Molecular analysis of CAG repeats at five different spinocerebellar ataxia loci: correlation and alternative explanations for disease pathogenesis

Spinocerebellar ataxias (SCAs) are caused by expansion of (CAG)n triplet repeats. These repeats occur as polymorphic forms in general population; however, beyond a threshold size they become pathogenic. The sizes and distributions of repeats at the SCA1, SCA2, SCA3, SCA7 and DRPLA loci were assessed by molecular analysis of 124 unrelated ataxia patients and 44 controls, and the association of larger normal (LN) alleles with disease prevalence was evaluated. Triplet repeat expansions in the disease range were detected in 8% (10/124) of the cases, with the majority having expansion at the SCA1 locus. Normal allele ranges in the cohort studied were similar to the Caucasian and North Indian populations but differed from the Korean and Japanese populations at various loci. The percentage of individuals with LN alleles at the SCA1 and SCA2 loci was higher than reported in Indians, Japanese and Caucasians. LN alleles showed a good correlation with the incidence of SCA1, indicating that SCA1 is the most prevalent ataxia in our population. The majority of cases with clinical symptoms of SCA could not be diagnosed by established CAG repeat criteria, suggesting that there may be an alternative basis for disease pathogenesis: (i) Repeats lower than the normal range may also result in abnormal phenotypes (ii) LN alleles at different loci in the same individual may contribute to symptoms (iii) Exogenous factors may play a role in triggering disease symptoms in individuals with LN alleles (iv) Triplet repeats may reach the disease range in the brain but not in the blood.     

Molecular analysis of Spinocerebellar ataxias in Koreans: frequencies and reference ranges of SCA1, SCA2, SCA3, SCA6, and SCA7.

Spinocerebellar ataxias (SCAs) are a heterogeneous group of neurodegenerative disorders. CAG repeat expansions in the causative genes have been identified as the basic cause of several types of SCAs, and have been used for the diagnoses and classifications of patients with ataxia. In order to assess the frequency and CAG repeat size ranges of SCAs, and to establish an effective strategy for molecular diagnosis, we performed a molecular analysis of SCA1, SCA2, SCA3, SCA6, and SCA7 in 76 patients. These patients were as follows: 32 with dominant inheritance, 39 sporadic cases, and 5 with unknown family histories. The normal and affected CAG repeat size ranges were established at five SCA loci in Koreans, which was consistent with previous reports. The total prevalence of the five types of SCAs was 39.5% in the 76 patients with ataxia, regardless of their family history. It was 75.0% in the 32 families with a dominant inheritance. The most frequent type was SCA3 (15.8%), followed by SCA2 (14.5%). Both types combined formed 76.7% of the 30 patients with CAG expansions. SCA1, SCA6, and SCA7 were less frequent, affecting 3.9%, 2.6%, and 2.6% of the cases, respectively. This mutation spectrum is quite different from a previous report concerning Koreans, but is similar to the distributions that are seen in several ethnic populations worldwide. For a correct and effective diagnosis of SCAs, we suggest that a molecular diagnosis be undertaken, even in patients without a family history, as well as those with a family history. A stepwise approach is also recommended. Patients with ataxia should be tested for SCA2 and SCA3. Individuals testing negative should be tested for SCA1, SCA6, and SCA7.     

Distribution of CAG repeat size in the dentatorubral and pallidoluysian atrophy (DRPLA) gene in a normal population in Taiwan

Dentatorubral and pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder with expansion of trinucleotide CAG repeats in the coding region of the gene. Expansion of the repeat tract beyond the normal range produces gene products with extended polyglutamine tracts. In this study, we analyzed the distribution of the CAG repeats in the DRPLA alleles in a normal Taiwanese population. We observed 15 different alleles and found that the range of the CAG repeat number was from 7-21. The most frequent allele contained 15 CAG repeats that represented 20% of the total analyzed alleles, followed by the 17 repeats (15.8%). The heterozygosity rate of this locus was 88%. Twelve parents-to-children transmissions of the DRPLA alleles in a Machado-Joseph disease family appeared to be normal without any alteration of the CAG repeat numbers. Phenotypes of DRPLA overlapped those of autosomal dominant cerebellar ataxia (ADCA). In order to identify DRPLA patients in Taiwan, we screened six autosomal dominant cerebellar ataxia patients without expansion in known spinocerebellar ataxia genes. All six patients had the repeat numbers within the normal range; thus, the possibility of DRPLA could be excluded.     

