Three-step site-directed mutagenesis screen identifies pathogenic <i>MLH1</i> variants associated with Lynch syndrome.

Abstract

Inactivating mutations in the MLH1 DNA mismatch repair (MMR) gene underlie 42% of Lynch syndrome (LS) cases. LS is a cancer predisposition causing early onset colorectal and endometrial cancer. Nonsense and frameshift alterations unambiguously cause LS. The phenotype of missense mutations that only alter a single amino acid is often unclear. These variants of uncertain significance (VUS) hinder LS diagnosis and family screening and therefore functional tests are urgently needed. We developed a functional test for MLH1 VUS termed 'oligonucleotide-directed mutation screening' (ODMS). The <i>MLH1</i> variant was introduced by oligonucleotide-directed gene modification in mouse embryonic stem cells that were subsequently exposed to the guanine analogue 6-thioguanine to determine whether the variant abrogated MMR. In a proof-of-principle analysis, we demonstrate that ODMS can distinguish pathogenic and non-pathogenic <i>MLH1</i> variants with a sensitivity of >95% and a specificity of <u>></u>91%. We subsequently applied the screen to 51 <i>MLH1</i> VUS and identified 31 pathogenic variants. ODMS is a reliable tool to identify pathogenic <i>MLH1</i> variants. Implementation in clinical diagnostics will improve clinical care of patients with suspected LS and their relatives.