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Multiplex and multimodal mapping of variant effects in secreted proteins via MultiSTEP

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Popp, Nicholas A., Powell, Rachel L., Wheelock, Melinda K., Holmes, Kristen J., Zapp, Brendan D., Sheldon, Kathryn M., Fletcher, Shelley N., Wu, Xiaoping +7 more·2025·Nature Structural & Molecular Biology
DOI 10.1038/s41594-025-01582-wPMC PMC12373428
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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.

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  • A study used fusion of secreted peptides retaining their own signal sequence to a transmembrane protein anchoring them to the plasma membrane.

    “A step towards this was recently provided by a study that used fusion of secreted peptides retaining their own signal sequence to a transmembrane protein anchoring them to the plasma membrane (117).”

    From Massively parallel functional genomic assays in endocrinology: from promise to delivery (2026) · cites this paper as [117]

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Authors
Popp, N. A., Powell, R. L., Wheelock, M. K., Holmes, K. J., Zapp, B. D., Sheldon, K. M., Fletcher, S. N., Wu, X., Fayer, S., Rubin, A. F., Lannert, K. W., Chang, A. T., Sheehan, J. P., Johnsen, J. M., & Fowler, D. M.
Affiliations
  • University of Washington (US)
  • Bloodworks Northwest (US)
  • Seattle Children's Hospital (US)
  • The University of Melbourne (AU)
  • University of Wisconsin–Madison (US)
Corresponding author
  • Johnsen, Jill M.
  • Fowler, Douglas M.
ORCID
Venue
Nature Structural & Molecular Biology
Publisher
Springer Science and Business Media LLC
Published
2025-06-13
Volume
32
Issue
10
Pages
2099-2111
ISSN
  • 1545-9985 (ISSN-L)
  • 1545-9993
DOI
10.1038/s41594-025-01582-w
PMC
PMC12373428
OpenAlex
W4411285761
Cited by
15
References
5
Citations by year
2026: 9 · 2025: 3 · 2024: 3
FWCI
7.95
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Structural & Molecular Biology (publishedVersion)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nd)
  • PubMed (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed Central (submittedVersion, cc-by-nd)
  • Minerva Access (University of Melbourne) (submittedVersion)
Funders
  • Biogen
  • National Institutes of Health (UL1TR002373)
  • National Heart, Lung, and Blood Institute (F30HL151075, R01HL152066, R01HL149855)
  • National Human Genome Research Institute (RM1HG010461, UM1HG011969)
  • National Cancer Institute (P30CA014520)
  • National Institute of General Medical Sciences (R01GM109110, T32GM007454)
  • National Center for Advancing Translational Sciences
Topics
  • Health Sciences › Medicine › Hematology › Hemophilia Treatment and Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genomics and Rare Diseases
  • Health Sciences › Medicine › Hematology › Platelet Disorders and Treatments
Keywords
secreted proteins, variant effect prediction, cell surface display, coagulation factor IX, hemophilia B, missense mutations, protein secretion, post-translational modification, signal peptide, variants of uncertain significance, F9 gene, serine protease

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