SCA8 repeat expansion: large CTA/CTG repeat alleles are more common in ataxic patients,including those with SCA6

We analyzed the SCA8 CTA/CTG repeat in a large group of Japanese subjects. The frequency of large alleles (85-399 CTA/CTG repeats) was 1.9% in spinocerebellar ataxia (SCA), 0.4% in Parkinson disease, 0.3% in Alzheimer disease, and 0% in a healthy control group; the frequency was significantly higher in the group with SCA than in the control group. Homozygotes for large alleles were observed only in the group with SCA. In five patients with SCA from two families, a large SCA8 CTA/CTG repeat and a large SCA6 CAG repeat coexisted. Age at onset was correlated with SCA8 repeats rather than SCA6 repeats in these five patients. In one of these families, at least one patient showed only a large SCA8 CTA/CTG repeat allele, with no large SCA6 CAG repeat allele. We speculate that the presence of a large SCA8 CTA/CTG repeat allele influences the function of channels such as alpha(1A)-voltage-dependent calcium channel through changing or aberrant splicing, resulting in the development of cerebellar ataxia, especially in homozygous patients.     

Expanded CAG repeats in spinocerebellar ataxia (SCA1) segregate with distinct haplotypes in South African families

The autosomal dominant late onset spinocerebellar ataxias (SCAs) are genetically heterogeneous. Three genes, SCA1 on 6p, SCA2 on 12q and MJD1 on 14q, have been isolated for SCA1, SCA2 and Machado-Joseph disease (MJD), respectively. In these three autosomal dominant disorders the mutation is an expanded CAG repeat. Evidence for heterogeneity in families not linked to the SCA1, SCA2 and MJD loci is provided by the mapping of SCA loci to chromosomes 16q, 11cen and 3p. A total of 14 South African kindreds and 22 sporadic individuals with SCA were investigated for the expanded SCA1 and MJD repeats. None of the families nor the sporadic individuals showed expansion of the MJD repeat. Expanded SCA1 and CAG repeats were found to cosegregate with the disorder in six of the families tested and were also observed in one sporadic individual with a negative family history of SCA. The use of the microsatellite markers D6S260, D6S89 and D6S274 provided evidence that the expanded SCA1 repeats segregated with three distinct haplotypes in the six families. Use of the highly polymorphic tightly linked microsatellite markers is still important as this stage, particularly where this coincides with the possibility of a homozygous genotype with the trinucleotide repeat marker. Importantly, our molecular findings indicate: (1) an absence of MJD expanded repeats underlying SCA; (2) the major disease in this group is due to mutations in the SCA1 gene; and (3) the familial disorder in the majority population group (i.e. mixed ancestry) in the Western Cape region of South Africa is most likely to be the result of two distinct founder events. Received: 4 November 1996 / Accepted: 6 February 1997     

Polymorphisms at 13 expressed human sequences containing CAG/CTG repeats and analysis in autosomal dominant cerebellar ataxia (ADCA) patients

