Department of Medical Biophysics and Radiation Biology, Faculty of Medical Sciences, Kyushu University, Japan; Department of Biology, School of Sciences, Kyushu University, Fukuoka, Japan.
Clinical Research Institute, Cancer Genetics Laboratory, National Kyushu Cancer Center, Fukuoka, Japan.
Exp Cell Res. 2019 Apr 15;377(1-2):24-35. doi: 10.1016/j.yexcr.2019.02.020. Epub 2019 Feb 22.
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.
重复不稳定性与各种人类疾病有关。在肿瘤疾病中，微卫星不稳定性（MSI）被认为仅仅反映了 DNA 错配修复（MMR）缺陷。然而，已经指出了几个差异。首先，人类肿瘤中的 MSI 表型并不统一。已建立的分类利用微卫星变化的频率，即 MSI-H（高）和 -L（低），前者被认为是一种真正的 MMR 缺陷表型。此外，我们观察到 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，在 MMR 基因敲除小鼠中发生的肿瘤没有 MSI-H 肿瘤中典型的剧烈微卫星变化（即 B 型模式），而是较小且更微妙的改变（即 A 型模式）。在本研究中，使用 CRISPR/Cas9 系统将 Lynch 综合征（LS）家系中报道的 MSH2 突变引入 HeLa 细胞。建立的突变克隆清楚地表现出 MMR 缺陷表型，具有烷化剂耐受性和增加的突变频率。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均匀的 A 型，这证实了小鼠中的结果。我们的发现表明，人类基因组中重复不稳定性的分子机制更加复杂。
