Multiplex, multimodal mapping of variant effects in secreted proteins
Nicholas A Popp1,2,3, Rachel L Powell1, Melinda K Wheelock1,3
1Department of Genome Sciences, University of Washington School of Medicine, Seattle, WA, USA.
Biorxiv : the Preprint Server for Biology
|February 20, 2025
Summary
We developed Multiplexed Surface Tethering of Extracellular Proteins (MultiSTEP), a new method to assess genetic variant effects in secreted proteins. This approach improves understanding of diseases like hemophilia B by analyzing protein secretion and modification.
Area of Science:
- Genomics
- Molecular Biology
- Biochemistry
Background:
- Understanding genetic variant function is crucial, but current methods struggle with secreted proteins.
- Multiplexed Assays of Variant Effect (MAVEs) are powerful but limited for ~10% of human genes encoding secreted proteins.
Purpose of the Study:
- To develop and validate a scalable method for assessing variant effects in secreted proteins.
- To investigate the functional consequences of genetic variation in coagulation factor IX (FIX) using the new method.
Main Methods:
- Developed Multiplexed Surface Tethering of Extracellular Proteins (MultiSTEP), a human cell surface display technique.
- Applied MultiSTEP to study missense variants in coagulation factor IX (FIX), measuring secretion and post-translational modification using antibody panels.
- Analyzed 44,816 variant effects for 436 synonymous and 8,528 missense variants in the *F9* gene.
Main Results:
- 49.6% of *F9* missense variants affected FIX secretion or post-translational modification.
- Identified functional constraints on secretion within the signal peptide and for cysteine-altering variants.
- Secretion scores correlated with FIX levels in hemophilia B, with secretion loss linked to severe disease.
- Reclassified 63.1% of *F9* variants of uncertain significance.
Conclusions:
- MultiSTEP is a versatile, scalable method for assessing variant effects in secreted proteins.
- The method provides insights into FIX function, disease mechanisms in hemophilia B, and variant interpretation.
- MultiSTEP is generalizable to various secreted proteins, addressing a key gap in functional genomics.
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