Analysis of SCA1, DRPLA,MJD, SCA2, and SCA6 CAG repeats in 48 portuguese ataxia families

The spinocerebellar ataxias (SCAs) are clinically and genetically a heterogeneous group of neurodegenerative disorders. To date, eight different loci causing SCA have been identified: SCA1, SCA2, Machado-Joseph disease (MJD)/SCA3, SCA4, SCA5, SCA6, SCA7, and dentatorubropallidoluysian atrophy (DRPLA). Expansion of a CAG repeat in the disease genes has been found in five of these disorders. To estimate the relative frequencies of the SCA1, DRPLA, MJD, SCA2, and SCA6 mutations among Portuguese ataxia patients, we collected DNA samples from 48 ataxia families and performed polymerase chain reaction (PCR) amplification of the CAG repeat mutations on chromosomes 6p, 12p, 14q, 12q, and 19p, respectively. Fifty-five individuals belonging to 34 dominant families (74%) had an expanded CAG repeat at the MJD gene. In five individuals from two kindreds with a dominant pattern of inheritance (4%), an expanded CAG repeat at the SCA2 gene was found. In MJD patients, the normal allele size ranged from 13 to 41, whereas the mutant alleles contained 65 to 80 repeats. For the SCA2 patients, normal alleles had 22 or 23, while expanded alleles had between 36 and 47 CAG units. We did not find the SCA1, DRPLA, or SCA6 mutations in our group of families. The MJD mutation remains the most common cause of SCA in Portugal, while a small number of cases are caused by mutations at the SCA2 gene, and 22% are due to still unidentified genes. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 81:134–138, 1998. © 1998 Wiley-Liss, Inc.     

Comparative studies of the CAG repeats in the spinocerebellar ataxia type 1 (SCA1) gene

The CAG repeat tract at the autosomal dominant spinocerebellar ataxia type 1 (SCA1) locus was analyzed in SCA1 families and French-Acadian, African-American, Caucasian, Greenland Inuit, and Thai populations. The normal alleles had 9–37 repeats, whereas disease alleles contained 44–64 repeats. The CAG repeat tract contained one or two CAT interruptions in 44 of 47 normal human chromosomes and in all five chimpanzees examined. In contrast, no CAT interruptions were found in Old World monkeys or expanded human alleles. The number and positions of CAT interruptions may be important in stabilizing CAG repeat tracts in normal chromosomes. At least five codons occupy the region corresponding to the polyglutamine tract at the SCA1 locus in mice, rats, and other rodents. They comprise three or four CCN (coding for proline) in addition to one or two CAG repeats. Am. J. Med. Genet. 74:488–493, 1997. © 1997 Wiley-Liss, Inc.     

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。     

Molecular features of the CAG repeats of spinocerebellar ataxia 6 (SCA6)

Spinocerebellar ataxia 6 (SCA6) is an autosomal dominant spinocerebellar degeneration caused by the expansion of the polymorphic CAG repeat in the human alpha1A voltage-dependent calcium channel subunit gene (CACNL1A4 gene). We have analyzed 60 SCA6 individuals from 39 independent SCA6 Japanese families and found that the CAG repeat length is inversely correlated with the age of onset (n = 58, r = - 0.51, P < 0.0001). SCA6 chromosomes contained 21-30 repeat units, whereas normal chromosomes displayed 6-17 repeats. There was no overlap between the normal and affected CAG repeat number. The anticipation of the disease was observed clinically in all eight parent-child pairs that we examined; the mean age of onset was significantly lower (P = 0.0042) in children than in parents. However, a parent-child analysis showed the increase in the expansion of CAG repeats only in one pair and no diminution in any affected cases. This result suggests that factors other than CAG repeats may produce the clinical anticipation. A homozygotic case could not demonstrate an unequivocal gene dosage effect on the age of onset.      

The calcium channel alpha2/delta1 subunit is involved in extracellular signalling

The α2/δ1 subunit forms part of the dihydropyridine receptor, an essential protein complex for excitation–contraction (EC) coupling in skeletal muscle. Because of the lack of a viable knock-out animal, little is known regarding the role of the α2/δ1 subunit in EC coupling or in other cell functions. Interestingly, the α2/δ1 appears before the α1 subunit in development and contains extracellular conserved domains known to be important in cell signalling and inter-protein interactions. These facts raise the possibility that the α2/δ1 subunit performs vital functions not associated with EC coupling. Here, we tested the hypothesis that the α2/δ1 subunit is important for interactions of muscle cells with their environment. Using confocal microscopy, we followed the immunolocalization of α2/δ1 and α1 subunits with age. We found that in 2-day-old myotubes, the α2/δ1 subunit concentrated towards the ends of the cells, while the α1 subunit clustered near the centre. As myotubes aged (6–12 days), the α2/δ1 became evenly distributed along the myotubes and co-localized with α1. When the expression of α2/δ1 was blocked with siRNA, migration, attachment and spreading of myoblasts were impaired while the L-type calcium current remained unaffected. The results suggest a previously unidentified role of the α2/δ1 subunit in skeletal muscle and support the involvement of this protein in extracellular signalling. This new role of the α2/δ1 subunit may be crucial for muscle development, muscle repair and at times in which myoblast attachment and migration are fundamental.     

