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

我们研究了导致亨廷顿病（HD）和遗传性共济失调等神经退行性疾病的CAG重复序列减数分裂和有丝分裂不稳定性的分子机制。首先，采用激光捕获显微切割（LCM），我们研究了亨廷顿病（HD）替代小鼠生殖系细胞中CAG重复序列的减数分裂不稳定性。 LCM 能够分离单谱系睾丸细胞以进行后续分子分析。我们发现精细胞中的 CAG 重复序列明显小于扩展等位基因纯合小鼠精母细胞中的重复序列（Mann-Whitney U 检验，P<0.05）。对于所检查的小鼠，CAG 重复序列倾向于随着细胞分化而收缩。其次，我们使用 LCM 研究了两名马查多-约瑟夫病 (MJD) 患者的可变脑细胞谱系中 CAG 重复序列的有丝分裂不稳定性。我们发现小脑皮层细胞（分子细胞、浦肯野细胞和颗粒细胞细胞）中的 CAG 重复序列大小明显小于 小脑白质细胞（Mann-Whitney U 检验，P<0.05）。小脑皮质细胞之间的CAG重复大小没有显着差异。第三，虽然与其他CAG重复疾病相比，CAG重复数量的代际稳定性被认为是SCA6的特定分子特征，但我们在两个日本SCA6家族中发现SCA6/CACNL1A基因中CAG重复的减数分裂不稳定性，包括从头扩增。在一个家族中，CAG_<20>等位基因在父系遗传期间扩展为CAG_<26>等位基因，而在另一个家族中，CAG_<19>等位基因在母系遗传期间扩展为CAG_<20>等位基因。这是第一例单倍型分析证实的 SCA6 从头扩增，证实了扩增等位基因源自一个正常等位基因。最后，我们检查了血细胞中 CAG 重复序列的出生后扩增是否发生，导致“CAG 重复疾病”。我们分析了成年 HD 和 MJD 的脐带血 (UCB) 和外周血 (PB) 细胞中的 CAG 重复序列。我们发现HD和MJD血细胞中的体细胞嵌合随着时间的推移显着增加（Mann-Whitney U检验，P<0.005），表明体细胞不稳定在患者的整个生命周期中持续存在。这是关于“CAG 重复疾病”血细胞中 CAG 重复体细胞不稳定性的第一份报告。较少的

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