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.
- lynch syndrome
- variants of uncertain significance
- DNA mismatch repair
- functional test
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Contributors HH: designed and performed experiments; wrote first draft. MD: designed and performed experiments. JL: performed experiments. WP: performed experiments; provided crucial information. TvR: performed experiments and optimised technology. SV: selected VUS and provided clinical information. RMWH: selected VUS and provided clinical information. HtR: raised funding, designed experiments, supervision; wrote and edited manuscript.
Funding This work was supported by the Dutch Cancer Society (grant NKI 2009-4477) and The Dutch Research Council (grant NWO-TTW 14888).
Competing interests None declared.
Patient consent for publication Not required.
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement All data relevant to the study are included in the article or uploaded as supplementary information.
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