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

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Abstract

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.

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Acknowledgements

This work was supported by Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ-Brazil) (Grant No. E26/170.026/2008), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq-Brazil) (Grant Nos. 573806/2008-0, 305873/2014-8), Instituto Nacional para Controle do Cancer (http://www.inctcancer-control.com.br) and Brazilian Ministry of Heath.

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Author notes

  1. Maria Isabel W. Achatz

    Present address: Division of Cancer Epidemiology and Genetics, National Cancer Institute (NCI)/National Institutes of Health (NIH), Bethesda, MD, USA

Authors and Affiliations

  1. Genetics Program, Instituto Nacional de Câncer, Andre Cavalcanti 37, Rio de Janeiro, RJ, 20231-050, Brazil

    Barbara Luísa Soares, Ayslan Castro Brant, Renan Gomes, Tatiane Pastor & Miguel Angelo Martins Moreira

  2. Genetic Pos-Graduation Program, Universidade Federal do Rio de Janeiro-UFRJ, Rio de Janeiro, RJ, Brazil

    Ayslan Castro Brant

  3. Laboratório de Genética Humana e Médica, Instituto de Ciências Biológicas, Universidade Federal do Pará, Belém, PA, Brazil

    Ândrea Ribeiro-dos-Santos

  4. Hospital Universitário João Barros Barreto, Universidade Federal do Pará, Belem, PA, Brazil

    Paulo Pimentel de Assumpção

  5. Departamento of Oncogenetics, Hospital A.C. Camargo, São Paulo, SP, Brazil

    Maria Isabel W. Achatz

  6. 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

    Naye Balzan Schneider & Patrícia Ashton-Prolla

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  1. Barbara Luísa Soares
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Correspondence to Miguel Angelo Martins Moreira.

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The authors declare that they have no conflict of interest.

Ethical approval

All procedures performed in the present work was approved by the Ethics Committee of each institution (CAAE-0254.1.001.007-11) in accordance with the ethical standards and with the Helsinki declaration.

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Informed consent was obtained from all patients included in the study.

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Soares, B.L., Brant, A.C., Gomes, R. et al. Screening for germline mutations in mismatch repair genes in patients with Lynch syndrome by next generation sequencing. Familial Cancer 17, 387–394 (2018). https://doi.org/10.1007/s10689-017-0043-5

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