Dynamic mutations in human genes: A review of trinucleotide repeat diseases

Dynamic mutations in human genes result from unstable trinucleotide 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-gene mutational events, e.g. point mutations, deletions, insertions and inversions, typically associated with Mendelian inheritance patterns. Intergenerational instability of dynamic mutations within families has provided an explanation for the genetic anticipation, 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 from 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 specific phenotype. Five disorders resulting from small-scale expansions of CAG repeats within the protein-coding region are known: spinobulbar muscular atrophy, Huntington’s disease, spinocerebellar ataxia type 1, dentatorubral-pallidoluysian atrophy (DRPLA) and Machado-Joseph disease. A sixth disorder, Haw River syndrome, is allelic to DRPLA. Five folate-sensitive chromosomal fragile sites characterized to date, viz. FRAXA, FRAXE, FRAXF, FRA11B and FRA16A, all have large-scale CGG repeat expansion. Two disorders, fragile X syndrome and FRAXE mental retardation, result from instability of CGG repeats in the 5’ untranslated region ofFMR1 andF M R2 genes respectively. FRA11B lies close to chromosome 1 1q deletion endpoints in many Jacobsen syndrome patients and may be related to the deletion event producing partial aneuploidy for 1lq. Expansion of FRAXF and FRA16A has not been associated with a phenotype. Myotonic dystrophy results from a large-scale CTG expansion in the 3’ untranslated region of the myotonin protein kinase gene while Friedreich’s ataxia has recently been found to have a large-scale GAA repeat in the first intron ofX25. This article reviews the characteristics of trinucleotide repeat disorders and summarizes current understanding of the molecular pathophysiology.     

Mouse models of triplet repeat diseases

Triplet repeat expansions were first discovered in 1991 and since then have been found to be the mutation underlying a range of neurodegenerative, neuromuscular, and cognitive disorders including fragile X syndrome, myotonic dystrophy, Friedreich's ataxia, and the polyglutamine disorders that include Huntington's disease. The repeats exert their detrimental effects through different molecular mechanisms dependent on whether they are located in coding or noncoding regions of the gene in question. During the past 10 yr, a wide range of strategies have been used to successfully establish mouse models for all of these disorders. This review presents an overview of these mouse models, discusses the insights into the molecular pathogenesis of these disorders that have been gained from their analysis and the strategies that are being used to uncover novel therapeutic options.     

Chromatin remodeling in the noncoding repeat expansion diseases

Friedreich ataxia, myotonic dystrophy type 1 and 3 forms of intellectual disability, fragile X syndrome, FRAXE mental retardation, and FRA12A mental retardation are repeat expansion diseases caused by expansion of CTG.CAG, GAA.TTC, or CGG.CCG repeat tracts. These repeats are transcribed but not translated. They are located in different parts of different genes and cause symptoms that range from ataxia and hypertrophic cardiomyopathy to muscle wasting, male infertility, and mental retardation, yet recent reports suggest that, despite these differences, the repeats may share a common property, namely the ability to initiate repeat-mediated epigenetic changes that result in heterochromatin formation.     

DNA structures, repeat expansions and human hereditary disorders

Expansions of simple DNA repeats are responsible for more than two dozen hereditary disorders in humans, including fragile X syndrome, myotonic dystrophy, Huntington's disease, various spinocerebellar ataxias, Friedreich's ataxia and others. During the past decade, it became clear that unusual structural features of expandable repeats greatly contribute to their instability and could lead to their expansion. Furthermore, DNA replication, repair and recombination are implicated in the formation of repeat expansions, as shown in various experimental systems. The replication model of repeat expansion stipulates that unusual structures of expandable repeats stall replication fork progression, whereas extra repeats are added during replication fork restart. It also explains the bias toward repeat expansion or contraction that was observed in different organisms.     

The fragile X syndromes

Fragile X syndrome is a leading cause of mental retardation worldwide, with an incidence of approximately one case in 2000 live births. It is amongst the most common of human genetic diseases, and was the first to be associated with an unstable trinucleotide (CGG) repeat sequence. It is also characterized by a chromosomal fragile site which was the first of (now) four such sites to be identified at the molecular level. Each shows very similar features suggesting that other representatives of this type of fragile site will likely involve similar sequences. As with the other unstable trinucleotide repeats, the sequence at the fragile X locus is found to be remarkably unstable upon genetic transmission, however many features differ from the other repeats. As repeat expansion at the fragile X locus results in loss of expression of the co-resident FMR1 gene, the basis for clinical features is best understood in this disorder. Two additional fragile sites in the vicinity have been identified, and at least one of these is associated with mental retardation.     

