遗传性脊髓小脑型共济失调的CAG三核苷酸突变检测

目的 评价ＳＣＡ１、ＳＣＡ２、ＳＣＡ３／ＭｊＤ、ＳＣＡ６、ＳＣＡ７和ＤＲＰＬＡ的ＣＡＧ三核苷酸异常扩增突变「（ＣＡＧ）ｎ」,在中国人遗传性脊髓小脑型共济失调（ｓｐｉｎｏｃｅｒｅｂｅｌｌａｒ ａｔａｘｉａ,ＳＣＡ）患者的分布频率。方法 经聚合酶链反应、变性聚丙烯酰按凝胶电泳和银染显带技术,检测分析了８５个中国人常染色体显性遗传ＳＣＡ家系（其中患者１６７例）和３７例散发ＳＣＡ患者的ＳＣＡ１、ＳＣＡ２、     

Trinucleotide expansion within the MJD1 gene presents clinically as spinocerebellar ataxia and occurs most frequently in German SCA patients

Autosomal dominant spinocerebellar ataxia (SCA) is a clinicallyand genetically heterogeneous neurodegenerative disorder whichleads to progressive cerebellar ataxia. A gene responsible forSCA type 3 has been mapped to human chromosome 14q, close tothe Machado-Joseph disease (MJD) locus. The MJD1 gene has recentlybeen cloned and the disease causing mutation has been identifiedas an unstable and expanded (CAG)_n trinucleotide repeat. Assome clinical features of MJD overlap with those of SCA we investigatedthe MJD mutation in 38 German families with dominantly inheritedSCA. The MJD1 (CAG)_n expansion was identified in 19 families.In contrast, the trinucleotide expansion was not observed in21 ataxia patients without family history of the disease. Analysisof the (CAG)_n repeat length in 30 patients revealed an inversecorrelation with the age of onset. The (CAG)_n stretch of theaffected allele varied between 67 and 78 trinucleotide units,the normal alleles carried between 12 and 28 simple repeats.These results demonstrate that the MJD mutation causes the diseasephenotype of most SCA patients in Germany.     

Uncloned expanded CAG/CTG repeat sequences in autosomal dominant cerebellar ataxia (ADCA) detected by the repeat expansion detection (RED) method.

In some neurodegenerative diseases, genetic anticipation correlates with expansions of the CAG/CTG repeat sequence above the normal range through the generations of a pedigree. Among these neurodegenerative diseases are late onset autosomal dominant cerebellar ataxias (ADCA). ADCA are genetically heterogeneous disorders with different cloned genes for spinocerebellar ataxia type 1 (SCA1), type 2 (SCA2), type 3 or Machado-Joseph disease (SCA3/MJD), and type 6 (SCA6). Another related dominant ataxia, dentatorubral-pallidoluysian atrophy (DRPLA), also shows CAG/CTG repeat expansions. Genetic anticipation has been reported for all of them except for the recently cloned SCA6 gene. Other, as yet undetected SCA genes may show the same features. We have used the repeat expansion detection (RED) method to detect repeat expansions directly in DNA samples from ADCA patients not resulting from known genes. Our sample consists of 19 affected index cases, corresponding to 52.8% of our ADCA families without CAG/CTG repeat expansions in the SCA1, SCA2, SCA3/MJD, SCA6, or DRPLA genes. Eighty-nine percent of the index cases had expansions of a CAG/CTG sequence greater than 40 repeats by RED, while these were observed in only 26.9% of 78 healthy subjects from the general population (p < 0.0001). The distribution of RED fragments in controls and ADCA patients also shows significant differences with the Mann-Whitney U test (U = 376.5, p = 0.0007). Moreover, there was a significant inverse correlation between the size of expansion and the age of onset (r = -0.54, p = 0.018). These results show CAG/CTG repeat expansions of over 40 repeats in our sample of ADCA families not resulting from known SCA genes.     

Genetic testing in spinocerebellar ataxia in Taiwan: expansions of trinucleotide repeats in SCA8 and SCA17 are associated with typical Parkinson's disease

DNA tests in normal subjects and patients with ataxia and Parkinson's disease (PD) were carried out to assess the frequency of spinocerebellar ataxia (SCA) and to document the distribution of SCA mutations underlying ethnic Chinese in Taiwan. MJD/SCA3 (46%) was the most common autosomal dominant SCA in the Taiwanese cohort, followed by SCA6 (18%) and SCA1 (3%). No expansions of SCA types 2, 10, 12, or dentatorubropallidoluysian atrophy (DRPLA) were detected. The clinical phenotypes of these affected SCA patients were very heterogeneous. All of them showed clinical symptoms of cerebellar ataxia, with or without other associated features. The frequencies of large normal alleles are closely associated with the prevalence of SCA1, SCA2, MJD/SCA3, SCA6, and DRPLA among Taiwanese, Japanese, and Caucasians. Interestingly, abnormal expansions of SCA8 and SCA17 genes were detected in patients with PD. The clinical presentation for these patients is typical of idiopathic PD with the following characteristics: late onset of disease, resting tremor in the limbs, rigidity, bradykinesia, and a good response to levodopa. This study appears to be the first report describing the PD phenotype in association with an expanded allele in the TATA-binding protein gene and suggests that SCA8 may also be a cause of typical PD.     

