Reevaluation of the exact CAG repeat length in hereditary cerebellar ataxias using highly denaturing conditions and long PCR

Hereditary cerebellar ataxias, including spinocerebellar ataxia type I (SCA1), dentato-rubro-pallidoluysian atrophy (DRPLA), and Machado-Joseph disease (MJD), have been associated with unstable CAG repeats. The length of the CAG repeat is a major factor in determining the age of onset of these diseases. In electrophoresis through acrylamide gels with formamide, the CAG repeat length following the polymerase chain reaction (PCR) coincides with the sequence-determined repeat length after subcloning. However, without formamide, PCR products with long CAG repeats appear 1–4 repeats shorter than when electrophoresed with formamide, and the repeat lengths are variable. In addition, the larger the CAG repeats are, the more difficult are the PCR reactions. A mixture containing thermostable Taq and Pwo DNA polymerases (so-called “long PCR”) is much more sensitive than that with Taq polymerase alone in detecting expanded CAG repeats. Therefore, highly denaturing conditions, especially formamide gel electrophoresis, and the “long PCR” protocol should be used to evaluate the exact CAG repeat length. We have used these principles to detect unstable CAG repeats. The normal ranges are 14–34 repeats for MJD, 6–31 repeats for DRPLA, and 21–32 repeats for SCA1. Received: 29 August 1995 / Revised: 12 October 1995     

Spinocerebellar ataxias in Spanish patients: genetic analysis of familial and sporadic cases

Autosomal dominant cerebellar ataxias (ADCA) are a clinically heterogeneous group of neurodegenerative disorders caused by unstable CAG repeat expansions encoding polyglutamine tracts. Five spinocerebellar ataxia genes (SCA1, SCA2, SCA3, SCA6 and SCA7) and another related dominant ataxia gene (DRPLA) have been cloned, allowing the genetic classification of these disorders. We present here the molecular analysis of 87 unrelated familial and 60 sporadic Spanish cases of spinocerebellar ataxia. For ADCA cases 15% were SCA2, 15% SCA3, 6% SCA1, 3% SCA7, 1% SCA6 and 1% DRPLA, an extremely rare mutation in Caucasoid populations. About 58% of ADCA cases remained genetically unclassified. All the SCA1 cases belong to the same geographical area and share a common haplotype for the SCA1 mutation. The expanded alleles ranged from 41 to 59 repeats for SCA1, 17 to 29 for SCA2, 67 to 77 for SCA3, and 38 to 113 for SCA7. One SCA6 case had 25 repeats and one DRPLA case had 63 repeats. The highest CAG repeat variation in meiotic transmission of expanded alleles was detected in SCA7, this being of +67 units in one paternal transmission and giving rise to a 113 CAG repeat allele in a patient who died at 3 years of age. Meiotic transmissions have also shown a tendency to more frequent paternal transmission of expanded alleles in SCA1 and maternal in SCA7. All SCA1 and SCA2 expanded alleles analyzed consisted of pure CAG repeats, whereas normal alleles were interrupted by 1–2 CAT trinucleotides in SCA1, except for three alleles of 6, 14 and 21 CAG repeats, and by 1–3 CAA trinucleotides in SCA2. No SCA or DRPLA mutations were detected in the 60 sporadic cases of spinocerebellar ataxia, but one late onset patient was identified as a recessive form due to GAA-repeat expansions in the Friedreich’s ataxia gene. Received: 6 January 1999 / Accepted: 18 March 1999     

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。     

Analysis of the allele polymorphism of (CTG)n and (GAG)n triplet repeats in DM,DRPLA, and SCA1 genes in various populations of Russia

Polymorphism of highly polymorphic triplet repeats CTG of the 3'-untranslated region of the myotonin protein kinase gene and CAG of the genes associated with dentatorobral-pallidoluysian atrophy (DRPLA, or Hew River syndrome) and spinocerebellar ataxia type 1 (SCA1) was analyzed in several ethnic populations of Russia. A difference in allele spectra of the three genes was demonstrated for populations differing in ethnic origin.     

