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Fam Cancer. 2018 Jul;17(3):387-394. doi: 10.1007/s10689-017-0043-5.

Screening for germline mutations in mismatch repair genes in patients with Lynch syndrome by next generation sequencing.

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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.
Laboratório de Medicina Genômica, Centro de Pesquisa Experimental - Hospital de Clínicas de Porto Alegre (HCPA) and Programa de Pós Graduação em Genética e Biologia Molecular, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Brazil.
Laboratório de Genética Humana e Médica, Instituto de Ciências Biológicas, Universidade Federal do Pará, Belém, PA, Brazil.
Hospital Universitário João Barros Barreto, Universidade Federal do Pará, Belem, PA, Brazil.
Departamento of Oncogenetics, Hospital A.C. Camargo, São Paulo, SP, Brazil.
Division of Cancer Epidemiology and Genetics, National Cancer Institute (NCI)/National Institutes of Health (NIH), Bethesda, MD, USA.
Genetics Program, Instituto Nacional de Câncer, Andre Cavalcanti 37, Rio de Janeiro, RJ, 20231-050, Brazil.


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.


Long-Range PCRs; Lynch syndrome; Multiplex PCRs; Next generation sequencing; PMS2 pseudogenes

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