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

被引:12
作者
Soares, Barbara Luisa [1 ]
Brant, Ayslan Castro [1 ,2 ]
Gomes, Renan [1 ]
Pastor, Tatiane [1 ]
Schneider, Naye Balzan [6 ,7 ]
Ribeiro-dos-Santos, Andrea [3 ]
de Assumpcao, Paulo Pimentel [4 ]
Achatz, Maria Isabel W. [5 ,8 ]
Ashton-Prolla, Patricia [6 ,7 ]
Martins Moreira, Miguel Angelo [1 ]
机构
[1] Inst Nacl Canc, Genet Program, Andre Cavalcanti 37, BR-20231050 Rio De Janeiro, RJ, Brazil
[2] Univ Fed Rio de Janeiro UFRJ, Genet Posgrad Program, Rio De Janeiro, RJ, Brazil
[3] Univ Fed Para, Lab Genet Humana & Med, Inst Ciencias Biol, Belem, PA, Brazil
[4] Univ Fed Para, Hosp Univ Joao Barros Barreto, Belem, PA, Brazil
[5] Hosp AC Camargo Fund Antonio Prudente, Dept Oncogenet, Sao Paulo, SP, Brazil
[6] Univ Fed Rio Grande do Sul, HCPA, Lab Med Genom, Ctr Pesquisa Expt, Porto Alegre, RS, Brazil
[7] Univ Fed Rio Grande do Sul, HCPA, Programa Posgrad Genet & Biol Mol, Porto Alegre, RS, Brazil
[8] NCI, Div Canc Epidemiol & Genet, NIH, Bethesda, MD 20892 USA
关键词
Lynch syndrome; Multiplex PCRs; Long-Range PCRs; PMS2; pseudogenes; Next generation sequencing; NONPOLYPOSIS COLON-CANCER; COLORECTAL-CANCER; HEREDITARY; PMS2; MSH2; PSEUDOGENES; DIAGNOSTICS; GUIDELINES; MANAGEMENT; FAMILIES;
D O I
10.1007/s10689-017-0043-5
中图分类号
R73 [肿瘤学];
学科分类号
100214 ;
摘要
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.
引用
收藏
页码:387 / 394
页数:8
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