Japanese families with autosomal dominant pure cerebellar ataxia map to chromosome 19p13.1-p13.2 and are strongly associated with mild CAG expansions in the spinocerebellar ataxia type 6 gene in chromosome 19p13.1.

Autosomal dominant cerebellar ataxia is a group of clinically and genetically heterogeneous disorders. We carried out genomewide linkage analysis in 15 families with autosomal dominant pure cerebellar ataxia (ADPCA). Evidence for linkage to chromosome 19p markers was found in nine families, and combined multipoint analysis refined the candidate region to a 13.3-cM interval in 19p13.1-p13.2. The remaining six families were excluded for this region. Analysis of CAG-repeat expansion in the alpha1A-voltage-dependent calcium channel (CACNL1A4) gene lying in 19p13.1, recently identified among 8 small American kindreds with ADPCA (spinocerebellar ataxia type 6 [SCA6]), revealed that 8 of the 15 families studied had similar, very small expansion in this gene: all affected individuals had larger alleles (range of CAG repeats 21-25), compared with alleles observed in neurologically normal Japanese (range 5-20 repeats). Inverse correlation between the CAG-repeat number and the age at onset was found in affected individuals with expansion. The number of CAG repeats in expanded chromosomes was completely stable within each family, which was consistent with the fact that anticipation was not statistically proved in the SCA6 families that we studied. We conclude that more than half of Japanese cases of ADPCA map to 19p13.1-p13.2 and are strongly associated with the mild CAG expansion in the SCA6/CACNL1A4 gene.     

Analysis of the Allele Polymorphism of (CTG)nand (CAG)n Triplet Repeats in Loci DM, DRPLA, and SCA1 in Several Populations of Russia

Polymorphism of highly polymorphic triplet repeats CTG of the 3"-untranslated region of the myotonin protein kinase gene and CAG of the genes associated with dentatorubral-pallidoluysian atrophy (DRPLA, or Hew River syndrome) and spinocerebellar ataxia type 1 (SCA1) was analyzed in several ethnic populations of Russia. A difference in allele spectra of the three genes was demonstrated for populations differing in ethnic origin.     

Marked Phenotypic Heterogeneity Associated with Expansion of a CAG Repeat Sequence at the Spinocerebellar Ataxia 3/Machado-Joseph Disease Locus

The spinocerebellar ataxia 3 locus (SCA3) for type I autosomal dominant cerebellar ataxia (ADCA type I), a clinically and genetically heterogeneous group of neuro-degenerative disorders, has been mapped to chromosome 14q32.1. ADCA type I patients from families segregating SCA3 share clinical features in common with those with Machado-Joseph disease (MJD), the gene of which maps to the same region. We show here that the disease gene segregating in each of three French ADCA type I kindreds and in a French family with neuropatho-logical findings suggesting the ataxochoreic form of dentatorubropallidoluysian atrophy carries an expanded CAG repeat sequence located at the same locus as that for MJD. Analysis of the mutation in these families shows a strong negative correlation between size of the expanded CAG repeat and age at onset of clinical disease. Instability of the expanded triplet repeat was not found to be affected by sex of the parent transmitting the mutation. Evidence was found for somatic and gonadal mosaicism for alleles carrying expanded trinucleotide repeats.     

