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

我们研究了CAG重复序列的减数分裂和有丝分裂不稳定性导致神经退行性疾病如亨廷顿病（HD）和遗传性共济失调的分子机制。首先，采用激光捕获显微解剖（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能够分离单系睾丸细胞，用于后续的分子分析。我们发现扩增等位基因的精细胞中的CAG重复数明显小于小鼠纯合子中的精细胞（Mann-Whitney U检验，P<0.05）。对于所检查的小鼠，CAG重复序列倾向于与细胞分化收缩。其次，我们利用LCM研究了两例Machado-Joseph病（MJD）患者可变脑细胞谱系中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）CAG重复序列的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CAG重复序列大小无显著差异。第三，尽管与其他CAG重复序列疾病相比，CAG重复序列数的代际稳定性被认为是SCA6的一个特定分子特征，但我们在两个日本SCA6家族中发现了CAG重复序列的减数分裂不稳定性，包括从头扩增。在一个家族中，CAG_<20>等位基因在父系传播中扩增为CAG_<26>等位基因，在另一个家族中，CAG_<19>等位基因在母系传播中扩增为CAG_<20>等位基因。这是第一例单倍型分析证实的sc6从头扩增，证实了从一个正常等位基因衍生出扩增等位基因。最后，我们检查了出生后血细胞中CAG重复序列的扩增是否会发生“CAG重复序列疾病”。我们分析了成年HD和MJD患者脐带血（UCB）和外周血（PB）细胞中CAG重复序列。我们发现HD和MJD血细胞中的体细胞嵌合体随着时间的推移显著增加（Mann-Whitney U检验，P<0.005），表明体细胞不稳定性在患者的一生中持续存在。本文首次报道了CAG重复序列在“CAG重复序列病”患者血细胞中的体细胞不稳定性。少

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