Abstract
A significant proportion of DNA-mismatch repair (MMR) variants are classified as of unknown significance, precluding diagnosis. The invitro MMR assay is used to assess their MMR capability, likely the most important function of an MMR protein. However, the robustness of the assay, crucial for its use in the clinical setting, has been rarely evaluated. The aim of the present work was to validate an invitro MMR assay approach to the functional characterization of MMR variants, as a first step to meeting quality standards of diagnostic laboratories. The MMR assay was optimized by testing a variety of reagents and experimental conditions. Reference materials and standard operating procedures were established. To determine the intra- and interexperimental variability of the assay and its reproducibility among centers, independent transfections of six previously characterized MLH1 variants were performed in two independent laboratories. Reagents and conditions optimal for performing the invitro MMR assay were determined. The validated assay demonstrated no significant intra- or interexperimental variability and good reproducibility between centers. We set up a robust invitro MMR assay that can provide relevant invitro functional evidence for MMR variant pathogenicity assessment, eventually improving the molecular diagnosis of hereditary cancer syndromes associated with MMR deficiency.
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