Genetic background of apparently idiopathic sporadic cerebellar ataxia

Disease-causing mutations have been identified in various entities of autosomal dominant ataxia and in Friedreich's ataxia. However, no molecular pathogenic factor is known to cause idiopathic cerebellar ataxias. We investigated the CAG/CTG trinucleotide repeats causing spinocerebellar ataxia types 1, 2, 3, 6, 7, 8 and 12, and the GAA repeat of the frataxin gene in 124 patients apparently suffering from idiopathic sporadic ataxia, including 20 patients with the clinical diagnosis of multiple system atrophy. Patients with a positive family history, a typical Friedreich phenotype, or symptomatic ataxia were excluded. Genetic analyses uncovered the most common Friedreich mutation in 10 patients with an age at onset between 13 and 36 years. The SCA6 mutation was present in nine patients with disease onset between 47 and 68 years of age. The CTG repeat associated with SCA8 was expanded in three patients. One patient had SCA2 attributable to a de novo mutation from a paternally transmitted, intermediate allele. We did not identify the SCA1, SCA3, SCA7 or SCA12 mutation in idiopathic sporadic ataxia patients. No trinucleotide repeat expansion was detected in the MSA subgroup. This study has revealed the genetic basis in 19% of apparently idiopathic ataxia patients. SCA6 is the most frequent mutation in late onset cerebellar ataxia. The frataxin trinucleotide expansion should be investigated in all sporadic ataxia patients with onset before age 40, even when the phenotype is atypical for Friedreich's ataxia.     

广西一脊髓小脑共济失调3型家系SCA3/MJD基因突变和多态性的分析

常染色体显性脊髓小脑型共济失调(Autosomal dominant spinocerebellar ataxias, ADCAs)是一种神经系统退行性疾病, 具有高度的遗传异质性, 其中脊髓小脑型共济失调3型(Spinocerebellar ataxias type 3, SCA3)是一种常见的类型。文章通过PCR扩增广西一个脊髓小脑共济失调家系SCA3/MJD基因片段, 用毛细管电泳和测序方法检测了SCA3/MJD基因的CAG重复序列大小、传递特点以及SCA3/MJD基因的变异。结果显示：家系的所有4名患者和3名无症状携带者(Asymptomatic carrier)的SCA3/MJD基因第10外显子中存在异常扩增的CAG重复序列, 重复次数为64~71次; CAG重复次数在具有cgg等位基因的正常个体间传递时保持不变, 提示cgg等位基因不是正常个体两代间CAG重复序列稳定性的影响因素。SCA3/MJD基因中另有两个单碱基点突变, 一个是内含子区的杂合性突变(IVS9-113 T>C), 另一个是外显子区域的错义突变(220 G>A, 220 Glu>Gly)。这两个点突变为首次报道, 但尚不能明确这两个新的点突变对SCA3表型的影响。     

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.     

Studies of the CAG repeat in the Machado-Joseph disease gene in Taiwan

Machado-Joseph disease (MJD) is an autosomal dominant spinocerebellar degeneration characterized by cerebellar ataxia and pyramidal signs associated in varying degrees with a dystonic-rigid extrapyramidal syndrome or peripheral amyotrophy. Unstable CAG trinucleotide repeat expansion in the MJD gene on the long arm of chromosome 14 has been identified as the pathological mutation for MJD. While investigating the distribution of CAG repeat lengths of the MJD gene in Taiwan’s population, we have identified 18 MJD-affected patients and 12 at-risk individuals in seven families. In addition, we have analyzed the range of CAG repeat lengths in 96 control individuals. The CAG repeat number ranged from 13 to 44 in the controls and 72–85 in the affected and at- risk individuals. Our results indicated that the CAG repeat number was inversely correlated with the age of onset. The differences in CAG repeat length between parent and child and between siblings are greater with paternal transmission than maternal transmission. Our data show a tendency towards the phenomenon of anticipation in the MJD families but do not support unidirectional expansion of CAG repeats during transmission. We also demonstrated that PCR amplification of the CAG repeats in the MJD gene from villous DNA was possible and might prove useful as a diagnostic tool for affected families in the future. Received: 4 December 1996 / Accepted: 5 March 1997     

Why is SCA12 different from other SCAs?

Spinocerebellar ataxia type 12 (SCA12), now described in European-American and Asian (Indian) pedigrees, is unique among the SCAs from clinical, pathological, and molecular perspectives. Clinically, the distinguishing feature is early and prominent action tremor with variability in other signs. Pathologically, brain MRIs also suggest variability, with prominent cortical as well as cerebellar atrophy. Genetically, SCA12 is caused by a CAG repeat expansion that does not encode polyglutamine; we speculate that the mutation may affect expression of the gene PPP2R2B, which encodes a brain-specific regulatory subunit of the protein phosphatase PP2A.     

