Molecular genetic analysis of autosomal dominant cerebellar ataxia with retinal degeneration (ADCA type II) caused by CAG triplet repeat expansion

Autosomal dominant cerebellar ataxia with retinal degeneration (ADCAII) was previously mapped by linkage analysis studies to chromosome 3p12- p21.1 (SCA7). Positional cloning efforts have recently identified a novel gene, SCA7 , containing a translated CAG repeat, expanded in SCA7 patients. We cloned the SCA7 gene from a yeast artificial chromosome (YAC) clone contig spanning the SCA7 candidate region. Using a combination of genomic sequencing and cosmid-based exon trapping, two expressed sequence tags were identified. Sequencing of the corresponding cDNA clones and RT-PCR analysis identified the full- length SCA7 cDNA. Together, our sequence data defined the intron/exon boundaries of the first two coding exons of the SCA7 gene, with the first exon containing the expanded CAG repeat. Further, sequence comparison with the published SCA7 cDNA identified one additional putative exon in the 5'-UTR region of the SCA7 gene. The SCA7 gene was mapped on the YAC contig in the 2.5 cM interval between D3S1600 and D3S1287. In one extended Belgian SCA7 pedigree the expanded alleles ranged from 38 to at least 55 repeats with allele lengths being inversely correlated with onset age of ADCAII symptoms. The SCA7 repeats increased in length in successive generations. Normal alleles had from four to 18 repeats, with 10 repeats being the most common allele.      

The repeat expansion detection method in the analysis of diseases with CAG/CTG repeat expansion: Usefulness and limitations

The repeat expansion detection (RED) method was described to detect expansions of trinucleotide repeats of unknown chromosomal location. We have improved the RED method by the use of 8-mer oligonucleotides and assessed its usefulness in 30 samples from patients with spinocerebellar ataxia type 1 (SCA1), Huntington's disease (HD), and Machado Joseph's disease (MJD), for which the number of CAG/CTG repeats was determined by sequencing. There was a good correlation between the number of repeats detected by sequencing and those identified by RED. However, in 17% of samples, the RED gave additional fragments for ligation products of different size than the CAG/CTG repeat expansion detected in the sample by sequencing. The same was observed in a group of control subjects (n = 78) without known clinical abnormalities in which products of more than 40 repeats were detected in 27% of them, indicating that CAG/CTG repeat expansions are common in the general population. Wether this corresponds to unidentified loci with expansions deserves further investigation. Hum Mutat 10:486–488, 1997. © 1997 Wiley-Liss, Inc.     

Physical map and haplotype analysis of 16q-linked autosomal dominant cerebellar ataxia (ADCA) type III in Japan

 Autosomal dominant cerebellar ataxia (ADCA) is a group of heterogeneous neurodegenerative disorders. We previously mapped a gene locus for ADCA with pure cerebellar syndrome (ADCA type III) to a 3-cM region in chromosome 16q, and found a common haplotype among affected individuals. This region was exactly within the locus for another ADCA, spinocerebellar ataxia type 4 (SCA4). To identify the gene causing 16q-linked ADCA type III, we constructed a contig with 38 bacterial artificial chromosome clones between D16S3043 and D16S3095. The size of this contig was estimated to be 4.8 Mb. We found more than 500 nucleotide tandem repeats, including 9 CAG/CTG repeats in this candidate region, although none of the 94 tandem repeats analyzed were expanded in affected individuals. However, we found 11 new polymorphic markers, giving 22 markers spanning the candidate region. By typing these markers on eight Japanese families with ADCA type III, including two new families, we found that a common “founder” haplotype is seen in a more restricted 3.8-Mb region, spanning markers GGAA05 and D16S3095. We present here a newly refined critical interval of 16q-ADCA type III/SCA4. Data of 11 new DNA markers on 16q22.1 would also be useful for other research of genes mapped to this region. Received: June 25, 2002 / Accepted: November 22, 2002 Correspondence to:H. Mizusawa     

