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Complement factor I functional assay for assessing CFI variants
Abstract   Open access   Peer reviewed

Complement factor I functional assay for assessing CFI variants

Renee Goodfellow, Nicole Meyer, Nicolo Ghiringhelli Borsa, Amanda Taylor, Carla Nester, Richard Smith and Yuzhou Zhang
Immunobiology (1979), Vol.230(4), p.152938
07/2025
DOI: 10.1016/j.imbio.2025.152938
url
https://doi.org/10.1016/j.imbio.2025.152938View
Published (Version of record) Open Access

Abstract

Complement Factor I (FI) is a serine protease essential for regulating complement activity across all pathways. With the assistance of cofactors—factor H (FH), membrane cofactor protein (MCP/CD46), soluble complement receptor 1 (sCR1), or C4 binding protein (C4BP)—FI inactivates C3b and C4b through proteolytic cleavage. Rare variants (RVs) in CFI can cause FI deficiency via two primary mechanisms: reduced protein expression (haploinsufficiency, type I) or impaired functional activity (type II). Differentiating type II defects is crucial, as these variants may influence therapeutic decisions in complement-mediated diseases. To evaluate functional defects independent of FI expression levels, we developed a standardized cell-based assay. C3b-coated sheep erythrocytes were incubated with patient serum or plasma (diluted 1:32), supplemented with purified cofactors (FH, MCP, or sCR1), and incubated at 37 °C for 15 minutes. Residual C3b was quantified using FB and FD with diluted rat serum EDTA as a source of C5-C9, enabling membrane attack complex-mediated hemolysis. Controls included sera/plasma from healthy donors (negative control) and a previously known loss-of-function CFI variant (c.1006C>G, p.Arg336Gly). Genetic analysis identified 59 patients with 42 distinct CFI missense RVs, including 10 novel variants. Of these, 20 RVs showed reduced plasma FI expression (type I), while 22 had normal FI levels. Among variants with normal expression, our functional assay identified five loss-of-function variants clustered in two regions of the serine protease domain. Our validated assay reliably identifies type II (functional) CFI variants, emphasizing the importance of integrating genetic and functional analyses for accurate classification and informed clinical decision-making in patients with complement-mediated diseases. Acknowledgements. Supported in part by National Institutes of Health R01 DK110023.

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