Analysis of CAG repeats in SCA1, SCA2, SCA3, SCA6, SCA7 and DRPLA loci in spinocerebellar ataxia patients and distribution of CAG repeats at the SCA1, SCA2 and SCA6 loci in nine ethnic populations of eastern India

To identify various subtypes of spinocerebellar ataxias (SCAs) among 57 unrelated individuals clinically diagnosed as ataxia patients we analysed the SCA1, SCA2, SCA3, SCA6, SCA7 and DRPLA loci for expansion of CAG repeats. We detected CAG repeat expansion in 6 patients (10.5%) at the SCA1 locus. Ten of the 57 patients (17.5%) had CAG repeat expansion at the SCA2 locus, while four had CAG expansion at the SCA3/MJD locus (7%). At the SCA6 locus there was a single patient (1.8%) with 21 CAG repeats. We have not detected any patient with expansion in the SCA7 and DRPLA loci. To test whether the frequencies of the large normal alleles in SCA1, SCA2 and SCA6 loci can reflect some light on prevalence of the subtypes of SCAs we studied the CAG repeat variation in these loci in nine ethnic sub-populations of eastern India from which the patients originated. We report here that the frequency of large normal alleles (>31 CAG repeats) in SCA1 locus to be 0.211 of 394 chromosomes studied. We also report that the frequency of large normal alleles (>22 CAG repeats) at the SCA2 locus is 0.038 while at the SCA6 locus frequency of large normal alleles (>13 repeats) is 0.032. We discussed our data in light of the distribution of normal alleles and prevalence of SCAs in the Japanese and white populations.     

Spinocerebellar ataxia type 1 and Machado-Joseph disease: incidence of CAG expansions among adult-onset ataxia patients from 311 families with dominant, recessive, or sporadic ataxia.

The ataxias are a complex group of diseases with both environmental and genetic causes. Among the autosomal dominant forms of ataxia the genes for two, spinocerebellar ataxia type 1 (SCA1) and Machado-Joseph disease (MJD), have been isolated. In both of these disorders the molecular basis of disease is the expansion of an unstable CAG trinucleotide repeat. To assess the frequency of the SCA1 and MJD trinucleotide repeat expansions among individuals diagnosed with ataxia we have collected DNA from individuals representing 311 families with adult-onset ataxia of unknown etiology and screened these samples for trinucleotide repeat expansions within the SCA1 and MJD genes. Within this group there are 149 families with dominantly inherited ataxia. Of these, 3% had SCA1 trinucleotide repeat expansions, whereas 21% were positive for the MJD trinucleotide expansion. Thus, together SCA1 and MJD represent 24% of the autosomal dominant ataxias in our group, and the frequency of MJD is substantially greater than that of SCA1. For the 57 patients with MJD trinucleotide repeat expansions, a strong inverse correlation between CAG repeat size and age at onset was observed (r = -.838). Among the MJD patients, the normal and affected ranges of CAG repeat size are 14-40 and 68-82 repeats, respectively. For SCA1 the normal and affected ranges are much closer, containing 19-38 and 40-81 CAG repeats, respectively.     

The prevalence and wide clinical spectrum of the spinocerebellar ataxia type 2 trinucleotide repeat in patients with autosomal dominant cerebellar ataxia.