Genetic anticipation – increasing severity and a decrease in the age of onset with successive generations of a pedigree – is clearly present in autosomal dominant cerebellar ataxia (ADCA). Anticipation is correlated with expansion of the CAG/CTG repeat sequence to sizes above those in the normal range through the generations of a pedigree. Genetic heterogeneity has been demonstrated for ADCA, with four cloned genes (SCA1, SCA2, SCA3/MJD, and SCA6) and three mapped loci (SCA4, SCA5 and SCA7). Another related dominant ataxia, dentatorubral-pallidoluysian atrophy (DRPLA), presents anticipation with CAG/CTG repeat expansions. We had previously analysed ADCA patients who had not shown repeat expansions in cloned genes for CAG/CTG repeat expansions by the repeat expansion detection method (RED) and had detected expansions of between 48 and 88 units in 17 unrelated familial cases. We present here an analysis of 13 genes and expressed sequence tags (ESTs) containing 10 or more CAG/ CTG repeat sequences selected from public databases in the 17 unrelated ADCA patients. Of the 13 selected genes and ESTs, 9 were found to be polymorphic with heterozygosities ranging between 0.09 and 0.80 and 2 to 17 alleles. In ADCA patients none of the loci showed expansions above the normal range of the CAG/CTG repeat sequences, excluding them as the mutation causing ADCA. Received: 28 May 1997 / Accepted: 30 June 1997     

High germinal instability of the (CTG)n at the SCA8 locus of both expanded and normal alleles

The autosomal dominant spinocerebellar ataxias (SCAs) are a group of late-onset, neurodegenerative disorders for which 10 loci have been mapped (SCA1, SCA2, SCA4-SCA8, SCA10, MJD, and DRPLA). The mutant proteins have shown an expanded polyglutamine tract in SCA1, SCA2, MJD/SCA3, SCA6, SCA7, and DRPLA; a glycine-to-arginine substitution was found in SCA6 as well. Recently, an untranslated (CTG)n expansion on chromosome 13q was described as being the cause of SCA8. We have now (1) assessed the repeat size in a group of patients with ataxia and a large number of controls, (2) examined the intergenerational transmission of the repeat, and (3) estimated the instability of repeat size in the sperm of one patient and two healthy controls. Normal SCA8 chromosomes showed an apparently trimodal distribution, with classes of small (15-21 CTGs), intermediate (22-37 CTGs), and large (40-91 CTGs) alleles; large alleles accounted for only0.7% of all normal-size alleles. No expanded alleles (>/=100 CTGs) were found in controls. Expansion of the CTG tract was found in five families with ataxia; expanded alleles (all paternally transmitted) were characterized mostly by repeat-size contraction. There was a high germinal instability of both expanded and normal alleles: in one patient, the expanded allele (152 CTGs) had mostly contraction in size (often into the normal range); in the sperm of two normal controls, contractions were also more frequent, but occasional expansions into the upper limit of the normal size range were also seen. In conclusion, our results show (1) no overlapping between control (15-91) and pathogenic (100-152) alleles and (2) a high instability in spermatogenesis (both for expanded and normal alleles), suggesting a high mutational rate at the SCA8 locus.     

Population Genetics and New Insight into Range of CAG Repeats of Spinocerebellar Ataxia Type 3 in the Han Chinese Population

Spinocerebellar ataxia type 3 (SCA3), also called Machado-Joseph disease (MJD), is one of the most common SCAs worldwide and caused by a CAG repeat expansion located in ATXN3 gene. Based on the CAG repeat numbers, alleles of ATXN3 can be divided into normal alleles (ANs), intermediate alleles (AIs) and expanded alleles (AEs). It was controversial whether the frequency of large normal alleles (large ANs) is related to the prevalence of SCA3 or not. And there were huge chaos in the comprehension of the specific numbers of the range of CAG repeats which is fundamental for genetic analysis of SCA3. To illustrate these issues, we made a novel CAG repeat ladder to detect CAG repeats of ATXN3 in 1003 unrelated Chinese normal individuals and studied haplotypes defined by three single nucleotide polymorphisms (SNPs) closed to ATXN3. We found that the number of CAG repeats ranged from 13 to 49, among them, 14 was the most common number. Positive skew, the highest frequency of large ANs and 4 AIs which had never been reported before were found. Also, AEs and large ANs shared the same haplotypes defined by the SNPs. Based on these data and other related studies, we presumed that de novo mutations of ATXN3 emerging from large ANs are at least one survival mechanisms of mutational ATXN3 and we can redefine the range of CAG repeats as: ANs≤44, 45 ≤AIs ≤49 and AEs≥50.     

