Sorbonne Université, Inserm, Centre de Recherche Saint-Antoine, CRSA, Paris, France; Equipe labellisée par la Ligue Nationale contre le Cancer, Paris, France.
Sorbonne Université, Inserm, Centre de recherche Saint-Antoine, UMS30-LUMIC, Plateforme de Cytométrie en Flux CISA, site Saint-Antoine, Paris, France.
Gastroenterology. 2019 Aug;157(2):421-431. doi: 10.1053/j.gastro.2019.03.071. Epub 2019 Apr 15.
BACKGROUND & AIMS: Approximately 75% of patients with suspected Lynch syndrome carry variants in MLH1 or MSH2, proteins encoded by these genes are required for DNA mismatch repair (MMR). However, 30% of these are variants of unknown significance (VUS). A assay that measures cell response to the cytotoxic effects of a methylating agent can determine the effects of VUS in MMR genes and identify patients with constitutional MMR-deficiency syndrome. We adapted this method to test the effects of VUS in MLH1 and MSH2 genes found in patients with suspected Lynch syndrome.
We transiently expressed MLH1 or MSH2 variants in MLH1- or MSH2-null human colorectal cancer cell lines (HCT116 or LoVo), respectively. The MMR process causes death of cells with methylation-damaged DNA bases, so we measured proportions of cells that undergo death following exposure to the methylating agent; cells that escaped its toxicity were considered to have variants that affect function of the gene product. Using this assay, we analyzed 88 variants (mainly missense variants), comprising a validation set of 40 previously classified variants (19 in MLH1 and 21 in MSH2) and a prospective set of 48 VUS (25 in MLH1 and 23 in MSH2). Prediction scores were calculated for all VUS according to the recommendations of the American College of Medical Genetics and Genomics, based on clinical, somatic, in silico, population, and functional data.
The assay correctly classified 39 of 40 variants in the validation set. The assay identified 12 VUS that did alter function of the gene product and 28 VUS that did not; the remaining 8 VUS had intermediate effects on MMR capacity and could not be classified. Comparison of assay results with prediction scores confirmed the ability of the assay to discriminate VUS that affected the function of the gene products from those that did not.
Using an assay that measures the ability of the cells to undergo death following DNA damage induction by a methylating agent, we were able to assess whether variants in MLH1 and MSH2 cause defects in DNA MMR. This assay might be used to help assessing the pathogenicity of VUS in MLH1 and MSH2 found in patients with suspected Lynch syndrome.
约 75%的疑似林奇综合征患者携带 MLH1 或 MSH2 中的变异，这些基因编码的蛋白是 DNA 错配修复（MMR）所必需的。然而，其中 30%的变异为意义不明的变异（VUS）。一种测量细胞对致甲基化剂细胞毒性反应的检测方法可确定 MMR 基因中 VUS 的影响，并识别出具有先天 MMR 缺陷综合征的患者。我们对这种方法进行了改编，以检测疑似林奇综合征患者中 MLH1 和 MSH2 基因中的 VUS 的影响。
我们分别在 MLH1 或 MSH2 缺失的人结直肠癌细胞系（HCT116 或 LoVo）中瞬时表达 MLH1 或 MSH2 的变异体。MMR 过程会导致具有甲基化损伤 DNA 碱基的细胞死亡，因此我们测量了暴露于致甲基化剂后发生死亡的细胞比例；逃脱其毒性的细胞被认为具有影响基因产物功能的变异体。我们使用该检测方法分析了 88 个变异体（主要为错义变异体），其中包括验证集的 40 个先前分类的变异体（MLH1 中有 19 个，MSH2 中有 21 个）和前瞻性集的 48 个 VUS（MLH1 中有 25 个，MSH2 中有 23 个）。根据美国医学遗传学和基因组学学院的建议，基于临床、体细胞、计算、人群和功能数据，为所有 VUS 计算了预测评分。
验证集的 40 个变异体中，该检测方法正确分类了 39 个。该检测方法确定了 12 个确实改变了基因产物功能的 VUS 和 28 个未改变基因产物功能的 VUS；其余 8 个 VUS 对 MMR 能力的影响处于中间，无法分类。检测结果与预测评分的比较证实了该检测方法能够区分影响基因产物功能的 VUS 与不影响基因产物功能的 VUS。
使用一种检测方法，测量细胞在致甲基化剂诱导的 DNA 损伤后发生死亡的能力，我们能够评估 MLH1 和 MSH2 中的变异是否导致 DNA MMR 缺陷。该检测方法可用于帮助评估疑似林奇综合征患者中 MLH1 和 MSH2 中发现的 VUS 的致病性。
