Soares Barbara Luísa, Brant Ayslan Castro, Gomes Renan, Pastor Tatiane, Schneider Naye Balzan, Ribeiro-Dos-Santos Ândrea, de Assumpção Paulo Pimentel, Achatz Maria Isabel W, Ashton-Prolla Patrícia, Moreira Miguel Angelo Martins
Genetics Program, Instituto Nacional de Câncer, Andre Cavalcanti 37, Rio de Janeiro, RJ, 20231-050, Brazil.
Genetic Pos-Graduation Program, Universidade Federal do Rio de Janeiro-UFRJ, Rio de Janeiro, RJ, Brazil.
Fam Cancer. 2018 Jul;17(3):387-394. doi: 10.1007/s10689-017-0043-5.
Lynch syndrome (LS) is an autosomal dominant disorder, with high penetrance that affects approximately 3% of the cases of colorectal cancer. Affected individuals inherit germline mutations in genes responsible for DNA mismatch repair, mainly at MSH2, MLH1, MSH6 and PMS2. The molecular screening of these individuals is frequently costly and time consuming due to the large size of these genes. In addition, PMS2 mutation detection is often a challenge because there are 16 different pseudogenes identified until now. In the present work we evaluate a molecular screening strategy based in next generation sequencing (NGS) in order to optimize the mutation detection in LS patients. We established 16 multiplex PCRs for MSH2, MSH6 and MLH1 and 5 Long-Range PCRs for PMS2, coupled with NGS. The strategy was validated by screening 66 patients who filled Bethesda and Amsterdam criteria for LS from health institutions of Brazil. The mean depth of coverage for MSH2, MSH6, MLH1 and PMS2 genes was 7.988, 36.313, 11.899 and 4.772 times, respectively. Ninety-four variants were found in exons and flanking intron/exon regions for the four MMR genes. Twenty-five were pathogenic or VUS and found in 32 patients (7 in MSH2, 5 in MSH6, 12 in MLH1 e 1 in PMS2). All variants were confirmed by Sanger sequencing. The strategy was efficient to reduce time consuming and costs to identify genetic changes at these MMR genes, reducing in three times the number of PCR reactions performed per patient and was efficient in identifying variants at PMS2 gene.
林奇综合征（LS）是一种常染色体显性疾病，具有高外显率，约影响3%的结直肠癌病例。受影响个体遗传了负责DNA错配修复的基因中的种系突变，主要发生在MSH2、MLH1、MSH6和PMS2基因。由于这些基因的大小较大，对这些个体进行分子筛查通常成本高昂且耗时。此外，PMS2突变检测往往具有挑战性，因为到目前为止已鉴定出16种不同的假基因。在本研究中，我们评估了一种基于下一代测序（NGS）的分子筛查策略，以优化LS患者的突变检测。我们为MSH2、MSH6和MLH1建立了16个多重PCR，为PMS2建立了5个长片段PCR，并结合NGS。通过对来自巴西医疗机构的66名符合LS的贝塞斯达和阿姆斯特丹标准的患者进行筛查，验证了该策略。MSH2、MSH6、MLH1和PMS2基因的平均覆盖深度分别为7.988、36.313、11.899和4.772倍。在四个错配修复（MMR）基因的外显子以及侧翼内含子/外显子区域中发现了94个变异。其中25个为致病性或意义未明的变异（VUS），在32名患者中被发现（MSH2中有7个，MSH6中有5个，MLH1中有12个，PMS2中有1个）。所有变异均通过桑格测序得到确认。该策略有效地减少了识别这些MMR基因遗传变化的时间和成本，将每位患者进行的PCR反应数量减少了三分之二，并且在识别PMS2基因的变异方面效率较高。
