Trinucleotide Repeat Instability: Genetic Features and Molecular Mechanisms

Trinucleotide repeat expansions are an important cause of inherited neurodegenerative disease. The expanded repeats are unstable, changing in size when transmitted from parents to offspring (inter-generational instability, "meiotic instability") and often showing size variation within the tissues of an affected individual (somatic mosaicism, "mitotic instability"). Repeat instability is a clinically important phenomenon, as increasing repeat lengths correlate with an earlier age of onset and a more severe disease phenotype. The tendency of expanded trinucleotide repeats to increase in length during their transmission from parent to offspring in these diseases provides a molecular explanation for anticipation (increasing disease severity in successive affected generations). In this review, I explore the genetic and molecular basis of trinucleotide repeat instability. Studies of patients and families with trinucleotide repeat disorders have revealed a number of factors that determine the rate and magnitude of trinucleotide repeat change. Analysis of trinucleotide repeat instability in bacteria, yeast, and mice has yielded additional insights. Despite these advances, the pathways and mechanisms underlying trinucleotide repeat instability in humans remain largely unknown. There are many reasons to suspect that this uniquely human phenomenon will significantly impact upon our understanding of development, differentiation and neurobiology.     

Trinucleotide repeats and other microsatellites in yeasts

Microsatellites are direct tandem DNA repeats found in all genomes. A particular class of microsatellites, called trinucleotide repeats, is responsible for a number of neurological disorders in humans. We review here our current state of knowledge on trinucleotide repeat instability, and discuss the molecular mechanisms that may be involved in trinucleotide repeat expansions leading to fatal diseases in humans. We also present original data on microsatellite distribution in several microbial genomes, and on the use of microsatellites as physical markers to accurately and easily genotype yeast strains.     

Pathogenesis of polyglutamine-induced disease: A model for SCA1

During the past 7 years several inheritable neurological disorders have been found to be due to the expansion of an unstable CAG trinucleotide repeat that leads to an increase in the length of a polyglutamine tract within a disease-specific protein. Based on pathological evidence obtained from the brains of affected individuals and transgenic mice expressing a mutant human gene, it was proposed that the formation of nuclear aggregates of the polyglutamine protein plays a critical role in pathogenesis. However, recent evidence indicates that this may not be the case. This review focuses on our results for one of these disorders, spinocerebellar ataxia type 1 (SCA1), and presents a model for SCA1 pathogenesis.     

The CAG/Polyglutamine Tract Diseases: Gene Products and Molecular Pathogenesis

In the past few years, a new type of genetic mutation, expansion of trinucleotide repeats, has been shown to cause neurologic disease. This new class of mutations was first identified in 1991 as the underlying genetic defect in spinal and bulbar muscular atrophy and the fragile X syndrome, and in recent years, trinucleotide repeat expansions have been found to be the causative mechanism in 10 other neurologic diseases. These mutations are produced by heritable unstable DNA and are termed “dynamic mutations” because of changes in the number of repeat units inherited from generation to generation. In the normal population, these repeat units, although polymorphic, are stably inherited. To date four types of trinucleotide repeat expansions have been identified: (1) long cytosine-guanine-guanine (CGG) repeats in the two fragile X syndromes (FRAXA and FRAXE), (2) long cytosine-thymine-guanine (CTG) repeat expansions in myotonic dystrophy, (3) long guanine-adenine-adenine repeat expansions in Friedreich's ataxia and (4) short cytosine-adenine-guanine repeat expansions (CAG) which are implicated in eight neurodegenerative disorders and are the focus of this review. Diseases that are caused by trinucleotide repeat expansions exhibit a phenomenon called anticipation that can not be explained by conventional Mendelian genetics. Anticipation is defined as increase in the severity of disease with an earlier age of onset of symptoms in successive generations [5, 50, 57, 106]. Anticipation is often influenced by the sex of the transmitting parent, and for most CAG repeat disorders, the disease is more severe when paternally transmitted. The severity and the age of onset of the disease have been correlated with the size of the repeats on mutant alleles, with the age of onset being inversely correlated with the size of the expansion. In all eight disorders caused by CAG repeat expansion, the repeat is located within the coding region of the gene involved and in all cases it is translated into a stretch of polyglutamines in the respective proteins. All the proteins are unrelated outside of the polyglutamine stretch and most are novel with exception of the androgen receptor and the voltage gated α1A calcium channel, which are mutated in spinal and bulbar muscular atrophy and spinocerebellar ataxia type 6. It is intriguing that the proteins are ubiquitously expressed in both peripheral and nervous tissue but in each disorder only a select population of nerve cells are targeted for degeneration as a consequence of the expanded CAG repeat. Current thinking among scientists working on the molecular mechanisms of neurodegeneration in these diseases is that the presence of an expanded polyglutamine confers a gain of function onto the involved protein. To understand the mechanisms underlying the pathogenesis of these diseases, investigators have turned to generating transgenic mice which recapitulate some of the features of the human disease and hence are excellent model systems to study the progression of the disease in vivo.     

