['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.', '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.', '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.'] Does expression defect size among unclassified MLH1 variants determine pathogenicity in Lynch syndrome diagnosis?