Fragiles-X Syndrom

Fragile X syndrome is an X-linked neurodevelopmental disorder affecting both males and females. Phenotypical characteristics include intellectual deficits, somatic symptoms and behavioural abnormalities caused by loss of the FMRP protein, which leads to destruction of synapses with metabotropic glutamate receptors. The FMR1 gene harbours a CGG repeat in the 5’-untranslated region. The vast majority of fragile-X syndrome patients have a largely expanded CGG repeat (220 or more triplets, designated “full mutation”) and an inactive gene. Full mutation alleles originate upon proliferation of oogonia in the fetal ovary of females who carry a mitotically unstable premutation (59–200 repeats). Premutation carriers have no symptoms of fragile X syndrome; they may, however, experience premature ovarian insufficiency and/or fragile X-associated tremor/ataxia syndrome. The diagnosis of both syndromes requires genetic testing to measure the number of CGG repeats. Prenatal diagnostics of fragile X syndrome is offered to females carrying a pre- or full mutation.     

A fragile X case with an amplification/deletion mosaic pattern

Fragile X syndrome is the most common cause of hereditary mental retardation. The FMR1 gene, which is involved in fragile X syndrome, contains a polymorphic CGG repeat, which expands in affected patients. Expanding triplet repeats have been shown to be a new type of mutation, termed "dynamic mutation", responsible for more than 12 genetic diseases. These mutations occur as multiple steps rather than as a single event. The first step leads to an unstable allele that then becomes increasingly unstable generally achieving further increases in copy or occasionally contraction. In this report, we describe a fragile X boy with both a hypermethylated full mutation and a deletion of 905 bp encompassing the CGG repeat. The upstream breakpoint is 438 bp 5' to the CGG repeat and the downstream breakpoint is 420 bp 3' of the triplet repeats. The deletion includes the ATG starting codon for translation of the FMR1 gene. This was confirmed by using FMRP immunocytochemistry both on blood smears and hair roots. The deleted region is flanked by a ccgg direct repeat next to the breakpoints; this may have had a critical role in the formation of a secondary DNA structure leading to the deletion.     

Genetic instabilities of triplet repeat sequences by recombination

The expansion of triplet repeat sequences is an initial step in the disease etiology of a number of hereditary neurological disorders in humans. Diseases such as myotonic dystrophy, Huntington's, several spinocerebellar ataxias, fragile X syndrome, and Friedreich's ataxia are caused by the expansions of CTG.CAG, CGG.CCG, or GAA.TTC repeats. The mechanisms of the expansion process have been investigated intensely in E. coli, yeast, transgenic mice, mammalian cell culture, and in human clinical cases. Whereas studies from 1994-1999 have implicated DNA replication and repair at the paused synthesis sites due to the unusual conformations of the triplet repeat sequences, recent work has shown that homologous recombination (gene conversion) is a powerful mechanism for generating massive expansions, in addition to, or in concert with, replication and repair.     

Stability of triplet repeats of myotonic dystrophy and fragile X loci in human mutator mismatch repair cell lines

At least nine human genetic diseases, including myotonic dystrophy (DM) and fragile X syndrome have been associated with the expansion of CTG or CGG trinucleotide repeats within the disease loci. Little is known about the molecular mechanisms or the genetic control of the expansion of triplet repeats. Mutations in human mismatch repair genes are associated with the increased polymorphism of many microsatellites, including dinucleotide repeats. The effect of mutations in two mismatch repair genes on the size of trinucleotide repeats in the DM and FRAXA loci has been analyzed. PCR and Southern analysis of the triplet repeat regions of the DM and fragile X mental retardation (FRAXA) loci in cell lines HTC116 and LoVo, which contain mutations in both alleles of the hMLH1 and hMSH2 genes, respectively, indicated that the size of the endogenous (CTG)_n and (CGG)_n tracts fall within the range observed in the normal population. This suggests that mutations in hMLH1 or hMSH2 do not result in the instability of CTG or CGG tracts to the levels observed in individuals with myotonic dystrophy or fragile X syndrome. Received: 4 December 1995 / Revised: 29 January 1996, 7 March 1996     

RNA-binding proteins hnRNP A2/B1 and CUGBP1 suppress fragile X CGG premutation repeat-induced neurodegeneration in a Drosophila model of FXTAS

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a recently described neurodegenerative disorder of older adult carriers of premutation alleles (60-200 CGG repeats) in the fragile X mental retardation gene (FMR1). It has been proposed that FXTAS is an RNA-mediated neurodegenerative disease caused by the titration of RNA-binding proteins by the CGG repeats. To test this hypothesis, we utilize a transgenic Drosophila model of FXTAS that expresses a premutation-length repeat (90 CGG repeats) from the 5' UTR of the human FMR1 gene and displays neuronal degeneration. Here, we show that overexpression of RNA-binding proteins hnRNP A2/B1 and CUGBP1 suppresses the phenotype of the CGG transgenic fly. Furthermore, we show that hnRNP A2/B1 directly interacts with riboCGG repeats and that the CUGBP1 protein interacts with the riboCGG repeats via hnRNP A2/B1.     

