Novel African American Colorectal Cancer MSH3 Variants Associate With Major Genomic Instability

Pathogenic variants of MSH3 can increase mutational load within colorectal cells, which may drive initiation and progression of colorectal cancer (CRC). We identified several variants within MSH3 among CRCs from African Americans (AA). To predict and assess the functional significance of these MSH3 variants, we employed a combination of in silico analyses and in vitro functionality assays. Our objective was to elucidate the correlation between computational predictions and functional outcomes.

A CRISPR-Cas9 knock-in approach was used to introduce and generate specific point mutations in Exons 21, 22, and 23 of MSH3 in the wild-type (WT) MSH3 CRC cell line SW620, which was confirmed with Sanger sequencing. We employed cell proliferation, microsatellite instability assays, and whole genome sequencing to assess biological and genetic consequences. We utilized immunofluorescence, Western blot, and coimmunoprecipitation methods to assess subcellular localization and differences in heterodimer MSH2 binding between WT and variant MSH3 proteins.

We previously identified six novel, potentially pathogenic nonsynonymous variants (c.G1237A, c.C2759T, c.G1397A, c.G2926A, c.C3028T, and c.G3241A) within six exons (Exons 8 (E413K), 9 (S466N), 20 (S920F), 21 (E976K), 22 (H1010Y), and 23 (E1081K), respectively) of MSH3 among AA CRCs as assessed by computational bioinformatic and molecular dynamic simulation analysis. We successfully knocked in three of the MSH3 variants (Exons 21, 22, and 23). Biological phenotypic assays revealed no observable changes in cell morphology or proliferation between WT and MSH3-variant knocked-in cells, and no differences were observed in microsatellite assays. Subcellular localization of variant MSH3 protein was unaffected compared with WT, whereas the interaction between MSH3 and MSH2 was not impacted. Short tandem repeats (STRs), or microsatellites, are short DNA motifs of two to six base pairs repeated consecutively, and they serve as powerful genetic markers for studying inheritance patterns and disease-associated repeat instability. In our analysis, STR profiling revealed both repeat expansions and contractions across multiple motifs, with tetranucleotide repeats showing the most pronounced alterations. Notably, loci flanking LINC00550 (ATTT), FBXL7 (AGAT), and DUSP28 (CTTT/GTTT) displayed consistent repeat expansions (+1.5 to +2), whereas intronic regions within GPC5, TMEM232, and ABCA13 exhibited contractions. Trinucleotide STRs revealed a mix of instability, with repeat gains at SLC25A12 and losses near GTF3C3 and AKAP12. Pentanucleotide and hexanucleotide motifs were more stable overall, but expansions were still noted at CNTN5, CPEB1, and CBSL, and contractions at SGO1 and FER. These findings highlight the bidirectional and motif-specific nature of STR instability driven by MSH3 deficiency. Our results support the utility of STR-based assays as sensitive tools to detect nonclassical MSI events and deepen our understanding of MSH3's role in preserving microsatellite integrity across the genome.

This study investigated the functional consequences of MSH3 variants identified among AA CRCs. While CRISPR-Cas9 knock-in of MSH3 variants (Exons 21, 22, and 23) in SW620 cells did not alter cell morphology, proliferation, protein localization, or MSH2 binding, we observed genetic changes that collectively underscore the bidirectional nature of STR instability in MSH3-deficient cells and reinforce this protein's pivotal role in suppressing slippage at longer repeat motifs. This study advances our understanding of how MSH3 deficiency contributes to genomic instability beyond canonically defined MSI loci, offering novel insights into the mutational landscapes of MMR-deficient tumors and how these MSH3 mutations can potentially contribute to the outcome of AA CRC patients.