Anticipation in schizophrenia: No evidence of expanded CAG/CTG repeat sequences in French families and sporadic cases

A decrease in age of onset of schizophrenia through consecutive family generations (anticipation) has been found in several studies. Anticipation is known to result from expansion of CAG repeats in genes that determine several neurodegenerative disorders. In a previous study we analysed 26 unilineal two-generation French pedigrees and found clinical evidence of anticipation. A 10-year mean reduction in age of onset of schizophrenia was found in the second generation compared with the parental generation. The repeat expansion detection method was used to screen for CAG expansion in 21 of the 26 families with evidence of anticipation for the disease and in 59 sporadic schizophrenics and 59 controls. Comparison of the frequency distributions of CAG/CTG repeat size observed in schizophrenics and controls showed no significant difference, even when we considered familial (P = 0.23) and sporadic (P = 0.25) affected individuals separately. These results do not support the association between long CAG repeats and schizophrenia. However, the possibility that expansions with fewer than 40 repeats are involved in schizophrenia cannot be excluded. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 81:342–346, 1998. © 1998 Wiley-Liss, Inc.     

Exclusion of CAG repeat expansion as the cause of disease in autosomal dominant retinitis pigmentosa families.

The involvement of genes with expanded tracts of (CAG)n in some neurodegenerative diseases is well established. Whether genes containing these motifs could also have a role in degenerative diseases affecting the retina, which is also neural in origin, is unknown. We investigated (CAG)n expansions as a cause of disease in a panel of eight autosomal dominant retinitis pigmentosa (ADRP) pedigrees, including families known to map to the RP9, RP11, and RP13 loci, using the technique known as "repeat expansion detection" (RED). An expansion was detected in one of the unlinked families, but it did not segregate with the disease and was thus nonpathogenic. Expansions were not detected in any other families. In conclusion, expanded (CAG)n repeats are not the cause of disease in the families we have studied, but given the high level of heterogeneity in RP and in retinal degenerations in general they remain strong candidates for involvement in other forms of retinal dystrophy.     

CAG repeat expansion in autosomal dominant familial spastic paraparesis: novel expansion in a subset of patients

Autosomal dominant familial spastic paraplegia (FSP) is a genetically heterogeneous neurodegenerative disorder displaying anticipation for which three loci have been mapped to the chromosomal positions 14q11.2- q24.3 (SPG3), 2p21-p24 (SPG4) and 15q11.1 (SPG6). The repeat expansion detection (RED) method has been used to demonstrate expanded CAG repeats in some FSP families that map to SPG4. We analyzed 20 FSP families, including four for which there is evidence for linkage to SPG4, and found that in most cases the repeat expansion detected by RED is due to non-pathogenic expansions of the chromosome 18q21.1 SEF2-1 or 17q21.3 ERDA1 locus. Polymorphic expansions at SEF2-1 and ERDA1 appear frequent and may confound RED studies in the search for genes causing disorders demonstrating anticipation. In six FSP families, however, CAG repeat expansion was detected in a subset of affected and at-risk individuals that did not result from expansion of the SEF2-1 and ERDA1 loci. Overall, 11 of 37 (30%) of the FSP patients with a CAG/CTG repeat expansion are unaccounted for by the SEF2-1 and ERDA1 loci, compared with two of 23 (9%) of the unaffected at-risk individuals and none of 19 controls. In the majority of cases these novel expansions were shorter than those previously reported.      