Myotonia caused by mutations in the muscle chloride channel gene CLCN1

Pure non-syndromic, non-dystrophic myotonia in humans is caused by mutations in the genes coding for the skeletal muscle sodium channel (SCN5A) or the skeletal muscle chloride channel (CLCN1) with similar phenotypes. Chloride-channel myotonia can be dominant (Thomsen-type myotonia) or recessive (Becker-type myotonia). More than 60 myotonia-causing mutations in the CLCN1 gene have been identified, with only a few of them being dominant. A common phenotype of dominant mutations is a dominant negative effect of mutant subunits in mutant-WT heterodimers, causing a large shift of the steady-state open probability voltage-dependence towards more positive, unphysiological voltages. The study of the properties of disease causing mutations has helped in understanding the functional properties of the CLC-1 channel that is part of a nine-member gene family of chloride channels. The large body of knowledge obtained for CLC-1 may also help to better understand the other CLC channels, three of which are also involved in genetic diseases.     

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.     

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

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

Structure and alternative splicing of the gene encoding alpha1I, a human brain T calcium channel alpha1 subunit.

The structure of CACNA1I, the gene encoding alpha1I, a human brain T Ca2+ channel alpha1 subunit, was determined by comparison of polymerase chain reaction-amplified brain cDNA and genomic sequences. The gene consists of at least 36 exons spanning at least 115,168 basepairs of chromosome 22q12.3-13.2. The predicted protein has 2016 amino acids and 28 potential phosphorylation sites. Alternative splicing of the gene occurs at two sites: cassette exon 9 and an alternative acceptor in exon 33. Molecular diversity generated by alternative splicing and post-translational modification of this and other members of the T alpha1 subunit gene family may account for the observed heterogeneity of T currents in central neurons.     

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.     

Proteolytic cleavage and cellular toxicity of the human alpha1A calcium channel in spinocerebellar ataxia type 6

Spinocerebellar ataxia type 6 (SCA6) is a neurodegenerative disease caused by small CAG repeat expansion in the alpha1A calcium channel gene. We found that the human alpha1A calcium channel protein expressed in human embryonic kidney 293T cells produces a 75 kDa C-terminal fragment. This fragment is more toxic to cells than the full-length alpha1A calcium channel, regardless of polyglutamine tract length. In cells stably transfected with plasmids of full-length alpha1A calcium channel cDNAs, the C-terminal fragment protein is present in the mutant transformant but not in the wild-type one, indicative that this C-terminal fragment with the expanded polyglutamine tract is more resistant to proteolysis than that with the normal sized polyglutamine tract. We speculate that the toxic C-terminal fragment, in which resistance to proteolysis is rendered by the expanded polyglutamine, has a key role in the pathological mechanism of SCA6.     

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

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

Dnmt1 deficiency promotes CAG repeat expansion in the mouse germline

Expanded CAG repeat tracts are the cause of at least a dozenneurodegenerative disorders. In humans, long CAG repeats tendto expand during transmissions from parent to offspring, leadingto an earlier age of disease onset and more severe symptomsin subsequent generations. Here, we show that the maintenanceDNA methyltransferase Dnmt1, which preserves the patterns ofCpG methylation, plays a key role in CAG repeat instabilityin human cells and in the male and female mouse germlines. SiRNAknockdown of Dnmt1 in human cells destabilized CAG triplet repeats,and Dnmt1 deficiency in mice promoted intergenerational expansionof CAG repeats at the murine spinocerebellar ataxia type 1 (Sca1)locus. Importantly, Dnmt1^+/– SCA1 mice, unlike their Dnmt1^+/+SCA1 counterparts, closely reproduced the intergenerationalinstability patterns observed in human SCA1 patients. In addition,we found aberrant DNA and histone methylation at sites withinthe CpG island that abuts the expanded repeat tract in Dnmt1-deficientmice. These studies suggest that local chromatin structure mayplay a role in triplet repeat instability. These results areconsistent with normal epigenetic changes during germline developmentcontributing to intergenerational instability of CAG repeatsin mice and in humans.     

Analysis of the dynamic mutation in the SCA7 gene shows marked parental effects on CAG repeat transmission

The gene for spinocerebellar ataxia 7 (SCA7) includes a transcribed, translated CAG tract that is expanded in SCA7 patients. We have determined expansions in 73 individuals from 17 SCA7 kindreds and compared them with repeat lengths of 180 unaffected individuals. Subjects with abnormal expansions comprise 59 clinically affected individuals and 14 at-risk currently unaffected individuals predicted to carry the mutation by haplotype analysis. For expanded alleles, CAG repeat length correlates with disease progression and severity and correlates inversely with age of onset. Increased repeat lengths are seen in generational transmission of the disease allele, consistent with the pattern of clinical anticipation seen in these kindreds. Repeat lengths in expanded alleles show somatic mosaicism in leukocyte DNA, suggesting that these alleles are unstable within individuals as well as between generations. Although dynamic repeat expansions from paternal transmissions are greater than those from maternal transmissions, maternal transmission of disease is more common, suggesting germline or embryonic effects of the repeat expansion.      

H1-receptor dependent increase in platelet aggregation is mediated by intracellular calcium

The influence of histamine on human platelet function was studied by measuring the uptake of [³H]-histamine and by evaluating the effect of histamine on Ca²⁺ influx and on intracellular Ca²⁺ levels. The uptake of histamine by platelets is significantly reduced by mepyramine and unaffected by cimetidine. Histamine was found to increase both the [⁴⁵Ca]²⁺ uptake by resting and stimulated platelets, and the intracellular Ca²⁺ levels in Fura 2-AM loaded platelets. H₁-antihistaminics significantly reduced these processes, suggesting a role for histamine, which in platelets promotes aggregation by acting through H₁-receptors modulating intracellular Ca²⁺ levels.