The dominant cerebellar ataxias (ADCAs) represent a clinically and genetically heterogeneous group of disorders linked by progressive deterioration in balance and coordination. The utility of genetic classification of the ADCAs has been highlighted by the striking variability in clinical phenotype observed within families and the overlap in clinical phenotype observed between those with different genotypes. The recent demonstration that spinocerebellar ataxia type 2 (SCA2) is caused by a CAG repeat expansion within the ataxin-2 gene has allowed us to determine the frequency of SCA2 compared with SCA1, SCA3/Machado-Joseph disease (MJD), and dentatorubropallidoluysian atrophy (DRPLA) in patients with sporadic and inherited ataxia. SCA2 accounts for 13% of patients with ADCA (without retinal degeneration), intermediate between SCA1 and SCA3/MJD, which account for 6% and 23%, respectively. Together, SCA1, SCA2, and SCA3/MJD constitute >40% of the mutations leading to ADCA I in our population. No patient without a family history of ataxia, or with a pure cerebellar or spastic syndrome, tested positive for SCA1, SCA2, or SCA3. No overlap in ataxin-2 allele size between normal and disease chromosomes, or intermediate-sized alleles, were observed. Repeat length correlated inversely with age at onset, accounting for approximately 80% of the variability in onset age. Haplotype analysis provided no evidence for a single founder chromosome, and diverse ethnic origins were observed among SCA2 kindreds. In addition, a wide spectrum of clinical phenotypes was observed among SCA2 patients, including typical mild dominant ataxia, the MJD phenotype with facial fasciculations and lid retraction, and early-onset ataxia with a rapid course, chorea, and dementia.     

TATA Box-Binding Protein gene is associated with risk for schizophrenia, age at onset and prefrontal function

Schizophrenia is a common polygenic disease in distinct populations, while spinocerebellar ataxia type 17 (SCA17) is a rare autosomal dominant neurodegenerative disorder. Both diseases involve psychotic symptoms. SCA17 is caused by an expanded polyglutamine tract in the TATA box-binding protein ( TBP ) gene. In the present study, we investigated the association between schizophrenia and CAG repeat length in common TBP alleles with fewer than 42 CAG repeats in a Japanese population (326 patients with schizophrenia and 116 healthy controls). We found that higher frequency of alleles with greater than 35 CAG repeats in patients with schizophrenia compared with that in controls ( p = 0.042). We also examined the correlation between CAG repeats length and age at onset of schizophrenia. We observed a negative correlation between the number of CAG repeats in the chromosome with longer CAG repeats out of two chromosomes and age at onset of schizophrenia ( p = 0.020). We further provided evidence that TBP genotypes with greater than 35 CAG repeats, which were enriched in patients with schizophrenia, were significantly associated with hypoactivation of the prefrontal cortex measured by near-infrared spectroscopy during the tower of Hanoi, a task of executive function (right PFC; p = 0.015, left PFC; p = 0.010). These findings suggest possible associations of the genetic variations of the TBP gene with risk for schizophrenia, age at onset and prefrontal function.     

A novel trinucleotide repeat expansion at chromosome 3q26.2 identified by a CAG/CTG repeat expansion detection array

CAG/CTG repeat expansions cause at least 12 different neurological disorders, and additional disorders of this type probably exist. Using the repeat expansion detection (RED) assay, we identified an expanded CAG/CTG repeat in a 50-year-old woman with an autosomal dominant syndrome with prominent progressive sensory neuropathy. The expansion could not be accounted for by any of the CAG/CTG repeats known to undergo expansion. To identify the locus of the expansion, we created a PCR array to assess the repeat length of all repeats of eight or more CAG or CTG triplets in the human genome. The expansion was localized to a repeat contained in an intron of a Genscan-predicted gene, 185 nt downstream of a predicted exon that is conserved through mouse. The closest experimentally verified gene in the region (TNIK, encoding a serine/threonine kinase) occurs approximately 63 Kb downstream from the repeat. The length of the expansion in the proband is 98 triplets. This repeat is not expanded in the proband’s cousin (the only other affected family member for whom DNA is currently available) and no expansions were detected in a set of 230 patients with movement disorders of unknown cause. An expanded allele containing 58 triplets was detected in a single control individual, and no other expansions were detected in a set of 255 controls. The normal repeat length ranges from 5 to 30 triplets, with 8 triplets the most common allele. Our results suggest that this new repeat expansion is probably not the direct cause of the phenotype in the proband. Whether the repeat contributes to the patient’s phenotype, or is associated with another phenotype, remains to be determined.Electronic Supplementary Material Supplementary material is available for this article at .