A calibrated cell-based functional assay to aid classification of MLH1 DNA mismatch repair gene variants

Abstract

Functional assays provide important evidence for classifying the disease significance of germline variants in DNA mismatch repair genes. Numerous laboratories, including our own, have developed functional assays to study mismatch repair gene variants. However, previous assays are limited due to the model system employed, the manner of gene expression, or the environment in which function is assessed. Here, we developed a human cell-based approach for testing the function of variants of uncertain significance (VUS) in the MLH1 gene. Using clustered regularly interspaced short palindromic repeats gene editing, we knocked in MLH1 VUS into the endogenous MLH1 loci in human embryonic stem cells. We examined their impact on RNA and protein, including their ability to prevent microsatellite instability and instigate a DNA damage response. A statistical clustering analysis determined the range of functions associated with known pathogenic or benign variants, and linear regression was performed using existing odds in favor of pathogenicity scores for these control variants to calibrate our functional assay results. By converting the functional outputs into a single odds in favor of pathogenicity score, variant classification expert panels can use these results to readily reassess these VUS. Ultimately, this information will guide proper diagnosis and disease management for suspected Lynch syndrome patients.

Keywords: CRISPR gene editing; DNA damage response; Lynch syndrome; functional assay; microsatellite instability; mismatch repair; variant of uncertain significance.

Figure 1

MLH1 variants under study. Representative image of the MLH1 variants in this study mapped to the N-terminal structure of human MLH1 (Protein Data Bank ID code 4P7A) and C-terminal structure of Saccharomyces cerevisiae MLH1 (Protein Data Bank ID code 4E4W), connected by a theoretical linker from the human protein. A: The known benign (green) and pathogenic (red) variants selected for functional evaluation. B: The missense variants of uncertain significance (VUS) examined in this study.

Figure 2

Steady state levels of MLH1 and PMS2 proteins and alternative splicing variants. A: Representative immunoblots show the steady state levels of MLH1 and PMS2 in benign, pathogenic, and VUS expressing cell lines. Quantification of the protein levels with respect to the WT levels are shown after normalizing for loading. WT, wild-type hESCs; KO, MLH1 knockout hESCs B: Reverse transcribed total RNA from p.R265S, p.S265R, p.A42V and p.S627T cells was PCR amplified to detect the presence of variant containing exons. Representative agarose gel images showing alternative splicing in indicated cell lines. CHX= Cycloheximide.

Figure 3

The mismatch repair-dependent damage response in variant cell lines. Cell survival after 48 hr treatment with 2 μM of the DNA alkylating agent N-methyl-N’-nitro-N-nitrosoguanidine. The values are represented as the mean ± SEM; n=3-5. A statistical clustering analysis was performed which grouped the variants into two populations based on their percent survival (blue and orange).