PO.PS01.08 · 人群科学
遗传性双等位基因 MSH3 变异致病性证据及其导致早发性结直肠癌的作用
Evidence for pathogenicity of inherited biallelic MSH3 variants causing early-onset colorectal cancer
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景。我们此前报道了全球第四个家系中的一名个体，其患有早发性结直肠腺癌并携带种系复合杂合 MSH3 变异（c.2436-1G>A / c.3265A>T）。该个体的肿瘤 DNA 表现出微卫星不稳定性低（MSI-L）以及在选定四核苷酸重复序列上的微卫星改变升高（EMAST），提示两个等位基因均具有致病性。此外，肿瘤细胞在细胞质中表现出 MSH3，部分细胞出现 MSH6 缺失，这可能提示至少有一个表达 MSH3 的等位基因对 MSH2 具有显性负效应，而 MSH2 与 MSH3 和 MSH6 均结合以维持其稳定性。在此，我们旨在证明这两个 MSH3 变异均具有致病性，并评估其对其他错配修复蛋白的影响。
方法。使用 CRISPR/Cas9 将两个 MSH3 变异 c.2436-1G>A 和 c.3265A>T 分别导入 SW620 细胞。通过 Sanger 测序确认每个变异的存在。对变异克隆进行 Western 印迹（WB）和片段分析，并与亲本 SW620 细胞进行比较。
结果。我们成功建立了具有纯合 c.2436-1G>A 的 MSH3 变异克隆以及具有纯合 c.3265A>T 的克隆。WB 分析显示，含纯合 c.2436-1G>A 的细胞中 MSH3 蛋白完全缺失。然而，在含纯合 c.3265A>T 的细胞中检测到变异型 MSH3 蛋白，尽管其蛋白量少于亲本 SW620 中表达的野生型（WT）MSH3。这些结果提示，变异 c.3265A>T 的转录速率可能降低，且/或变异产生的蛋白可能比 WT MSH3 稳定性更差。这两个变异细胞系中 MSH2、MSH6 和 MLH1 的表达水平与亲本 SW620 细胞中的表达相似。在从每个变异分离的亚克隆中，18.8%（6/32）检测到 MSI-L/EMAST，表明 MMR 功能丧失，而来自 SW620 的亚克隆（0/32）均未表现出 MSI-L/EMAST。
结论。我们建立了在一名早发性结直肠癌患者中鉴定出的两个种系 MSH3 变异（c.2436-1G>A 和 c.3265A>T）的细胞模型。携带每个变异的细胞均表现出 MSI-L/EMAST，表明 MSH3 功能丧失，证明这两个种系变异具有致病性。由于我们未观察到细胞 MSH6 表达发生任何变化，进一步的研究正在进行中，以生成对这两个变异呈复合杂合的细胞，并进一步阐明这些变异组合的病理效应。
查看英文原文 English abstract
Background. We previously reported on an individual from a fourth family worldwide with early-onset colorectal adenocarcinoma with germline compound heterozygous MSH3 variants (c.2436-1G>A / c.3265A>T). Tumor DNA from this individual demonstrated microsatellite instability-low (MSI-L) and elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) suggestive that both alleles were pathogenic. Additionally, tumor cells exhibited MSH3 in the cytoplasm, with some cells showing loss of MSH6 that might be suggestive of at least one MSH3-expressing allele having a dominant-negative effect on MSH2, which binds to both MSH3 and MSH6 for their stability. Here, we aimed to prove that both MSH3 variants are pathogenic as well as to assess effects on other mismatch repair proteins.
Methods. Both MSH3 variants, c.2436-1G>A and c.3265A>T, were introduced individually into SW620 cells using CRISPR/Cas9. Presence of each variant was confirmed by Sanger sequencing. Western blotting (WB) and fragment analysis were performed on variant clones and compared with parental SW620 cells.
Results. We successfully established MSH3 variant clones with homozygous c.2436-1G>A and clones with homozygous c.3265A>T. WB analysis revealed complete loss of MSH3 protein in cells containing homozygous c.2436-1G>A. However, variant MSH3 protein was detected in cells containing homozygous c.3265A>T, although the amount of protein was less than that of wild-type (WT) MSH3 expressed in parental SW620. These results suggest that the transcription rate of variant c.3265A>T may be reduced and/or variant-generated protein may be less stable than WT MSH3. Expression levels of MSH2, MSH6 and MLH1 in these 2 variant cell lines were similar to expression in parental SW620 cells. MSI-L/EMAST was detected in 18.8% (6/32) of subclones isolated from each variant indicating loss of MMR function, whereas no subclones (0/32) from SW620 showed MSI-L/EMAST.
Conclusions. We established cell models of two germline MSH3 variants (c.2436-1G>A and c.3265A>T) which had been identified in an early-onset colorectal cancer patient. Cells with each variant exhibited MSI-L/EMAST indicating loss of MSH3 function, proving that these two germline variants are pathogenic. As we did not observe any change in cell MSH6 expression, further studies are in progress to generate cells that are compound heterozygous for these two variants and to further elucidate the pathological effects of the combinations of these variants.
利益披露 Disclosure
Y. Aisu, None..
M. Koi, None..
J. M. Carethers, None.