Genetic polymorphism at spinocerebellar ataxia 1 and 2 loci in Brazil

Dynamic mutation involves the expansion of a tandem arrayed DNA sequence that is polymorphic in the population. This mechanism is associated with neurological/neuromuscular disorders and the pathology depends on the extension of the repeated tract, with a specific threshold for each disease. We made a PCR-based characterization of allelic polymorphism of SCA1 and SCA2 loci in a sample of 200 pairs of chromosomes in a population in Rio de Janeiro and found 23 different alleles at the SCA1 locus, varying from 10 to 39 CAG repeats (mean 27.7 +/- 3.3, mode 28) and 10 different alleles ranging from 19 to 29 CAG (mean 22.1 +/- 1.0, mode 22) at the SCA2 locus. The level of heterozygosis was 53% (SCA1) and 8% (SCA2).     

Molecular analysis of autosomal dominant hereditary ataxias in the Indian population: high frequency of SCA2 and evidence for a common founder mutation

Expansion of CTG/CAG trinucleotide repeats has been shown to cause a number of autosomal dominant cerebellar ataxias (ADCA) such as SCA1, SCA2, SCA3/ MJD, SCA6, SCA7, SCA8 and DRPLA. There is a wide variation in the clinical phenotype and prevalence of these ataxias in different populations. An analysis of ataxias in 42 Indian families indicates that SCA2 is the most frequent amongst all the ADCAs we have studied. In the SCA2 families, together with an intergenerational increase in repeat size, a horizontal increase with the birth order of the offspring was also observed, indicating an important role for parental age in repeat instability. This was strengthened by the detection of a pair of dizygotic twins with expanded alleles showing the same repeat number. Haplotype analysis indicates the presence of a common founder chromosome for the expanded allele in the Indian population. Polymorphism of CAG repeats in 135 normal individuals at the SCA loci studied showed similarity to the Caucasian population but was significantly different from the Japanese population.     

Elongated CAG repeats of the B37 gene in a Danish family with dentato-rubro-pallido-luysian atrophy

Dentato-rubro-pallido-luysian atrophy (DRPLA) is considered to be rare in Europe. We describe a Danish family in which affected individuals in at least three generations have been diagnosed as suffering from Huntington's disease. Because analysis of the Huntingtin gene revealed normal alleles and various of the patients had seizures, we analysed the B37 gene and found significantly elongated CAG repeats as have been reported in DRPLA. Affected individuals with almost identical repeat lengths presented very different symptoms. Both expansion and contraction in paternal transmission was encountered.     

Polymorphism of trinucleotide repeats in non-translated regions of SCA8 and SCA12 genes: allele distribution in a Polish control group

Spinocerebellar ataxias are a group of neurodegenerative disorders caused by dynamic mutations of microsatellite repeats. Two novel forms of SCAs have been described recently: SCA8, with expansions of CTA/CTG repeats in 3'UTR of the SCA8 gene, and SCA12, caused by expansion of the CAG tract in 5'UTR of the SCA12/PP2R2B gene. Analysis of CTA/CTG and CAG polymorphism in those two genes was performed in a Polish control group consisting of 100 individuals without any neurological signs. The distribution and ranges of the number of non-pathogenic repeats were similar to those observed in other populations described previously. Expansion of CTA/CTG repeats in the SCA8 locus was found in 2 of 100 controls and in 5 probands among 150 pedigrees affected with unidentified ataxias. As such expanded alleles were also observed in their healthy relatives, the pathogenic role of expansions in the SCA8 gene remains uncertain.     

