A functional assay–based procedure to classify mismatch repair gene variants in Lynch syndrome

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Abstract

Purpose

To enhance classification of variants of uncertain significance (VUS) in the DNA mismatch repair (MMR) genes in the cancer predisposition Lynch syndrome, we developed the cell-free in vitro MMR activity (CIMRA) assay. Here, we calibrate and validate the assay, enabling its integration with in silico and clinical data.

Methods

Two sets of previously classified MLH1 and MSH2 variants were selected from a curated MMR gene database, and their biochemical activity determined by the CIMRA assay. The assay was calibrated by regression analysis followed by symmetric cross-validation and Bayesian integration with in silico predictions of pathogenicity. CIMRA assay reproducibility was assessed in four laboratories.

Results

Concordance between the training runs met our prespecified validation criterion. The CIMRA assay alone correctly classified 65% of variants, with only 3% discordant classification. Bayesian integration with in silico predictions of pathogenicity increased the proportion of correctly classified variants to 87%, without changing the discordance rate. Interlaboratory results were highly reproducible.

Conclusion

The CIMRA assay accurately predicts pathogenic and benign MMR gene variants. Quantitative combination of assay results with in silico analysis correctly classified the majority of variants. Using this calibration, CIMRA assay results can be integrated into the diagnostic algorithm for MMR gene variants.

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Acknowledgements

We appreciate the assistance of John-Paul Plazzer, curator of the International Society for Gastrointestinal Hereditary Tumors (InSiGHT) Database. A.B.S., D.E.G., L.J.R., M.S.G., R.H.S., N.D.W., S.S.W., and S.V.T. are supported by US National Institutes of Health (NIH) National Cancer Institute (NCI) grant R01 CA164944. A.B.S. is supported by an Australian National Health and Medical Research Council (NHMRC) Senior Research Fellowship (ID1061779). B.A.T. is supported by an NHMRC CJ Martin Early Career Fellowship. D.G. was supported in part by an NHMRC grant (ID1109286) G.K. is supported by Harboefonden (grant number 15292), Familien Spogárds Fond, and Fabrikant Einer Willumsens Mindelegat. K.M.B., L.P., and S.V.T. are supported by US NIH NCI grant P30 CA042014. L.J.R. is funded by Nordea-fonden and the Olav Thon Foundation. N.D.W. is funded by the Dutch Digestive Foundation grant FP 16-012.

Author information

Correspondence to Lene J. Rasmussen PhD or Marc S. Greenblatt MD or Niels de Wind PhD or Sean V. Tavtigian PhD.

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The authors declare no conflicts of interest.

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Keywords

  • Lynch syndrome
  • variants of uncertain significance
  • functional assay
  • variant classification
  • assay calibration

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