{"database":"biostudies-literature","file_versions":[],"scores":null,"additional":{"submitter":["Popp NA"],"funding":["NCATS NIH HHS","NHLBI NIH HHS","NHGRI NIH HHS","NCI NIH HHS","NIGMS NIH HHS"],"pagination":["2099-2111"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/S-EPMC12373428"],"repository":["biostudies-literature"],"omics_type":["Unknown"],"volume":["32(10)"],"pubmed_abstract":["Despite widespread advances in DNA sequencing, the functional consequences of most genetic variants remain poorly understood. Multiplexed assays of variant effect can measure the function of variants at scale but cannot readily be applied to the ~10% of human genes encoding secreted proteins. Here we develop a flexible, scalable human cell surface display method, multiplexed surface tethering of extracellular proteins (MultiSTEP), to study the consequences of missense variation in coagulation factor IX (FIX), a serine protease in which genetic variation can cause hemophilia B. We combine MultiSTEP with a panel of antibodies to detect FIX secretion and post-translational modification (PTM), measuring 44,816 variant effects for 436 synonymous variants and 8,528 of the 8,759 possible F9 missense variants. Almost half of missense variants impact secretion, PTM or both. We also identify functional constraints on secretion within the signal peptide and for nearly all gain or loss of cysteine variants. Secretion scores correlate strongly with FIX levels in hemophilia B and reveal that loss-of-secretion variants are more often associated with severe disease. Integration of the secretion and PTM scores enables reclassification of 63.1% of F9 variants of uncertain significance in the My Life, Our Future hemophilia genotyping project. Lastly, we show that MultiSTEP can be applied to other secreted proteins, thus demonstrating that MultiSTEP is a multiplexed, multimodal and generalizable method for systematically assessing variant effects in secreted proteins at scale."],"journal":["Nature structural & molecular biology"],"pubmed_title":["Multiplex and multimodal mapping of variant effects in secreted proteins via MultiSTEP."],"pmcid":["PMC12373428"],"funding_grant_id":["R01 HL152066","F30 HL151075","UM1 HG011969","UL1 TR002373","RM1 HG010461","R01 HL149855","P30 CA014520","R01 GM109110"],"pubmed_authors":["Chang AT","Fletcher SN","Lannert KW","Powell RL","Fayer S","Sheehan JP","Popp NA","Holmes KJ","Zapp BD","Rubin AF","Fowler DM","Wheelock MK","Sheldon KM","Wu X","Johnsen JM"],"additional_accession":[]},"is_claimable":false,"name":"Multiplex and multimodal mapping of variant effects in secreted proteins via MultiSTEP.","description":"Despite widespread advances in DNA sequencing, the functional consequences of most genetic variants remain poorly understood. Multiplexed assays of variant effect can measure the function of variants at scale but cannot readily be applied to the ~10% of human genes encoding secreted proteins. Here we develop a flexible, scalable human cell surface display method, multiplexed surface tethering of extracellular proteins (MultiSTEP), to study the consequences of missense variation in coagulation factor IX (FIX), a serine protease in which genetic variation can cause hemophilia B. We combine MultiSTEP with a panel of antibodies to detect FIX secretion and post-translational modification (PTM), measuring 44,816 variant effects for 436 synonymous variants and 8,528 of the 8,759 possible F9 missense variants. Almost half of missense variants impact secretion, PTM or both. We also identify functional constraints on secretion within the signal peptide and for nearly all gain or loss of cysteine variants. Secretion scores correlate strongly with FIX levels in hemophilia B and reveal that loss-of-secretion variants are more often associated with severe disease. Integration of the secretion and PTM scores enables reclassification of 63.1% of F9 variants of uncertain significance in the My Life, Our Future hemophilia genotyping project. Lastly, we show that MultiSTEP can be applied to other secreted proteins, thus demonstrating that MultiSTEP is a multiplexed, multimodal and generalizable method for systematically assessing variant effects in secreted proteins at scale.","dates":{"release":"2025-01-01T00:00:00Z","publication":"2025 Oct","modification":"2026-06-06T02:14:22.179Z","creation":"2026-05-24T03:12:35.964Z"},"accession":"S-EPMC12373428","cross_references":{"pubmed":["40514537"],"doi":["10.1038/s41594-025-01582-w"]}}