脊髓小脑共济失调第7型的临床特征及基因突变研究

对一脊髓小脑性共济失调(Spinocerebellar ataxia, SCA)家系的患者进行临床特征及相关基因突变研究。对该家系进行详细的病史采集, 并对患者行视力、眼底血管造影、眼底拍照、视觉诱发电位、视网膜电图以及头颅MRI等辅助检查; 采用聚合酶链反应分别扩增SCA1、SCA2、SCA3、SCA6、SCA7、SCA17及DRPLA基因的CAG重复序列, 用8%变性聚丙烯酰胺凝胶电泳及直接测序进行突变分析。结果2名患者主要表现为小脑性共济失调、视力下降、眼底视网膜色素变性、小脑和脑干萎缩; 并存在SCA7基因的突变, 而未发现SCA1、SCA2、SCA3、SCA6、SCA17及DRPLA基因突变。说明该家系为SCA7突变家系, SCA7基因中CAG三核苷酸重复拷贝数的异常扩增是其致病原因。     

Patterns of instability of expanded CAG repeats at the ERDA1 locus in general populations.

A highly polymorphic CAG repeat locus, ERDA1, was recently described on human chromosome 17q21.3, with alleles as large as 50-90 repeats and without any disease association in the general population. We have studied allelic distribution at this locus in five human populations and have characterized the mutational patterns by direct observation of 731 meioses. The data show that large alleles (>/=40 CAG repeats) are generally most common in Asian populations, less common in populations of European ancestry, and least common among Africans. We have observed a high intergenerational instability (46. 3%+/-5.1%) of the large alleles. Although the mutation rate is not dependent on parental sex, paternal transmissions have predominantly resulted in contractions, whereas maternal transmissions have yielded expansions. Within this class of large alleles, the mutation rate increases concomitantly with increasing allele size, but the magnitude of repeat size change does not depend on the size of the progenitor allele. Sequencing of specific alleles reveals that the intermediate-sized alleles (30-40 repeats) have CAT/CAC interruptions within the CAG-repeat array. These results indicate that expansion and instability of trinucleotide repeats are not exclusively disease-associated phenomena. The implications of the existence of massively expanded alleles in the general populations are not yet understood.     

An unstable trinucleotide-repeat region on chromosome 13 implicated in spinocerebellar ataxia: a common expansion locus

Larger CAG/CTG trinucleotide-repeat tracts in individuals affected with schizophrenia (SCZ) and bipolar affective disorder (BPAD) in comparison with control individuals have previously been reported, implying a possible etiological role for trinucleotide repeats in these diseases. Two unstable CAG/CTG repeats, SEF2-1B and ERDA1, have recently been cloned, and studies indicate that the majority of individuals with large repeats as detected by repeat-expansion detection (RED) have large repeat alleles at these loci. These repeats do not show association of large alleles with either BPAD or SCZ. Using RED, we have identified a BPAD individual with a very large CAG/CTG repeat that is not due to expansion at SEF2-1B or ERDA1. From this individual's DNA, we have cloned a highly polymorphic trinucleotide repeat consisting of (CTA)n (CTG)n, which is very long ( approximately 1,800 bp) in this patient. The repeat region localizes to chromosome 13q21, within 1.2 cM of fragile site FRA13C. Repeat alleles in our sample were unstable in 13 (5.6%) of 231 meioses. Large alleles (>100 repeats) were observed in 14 (1. 25%) of 1,120 patients with psychosis, borderline personality disorder, or juvenile-onset depression and in 5 (.7%) of 710 healthy controls. Very large alleles were also detected for Centre d'Etude Polymorphisme Humaine (CEPH) reference family 1334. This triplet expansion has recently been reported to be the cause of spinocerebellar ataxia type 8 (SCA8); however, none of our large alleles above the disease threshold occurred in individuals either affected by SCA or with known family history of SCA. The high frequency of large alleles at this locus is inconsistent with the much rarer occurrence of SCA8. Thus, it seems unlikely that expansion alone causes SCA8; other genetic mechanisms may be necessary to explain SCA8 etiology.     