SCA6 is caused by moderate CAG expansion in the alpha1A-voltage- dependent calcium channel gene

Recently, moderate (CAG)>20 repeat expansions in the alpha1A-voltage- dependent calcium channel gene (CACNL1A4) have been identified in a previously unmapped type of SCA which has been named SCA6. We investigated the (CAG)n repeat length of the CACNL1A4 gene in 733 patients with sporadic ataxia and in 46 German families with dominantly inherited SCA which do not harbor the SCA1, SCA2, or MJD1/SCA3 mutation, respectively. The SCA6 (CAG)n expansion was identified in 32 patients most frequently with late manifestation of the disease. The (CAG)n stretch of the affected allele varied between 22 and 28 trinucleotide units and is therefore the shortest trinucleotide repeat expansion causing spinocerebellar ataxia. The (CAG)n repeat length is inversely correlated with the age at onset. In 11 parental transmissions of the expanded allele no repeat instability has been observed. Repeat instability was also not found for the normal allele investigating 431 meioses in the CEPH families. Analyzing 248 apparently healthy octogenerians revealed one allele of 18 repeats which is the longest normal CAG repeat in the CACNL1A4 gene reported. The SCA6 mutation causes the disease in approximately 10% of autosomal dominant SCA in Germany. Most importantly, the trinucleotide expansion was observed in four ataxia patients without obvious family history of the disease which necessitates a search for the SCA6 (CAG)n expansion even in sporadic patients.      

Evidence of a Common Founder for SCA12 in the Indian Population

Spinocerebellar ataxia type 12 (SCA12) is an autosomal dominant cerebellar ataxia associated with the expansion of an unstable CAG repeat in the 5′ region of the PPP2R2B gene on chromosome 5q31–5q32. We found that it accounts for ～16% (20/124) of all the autosomal dominant ataxia cases diagnosed in AIIMS, a major tertiary referral centre in North India. The length of the expanded allele in this population ranges from 51–69 CAG triplets. Interestingly, all the affected families belong to an endogamous population, which originated in the state of Haryana, India. We identified four novel SNPs and a dinucleotide marker spanning ～137 kb downstream of CAG repeat in the PPP2R2B gene. Analysis of 20 Indian SCA12 families and ethnically matched normal unrelated individuals revealed one haplotype to be significantly associated with the affected alleles (P= 0.000), clearly indicating the presence of a common founder for SCA12 in the Indian population. This haplotype was not shared by the American pedigree with SCA12. Therefore, the SCA12 expansion appears to have originated at least twice.     

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.     

一个遗传性共济失调3型家系中致病ATXN3中间类型等位基因分析

目的 研究遗传性共济失调3型中间类型等位基因致病表型的临床表现与基因突变特点.方法 应用PCR、毛细微管电泳、分子克隆及测序等方法 对1个临床诊断为遗传性共济失调家系进行ATXN3基因检测,对异常片段进行分子克隆测序.结果 证实该家系为遗传性共济失调3型家系,先证者异常片段CAG重复次数为43次;患者两个儿子异常片段重复分别为41、64次.结论 中间类型等位基因在两代间遗传是不稳定的,重复次数的改变是双向的,43次CAG重复是目前报道的遗传性共济失调3型发病患者最小不稳定重复次数.本家系的研究结果 进一步缩短了正常CAG重复次数与异常重复次数之间的差距.     

一个遗传性共济失调3型家系中致病ATXN3中间类型等位基因分析

目的 研究遗传性共济失调3型中间类型等位基因致病表型的临床表现与基因突变特点.方法 应用PCR、毛细微管电泳、分子克隆及测序等方法 对1个临床诊断为遗传性共济失调家系进行ATXN3基因检测,对异常片段进行分子克隆测序.结果 证实该家系为遗传性共济失调3型家系,先证者异常片段CAG重复次数为43次;患者两个儿子异常片段重复分别为41、64次.结论 中间类型等位基因在两代间遗传是不稳定的,重复次数的改变是双向的,43次CAG重复是目前报道的遗传性共济失调3型发病患者最小不稳定重复次数.本家系的研究结果 进一步缩短了正常CAG重复次数与异常重复次数之间的差距.     

