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However, for functionally annotating the thousands of cancer-associated missense mutations, or variants of uncertain significance (VUS), purifying variant proteins for biochemical and functional analysis is cost-prohibitive and inefficient. We describe parallel functional annotation (PFA) of large numbers of VUS using small cultures and crude extracts in 96-well plates. Using members of a histone methyltransferase family, we demonstrate high-throughput structural and functional annotation of cancer-associated mutations. By combining functional annotation of paralogs, we discovered two phylogenetic and clustering parameters that improve the accuracy of sequence-based functional predictions to over 90%. Our results demonstrate the value of PFA for defining oncogenic\/tumor suppressor functions of histone methyltransferases as well as enhancing the accuracy of sequence-based algorithms in predicting the effects of cancer-associated mutations.<\/jats:p>","DOI":"10.1038\/s41598-022-23229-2","type":"journal-article","created":{"date-parts":[[2022,11,2]],"date-time":"2022-11-02T12:03:49Z","timestamp":1667390629000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Parallel functional annotation of cancer-associated missense mutations in histone methyltransferases"],"prefix":"10.1038","volume":"12","author":[{"given":"Ashley J.","family":"Canning","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Susan","family":"Viggiano","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Martin E.","family":"Fernandez-Zapico","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Michael S.","family":"Cosgrove","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2022,11,2]]},"reference":[{"key":"23229_CR1","doi-asserted-by":"publisher","first-page":"D1005","DOI":"10.1093\/nar\/gky1120","volume":"47","author":"A Buniello","year":"2019","unstructured":"Buniello, A. et al. 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