Logo image
Back
Comprehensive functional analyses of 110 complement factor B variants provide novel diagnostic and structural insights
Abstract   Open access   Peer reviewed

Comprehensive functional analyses of 110 complement factor B variants provide novel diagnostic and structural insights

Hector Martin Merinero, Andrea Reparaz Suevos, Renee X. Goodfellow, Samantha Blain, Laura Garcia Robles, Juan Guzman Caldentey, Sonsoles Martin Santamaria, Santiago Rodriguez de Corodba, Richard J.H. Smith and Yuzhou Zhang
Immunobiology (1979), Vol.230(4), 153028
07/2025
DOI: 10.1016/j.imbio.2025.153028
url
https://doi.org/10.1016/j.imbio.2025.153028View
Published (Version of record) Open Access

Abstract

Complement-mediated hemolytic uremic syndrome (CM-HUS) is a life-threatening thrombotic microangiopathy driven by complement dysregulation, primarily due to pathogenic variants in genes encoding complement components and regulators. Accurate classification of these variants is critical to optimize the medical utility of genetic analyses. Among all CM-HUS-associated genes, variants in CFB, encoding factor B (FB), are particularly challenging due to their structural and functional complexity. We collected 110 CFB variants found exclusively in gnomAD (n = 39), CM-HUS patients (n = 16), or both (n = 55), and expressed them with a C-terminal His-tag in mammalian cells. Using purified FB protein, assembled C3-convertases were tested for overall on-surface and fluid-phase activity, affinity for C3b, and stability/resistance to FH-regulation using ELISA, immunofixation electrophoresis and hemolytic assays. In-silico predictions (CADD and REVEL) were compared to functional data to assess accuracy in classifying novel CFB variants. We validated the robustness of our methodology by finding the same functional consequences for 16 CFB variants reported previously. In total, of the 110 variants, 63 (57.3%) altered FB expression or function in-vitro and were classified as pathogenic (gain-of-function, n = 14; loss-of-function, n = 29; no expression, n = 20). Of the 16 variants reported only in CM-HUS patients, gain-of-function was overrepresented (43.8%) as compared to gnomAD-exclusive variants where loss-of-function (41.0%) and no-expression (23.1%) were more frequent. We describe the mechanisms by which variants affect FB function, including interference with C3 pro-convertase formation, impediments to FD cleavage, and increased C3-convertase stability/resistance to regulation by complement regulators. Notably, functional data on variants located in the serine protease domain, combined with detailed in-silico molecular dynamic analyses, provided novel structural insights into the interaction between the C3-convertase and its C3-substrate. We provide the most comprehensive functional report of FB variants to date, demonstrate that gain-of-function variants in CFB predispose to CM-HUS, and show that evidence of association with CM-HUS cannot be predicted with current in-silico software. Notably, the location of these variants in FB supports current understanding of the conformational changes and interactions described during C3-convertase assembly and activity but suggests there is a knowledge gap with respect to the mechanism by which complement regulators accelerate C3-convertase decay. This work was supported in part by funds from Kyle and Kelsey Duncan (Wyatt’s White Christmas); we thank those patients who have made this research possible. The work was also supported by Alexion Pharmaceuticals, Inc. We acknowledge the contribution to this work of Guillermo del Angel and William Mowrey.

Details

Metrics

12 Record Views
Logo image