一个遗传性共济失调3型家系中致病ATXN3中间类型等位基因分析

目的 研究遗传性共济失调3型中间类型等位基因致病表型的临床表现与基因突变特点.方法 应用PCR、毛细微管电泳、分子克隆及测序等方法 对1个临床诊断为遗传性共济失调家系进行ATXN3基因检测,对异常片段进行分子克隆测序.结果 证实该家系为遗传性共济失调3型家系,先证者异常片段CAG重复次数为43次;患者两个儿子异常片段重复分别为41、64次.结论 中间类型等位基因在两代间遗传是不稳定的,重复次数的改变是双向的,43次CAG重复是目前报道的遗传性共济失调3型发病患者最小不稳定重复次数.本家系的研究结果 进一步缩短了正常CAG重复次数与异常重复次数之间的差距.     

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.      

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.     

Frequency analysis of autosomal dominant cerebellar ataxias in Japanese patients and clinical characterization of spinocerebellar ataxia type 6

Using a molecular diagnostic approach, we investigated 101 kindreds with autosomal dominant cerebellar ataxias (ADCAs) from the central Honshu island of Japan, including spinocerebellar ataxia type 1 (SCA1), spinocerebellar ataxia type 2 (SCA2), Machado–Joseph disease (MJD), dentatorubral and pallidoluysian atrophy (DRPLA) and spinocerebellar ataxia type 6 (SCA6). In our unselected series, MJD was the most common type of ADCA, accounting for 33.7% followed by DRPLA (19.8%), SCA2 (5.9%) and SCA6 (5.9%). No SCA1 mutations were identified. We analysed the clinical features of six molecular confirmed SCA6 kindreds: in each family, there was an expanded allele in the α1A-voltage dependent calcium channel comprising between 23 and 25 CAG repeats. The mean age at onset of symptoms was 43±13 years. The clinical features consisted predominantly of cerebellar ataxia, dysarthria and horizontal nystagmus, which was generally consistent with ADCA type 3. However several new clinical features were found in some patients: dramatic anticipation, rapid disease progression, severe ataxia associated with action tremor or action myoclonus, and very early onset, which are not described as the classical features of ADCA type 3.     

东北地区正常汉族人群 SCA1及 SCA3/MJD基因内CAG重复变异研究

目的　对东北地区 110名汉族正常人 SCA1及 SCA3/ MJD基因 (CAG) n拷贝进行检测 ,探讨其正常变异范围 ,并对临床诊断为遗传型脊髓小脑共济失调的 8个家系的 2 5例患者和 6个散发病例进行基因分型评价和症状前及产前诊断。方法　应用荧光 - PCR方法测定不同基因型片段长度 ,并进行 DNA序列分析。结果　 SCA3/ MJD基因 (CAG) n正常变异范围为 14～ 38个拷贝 ,集中于 14个拷贝 ,其等位基因频率为 39.5 5 % ,杂合频率为 78.18% ,共 13种等位基因。检出一个家系先证者携带有 (CAG) 6 8的 SCA3/ MJD基因 ,并对该家系成员进行了症状前诊断 ,没有发现 (CAG) n拷贝异常突变 ;SCA1基因内 (CAG) n正常变异范围 2 0～ 39拷贝 ,集中于 2 6及 2 7次 ,等位基因频率分别为 34.0 9%和 2 0 .91% ,杂合频率为 84 .5 5 % ,共 13种等位基因 ;散发病例未检出 CAG扩展性突变。结论　 SCA1及 SCA3/ MJD基因中 (CAG) n正常变异范围存在地区和种族差异 ,SCAs基因分型是该病症状前及产前诊断的首选策略。     

Genetics of the SCA6 gene in a large family segregating an autosomal dominant "pure" cerebellar ataxia

Spinocerebellar ataxia type 6 (SCA6) is an autosomal dominant cerebellar degeneration caused by the expansion of a CAG trinucleotide repeat in the CACNA1A gene. Mutations in patients are characterised by expanded alleles of between 21 and 30 repeat units and by extreme gonadal stability when transmitted from parents to children. We have investigated the SCA6 mutation in a large Spanish kindred in which previously reported spinocerebellar SCA genes and loci had been excluded. We observed a 23 CAG repeat expanded allele in the 13 clinically affected subjects and in three out of 10 presymptomatic at risk subjects. Transmission of the mutant allele was stable in six parent to child pairs and in 29 meioses through the pedigree. Linkage analysis with the SCA6-CAG polymorphism and marker D19S221 confirmed the location of SCA6 on chromosome 19p13. The molecular findings in this large family confirm the expansion of the CAG repeat in the CACNA1A gene as the cause of SCA6 and the high meiotic stability of the repeat.     

