The large majority of germline alterations identified in the DNA mismatch repair (MMR) gene PMS2, a low-penetrance gene for the cancer predisposition Lynch syndrome, represent variants of uncertain... Show moreThe large majority of germline alterations identified in the DNA mismatch repair (MMR) gene PMS2, a low-penetrance gene for the cancer predisposition Lynch syndrome, represent variants of uncertain significance (VUS). The inability to classify most VUS interferes with personalized healthcare. The complete in vitro MMR activity (CIMRA) assay, that only requires sequence information on the VUS, provides a functional analysis-based quantitative tool to improve the classification of VUS in MMR proteins. To derive a formula that translates CIMRA assay results into the odds of pathogenicity (OddsPath) for VUS in PMS2 we used a set of clinically classified PMS2 variants supplemented by inactivating variants that were generated by an in cellulo genetic screen, as proxies for cancer-predisposing variants. Validation of this OddsPath revealed high predictive values for benign and predisposing PMS2 VUS. We conclude that the OddsPath provides an integral metric that, following the other, higher penetrance, MMR proteins MSH2, MSH6 and MLH1 can be incorporated as strong evidence type into the upcoming criteria for MMR gene VUS classification of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP). Show less
Houlleberghs, H.; Dekker, M.; Lusseveld, J.; Pieters, W.; Ravesteyn, T. van; Verhoef, S.; ... ; Riele, H. te 2020
BackgroundInactivating 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... Show moreBackgroundInactivating 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).MethodsThe 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.ResutsIn 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.ConclusionODMS 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. Show less
Drost, M.; Tiersma, Y.; Glubb, D.; Kathe, S.; Hees, S. van; Calleja, F.; ... ; Wind, N. de 2020
Purpose Variants in the DNA mismatch repair (MMR) gene MSH6, identified in individuals suspected of Lynch syndrome, are difficult to classify owing to the low cancer penetrance of defects in that... Show morePurpose Variants in the DNA mismatch repair (MMR) gene MSH6, identified in individuals suspected of Lynch syndrome, are difficult to classify owing to the low cancer penetrance of defects in that gene. This not only obfuscates personalized health care but also the development of a rapid and reliable classification procedure that does not require clinical data. Methods The complete in vitro MMR activity (CIMRA) assay was calibrated against clinically classified MSH6 variants and, employing Bayes' rule, integrated with computational predictions of pathogenicity. To enable the validation of this two-component classification procedure we have employed a genetic screen to generate a large set of inactivating Msh6 variants, as proxies for pathogenic variants. Results The genetic screen-derived variants established that the two-component classification procedure displays high sensitivities and specificities. Moreover, these inactivating variants enabled the direct reclassification of human variants of uncertain significance (VUS) as (likely) pathogenic. Conclusion The two-component classification procedure and the genetic screens provide complementary approaches to rapidly and cost-effectively classify the large majority of human MSH6 variants. The approach followed here provides a template for the classification of variants in other disease-predisposing genes, facilitating the translation of personalized genomics into personalized health care. Show less
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 cellfree in vitro... Show morePurpose: 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 cellfree 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. Show less