The "flap" endonuclease gene FEN1 is excluded as a candidate gene implicated in the CAG repeat expansion underlying Huntington disease

At least 12 disorders including Huntington disease (HD) are associated with expansion of a trinucleotide repeat (TNR). Factors contributing to the risk of expansion of TNRs and the mechanism of expansion have not been elucidated. Data from Saccharomyces cerevisiae suggest that the flap endonuclease FEN1 plays a role in expansion of repetitive DNA tracts. It has been hypothesized that insufficiency of FEN1 or a mutant FEN1 might contribute to the occurrence of expansion events of long repetitive DNA tracts after polymerase slippage events during lagging strand synthesis. The expression pattern of FEN1 was determined, and ubiquitous tissue expression, including germ cells, suggested that FEN1 has the potential to be involved in HD. Fifteen HD parent/child pairs that demonstrated intergenerational increases in CAG length of greater than 10 repeats were examined for possible mutations or polymorphisms within the FEN1 gene that could underlie the saltatory repeat expansions seen in these individuals. No alterations were observed compared to 50 controls, excluding FEN1 as a trans-acting factor underlying TNR expansion. The identification of a candidate gene(s) in HD or other CAG-expansion disorders implicated in TNR instability will elucidate the mechanism of expansion for this growing family of neurological disorders.     

Immunogenicity of P/Q-type calcium channel in small cell lung cancer: investigation of alpha1 subunit polyglutamine expansion

The ectopic expression of neuronal P/Q-type voltage-gated calcium channels in small cell lung carcinoma (SCLC) is thought to induce antisynaptic autoimmunity in the paraneoplastic Lambert-Eaton myasthenic syndrome. The gene CACNL1A4, encoding the principal (alpha1A) subunit of this calcium channel, is mutated in several inherited neurological disorders. One of these disorders (spinocerebellar ataxia, type 6, or SCA-6) involves the expansion of a trinucleotide (CAG) repeat unit. We hypothesized that a somatic CAG repeat instability of this gene in neoplastic cells might generate a non-self epitope capable of initiating autoimmunity to P/Q-type calcium channels. We therefore analyzed the CACNL1A4 gene in SCLC lines established from metastases derived from seven individual patients (four associated with Lambert-Eaton myasthenic syndrome, one associated with myasthenia gravis, and two not associated with neurological autoimmunity). We compared their CAG repeat numbers (determined by polymerase chain reaction (PCR) amplification followed by separation of products on a 6% polyacrylamide/8M urea gel) to published norms and to DNA from a patient with SCA-6. The number of CAG repeats in SCLC DNA fell within a normal range whether or not the neoplasm was complicated by neurological autoimmunity. Therefore, it is unlikely that somatically unstable CAG repeat units in the gene encoding the P/Q-type voltage-gated calcium channel account for this tumor protein's immunogenicity in the Lambert-Eaton myasthenic syndrome.     

Meiotic instability associated with the CAGR1 trinucleotide repeat at 13q13.

CAGR1 is a recently characterised polymorphic trinucleotide repeat localised to 13q13, which has been suggested as a possible candidate gene for neurological disorders that manifest genetic anticipation. To provide evidence in support of this hypothesis, a large number of chromosomes (n = 928) from patients with a wide variety of neurological diseases were screened for evidence of repeat expansion and meiotic instability. One person with a CAGR1 repeat number of 50 was identified (normal range 9-29). Subsequent molecular analyses of CAGR1 repeat number in additional family members showed meiotic instability of a (CAG)45 allele through three generations. While CAGR1 repeat number did not correlate with a readily discernible phenotype in this family, the finding of meiotic stability and mendelian inheritance of normal CAG alleles and meiotic instability of larger repeats fulfil several criteria thought essential for pathologically relevant mutations of this type. Thus, these data strengthen the hypothesis for a role of CAGR1 in the development of an as yet molecularly uncharacterised human neurological disease.     