CAG repeat analyses in frozen and formalin-fixed tissues following primer extension preamplification for evaluation of mitotic instability of expanded SCA1 alleles

Neurodegenerative disorders, including spinocerebellar ataxias (SCA), Huntington disease (HD) and dentatorubral-pallidoluysian atrophy (DRPLA), are associated with unstable CAG repeats. To investigate the mitotic stability of the repetitive element in the genes for SCA1, SCA3, HD, and DRPLA we extracted DNA from up to 13 tissue samples from four deceased individuals with progressive neurological disorders and neuropathological signs. Due to the formalin fixation of some tissues the genomic DNA was highly degraded and unsuitable for amplification of fragments longer than 150 bp. After size selection and primer extension preamplification, specific analyses could be performed even for expanded alleles. In all four patients the SCA1 mutation could be demonstrated, in one case with remarkable somatic heterogeneity of the elongated allele, whereas alleles of the normal range were stable in all tissues examined. Received: 3 February 1997 / Accepted: 9 April 1997     

Mitotic and meiotic instability of the CAG trinucleotide repeat in spinocerebellar ataxia type 1

Spinocerebellar ataxia type 1 (SCA1) is an autosomal, dominantly inherited neurodegenerative disease caused by an unstable CAG trinucleotide repeat expansion in the ataxin-1 gene located on chromosome 6p22-p23. The expanded CAG repeat is unstable during transmission, and a variation in the CAG repeat length has been found in different tissues, including sperm samples from affected males. In order further to examine the mitotic and meiotic instability of the (CAG)_n stretch we have performed single sperm and low-copy genome analysis in SCA1 patients and asymptomatic carriers. A pronounced variation in the size of the expanded allele was found in sperm cells and peripheral blood leucocytes, with a higher degree of instability seen in the sperm cells, where an allele with 50 repeat units was contracted in 11.8%, further expanded in 63.5% and unchanged in 24.6% of the single sperm analysed. We found a low instability of the normal alleles; the normal alleles from the individuals carrying a CAG repeat expansion were significantly more unstable than the normal alleles from the control individuals (P<0.001), indicating an interallelic interaction between the expanded and the normal alleles. Received: 8 June 1998 / Accepted: 10 September 1998     

Somatic mosaicism of expanded CAG repeats in brains of patients with dentatorubral-pallidoluysian atrophy: cellular population-dependent dynamics of mitotic instability.

Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disease caused by unstable expansion of a CAG repeat in the DRPLA gene. We performed detailed quantitative analysis of the size and the size distribution (range) of the expanded CAG repeats in various regions of the CNS of eight autopsied patients with DRPLA. Expanded alleles (AE) showed considerable variations in size, as well as in range, depending on the region of the CNS, whereas normal alleles did not show such variations, which indicates the occurrence of somatic mosaicism of AE in the CNS. The AE in the cerebellar cortex were consistently smaller by two to five repeat units than those in the cerebellar white matter. Moreover, the AE in the cerebral cortex were smaller by one to four repeat units than those in the cerebral white matter. These results suggest that the smaller AE in the cerebellar and cerebral cortices represent those of neuronal cells. The ranges of the AE in the cerebral cortex, cerebral white matter, and cerebellar white matter showed considerable variation ranging from 9 to 23 repeat units, whereas those in the cerebellar cortex showed little variance and were approximately 7 repeat units. The ranges of the AE in the cerebral cortex, cerebral white matter, and cerebellar white matter were much broader in patients with higher ages at death than they were in patients with lower ages at death, raising the possibility that the range of AE increases with time, as the result of mitotic instability of AE.