Detection of CAG repeats in pre-eclampsia/eclampsia using the repeat expansion detection method

Pre-eclampsia/eclampsia is a serious disorder of human pregnancy that has a worldwide incidence of 2-10% and carries a severe morbidity and mortality risk for both mother and child. Its precise cause remains unknown. However, there is increasing evidence of an underlying complex maternal genetic susceptibility. Its high population incidence in the face of strong negative selection pressure suggests that the gene(s) involved have a selective advantage and/or a high mutation rate. One class of genetic diseases that involve a high mutation rate are the trinucleotide repeat expansion diseases. Thus, the aim of this study was to determine whether there is an association between a trinucleotide (CAG) repeat expansion and pre-eclampsia/eclampsia. We have used the repeat expansion detection (RED) method, which was developed to directly identify clinically significant repeat expansions, to analyse genomic DNA from an Australian and New Zealand population. The maximal CAG repeat length for each individual was recorded and the Mann-Whitney U and Wilcoxon rank sum test for independent samples were used to compare distributions for CAG/CTG repeats between two populations. There were no statistically significant differences between the distribution of CAG repeats in normotensive (n = 59) and severe pre-eclampsia (n = 69) (Mann-Whitney U = 1732; P = 0.14), and normotensive (n = 59) and eclamptic (n = 15) populations (Mann-Whitney U = 417, P = 0.726). Therefore, these RED results do not support a role for a large CAG expansion in pre-eclampsia/eclampsia. However, these data do not preclude the possibility that a small CAG expansion is associated with the disorder nor do they negate the hypothesis that a highly mutable gene contributes to the genetic component of pre-eclampsia/eclampsia.     

Preimplantation genetic diagnosis of spinocerebellar ataxia 3 by (CAG)(n) repeat detection

Spinocerebellar ataxia 3 (SCA3) is an autosomal dominant neurodegenerative disorder characterized by variable expression and a variable age of onset. SCA3/MJD (Machado-Joseph disease) is caused by an expansion of a (CAG)(n) repeat in the MJD1 gene on chromosome 14q32.1. A single cell PCR protocol has been developed for preimplantation genetic diagnosis (PGD) of SCA3 to select unaffected embryos on the basis of the CAG genotype. Single leukocytes and blastomeres served as a single cell amplification test system to determine the percentage of allelic drop-out (ADO) and PCR efficiency. Out of 105 tested heterozygous single leukocytes, 103 (98.1%) showed a positive amplification signal, while five cells (4.9%) showed ADO. Amplification in single blastomeres was obtained in 13 out of a total of 14, and ADO was observed in two out of the 13 single blastomeres. PGD of SCA3 was performed in a couple with paternal transmission of the SCA3 allele. Seven embryos were available for biopsy, all biopsied blastomeres showed amplification and no ADO occurred. One embryo was diagnosed as affected whereas six embryos were diagnosed as unaffected. Two unaffected embryos were transferred and resulted in a singleton pregnancy and the birth of a healthy girl.     

CAG repeat expansion in autosomal dominant pure spastic paraplegia linked to chromosome 2p21-p24

CAG repeat expansions have been identified as the disease-causing dynamic mutations in the coding regions of genes in several dominantly inherited neurodegenerative disorders, including spinobulbar muscular atrophy, Huntington's disease, dentatorubral-pallidoluysian atrophy, spinocerebellar ataxia type 1, 2 and 6 and Machado-Joseph disease. The CAG repeat expansions are translated to elongated polyglutamine tracts and an increased size of the polyglutamine tract correlates with anticipation, the cardinal feature, seen in all these diseases. Autosomal dominant pure spastic peraplegia (ADPSP) is a degenerative disorder of the central motor system clinically characterized by slowly progressive and unremitting spasticity of the legs, hyperreflexia and Babinski's sign. Like the established CAG repeat diseases ADPSP is characterized by both inter- and intrafamilial variation and anticipation. Using the Repeat Expansion Detection (RED) method, we have analyzed 21 affected individuals from six Danish families with the disease linked to chromosome 2p21-p24. We found that 20 of 21 affected individuals showed CAG repeat expansions versus two of 21 healthy spouses, demonstrating a strongly statistically significant association between the occurrence of the repeat expansion and the disease (Fisher's test, P < 10(-5)) suggesting that a CAG repeat expansion is involved presumably as a dynamic mutation in ADPSP linked to chromosome 2p21-p24. The size of the expansion is estimated to be > or = 60 CAG repeat copies in the affected individuals. The CAG repeat expansion is very likely translated and expressed as indicated by the detection of a polyglutamine-containing protein in an ADPSP patient.      

