Germline pathogenic variants in the DNA mismatch repair genes (MMR): MLH1, MSH2, MSH6, and PMS2, are causative of Lynch syndrome (LS). However, many of the variants mapping outside the invariant splice site positions (IVS ± 1, IVS ± 2) are classified as variants of unknown significance (VUS). Three such variants (MLH1 c.588+5G>C, c.588+5G>T and c.677+5G>A) were identified in 8 unrelated LS families from Argentina, Brazil and Chile. Herein, we collected clinical information on these families and performed segregation analysis and RNA splicing studies to assess the implication of these VUS in LS etiology. Pedigrees showed a clear pattern of variant co-segregation with colorectal cancer and/or other LS-associated malignancies. Tumors presented deficient expression of MLH1-PMS2 proteins in 7/7 of the LS families, and MSI-high status in 3/3 cases. Moreover, RNA analyses revealed that c.588+5G>C and c.588+5G>T induce skipping of exon 7 whereas c.677+5G>A causes skipping of exon 8. In sum, we report that the combined clinical findings in the families and the molecular studies provided the evidences needed to demonstrate that the three MLH1 variants are causative of LS and to classify c.588+5G>C and c.677+5G>A as class 5 (pathogenic), and c.588+5G>T as class 4 (likely-pathogenic). Our findings underline the importance of performing clinical and family analyses, as well as RNA splicing assays in order to determine the clinical significance of intronic variants, and contribute to the genetic counseling and clinical management of patients and their relatives.

中文翻译：

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MLH1内含子变体映射到剪接供体位点的+ 5位导致对RNA剪接的有害作用。

DNA错配修复基因（MMR）中的生殖系致病变异：MLH1，MSH2，MSH6和PMS2是林奇综合征（LS）的病因。但是，许多映射在恒定剪接位点位置（IVS±1，IVS±2）之外的变体被分类为未知重要性（VUS）的变体。三种此类变体（MLH1在来自阿根廷，巴西和智利的8个无关的LS家族中鉴定出c.588 + 5G> C，c.588 + 5G> T和c.677 + 5G> A）。在这里，我们收集了这些家族的临床信息，并进行了分离分析和RNA剪接研究，以评估这些VUS在LS病因中的意义。家谱显示出与结肠直肠癌和/或其他与LS相关的恶性肿瘤的变体共分离的清晰模式。肿瘤在LS家族的7/7中显示MLH1-PMS2蛋白表达不足，在3/3例中显示MSI高状态。此外，RNA分析显示c.588 + 5G> C和c.588 + 5G> T导致外显子7跳过，而c.677 + 5G> A导致外显子8跳过。变体是LS的原因，并将c.588 + 5G> C和c.677 + 5G> A归类为5类（致病性），而c.588 + 5G> T归类为4类（可能致病性）。我们的发现强调了进行临床和家庭分析以及RNA剪接测定对确定内含子变体的临床意义，并有助于患者及其亲属的遗传咨询和临床管理的重要性。