Constitutional MLH1 methylation (epimutation) is a rare cause of Lynch syndrome. Low-level methylation (≤ 10%) has occasionally been described. This study aimed to identify low-level constitutional MLH1 epimutations and determine its causal role in patients with MLH1-hypermethylated colorectal cancer. Eighteen patients with MLH1-hypermethylated colorectal tumors in whom MLH1 methylation was previously undetected in blood by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) were screened for MLH1 methylation using highly sensitive MS-melting curve analysis (MS-MCA). Constitutional methylation was characterized by different approaches. MS-MCA identified one patient (5.6%) with low-level MLH1 methylation (~ 1%) in blood and other normal tissues, which was confirmed by clonal bisulfite sequencing in blood. The patient had developed three clonally related gastrointestinal MLH1-methylated tumor lesions at 22, 24, and 25 years of age. The methylated region in normal tissues overlapped with that reported for other carriers of constitutional MLH1 epimutations. Low-level MLH1 methylation and reduced allelic expression were linked to the same genetic haplotype, whereas the opposite allele was lost in patient’s tumors. Mutation screening of MLH1 and other hereditary cancer genes was negative. Herein, a highly sensitive MS-MCA-based approach has demonstrated its utility for the identification of low-level constitutional MLH1 epigenetic mosaicism. The eventual identification and characterization of additional cases will be critical to ascertain the cancer risks associated with constitutional MLH1 epigenetic mosaicism.

中文翻译：

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血液中高度敏感的MLH1甲基化分析可鉴定出患有低水平镶嵌MLH1突变的癌症患者。

体质性MLH1甲基化（表位突变）是林奇综合征的罕见原因。低级甲基化（≤10％）已被描述。这项研究旨在确定低水平的体质性MLH1突变，并确定其在MLH1高甲基化结直肠癌患者中的因果作用。使用高度敏感的MS熔解曲线分析（MS-MCA）筛选了18例先前未通过甲基化特异性多重连接依赖探针扩增（MS-MLPA）在血液中检测到MLH1甲基化的MLH1高甲基化结直肠肿瘤患者。体质甲基化的特征在于不同的方法。MS-MCA鉴定出一名患者（5.6％）的血液和其他正常组织中的MLH1甲基化水平较低（〜1％），这已通过血液中亚硫酸氢盐的克隆测序得到了证实。该患者在22、24和25岁时出现了3个与克隆相关的胃肠道MLH1甲基化肿瘤病变。正常组织中的甲基化区域与报告的其他构成性MLH1突变携带者的甲基化区域重叠。低水平的MLH1甲基化和减少的等位基因表达与相同的基因单倍型相关，而相反的等位基因在患者的肿瘤中丢失。MLH1和其他遗传性癌症基因的突变筛查是阴性的。在这里，基于MS-MCA的高度敏感的方法已证明其可用于识别低级结构性MLH1表观遗传镶嵌。其他病例的最终鉴定和表征对于确定与体质性MLH1表观遗传镶嵌相关的癌症风险至关重要。