三核苷酸重复序列扩增与精神系统疾病

三核苷酸重复序列扩增是三核苷酸重复序列的拷贝数在世代传递中逐渐增加的一种基因动态突变,现已发现近20种疾病的发生与这一突变相关。本文就近年来国际上关于三核苷酸重复序列扩增与精神系统疾病研究的一些成果进行综述,主要内容包括：①三核苷酸重复序列扩增相关疾病的分类;②三核苷酸重复序列扩增与疾病的早现遗传现象;③三核苷酸重复序列扩增与精神系统疾病的发病机制研究;④精神系统疾病相关基因内三核苷酸重复序列的研究。     

Correlation between magnitude of CAG repeat length alterations and length of the paternal repeat in paternally inherited Huntington's disease

An increasing number of diseases are being found to be due to elongation of specific trinucleotide repeat sequences. Inverse correlation between the age at onset and the length of the repeat has been found in most of these. The elongated CAG repeat causing Huntington's disease is highly unstable when inherited from an affected father. In this study we found an average parent-to-offspring difference of +0.08 repeat units in maternally inherited repeats, significantly less than the average difference of +2.92 repeat units with paternal transmission. Large repeat expansions, of more than 5 repeat units, were seen only in paternally inherited cases. With paternal transmission the magnitude of repeat length alterations was directly correlated to increasing paternal repeat length. Increasing variation in repeat length among siblings was correlated to increasing average repeat length in the sibship in both maternally and paternally inherited HD. Comparison of the magnitude of repeat length alterations to parental age at the time of birth of the offspring showed no correlation.     

Autosomal dominant cerebellar ataxias in the Kinki area of Japan

Summary The autosomal dominant cerebellar ataxias are a heterogeneous group of neurodegenerative disorders characterized by slowly progressive cerebellar ataxia. Recently, among the ataxias, spinocerebellar ataxia type 1 (SCA1), Machado-Joseph disease (MJD) and dentatorubral-pallidoluysian atrophy have been found to be caused by expansion of a CAG trinucleotide repeat in the coding region of the disease genes. We have analyzed the CAG repeats of 67 patients from 47 families with dominantly inherited ataxia who lived in the Kinki area of Japan. The following results were obtained. First, 31 patients from 22 families were found to be positive for the MJD repeat expansion, indicating that MJD is the most common dominantly inherited ataxia in the Kinki area of Japan. Second, no SCA1 repeat expansion was found among the families studied. This presents a striking contrast to the fact that there are many families with SCA1 in Hokkaido and the Tohoku area of Japan. These findings suggest geographic variation in autosomal dominant cerebellar ataxias in Japan.     

Fragile X syndrome: the FMR1 CGG repeat distribution among world populations

Fragile X syndrome (FXS) is characterized by moderate to severe intellectual disability, which is accompanied by macroorchidism and distinct facial morphology. FXS is caused by the expansion of the CGG trinucleotide repeat in the 5' untranslated region of the fragile X mental retardation 1 (FMR1) gene. The syndrome has been studied in ethnically diverse populations around the world and has been extensively characterized in several populations. Similar to other trinucleotide expansion disorders, the gene-specific instability of FMR1 is not accompanied by genomic instability. Currently we do not have a comprehensive understanding of the molecular underpinnings of gene-specific instability associated with tandem repeats. Molecular evidence from in vitro experiments and animal models supports several pathways for gene-specific trinucleotide repeat expansion. However, whether the mechanisms reported from other systems contribute to trinucleotide repeat expansion in humans is not clear. To understand how repeat instability in humans could occur, the CGG repeat expansion is explored through molecular analysis and population studies which characterized CGG repeat alleles of FMR1. Finally, the review discusses the relevance of these studies in understanding the mechanism of trinucleotide repeat expansion in FXS.     

An integrated map of chromosome 18 CAG trinucleotide repeat loci

Expansions of trinucleotide CAG repeats have been demonstrated in at least eight neurodegenerative disorders, and suggested to occur in several others, including bipolar disorder and schizophrenia. Chromosome 18 loci have been implicated in bipolar disorder pedigrees by linkage analysis. To address this putative link between chromosome 18 CAG trinucleotide repeats and neuropsychiatric illness, we have screened a chromosome 18 cosmid library (LL18NCO2" AD") and identified 14 novel candidate loci. Characterisation of these loci involved repeat flank sequencing, estimation of polymorphism frequency and mapping using FISH as well as radiation hybrid panels. These mapped trinucleotide loci will be useful in the investigation of chromosome 18 in neurodegenerative or psychiatric conditions, and will serve to integrate physical and radiation hybrid maps of chromosome 18.     

Intergenerational CAG repeat expansion at ERDA1 in a family with childhood-onset depression, schizoaffective disorder, and recurrent major depression

Reported evidence of anticipation for schizophrenia and bipolar disorder has recently precipitated a search for unstable trinucleotide repeats for these diseases. Several initial studies suggested an increase in the frequency of large CAG/CTG repeats in the genomes of schizophrenic and bipolar individuals. Published reports do not demonstrate expansion per se, and may be suggestive of allelic association with the disease rather than actual dynamic DNA mutations. This report documents evidence of a significant expansion of CAG/CTG repeats from one generation to the next in a family demonstrating evidence of anticipation for psychiatric disorders. Using the repeat expansion detection (RED) technique, we observed that a proband with multiple psychiatric diagnoses, including childhood-onset depression, inherited a larger CAG/CTG repeat than either parent. Analysis of the ERDA1 locus on 17q21.3 revealed that the proband inherited a very large allele from the father which increased in repeat number through transmission. The mother was diagnosed with schizoaffective disorder and the father with depression. While this DNA mutation may be a stochastic event unconnected with the disease, it could represent DNA instability as an etiologic factor in psychiatric diseases.     

