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

Mark Drost; Yvonne Tiersma; Bryony A. Thompson; Jane H. Frederiksen; Guido Keijzers; Dylan Glubb; Scott Kathe; Jan Osinga; Helga Westers; Lisa Pappas; Kenneth M. Boucher; Siska Molenkamp; José B. Zonneveld; Christi J. van Asperen; David E. Goldgar; Susan S. Wallace; Rolf H. Sijmons; Amanda B. Spurdle; Lene J. Rasmussen; Marc S. Greenblatt; Niels de Wind; Sean V. Tavtigian

doi:10.1038/s41436-018-0372-2

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

Mark Drost, Yvonne Tiersma, Bryony A. Thompson, Jane H. Frederiksen, Guido Keijzers, Dylan Glubb, Scott Kathe, Jan Osinga, Helga Westers, Lisa Pappas, Kenneth M. Boucher, Siska Molenkamp, José B. Zonneveld, Christi J. van Asperen, David E. Goldgar, Susan S. Wallace, Rolf H. Sijmons, Amanda B. Spurdle, Lene J. Rasmussen, Marc S. GreenblattNiels de Wind^*, Sean V. Tavtigian

^*Corresponding author for this work

11 Citations (Scopus)

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.

Original language	English
Journal	Genetics in Medicine
ISSN	1098-3600
DOIs	https://doi.org/10.1038/s41436-018-0372-2
Publication status	Published - 1 Jul 2019

Keywords

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

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Drost, M., Tiersma, Y., Thompson, B. A., Frederiksen, J. H., Keijzers, G., Glubb, D., Kathe, S., Osinga, J., Westers, H., Pappas, L., Boucher, K. M., Molenkamp, S., Zonneveld, J. B., van Asperen, C. J., Goldgar, D. E., Wallace, S. S., Sijmons, R. H., Spurdle, A. B., Rasmussen, L. J., ... Tavtigian, S. V. (2019). A functional assay–based procedure to classify mismatch repair gene variants in Lynch syndrome. Genetics in Medicine. https://doi.org/10.1038/s41436-018-0372-2

Drost, M, Tiersma, Y, Thompson, BA, Frederiksen, JH, Keijzers, G, Glubb, D, Kathe, S, Osinga, J, Westers, H, Pappas, L, Boucher, KM, Molenkamp, S, Zonneveld, JB, van Asperen, CJ, Goldgar, DE, Wallace, SS, Sijmons, RH, Spurdle, AB, Rasmussen, LJ, Greenblatt, MS, de Wind, N & Tavtigian, SV 2019, 'A functional assay–based procedure to classify mismatch repair gene variants in Lynch syndrome', Genetics in Medicine. https://doi.org/10.1038/s41436-018-0372-2

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title = "A functional assay–based procedure to classify mismatch repair gene variants in Lynch syndrome",

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.",

keywords = "assay calibration, functional assay, Lynch syndrome, variant classification, variants of uncertain significance",

author = "Mark Drost and Yvonne Tiersma and Thompson, {Bryony A.} and Frederiksen, {Jane H.} and Guido Keijzers and Dylan Glubb and Scott Kathe and Jan Osinga and Helga Westers and Lisa Pappas and Boucher, {Kenneth M.} and Siska Molenkamp and Zonneveld, {Jos{\'e} B.} and {van Asperen}, {Christi J.} and Goldgar, {David E.} and Wallace, {Susan S.} and Sijmons, {Rolf H.} and Spurdle, {Amanda B.} and Rasmussen, {Lene J.} and Greenblatt, {Marc S.} and {de Wind}, Niels and Tavtigian, {Sean V.}",

year = "2019",

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doi = "10.1038/s41436-018-0372-2",

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T1 - A functional assay–based procedure to classify mismatch repair gene variants in Lynch syndrome

AU - Drost, Mark

AU - Tiersma, Yvonne

AU - Thompson, Bryony A.

AU - Frederiksen, Jane H.

AU - Keijzers, Guido

AU - Glubb, Dylan

AU - Kathe, Scott

AU - Osinga, Jan

AU - Westers, Helga

AU - Pappas, Lisa

AU - Boucher, Kenneth M.

AU - Molenkamp, Siska

AU - Zonneveld, José B.

AU - van Asperen, Christi J.

AU - Goldgar, David E.

AU - Wallace, Susan S.

AU - Sijmons, Rolf H.

AU - Spurdle, Amanda B.

AU - Rasmussen, Lene J.

AU - Greenblatt, Marc S.

AU - de Wind, Niels

AU - Tavtigian, Sean V.

PY - 2019/7/1

Y1 - 2019/7/1

N2 - 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.

AB - 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.

KW - assay calibration

KW - functional assay

KW - Lynch syndrome

KW - variant classification

KW - variants of uncertain significance

U2 - 10.1038/s41436-018-0372-2

DO - 10.1038/s41436-018-0372-2

M3 - Journal article

C2 - 30504929

AN - SCOPUS:85058010232

SN - 1098-3600

JO - Genetics in Medicine

JF - Genetics in Medicine

ER -

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

Abstract

Keywords

UN SDGs

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