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Although this minisatellite harbors extraordinary allelic diversity in humans, the functional consequences of most naturally occurring variants remain unknown. Here we functionally characterize 80 human PRDM9 alleles using genome-wide chromatin profiling. Despite extensive sequence diversity within the ZNF array, most alleles function indistinguishably from common A and C hotspot-specifying alleles, revealing that human PRDM9 function is more constrained than its sequence variation predicts. In contrast, rare variants identified in infertile individuals occupy two functional extremes: abundant and novel DNA binding specificity or minimal DNA binding, suggesting that both gain- and loss-of-function alleles may disrupt symmetric hotspot specification during meiosis, plausibly contributing to human infertility. Together, our findings define the functional landscape of human PRDM9 variation and provide a framework for interpreting the impact of newly discovered PRDM9 alleles.<\/jats:p>","DOI":"10.64898\/2026.09.10.750150","type":"posted-content","created":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T01:10:29Z","timestamp":1789521029000},"source":"Crossref","is-referenced-by-count":0,"title":["PRDM9-mediated meiotic hotspot specification is constrained in humans despite extensive sequence diversity"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2696-5575","authenticated-orcid":false,"given":"Rachel L.","family":"Cosby","sequence":"first","affiliation":[{"name":"The Eunice Kennedy Shriver National Institute of Child Health and Human Development, the National Institutes of Health, Bethesda, MD, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8815-3364","authenticated-orcid":false,"given":"Marja Brolinson","family":"King","sequence":"additional","affiliation":[{"name":"The Eunice Kennedy Shriver National Institute of Child Health and Human Development, the National Institutes of Health, Bethesda, MD, USA"},{"name":"Institute for Reproductive Health, Cincinnati, OH, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4815-970X","authenticated-orcid":false,"given":"Alexandra M.","family":"Poch","sequence":"additional","affiliation":[{"name":"The Eunice Kennedy Shriver National Institute of Child Health and Human Development, the National Institutes of Health, Bethesda, MD, USA"},{"name":"Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, Fertility Center of the Carolinas, University of South Carolina School of Medicine\/Prisma Health Upstate, Greenville, South Carolina, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4144-7891","authenticated-orcid":false,"given":"M. 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