Expression Defect Size among Unclassified MLH1 Variants Determines Pathogenicity in Lynch Syndrome Diagnosis

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.

Application of a 5-tiered scheme for standardized classification of 2,360 unique mismatch repair gene variants in the InSiGHT locus-specific database

“…Assays reviewed for classification are shown in Supplementary Table 4, and the values used to define abrogated or normal function are shown in Supplementary Table 5. The cut-offs <25% and >75% set for protein expression, as used in previous publications 47,48 , are very conservative given reported abrogated function associated with MLH1 expression defects of ~50% or lower 49 . For variants that had normal/inconclusive/intermediate MMR activity in 2 independent assays, but deficient protein function in 2 independent assays, abrogated function was assigned.…”

A founder MLH1 mutation in Lynch syndrome families from Piedmont, Italy, is associated with an increased risk of pancreatic tumours and diverse immunohistochemical patterns

“…This has been shown for some missense mutations in the N-terminal ATPase pocket or in the C-terminal PMS2 binding region, which show an impaired MMR activity and no decrease in protein expression [52][53][54]. Normal MLH1 expression, however, has also been described in tumours from subjects with small and large in-frame deletions and truncating mutations [51,55].…”

Classifying MMR Variants: Time for Revised Nomenclature in Lynch Syndrome

“…Relative to the wild-type, the MLH1 expression levels of the putative pathogenic variants were 52% or lower, while those of the putative neutral variants were 65% or higher. (1) To further corroborate this threshold, MMR repair function was shown to be compromised when intracellular MLH1 level falls below 50%. Hinrichsen et al proposed to use MLH1 expression level of 52% (vs. wild-type) as the first cutoff for pathogenicity, and to use functional assays only to distinguish among high-expressing variants.…”

“…In this issue of Clinical Cancer Research, Hinrichsen and colleagues (1) report a critical threshold level of compromise in the expression of MLH1 , a major DNA mismatch repair (MMR) gene, that correlated with greater protein instability and clinical data, thereby providing a means for classifying the pathogenicity of missense variants in MLH1 .…”