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

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.

Optimization of the diagnosis of inherited colorectal cancer using NGS and capture of exonic and intronic sequences of panel genes

“…In conclusion, our NGS workflow based on the capture of exonic and intronic sequences, and a double pipeline with complementary algorithms allows a high disease-causing variant detection rate and, in a single step, the detection of the different types of gene alterations including SVs of the different CRC genes, and therefore a reduction of result delay for the patient benefit. Several recent reports performed on smaller series and not based on capture of exonic and intronic sequences have already shown the efficiency of NGS in the diagnosis of inherited CRC, but the reported disease-causing variant detection rate was lower or the detected variants did not include the mosaic alterations or complex SVs [13][14][15][16]. The panel that we used in this study can easily be extended to other genes, and we recently added to the panel POLE, POLD1, NTHL1 and MSH3 genes.…”

Characterisation of heterozygous PMS2 variants in French patients with Lynch syndrome

“…Pathogenic variants in MSH6 are less common and found in about 10%–15% patients. For PMS2, approximately 5% of heterozygous pathogenic variants are estimated to be involved in LS 4–7. However, the frequency for PMS2 gene alteration is most likely underestimated, not only because of a low penetrance of PMS2 -related LS making the clinical-based detection more difficult but also because of complications in molecular screening.…”

Molecular testing for colorectal cancer: Clinical applications

“…IHC analysis of selective loss of MMR protein expression was used in the past to guide the identification of the involved gene. Nowadays, testing for Lynch syndrome genes is almost always done by NGS, except for communities with a pronounced founder effect[ 85 , 86 ].…”