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.

Additional Metadata
Keywords DNA mismatch repair, functional test, lynch syndrome, MLH1, variants of uncertain significance
Persistent URL dx.doi.org/10.1136/jmedgenet-2019-106520, hdl.handle.net/1765/122396
Journal Journal of Medical Genetics
Citation
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