MLH1 p. His112Arg Variant Is Associated With Increased Cancer Susceptibility

ABSTRACT Lynch syndrome, a prevalent hereditary cancer predisposition, is frequently caused by pathogenic variants in the MutL homolog 1 (MLH1) mismatch repair gene. However, a significant proportion of MLH1 missense variants remain classified as variants of uncertain significance (VUS), creating clinical ambiguity. Functional validation assays in vertebrate models are crucial to assess the pathogenicity of these VUS. We previously reported the rare MLH1 NM_000249.4:c.335A>G (p.His112Arg) variant in individuals with cancer histories within the Qatar Genome Program. To assess its functional impact, we established the MLH1 p.His112Arg variant in the human colorectal cancer cell line, COLO205, using CRISPR‐Cas9 technology. Isogenic wild‐type (WT) and variant‐carrying cells were xenotransplanted into the zebrafish. Tumor growth was quantified via fluorescence microscopy at 24 and 72 h post‐injection. Zebrafish xenografted with MLH1 p.His112Arg cells exhibited a significant increase in tumor area compared to WT. This accelerated growth was associated with a significant upregulation of the proliferation marker MKI67 in variant‐carrying tumors, while cyclin D1 (CCND1) and proliferating cell nuclear antigen (PCNA) (key regulators of cell cycle progression and DNA replication, respectively) expression remained unchanged, despite a tendency for increased expression. Furthermore, our functional characterization of the MLH1 p.His112Arg variant in COLO205 cells showed an enhanced proliferation capacity compared to the WT cell line, as well as increased cell viability and decreased apoptotic susceptibility when subjected to methylation‐induced DNA damage. The MLH1 p.His112Arg variant confers a tumor‐promoting phenotype in an in vivo zebrafish model, providing functional evidence that supports its reclassification from VUS to likely pathogenic.