レーザーマイクロダイセクションを用いたCAGリピートの不安定化機構の研究

激光显微切割研究CAG重复序列的失稳机制

• 批准号: 11470148
• 负责人: TAKIYAMA Yoshihisa
• 金额: $ 9.02万
• 依托单位: Jichi Medical School
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11470148/
• 关键词: Laser microdissection; CAG repeats; Meiotic instability; Mitotic instability; MJD; SCA6; Umbilical cord blood; Huntington disease

We investigated the molecular mechanism on the meiotic and mitotic instability of CAG repeats causing neurodegenerative disorders such as Huntington disease (HD) and hereditary ataxias.First, employing a laser-captured microdissection (LCM), we investigated the meiotic instability of CAG repeats in the germ-line cells in the Huntington disease (HD) replacement mouse. LCM enables the isolation of single lineage testicular cells for subsequent molecular analysis. We found that CAG repeats in the spermatid are significantly smaller than the spermatocyte in the mouse homozygous for the expanded allele (Mann-Whitney U test, P<0.05). With regard to the mouse examined, the CAG repeats tended to contract with the cell differentiation.Sccond, we investigated the mitotic instability of CAG repeats in the variable brain cell lineage in two patients with Machado-Joseph disease (MJD) using LCM.We found that CAG repeat size in the cells of cerebellar cortex (molecular, Purkinje, and granular cell la … More yers) is significantly smaller than that in the cells of cerebellar white matter (Mann-Whitney U test, P<0.05). There was no significant differences in the CAG repeat size among the cells of the cerebellar cortex.Third, although the intergenerational stability of the CAG repeat number has been considered to be a specific molecular feature of SCA6 compared with other CAG repeat diseases, we showed meiotic instability of the CAG repeats in the SCA6/CACNL1A gene in two Japanese SCA6 families, including de novo expansion. In one family, the CAG _<20> allele expanded to the CAG_<26> one during paternal transmission, and in the other family, the CAG_<19> allele expanded to the CAG_<20> one during maternal transmission. This is the first case of haplotype analysis-proven de novo expansion in SCA6, confirming the derivation of an expanded allele from one normal allele.Finally, we examined whether the postnatal expansion of the CAG repeats in the blood cells for 'CAG repeat diseases' occurs. We analyzed the CAG repeats in the umbilical cord blood (UCB) and peripheral blood (PB) cells with HD and MJD in adulthood. We found that somatic mosaicism in the blood cells of HD and MJD significantly increases over time (Mann-Whitney U test, P<0.005), indicating that somatic instability is continuous throughout the life of patients. This is the first report on the somatic instability of CAG repeats in the blood cells of 'CAG repeat diseases'. Less

为了研究CAG重复序列在神经退行性疾病如亨廷顿病（Huntington disease，HD）和遗传性共济失调中的减数分裂和有丝分裂不稳定性的分子机制，我们首先采用激光捕获显微切割技术（laser-captured microdissection，LCM）研究了亨廷顿病（Huntington disease，HD）替代小鼠生殖细胞中CAG重复序列的减数分裂不稳定性。LCM能够分离单系睾丸细胞用于随后的分子分析。我们发现，在扩展等位基因纯合子小鼠中，精细胞中的CAG重复序列显著小于精母细胞中的CAG重复序列（Mann-Whitney U检验，P<0.05）。在所研究的小鼠中，CAG重复序列随着细胞分化而趋于收缩。第二，我们用LCM研究了两名Machado-Joseph病（MJD）患者不同脑细胞谱系中CAG重复序列的有丝分裂不稳定性。我们发现小脑皮质细胞（分子细胞、浦肯野细胞和颗粒细胞）中CAG重复序列的大小在有丝分裂不稳定性中呈负相关。在小脑皮质细胞（分子细胞、颗粒细胞和颗粒细胞）中，CAG重复序列的大小在有丝分裂不稳定性中呈负相关。在小脑皮质细胞（分子细胞、颗粒细胞和浦肯野细胞）中，CAG重复序列的大小在有丝分裂不稳定性中呈负相关。在小脑皮质细胞（分子细胞、颗粒细胞和颗粒细胞）中，CAG重复序列的大小在有丝分裂不稳定性中呈负相关。在小脑皮质细胞（分子细胞、颗粒细胞和颗粒细胞）中，CAG重复序列的大小在有丝分裂不稳定性中呈负相关，在有丝分裂不稳定性中呈负相关，在有丝分裂不稳定性中呈负相关。 ...更多信息 yers）显著小于小脑白色质细胞（Mann-Whitney U检验，P<0.05）。小脑皮质细胞中CAG重复序列大小无显著差异。第三，尽管CAG重复序列数的代际稳定性被认为是SCA 6与其他CAG重复序列疾病相比的一个特定分子特征，但我们在两个日本SCA 6家族中发现了SCA 6/CACNL 1A基因中CAG重复序列的减数分裂不稳定性，包括从头扩增。在一个家系中，CAG _<20>等位基因在父系传播时扩增为CAG_<26>1，在另一个家系中，CAG_<19>等位基因在母系传播时扩增为CAG_<20>1。这是第一例单倍型分析证实的SCA 6从头扩增，证实了一个扩增的等位基因来自一个正常的等位基因。最后，我们检查了是否出生后扩增的CAG重复在血细胞中的“CAG重复疾病”发生。我们分析了成人HD和MJD患者的脐带血（UCB）和外周血（PB）细胞中CAG重复序列。我们发现HD和MJD的血细胞中的体细胞嵌合体随时间显著增加（Mann-Whitney U检验，P&lt;0.005），表明体细胞不稳定性在患者的整个生命中是连续的。这是第一次报道CAG重复序列在“CAG重复疾病”血细胞中的体细胞不稳定性。少

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