Evidence for pathogenicity of inherited biallelic MSH3 variants causing early-onset colorectal cancer

Background. We previously reported on an individual from a fourth family worldwide with early-onset colorectal adenocarcinoma with germline compound heterozygous MSH3 variants (c.2436-1G>A / c.3265A>T). Tumor DNA from this individual demonstrated microsatellite instability-low (MSI-L) and elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) suggestive that both alleles were pathogenic. Additionally, tumor cells exhibited MSH3 in the cytoplasm, with some cells showing loss of MSH6 that might be suggestive of at least one MSH3-expressing allele having a dominant-negative effect on MSH2, which binds to both MSH3 and MSH6 for their stability. Here, we aimed to prove that both MSH3 variants are pathogenic as well as to assess effects on other mismatch repair proteins. Methods. Both MSH3 variants, c.2436-1G>A and c.3265A>T, were introduced individually into SW620 cells using CRISPR/Cas9. Presence of each variant was confirmed by Sanger sequencing. Western blotting (WB) and fragment analysis were performed on variant clones and compared with parental SW620 cells. Results. We successfully established MSH3 variant clones with homozygous c.2436-1G>A and clones with homozygous c.3265A>T. WB analysis revealed complete loss of MSH3 protein in cells containing homozygous c.2436-1G>A. However, variant MSH3 protein was detected in cells containing homozygous c.3265A>T, although the amount of protein was less than that of wild-type (WT) MSH3 expressed in parental SW620. These results suggest that the transcription rate of variant c.3265A>T may be reduced and/or variant-generated protein may be less stable than WT MSH3. Expression levels of MSH2, MSH6 and MLH1 in these 2 variant cell lines were similar to expression in parental SW620 cells. MSI-L/EMAST was detected in 18.8% (6/32) of subclones isolated from each variant indicating loss of MMR function, whereas no subclones (0/32) from SW620 showed MSI-L/EMAST. Conclusions. We established cell models of two germline MSH3 variants (c.2436-1G>A and c.3265A>T) which had been identified in an early-onset colorectal cancer patient. Cells with each variant exhibited MSI-L/EMAST indicating loss of MSH3 function, proving that these two germline variants are pathogenic. As we did not observe any change in cell MSH6 expression, further studies are in progress to generate cells that are compound heterozygous for these two variants and to further elucidate the pathological effects of the combinations of these variants.