Tumor-independent Detection of Inherited Mismatch Repair Deficiency for the Diagnosis of Lynch Syndrome with High Specificity and Sensitivity.

Lynch syndrome (LS) is the most common hereditary cancer syndrome. Early diagnosis improves prognosis and reduces health care costs, through existing cancer surveillance methods. The problem is finding and diagnosing the cancer predisposing genetic condition. The current workup involves a complex array of tests that combines family cancer history and clinical phenotypes with tumor characteristics and sequencing data, followed by a challenging task to interpret the found variant(s). On the basis of the knowledge that an inherited mismatch repair (MMR) deficiency is a hallmark of LS, we have developed and validated a functional MMR test, DiagMMR, that detects inherited MMR deficiency directly from healthy tissue without need of tumor and variant information. The validation included 119 skin biopsies collected from clinically pathogenic MMR variant carriers (MSH2, MSH6) and controls, and was followed by a small clinical pilot study. The repair reaction was performed on proteins extracted from primary fibroblasts and the interpretation was based on the MMR capability of the sample in relation to cutoff, which distinguishes MMR proficient (non-LS) from MMR deficient (LS) function. The results were compared with the reference standard (germline NGS). The test was shown to have exceptional specificity (100%) with high sensitivity (89%) and accuracy (97%). The ability to efficiently distinguish LS carriers from controls was further shown with a high area under the receiving operating characteristic (AUROC) value (0.97). This test offers an excellent tool for detecting inherited MMR deficiency linked to MSH2 or MSH6 and can be used alone or with conventional tests to recognize genetically predisposed individuals. Significance: Clinical validation of DiagMMR shows high accuracy in distinguishing individuals with hereditary MSH2 or MSH6 MMR deficiency (i.e., LS). The method presented overcomes challenges faced by the complexity of current methods and can be used alone or with conventional tests to improve the ability to recognize genetically predisposed individuals.

PMS2 expression decrease causes severe problems in mismatch repair.

PMS2 is one of the four susceptibility genes in Lynch syndrome (LS), the most common cancer syndrome in the world. Inherited mutations in DNA mismatch repair (MMR) genes, MLH1, MSH2, and MSH6, account for approximately 90% of LS, while a relatively small number of LS families segregate a PMS2 mutation. This and the low cancer penetrance in PMS2 families suggest that PMS2 is only a moderate or low-risk susceptibility gene. We have previously shown that even a partial expression decrease in MLH1, MSH2, or MSH6 suggests that heterozygous LS mutation carriers have MMR malfunction in constitutive tissues. Whether and how PMS2 expression decrease affects the repair capability is not known. Here, we show that PMS2 knockdown cells retaining 19%, 33%, or 53% of PMS2 expression all have significantly reduced MMR efficiency. Surprisingly, the cells retaining expression levels comparable to PMS2 mutation carriers indicate the lowest repair efficiency.

Assessment of the InSiGHT Interpretation Criteria for the Clinical Classification of 24 MLH1 and MSH2 Gene Variants.

Pathogenicity assessment of DNA variants in disease genes to explain their clinical consequences is an integral component of diagnostic molecular testing. The International Society for Gastrointestinal Hereditary Tumors (InSiGHT) has developed specific criteria for the interpretation of mismatch repair (MMR) gene variants. Here, we performed a systematic investigation of 24 MLH1 and MSH2 variants. The assessments were done by analyzing population frequency, segregation, tumor molecular characteristics, RNA effects, protein expression levels, and in vitro MMR activity. Classifications were confirmed for 15 variants and changed for three, and for the first time determined for six novel variants. Overall, based on our results, we propose the introduction of some refinements to the InSiGHT classification rules. The proposed changes have the advantage of homogenizing the InSIGHT interpretation criteria with those set out by the Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium for the BRCA1/BRCA2 genes. We also observed that the addition of only few clinical data was sufficient to obtain a more stable classification for variants considered as "likely pathogenic" or "likely nonpathogenic." This shows the importance of obtaining as many as possible points of evidence for variant interpretation, especially from the clinical setting.

Assessing how reduced expression levels of the mismatch repair genes MLH1, MSH2, and MSH6 affect repair efficiency.

Lynch syndrome (LS), the most common familial colon cancer, is associated with mismatch repair (MMR) malfunction. As mutation carriers inherit one normal and one defected MMR gene allele, cancer risk can be considered as limited amount of normal MMR gene product. How reductions in different MMR gene expressions affect MMR capability is, however, not known. The in vitro MMR assay is a method for the pathogenicity assessment of MMR gene variants causing functional or expressional defects and thus also suitable to evaluate the effects of reduced expression of normal mRNA. Here, the assay was applied to quantify repair efficiencies of human cells retaining varying expression levels (25%/50%/75%) of the main LS susceptibility genes MLH1, MSH2, or MSH6. Compared with the shRNA knockdown control, already a 50% reduction in mRNA levels could be detected as decreased MMR function although without statistical significance in MLH1. In MSH2 and MLH1, total loss of MMR was achieved with 25% expression, whereas in MSH6 and MSH2, the repair capability decreased significantly already with 75% expression. Our results provide a preliminary indication of relative expressions required for wild-type function and suggest that the in vitro MMR assay could be used to recognize expression levels indicative of LS.