Clinical description of an adult male with psychosis who showed FMR1 gene methylation mosaicism

Unstable trinucleotide repeat DNA contained in numerous genes has been proposed as the underlying mechanism in the clinical phenomenon of genetic anticipation in fragile X syndrome and other neurodegenerative diseases. No clear evidence has been found for the role of these abnormal trinucleotide repeat expansion-containing genes in schizophrenia or other psychiatric disorders. This report describes an adult male with psychosis who was later found to have methylation mosaicism of the FMR1 gene. We discuss history, examination, and investigation which led to the diagnosis and treatment response of this patient. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 81:222–224, 1998. © 1998 Wiley-Liss, Inc.     

Somatic stability of the expanded CAG trinucleotide repeat in X-linked spinal and bulbar muscular atrophy

Expansion of trinucleotide repeats has now been associated with eight inherited diseases: X-linked spinal and bulbar muscular atrophy, two fragile X syndromes, myotonic dystrophy, Huntington's disease, spinocerebellar ataxia type I, dentatorubral pallidoluysian atrophy and Machado-Joseph disease. It has been shown that these expanded DNA repeats are unstable in number when transmitted from parents to offspring (“meiotic instability”), while somatic variation in repeat number has also been found in the fragile X syndrome and myotonic dystrophy. Moderate meiotic instability has been demonstrated in X-linked spinal and bulbar muscular atrophy (SBMA, Kennedy's disease). In order to determine if the expanded CAG repeat in SBMA also shows somatic instability, we compared different tissues from two patients with SBMA. We then examined the in vitro stability of the CAG repeat expansion by analyzing fibroblast cell cultures. Length comparison of expanded CAG repeats from all these materials clearly demonstrates that the CAG trinucleotide repeat in SBMA does not exhibit somatic variation. © 1996 Wiley-Liss, Inc.     

Myotonic dystrophy--no evidence for preferential transmission of the mutated allele: a prenatal analysis

Myotonic dystrophy is the commonest autosomal dominant type of muscular dystrophy in adults. It is one of the trinucleotide repeat expansion disorders, and its severity correlates with the number of CTG repeats in the myotonic dystrophy gene. It has been suggested that myotonic dystrophy exhibits the phenomenon of preferential transmission of the larger mutated alleles that has been described in other trinucleotide repeat disorders. Several authors have reported that the frequency of transmission of the mutated alleles is higher than 50%--a finding that, if true, does not comply with the Mendelian laws of segregation. However, these studies were based on data from the analysis of pedigrees with ascertainment bias. In our study, we determined the frequency of transmission of mutated alleles using data from prenatal molecular studies, which are not subject to ascertainment bias. This is the first study to examine the segregation of the mutated alleles in myotonic dystrophy in pregnancy. Eighty-three fetuses were examined, 30 of 62 mothers (48.38%) and 8 of 21 fathers (38.09%) transmitted the mutated allele, giving an overall transmission rate of 45.78%. We found no evidence of statistically significant deviation of the frequency of transmission of the mutated alleles from the 50% expected in autosomal dominant disorders. This study, unlike previous ones, excludes preferential transmission in myotonic dystrophy, a finding that may be attributable to the lack of correction for ascertainment bias in previous studies and to the use of prenatal data in this study.     

Analysis of the trinucleotide CAG repeat from the human mitochondrial DNA polymerase gene in healthy and diseased individuals

The human nuclear gene (POLG) for the catalytic subunit of mitochondrial DNA polymerase (DNA polymerase gamma) contains a trinucleotide CAG microsatellite repeat within the coding sequence. We have investigated the frequency of different repeat-length alleles in populations of diseased and healthy individuals. The predominant allele of 10 CAG repeats was found at a very similar frequency (approximately 88%) in both Finnish and ethnically mixed population samples, with homozygosity close to the equilibrium prediction. Other alleles of between 5 and 13 repeat units were detected, but no larger, expanded alleles were found. A series of 51 British myotonic dystrophy patients showed no significant variation from controls, indicating an absence of generalised CAG repeat instability. Patients with a variety of molecular lesions in mtDNA, including sporadic, clonal deletions, maternally inherited point mutations, autosomally transmitted mtDNA depletion and autosomal dominant multiple deletions showed no differences in POLG trinucleotide repeat-length distribution from controls. These findings rule out POLG repeat expansion as a common pathogenic mechanism in disorders characterised by mitochondrial genome instability.