOMERS SS: ‘Cryptic’ Repeating Triplets

. of Purines and Pyrimidines (cRRY (i)) are Frequent and Polymorphic: Analysis of Coding cRRY(i) in the Proopi- omelanocortin (POMC) and TATA-Binding Protein (TBP) Genes. Am J Hum Genet 1993, 52:1182-1190.

An interesting study of trinucleotide repeats which suggests that con- selved perfect triplet repeats within genes may be derived from cryptic rep&,&.

64. RIGGINS GJ, LOKEY LK, CFIASTAIN JL, LEINER HA, SHEKMAN SL, . WILKINSON KD, WAJUEN ST: Human Genes Containing

Polymorphic Trinucleotide Repeats. Nature Genet 1992, 2:186191.

Through cDNA library screening with triplet repeat probes and database searches, numerous examples of human genes containing normally polymorphic exonic repeats were identified, indicating that other loci exist, which potentially could undergo similar mutational changes.

65. SCHALUNG M, HUDSON TJ, BUETOW KH, HOUSMAN DE: Direct . Detection of Novel Expanded Trinucleotide Repeats in the

Human Genome. Nature Genet 1993, 4:13%139. A method of detecting expanded trinucleotide repeats in genomic DNA that should prove useful in screening families with suspected expansion mutations. Unfortunately, the present method does not allow identification of flanking sequences to specifically clone the locus.

ST Warren, Howard Hughes Medical Institute, Emoty University School of Medicine, Rollins Research Centre, Atlanta, Georgia 30322, USA DL Nelson, Institute for Molecular Genetics and The Human Genome Center, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA

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