Background: 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). Methods: The MLH1 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. Resuts: In a proof-of-principle analysis, we demonstrate that ODMS can distinguish pathogenic and non-pathogenic MLH1 variants with a sensitivity of >95% and a specificity of >91%. We subsequently applied the screen to 51 MLH1 VUS and identified 31 pathogenic variants. Conclusion: ODMS is a reliable tool to identify pathogenic MLH1 variants. Implementation in clinical diagnostics will improve clinical care of patients with suspected LS and their relatives.

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Keywords DNA mismatch repair, functional test, lynch syndrome, MLH1, variants of uncertain significance
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Journal Journal of Medical Genetics
Houlleberghs, H, Dekker, M, Lusseveld, J. (Jarnick), Pieters, W. (Wietske), Van Ravesteyn, T. (Thomas), Verhoef, S. (Senno), … te Riele, H.P.J. (2019). Three-step site-directed mutagenesis screen identifies pathogenic MLH1 variants associated with Lynch syndrome. Journal of Medical Genetics. doi:10.1136/jmedgenet-2019-106520