Apparent regression of the CGG repeat in FMR1 to an allele of normal size

The fragile X syndrome is the result of amplification of a CGG trinucleotide repeat in the FMR1 gene and anticipation in this disease is caused by an intergenerational expansion of this repeat. Although regression of a CGG repeat in the premutation range is not uncommon, regression from a full premutation (>200 repeats) or premutation range (50–200 repeats) to a repeat of normal size (<50 repeats) has not yet been documented. We present here a family in which the number of repeats apparently regressed from approximately 110 in the mother to 44 in her daughter. Although the CGG repeat of the daughter is in the normal range, she is a carrier of the fragile X mutation based upon the segregation pattern of Xq27 markers flanking FMR1. It is unclear, however, whether this allele of 44 repeats will be stably transmitted, as the daughter has as yet no progeny. Nevertheless, the size range between normal alleles and premutation alleles overlap, a factor that complicates genetic counseling.     

Screening for FXTAS in 95 Spanish patients negative for Huntington disease

Fragile X syndrome is the most common form of hereditary mental retardation. The molecular basis of this syndrome is mainly a CGG expansion in the 5' untranslated region of the FMR1 gene. Expansions with more than 200 CGG repeats abolish gene expression causing the classical fragile X phenotype. Premutation carriers (55-200 CGG) have normal cognitive function with increased risk of developing premature ovarian failure and fragile X-associated tremor-ataxia syndrome (FXTAS). Some clinical features associated with FXTAS, such as tremor, gait ataxia, cognitive decline, and generalized brain atrophy, are also seen in other movement disorders. Ninety-five patients referred for HD, who tested negative for the expansion in the IT15 gene, were screened for FMR1 CGG-repeat expansion. One FMR1 premutation male carrier was detected, giving an FXTAS frequency of 1.6%. Our results highlight that FXTAS is still not well diagnosed; therefore, we recommend FMR1 premutation screenings in all patients with late-onset tremor, ataxia, and cognitive dysfunction.     

脆性X综合征的分子遗传学研究进展

脆性X综合征是常见的遗传性智力低下性疾病,其发病率高,临床表现复杂,遗传规律独特,对脆性X 综合征的发病机理和脆性X综合征筛查与诊断方法等方面的一些研究进展进行了综述.     

Linkage disequilibrium between the fragile X mutation and two closely linked CA repeats suggests that fragile X chromosomes are derived from a small number of founder chromosomes

In order to investigate the origin of mutations responsible for the fragile X syndrome, two polymorphic CA repeats, one at 10 kb (FRAXAC2) and the other at 150 kb (DXS548) from the mutation target, were analyzed in normal and fragile X chromosomes. Contrary to observations made in myotonic dystrophy, fragile X mutations were not strongly associated with a single allele at the marker loci. However, significant differences in allelic and haplotypic distributions were observed between normal and fragile X chromosomes, indicating that a limited number of primary events may have been at the origin of most present-day fragile X chromosomes in Caucasian populations. We propose a putative scheme with six founder chromosomes from which most of the observed fragile X–linked haplotypes can be derived directly or by a single event at one of the marker loci, either a change of one repeat unit or a recombination between DXS548 and the mutation target. Such founder chromosomes may have carried a number of CGG repeats in an upper-normal range, from which recurrent multistep expansion mutations have arisen.     

Polarity of mutations in tumor-associated microsatellite instability

Many factors have been implicated in influencing the rate of microsatellite mutations, including the length and base composition of the repeat motif, number of repeats, base composition of flanking sequences and, perhaps most importantly, degree of perfection of the repeats. The latter is of clinical relevance, since in both spino-cerebellar ataxia and fragile X syndrome, alleles with imperfect repeats appear to be much more stable than perfect ones. As yet, the relative importance of increased replication slippage and decreased mismatch repair efficiency in the preference of mutations to occur within perfect repeats has not been fully determined. D13S308E is an asymmetric trinucleotide repeat microsatellite with the sequence (CAT)₃CAC(CAT)CAC(CAT)₂CAC(CAT)CAC(CAT) ₁₅ , thus containing two parts: an 11-repeat imperfect portion (underlined above) and a 15-repeat perfect one (bold). We sequenced eight new mutant alleles of D13S308E from three human gastric tumors with instability in this and other microsatellites. In all mutations the size variation occurred exclusively in the perfect part of the microsatellite. These results constitute direct evidence that the molecular basis of microsatellite alterations seen in normal cells is similar to those that occur in human tumors with extensive microsatellite instability. The investigation of mechanisms involved in microsatellite mutations has been handicapped by the fact that they are rare events. The microsatellite instability observed in malignant tumors provides us with a useful general system to study these mechanisms. Received: 24 June 1997 / Accepted 7 October 1997