Spinocerebellar ataxia type 2 (SCA 2) in an infant with extreme CAG repeat expansion

Autosomal dominant cerebellar ataxias are a heterogeneous group of neurodegenerative disorders that generally present in adulthood. Spinocerebellar ataxia type 2 typically presents with progressive cerebellar symptoms, slow ocular saccades, and peripheral neuropathy. The onset of symptoms is usually between 20 and 40 years. We describe an infant who presented with neonatal hypotonia, developmental delay, and dysphagia. Ocular findings of retinitis pigmentosa were noted at 10 months. Her father had mild spinocerebellar ataxia first noted at age 22 years. Molecular studies of the SCA2 gene showed a CAG expansion of 43 repeats in the father and an extreme CAG repeat expansion of more than 200 in the baby. Our report expands the known phenotype and genotype of SCA2. Testing for dominant ataxias should be included in the evaluation of infants with nonspecific progressive neurologic symptoms and retinitis pigmentosa, especially in cases with a positive family history for spinocerebellar ataxia. Am. J. Med. Gen. 79:383–387, 1998. © 1998 Wiley-Liss, Inc.     

Somatic mosaicism of the CAG repeat expansion in spinocerebellar ataxia type 3/Machado-Joseph disease

An expanded and unstable CAG repeat in the coding region of the MJD1 gene is the mutation responsible for spinocerebellar ataxia 3/Machado-Joseph disease. In order to determine whether there was a higher degree of instability in affected regions, the size of the expanded CAG repeat was analyzed in different regions of the central nervous system, in two unrelated SCA3/MJD patients. The degree of somatic mosaicism was quantified and compared to that in a SCA1 patient. Instability of the expanded CAG repeat was observed in peripheral tissues as well as in CNS of the three patients, but there was no correlation between the degree of mosaicism and the selective vulnerability of CNS structures. As in the other diseases caused by expanded CAG repeats, a lower degree of mosaicism was found in the cerebellar cortex of both SCA1 and SCA3/MJD patients, probably reflecting specific properties of this structure. In SCA3/MJD, the degree of mosaicism seemed to correlate with age at death rather than with the size of the expanded CAG repeat. Finally, somatic instability was more pronounced in SCA1 than in SCA3/MJD patients. Hum Mutat 11:23–27, 1998. © 1998 Wiley-Liss, Inc.     

Analysis of ERDA1, CTG18.1, and uncloned CAG/CTG repeat sequences in familial Parkinson's disease with anticipation

In several neurodegenerative diseases, anticipation or increase in disease severity in succeeding generations within families correlates with expansions of an intragenic CAG/CTG repeat sequence above the normal range through the generations of a pedigree. Some kindreds of familial Parkinson's disease (PD) exhibit genetic anticipation. We used the repeat expansion detection (RED) method to detect repeat expansions directly in DNA samples from the index cases of 34 different PD families with anticipation. The mean age at onset of the younger probands was 48.8 +/- 10.8 years and the mean intergenerational difference was 19.2 +/- 10 years. The distribution of the RED products greater than 40 repeats was not significantly different between patients and controls with the Mann-Whitney U test (U = 510.5, p = 0.67). The samples were then screened for the two expanded-repeat loci, ERDA1 and CTG18.1. We found that in all cases the repeat expansion detected by the RED method may be accounted for by an expansion at these loci. Our results demonstrate that unstable CAG/CTG expansions corresponding to uncloned or cloned sequences (ERDA1, CTG18.1) are not involved in the etiology of rare familial case of PD with genetic anticipation. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 88:738-741, 1999 Copyright 1999 Wiley-Liss, Inc.     

