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Original research
Three-step site-directed mutagenesis screen identifies pathogenic MLH1 variants associated with Lynch syndrome
  1. Hellen Houlleberghs1,
  2. Marleen Dekker1,
  3. Jarnick Lusseveld1,
  4. Wietske Pieters1,
  5. Thomas van Ravesteyn1,
  6. Senno Verhoef2,
  7. Robert M W Hofstra3,
  8. Hein te Riele1
  1. 1 Division of Tumor Biology and Immunology, The Netherlands Cancer Institute, Amsterdam, The Netherlands
  2. 2 Department of Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands
  3. 3 Department of Clinical Genetics, Erasmus MC, Erasmus University Rotterdam, Rotterdam, The Netherlands
  1. Correspondence to Professor Hein te Riele, Division of Tumor Biology and Immunology, The Netherlands Cancer Institute, Amsterdam 1066 CX, The Netherlands; h.t.riele{at}


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
  • MLH1
  • 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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