リンチ症候群の分子診断における次世代シーケンシング（NGS）およびMLPA法の応用

Background: Lynch syndrome (LS) is a cancer-susceptibility syndrome associated with autosomal dominant predisposition to a spectrum of cancers, primarily of the colorectum and endometrium, which exhibit impaired DNA mismatch repair (MMR) activity. LS is caused by a hereditary (germline) pathogenic (PV) or likely pathogenic variant (LPV) in one of the mismatch repair (MMR) genes-MLH1, MSH2, MSH6, PMS2, or EPCAM. Although point mutations are the most common genetic changes in MMR genes, >20% are large genomic rearrangements. We hypothesized that a two-tier diagnostic strategy for Lynch syndrome (LS) using next generation sequencing (NGS) and multiplex ligation-dependent probe amplification (MLPA) can increase diagnostic yield of patients with Lynch syndrome. Methods: This study included 60 patients suspected of LS. After genetic counseling, they were referred to genetic testing. Genomic DNA was extracted from peripheral blood and sequenced using NGS multigene panel testing covering 113 cancer susceptibility genes, including MMR genes. Regarding limitations of NGS analysis, which cannot reliably detect genomic alterations larger than 50 base pairs in length, the MLPA method was used for NGS negative DNA samples in order to identify larger deletions and duplications, commonly referred to as copy number variations (CNVs). Results: Different PVs were detected by NGS in 10 patients and CNVs were detected by MLPA in 7 more patients: 3xMLH1 del ex9-15, 2xMSH2 del ex1 and upstream, 1xMSH2 del ex9, and 1xMSH2 del ex1. We did not detect LPVs or variants of uncertain significance (VUS). In our cohort, the addition of MLPA provided an incremental yield of seven pathogenic CNVs, representing an 11.6% absolute increase in diagnostic sensitivity (from 16.7% to 28.3%) over the NGS-alone workflow, with CNVs accounting for 41% of all pathogenic findings. Conclusions: Our results show that MLPA is a very useful method in molecular diagnostics of LS and its implementation in routine genetic testing in combination with NGS using multigene panel testing would benefit both patients and health care providers.