Inactivation of DNA mismatch repair by variants of uncertain significance in the PMS2 gene

Abstract

Lynch syndrome (LS) is a common cancer predisposition caused by an inactivating mutation in one of four DNA mismatch repair (MMR) genes. Frequently a variant of uncertain significance (VUS), rather than an obviously pathogenic mutation, is identified in one of these genes. The inability to define pathogenicity of such variants precludes targeted healthcare. Here, we have modified a cell-free assay to test VUS in the MMR gene PMS2 for functional activity. We have analyzed nearly all VUS in PMS2 found thus far and describe loss of MMR activity for five, suggesting the applicability of the assay for diagnosis of LS.

Keywords: DNA mismatch repair; Lynch syndrome; PMS2; VUS.

Figure 1. Mismatch repair activity of PMS2 VUS

(A) Production of variant PMS2 alleles and proteins. All alleles, including template vector-derived T7 promoter and CITE sequences that are required for efficient transcription/translation in vitro, are generated by two sequential site-specific mutagenic PCR reactions. Variant PMS2 alleles are then used as a template in an in vitro transcription/translation reaction to produce variant PMS2 proteins. (B) Flow scheme of the cell-free assay. Left: Fluorescently labelled (light bulb) substrate pJHGT3’lnFAM is incubated in HCT-116 nuclear extract and in vitro produced heterodimeric variant PMS2/wild type MLH1 protein. Middle: After incubation, the substrate is purified and digested. Right: Repair products are visualized by automated fragment analysis and quantified. (C) Representative expression of 35S-Methionine-labeled variant PMS2 proteins, visualized after SDS-PAGE gel electrophoresis and autoradiography. Arrow: full-length PMS2 variants. (D) Relative repair efficiencies for PMS2 VUS. (−): Repair-deficient control, (+): Repair-proficient controls. Results are shown as mean±S.E.M. of 3–4 independent experiments for all VUS and >6 experiments for controls. Mock: Mock expression. Asterisks: Significantly higher than repair-deficient control E705K. * p<0.05; ** p>0.01; *** p<0.001 (Student’s one-tailed t-test). For the “Mock” and “PMS2 only” reactions, no repair was detected in any of the experiments.