Differential genomic destabilisation in human cells with pathogenic MSH2 mutations introduced by genome editing
Differential genomic destabilisation in human cells with pathogenic MSH2 mutations introduced by genome editing
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DOI：
10.1016/j.yexcr.2019.02.020
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发表时间：
2019-04-15
影响因子：
3.7
通讯作者：
Nakatsu, Yoshimichi
中科院分区：
文献类型：
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作者：
Hayashida, Genki;Shioi, Seijiro;Nakatsu, Yoshimichi
重复的不稳定与人类疾病有着不同的联系。在肿瘤性疾病中，微卫星不稳定性（MSI）被认为是DNA错配修复（MMR）缺陷的简单反映。然而，有人指出了一些不一致之处。首先，MSI+表型在人类肿瘤中是不均匀的。已建立的分类利用微卫星变化的频率，即MSI-H（高）和-L（低），前者被认为是真正的MMR缺陷型。此外，我们还观察到MSI的定性不同模式，即A型和B型。我们之前指出的一个差异是，MMR基因敲除小鼠中发生的肿瘤没有表现出MSI-H肿瘤中典型的剧烈微卫星变化（即B型模式），而是表现出较小和更微妙的变化（即A型模式）。在本研究中，林奇综合征（LS）家族中报告的MSH 2突变已使用CRISPR/Cas9系统引入HeLa细胞。建立的突变体克隆清楚地表现出MMR缺陷的表型与烷化剂耐受性和突变频率升高。然而，微卫星并没有像LS患者中发生的MSI-H肿瘤那样明显不稳定，并且所有观察到的改变都是一致的A型，这证实了小鼠中的结果。我们的研究结果表明，人类基因组中重复不稳定的分子机制更加复杂。
Repeat destabilisation is variously associated with human disease. In neoplastic diseases, microsatellite instability (MSI) has been regarded as simply reflecting DNA mismatch repair (MMR) deficiency. However, several discrepancies have been pointed out. Firstly, the MSI+ phenotype is not uniform in human neoplasms. Established classification utilises the frequency of microsatellite changes, i.e. MSI-H (high) and -L (low), the former regarded as an authentic MMR-defective phenotype. In addition, we have observed the qualitatively distinct modes of MSI, i.e. Type A and Type B. One discrepancy we previously pointed out is that tumours occurring in MMR gene knockout mice exhibited not drastic microsatellite changes typical in MSI-H tumours (i.e. Type B mode) but minor and more subtle alterations (i.e. Type A mode). In the present study, MSH2 mutations reported in Lynch syndrome (LS) kindred have been introduced into HeLa cells using the CRISPR/Cas9 system. The established mutant clones clearly exhibited MMR-defective phenotypes with alkylating agent-tolerance and elevated mutation frequencies. Nevertheless, microsatellites were not markedly destabilised as in MSI-H tumours occurring in LS patients, and all the observed alterations were uniformly Type A, which confirms the results in mice. Our findings suggest added complexities to the molecular mechanisms underlying repeat destabilisation in human genome.