Molecular and clinical correlations in autosomal dominant cerebellar ataxia with progressive macular dystrophy (SCA7)

Spinocerebellar ataxia 7 (SCA7) is caused by the expansion of an unstable CAG repeat in the first exon of the SCA7 gene. We have analyzed the SCA7 mutation in 19 families and one isolated case of various geographical origins, presenting with autosomal dominant cerebellar ataxia with progressive macular dystrophy. The SCA7 CAG repeat was expanded in 77 patients and in 11 at-risk individuals, with alleles containing from 37 to 130 repeats, demonstrating that SCA7 is genetically homogeneous. Repeats on normal alleles contained from 7 to 35 CAGs. There was a strong negative correlation (r = -0.84) between the age at onset and the size of the CAG repeat expansion in SCA7 patients. Larger expansions were associated with earlier onset, a more severe and rapid clinical course, and a higher frequency of decreased vision, ophthalmoplegia, extensor plantar response and scoliosis. The frequency of other clinical signs such as dysphagia and sphincter disturbances increased with disease duration. The mutation was highly unstable during transmission, with a mean increase of 10 +/- 16 CAG repeats, which was significantly greater in paternal (15 +/- 20) than in maternal (5 +/- 5) transmissions. This correlated well with the marked anticipation (19 +/- 13 years) observed in the families. Gonadal mosaicism, observed in the sperm of a patient, was particularly important, with expanded alleles ranging from 42 to >155 CAG repeats. The degree of instability during transmission, resulting mostly in expansions, is greater than in the seven other neurodegenerative disorders caused by polyglutamine expansions.      

脊髓小脑共济失调患者CAG病理重复次数检测

目的 研究中国汉族人群脊髓小脑性共济失调(spinocerebellar ataxia,SCA)1、2、3、6、7、12、17亚型致病基因的CAG三核苷酸病理重复次数范围.方法 应用聚合酶链反应、琼脂糖凝胶电泳、T载体克隆重组DNA技术并结合直接测序等技术对559例临床诊断为SCA的患者(363例常染色体显性遗传先证者,196例散发患者)进行SCA1、SCA2、SCA3/马查多-约瑟夫病(Machado-Joseph disease,MJD)、SCA6、SCA7、SCA12和SCA17致病基因CAG三核苷酸病理重复次数突变分析.结果 在559例SCA患者中,共检测出SCA1患者23例,CAG病理重复次数范围39～60次,平均(51.09±4.88)次;SCA2患者32例,CAG病理重复次数范围36～51次,平均(40.34±4.40)次;SCA3/MJD患者305例,CAG病理重复次数范围49～86次,平均(73.84±5.07)次;SCA6患者9例,CAG病理重复次数范围23～29次,平均(25.56±1.94)次;SCA7患者27例,CAG病理重复次数范围38～71次,平均(58.22±10.90)次;SCA12患者3例,CAG病理重复次数范围51～52次,平均(51.33±0.58)次;SCA17患者2例,CAG病理重复次数范围53～55次,平均(54.00±1.41)次.结论 SCA1的39次CAG病理重复、SCA3/MJD的49次CAG病理重复和SCA12的51次CAG病理重复为国内或国外报道的最小CAG病理重复次数;SCA3/MJD的86次CAG病理重复为国内外报道的最大CAG病理重复次数;SCA17为国内首次发现的SCA亚型;首次建立中国汉族人群不同SCA亚型CAG三核苷酸病理重复次数范围标准.     

Spinocerebellar ataxia 1 (SCA1) in the Japanese: Analysis of CAG trinucleitide repeat expansion and instability of the repeat for paternal transmission

Summary SCA1 is caused by expansion of an unstable CAG triplet repeat in a novel gene located on the short arm of chromosome 6. In 126 Japanese individuals from 12 pedigrees with SCA1, studies were done to determine if they carried this mutant gene. All the affected and presymptomatic individuals, determined by haplotype segregation analyses, carried an abnormally expanded allele with the range of 39–63 repeat units. This repeat size inversely correlated with the age at onset. However, contrary to reported results, size of the repeat did not correlate with gender of the transmitting parent. Therefore, the CAG triplet repeat instability on paternal transmission is not likely to be fundamental to SCA1.     

