PDF file - 81 KB, MAPP-MMR evaluation of the investigated MLH1 variants. MAPP-MMR (Multivariate Analysis of Protein Polymorphism-Mismatch Repair) values were calculated for all variants investigated in this study. Their individual dots in the Repair-Expression diagram (Figure 1C) were colored according to the calculated values, from green (low MAPP-MMR values corresponding to a low probability of the variant being deleterious) to red (high MAPP-MMR values corresponding to a high probability of the variant being deleterious). The mean score for variants classified deleterious by the authors was 16.5, the mean score for variants with uncertain significance was 10.8, and the mean score for neutral variants was 3.5. Variants with scores from 3.0 to 5.0 are considered as "borderline".
ARTICLE ABSTRACT
Purpose: Lynch syndrome is caused by a germline mutation in a mismatch repair gene, most commonly the MLH1 gene. However, one third of the identified alterations are missense variants with unclear clinical significance. The functionality of these variants can be tested in the laboratory, but the results cannot be used for clinical diagnosis. We therefore aimed to establish a laboratory test that can be applied clinically.Experimental Design: We assessed the expression, stability, and mismatch repair activity of 38 MLH1 missense variants and determined the pathogenicity status of recurrent variants using clinical data.Results: Four recurrent variants were classified as neutral (K618A, H718Y, E578G, V716M) and three as pathogenic (A681T, L622H, P654L). All seven variants were proficient in mismatch repair but showed defects in expression. Quantitative PCR, pulse-chase, and thermal stability experiments confirmed decreases in protein stability, which were stronger in the pathogenic variants. The minimal cellular MLH1 concentration for mismatch repair was determined, which corroborated that strongly destabilized variants can cause repair deficiency. Loss of MLH1 tumor immunostaining is consistently reported in carriers of the pathogenic variants, showing the impact of this protein instability on these tumors.Conclusions: Expression defects are frequent among MLH1 missense variants, but only severe defects cause Lynch syndrome. The data obtained here enabled us to establish a threshold for distinguishing tolerable (clinically neutral) from pathogenic expression defects. This threshold allows the translation of laboratory results for uncertain MLH1 variants into pathogenicity statements for diagnosis, thereby improving the targeting of cancer prevention measures in affected families. Clin Cancer Res; 19(9); 2432–41. ©2013 AACR.
