The germline MLH1 c.2054 C>T mutation disrupts DNA mismatch repair and is detectable by digital PCR

Lynch syndrome significantly increases the risk of developing colorectal cancer (CRC) due to an inherited defect in mismatch repair (MMR). Early detection relies on the identification of pathogenic mutations in patients with a family history of cancer, but few canonical Lynch mutations exist. Here, we describe four CRC patients found to carry an identical mutation in the MLH1 gene. Despite a strong family history of cancer, the MLH1 mutation was labeled discordantly regarding pathogenic potential. This highlights the need for improved diagnostics to screen for non-canonical Lynch variants. In addition to designing a novel digital PCR (dPCR) assay to rapidly detect MLH1 gene variants, we conducted in-depth analyses via molecular modeling, mutational signature analyses, and functional genetic assays to demonstrate that the MLH1 mutation results in a definitive MMR defect that increases cancer risk. This study emphasizes the need for improved diagnostic tools to identify pathogenic mutations in diverse populations.