Expression defect size among unclassified MLH 1 variants 5 determines pathogenicity 6 in Lynch syndrome diagnosis

45 Purpose: Lynch syndrome is caused by a germline mutation in a mismatch repair gene, most 46 commonly the MLH1 gene. However, one-third of the identified alterations are missense variants with 47 unclear clinical significance. The functionality of these variants can be tested in the laboratory, but the 48 results cannot be used for clinical diagnosis. We therefore aimed to establish a laboratory test that can 49 be applied clinically. 50 Experimental design: We assessed the expression, stability and mismatch repair activity of 38 MLH1 51 missense variants and determined the pathogenicity status of recurrent variants using clinical data. 52 Results: Four recurrent variants were classified as neutral (K618A, H718Y, E578G, V716M) and 53 three as pathogenic (A681T, L622H, P654L). All seven variants were proficient in mismatch repair 54 but showed defects in expression. qPCR, pulse-chase and thermal stability experiments confirmed 55 decreases in protein stability, which were stronger in the pathogenic variants. The minimal cellular 56 MLH1 concentration for mismatch repair was determined, which corroborated that strongly 57 destabilized variants can cause repair deficiency. Loss of MLH1 tumor immunostaining is consistently 58 reported in carriers of the pathogenic variants, demonstrating the impact of this protein instability on 59 these tumors. 60 Conclusions: Expression defects are frequent among MLH1 missense variants, but only severe defects 61 cause Lynch syndrome. The data obtained here enabled us to establish a threshold for distinguishing 62 tolerable (clinically neutral) from pathogenic expression defects. This threshold allows the translation 63 of laboratory results for uncertain MLH1 variants into pathogenicity statements for diagnosis, thereby 64 improving the targeting of cancer prevention measures in affected families. 65 66 Research. on June 1, 2017. © 2013 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 12, 2013; DOI: 10.1158/1078-0432.CCR-12-3299