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.


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.


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.


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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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.

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Author notes

  1. These authors contributed equally: Lene J. Rasmussen, Marc S. Greenblatt, Niels de Wind, and Sean V. Tavtigian


  1. Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands

    • Mark Drost PhD
    • , Yvonne Tiersma MS
    •  & Niels de Wind PhD
  2. Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT, USA

    • Bryony A. Thompson PhD
    •  & Sean V. Tavtigian PhD
  3. Centre for Epidemiology and Biostatistics, School of Population and Global Health, University of Melbourne, Melbourne, Australia

    • Bryony A. Thompson PhD
  4. Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen, Denmark

    • Jane H. Frederiksen PhD
    • , Guido Keijzers PhD
    •  & Lene J. Rasmussen PhD
  5. Department of Genetics and Computational Biology, QIMR Berghofer Medical Research Institute, Brisbane, Australia

    • Dylan Glubb PhD
    •  & Amanda B. Spurdle PhD
  6. Department of Microbiology and Molecular Genetics, University of Vermont Robert Larner, M.D. College of Medicine, Burlington, VT, USA

    • Scott Kathe PhD
    •  & Susan S. Wallace PhD
  7. Department of Genetics, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands

    • Jan Osinga BS
    • , Helga Westers PhD
    •  & Rolf H. Sijmons MD, PhD
  8. Department of Medicine, Division of Epidemiology, Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT, USA

    • Lisa Pappas MS
    •  & Kenneth M. Boucher PhD
  9. Department of Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands

    • Siska Molenkamp BS
    • , José B. Zonneveld BS
    •  & Christi J. van Asperen MD, PhD
  10. Department of Dermatology, Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT, USA

    • David E. Goldgar PhD
  11. Department of Medicine and University of Vermont Cancer Center, University of Vermont Robert Larner, M.D. College of Medicine, Burlington, VT, USA

    • Marc S. Greenblatt MD


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The authors declare no conflicts of interest.

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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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