{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,5]],"date-time":"2026-06-05T07:02:37Z","timestamp":1780642957417,"version":"3.54.1"},"reference-count":51,"publisher":"Wiley","issue":"1","license":[{"start":{"date-parts":[[2026,6,4]],"date-time":"2026-06-04T00:00:00Z","timestamp":1780531200000},"content-version":"vor","delay-in-days":154,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,1,1]],"date-time":"2026-01-01T00:00:00Z","timestamp":1767225600000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R01 CA258519"],"award-info":[{"award-number":["R01 CA258519"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Human Mutation"],"published-print":{"date-parts":[[2026,1]]},"abstract":"<jats:sec>\n                    <jats:label\/>\n                    <jats:p>\n                      Pathogenic variants of\n                      <jats:italic>MSH3<\/jats:italic>\n                      can increase mutational load within colorectal cells, which may drive initiation and progression of colorectal cancer (CRC). We identified several variants within\n                      <jats:italic>MSH3<\/jats:italic>\n                      among CRCs from African Americans (AA). To predict and assess the functional significance of these\n                      <jats:italic>MSH3<\/jats:italic>\n                      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.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      A CRISPR\u2010Cas9 knock\u2010in approach was used to introduce and generate specific point mutations in Exons 21, 22, and 23 of\n                      <jats:italic>MSH3<\/jats:italic>\n                      in the wild\u2010type (WT)\n                      <jats:italic>MSH3<\/jats:italic>\n                      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.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      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\n                      <jats:italic>MSH3<\/jats:italic>\n                      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\n                      <jats:italic>MSH3<\/jats:italic>\n                      \u2010variant knocked\u2010in 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\u2010associated 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\n                      <jats:italic>LINC00550<\/jats:italic>\n                      (ATTT),\n                      <jats:italic>FBXL7<\/jats:italic>\n                      (AGAT), and\n                      <jats:italic>DUSP28<\/jats:italic>\n                      (CTTT\/GTTT) displayed consistent repeat expansions (+1.5 to +2), whereas intronic regions within\n                      <jats:italic>GPC5<\/jats:italic>\n                      ,\n                      <jats:italic>TMEM232<\/jats:italic>\n                      , and\n                      <jats:italic>ABCA13<\/jats:italic>\n                      exhibited contractions. Trinucleotide STRs revealed a mix of instability, with repeat gains at\n                      <jats:italic>SLC25A12<\/jats:italic>\n                      and losses near\n                      <jats:italic>GTF3C3<\/jats:italic>\n                      and\n                      <jats:italic>AKAP12<\/jats:italic>\n                      . Pentanucleotide and hexanucleotide motifs were more stable overall, but expansions were still noted at\n                      <jats:italic>CNTN5<\/jats:italic>\n                      ,\n                      <jats:italic>CPEB1<\/jats:italic>\n                      , and\n                      <jats:italic>CBSL<\/jats:italic>\n                      , and contractions at\n                      <jats:italic>SGO1<\/jats:italic>\n                      and\n                      <jats:italic>FER<\/jats:italic>\n                      . These findings highlight the bidirectional and motif\u2010specific nature of STR instability driven by\n                      <jats:italic>MSH3<\/jats:italic>\n                      deficiency. Our results support the utility of STR\u2010based assays as sensitive tools to detect nonclassical MSI events and deepen our understanding of\n                      <jats:italic>MSH3<\/jats:italic>\n                      \u2032s role in preserving microsatellite integrity across the genome.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>\n                      This study investigated the functional consequences of MSH3 variants identified among AA CRCs. While CRISPR\u2010Cas9 knock\u2010in 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\n                      <jats:italic>MSH3<\/jats:italic>\n                      \u2010deficient cells and reinforce this protein\u2032s pivotal role in suppressing slippage at longer repeat motifs. This study advances our understanding of how\n                      <jats:italic>MSH3<\/jats:italic>\n                      deficiency contributes to genomic instability beyond canonically defined MSI loci, offering novel insights into the mutational landscapes of MMR\u2010deficient tumors and how these\n                      <jats:italic>MSH3<\/jats:italic>\n                      mutations can potentially contribute to the outcome of AA CRC patients.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1155\/humu\/5588764","type":"journal-article","created":{"date-parts":[[2026,6,5]],"date-time":"2026-06-05T06:28:16Z","timestamp":1780640896000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Novel African American Colorectal Cancer\n                    <i>MSH3<\/i>\n                    Variants Associate With Major Genomic Instability"],"prefix":"10.1155","volume":"2026","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9342-6243","authenticated-orcid":false,"given":"Mudasir","family":"Rashid","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hassan","family":"Brim","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shaolei","family":"Teng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-7595-0422","authenticated-orcid":false,"given":"Adebiyi","family":"Sobitan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-8400-5721","authenticated-orcid":false,"given":"Ruth","family":"Cruz-Cosme","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tang","family":"Qiyi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Minoru","family":"Koi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-6606-615X","authenticated-orcid":false,"given":"Katherine","family":"Casazza","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4243-0933","authenticated-orcid":false,"given":"Jennifer A.","family":"Surtees","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John M.","family":"Carethers","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4048-4666","authenticated-orcid":false,"given":"Hassan","family":"Ashktorab","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,6,4]]},"reference":[{"key":"e_1_2_11_1_2","doi-asserted-by":"publisher","DOI":"10.5858\/arpa.2019-0476-RA"},{"key":"e_1_2_11_2_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.tibtech.2017.12.001"},{"key":"e_1_2_11_3_2","doi-asserted-by":"publisher","DOI":"10.14573\/altex.2001241"},{"key":"e_1_2_11_4_2","unstructured":"VerliH. 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