Cis-acting modifiers of expanded CAG/CTG triplet repeat expandability: associations with flanking GC content and proximity to CpG islands.

An increasing number of human genetic disorders are associated with the expansion of trinucleotide repeats. The majority of these diseases are associated with CAG/CTG expansions, including Huntington's disease, myotonic dystrophy and many of the spinocerebellar ataxias. Recently, two new expanded CAG/CTG repeats have been identified that are not associated with a phenotype. Expanded alleles at all of these loci are unstable, with frequent length changes during intergenerational transmission. However, variation in the relative levels of instability, and the size and direction of the length change mutations observed, between the CAG/CTG loci is apparent. We have quantified these differences, taking into account effects of progenitor allele length, by calculating the relative expandability of each repeat. Since the repeat motifs are the same, these differences must be a result of flanking sequence modifiers. We present data that indicate a strong correlation between the relative expandability of these repeats and the flanking GC content. Moreover, we demonstrate that the most expandable loci are all located within CpG islands. These data provide the first insights into the molecular bases of cis -acting flanking sequences modifying the relative mutability of dispersed expanded human triplet repeats.     

Pms2 is a genetic enhancer of trinucleotide CAG.CTG repeat somatic mosaicism: implications for the mechanism of triplet repeat expansion

The expansion of CAG.CTG repeat sequences is the cause of several inherited human disorders. Longer alleles are associated with an earlier age of onset and more severe symptoms, and are highly unstable in the germline and soma with a marked tendency towards repeat length gains. Germinal expansions underlie anticipation; whereas age-dependent, tissue-specific, expansion-biased somatic instability probably contributes toward the progressive nature and tissue-specificity of the symptoms. The mechanism(s) of repeat instability is not known, but recent data have implicated mismatch-repair (MMR) gene mutS homologues in driving expansion. To gain further insight into the expansion mechanism, we have determined the levels of somatic mosaicism of a transgenic expanded CAG.CTG repeat in mice deficient for the Pms2 MMR gene. Pms2 is a MutL homologue that plays a critical role in the downstream processing of DNA mismatches. The rate of somatic expansion was reduced by approximately 50% in Pms2-null mice. A higher frequency of rare, but very large, deletions was also detected in these animals. No significant differences were observed between Pms2(+/+) and Pms2(+/-) mice, indicating that a single functional Pms2 allele is sufficient to generate normal levels of somatic mosaicism. These findings reveal that as well as MMR enzymes that directly bind mismatched DNA, proteins that are subsequently recruited to the complex also play a central role in the accumulation of repeat length changes. These data suggest that somatic expansion results not by replication slippage, single stranded annealing or simple MutS-mediated stabilization of secondary structures, but by inappropriate DNA MMR.     

Expanded RED products and loci containing CAG/CTG repeats on chromosome 17 (ERDA1) and chromosome 18 (CTG18.1) in trans-generational pairs with bipolar affective disorder.

The purpose of the present study was to further test if expanded CAG repeats detected by the repeat expansion detection (RED) method in bipolar affective disorder (BPAD) are correlated with ERDA1 (17q21.3) and/or CTG18.1 (18q21.1) loci expansions, and changes of phenotype severity in successive generations (anticipation). The sample was designed to analyze ERDA1 and CTG18.1 expansions in trans-generational pairs of affected individuals (parent-offspring pairs: G1 and G2). Clinical and genetic information was available on 95 two-generations pairs. We found in our sample no one patient carrying an expanded allele at the CTG18.1 locus. This observation is true for all individuals in G1 and G2. Using the conditional logistic regression, no statistical difference was observed between the two generations for ERDA1 alleles (chi(2) = 0.2, P = 0.65). These data do not support the correlation between expanded RED products (RED fragments >120) and expanded alleles at ERDA1 in trans-generational pairs with BPAD. We were not able to detect any correlation for CTG18.1. Earlier age at onset in offspring generation was also not associated with expanded RED products explained by expanded ERDA1 alleles.