Mendelian segregation of normal CAG trinucleotide repeat alleles at three autosomal loci

Segregation ratio distortion (SRD) with preferential transmission of expanded CAG alleles has been reported in Machado-Joseph disease (MJD/SCA3), spinocerebellar ataxia type I (SCA1), and dentatorubral-pallidoluysian atrophy (DRPLA). We have examined the transmission frequencies of alleles in normal heterozygotes at these disease loci in 377 pairs of twins and their parents and find no evidence for SRD.     

Portuguese families with dentatorubropallidoluysian atrophy (DRPLA) share a common haplotype of Asian origin

Dentatorubropallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder characterized by a variable combination of progressive ataxia, epilepsy, myoclonus, choreoathetosis and dementia. This disease is caused by a (CAG)(n) expansion in the DRPLA gene, on chromosome 12p13. DRPLA is prevalent in Japan, but several families of non-Japanese ancestry have already been published. To identify the origin of expanded alleles in Portuguese families with DRPLA, we studied two previously reported intragenic SNPs in introns 1 and 3, in addition to the CAG repeat of the DRPLA gene. The results showed that all four Portuguese DRPLA families shared the same haplotype, which is also common to that reported for Japanese DRPLA chromosomes. This haplotype is also the most frequent in Japanese normal alleles, whereas it was rare in Portuguese control chromosomes. Thus, our findings support that a founder DRPLA haplotype of Asian origin was introduced in Portugal, being responsible for the frequency of the disease in this country.     

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.     

CAG repeat instability at SCA2 locus: anchoring CAA interruptions and linked single nucleotide polymorphisms.

Spinocerebellar ataxia 2 (SCA2) is an autosomal dominant neurodegenerative disorder that results from the expansion of a cryptic CAG repeat within the exon 1 of the SCA2 gene. The CAG repeat in normal individuals varies in length from 14 to 31 repeats and is frequently interrupted by one or more CAA triplets, whereas the expanded alleles contain a pure uninterrupted stretch of 34 to 59 CAG repeats. We have previously reported the presence of a limited pool of 'ancestral' or 'at risk' haplotypes for the expanded SCA2 alleles in the Indian population. We now report the identification of two novel single nucleotide polymorphisms (SNPs) in exon 1 of the SCA2 gene and their characterization in 215 normal and 64 expanded chromosomes. The two biallelic SNPs distinguished two haplotypes, GT and CC, each of which formed a predominant haplotype associated with normal and expanded SCA2 alleles. All the expanded alleles segregated with CC haplotype, which otherwise was associated with only 29.3% of the normal chromosomes. CAA interspersion analysis revealed that majority of the normal alleles with CC haplotype were either pure or lacked the most proximal 5' CAA interruption. The repeat length variation at SCA2 locus also appeared to be polar with changes occurring mostly at the 5' end of the repeat. Our results demonstrate that CAA interruptions play an important role in conferring stability to SCA2 repeat and their absence predisposes alleles towards instability and pathological expansion. Our study also provides new haplotypes associated with SCA2 that should prove useful in further understanding the mutational history and mechanism of repeat instability at the SCA2 locus.     

Clinicopathology of spinocerebellar degeneration: Its correlation to the unstable CAG repeat of the affected gene

The recent advances in gene analysis have greatly facilitated the classification of autosomal dominant spinocerebellar ataxia (SCA). Analyses of linkage in large families with SCA have assigned gene foci to at least 8 chromosomes. One gene is located in the short arm of chromosome 6 (6p22-p23) and causes spinocerebellar ataxia type 1 (SCA1). A gene in the long arm of chromosome 14 (14q24.3-q32) underlies Machado-Joseph disease (MJD). A third gene locus is assigned to the short arm of chromosome 12 (12p2-pter) causing dentatorubropallidoluysian atrophy (DRPLA). The gene for spinocerebellar ataxia type 2 (SCA2) is located in the 12q23–24. Subsequently, a sporadic counterpart of hereditary olivopontocerebellar atrophy of the Menzel type is clearly defined, and all the syndromes (non-hereditary olivopontocerebellar atrophy, striatonigral degeneration and Shy-Drager syndrome) are now lumped under the term of multiple system atrophy (MSA). Oligodendroglial cytoplas-mic inclusions appear to be specific for and diagnostic of MSA. As the clinical features in SCA are variable and often appear to overlap with one another, which makes accurate classification difficult if not possible, the genotype is required for their unequivocal classification. However, major neuropathological features clearly distinguish SCA1 from SCA3/ MJD cases; the medial segment of the globus pallidus and intermediolateral column lesions in SCA3/MJD, and inferior olive and cerebellar cortical degeneration in SCA1. It has been stated that neurodegeneration in SCA3/MJD is more homogeneous than in SCA1 or SCA2 and that degeneration of the pallidoluysian system is not present in the latter. The pertinent pathology in each of the three types of SCA is illustrated. The background of clinicopathology and genetic analysis of dentatorubropallidoluysian atrophy is also reviewed.