Non-Coding Aberrations in Mismatch Repair Genes Underlie a Substantial Part of the Missing Heritability in Lynch Syndrome

Individuals with Lynch syndrome (LS) are prone to develop early-onset mismatch repair deficient (dMMR) colorectal- and endometrial cancers due to germline pathogenic variants (PVs) in one of the mismatch repair (MMR) genes MLH1, MSH2, MSH6, PMS2, or deletions affecting the 3’ region of EPCAM. Current germline diagnostics for LS, after the exclusion of somatic MLH1-promoter hypermethylation, include targeted short-read sequencing and multiplex ligation-dependent probe amplification of the coding regions of the MMR genes. In the absence of a germline PV in an MMR gene, the presence of somatic dMMR is investigated. This aforementioned strategy results in a genetic diagnosis of LS in approximately 54% of dMMR cancers before the age of 70 years. In individuals with a dMMR cancer without a germline PV, we detect two somatic nonepigenetic MMR aberrations in about 32% of cancers. Individuals who remain genetically unresolved after germline and somatic analysis (approximately 15% of individuals with dMMR cancers) are considered to have Lynch-like syndrome (LLS). For individuals with LLS and their relatives, treatment options and surveillance are yet unclear. We applied targeted long-read sequencing of the MMR genes to screen for non-coding PVs to explain the observed “missing heritability” in individuals with LLS (n = 32). This approach identified nine noncoding potential PVs in nine patients. Using functional assays, we show that six of these noncoding potential PVs cause splice-site defects. In conclusion, our studies show that 18.8% of individuals with LLS have a germline noncoding PV in an MMR gene that remained undetected by current routine diagnostics. These findings warrant that individual with LLS, who remain unresolved after germline and somatic analyses, should undergo germline sequencing of the complete loci of the MMR genes, preferably by long-read sequencing approaches, to improve LS diagnostics, treatment and cancer surveillance in patients and relatives.
 

Learning Objectives: 

1. Evaluate the diagnostic route for the detection of Lynch syndrome in individuals with a mismatch-repair deficient colorectal- or endometrial cancer.
2. Classify the application of short- and long-read sequencing approaches to detect pathogenic variants in mismatch repair genes.
3. Explain how to use functional assays, such as mini-gene assays, to assess the pathogenicity of noncoding aberrations and their effect on splicing in the mismatch repair genes.