Genetic coding variant in complement factor B (CFB) is associated with increased risk for perianal Crohn's disease and leads to impaired CFB cleavage and phagocytosis

Abstract

Objective: Perianal Crohn's disease (pCD) occurs in up to 40% of patients with CD and is associated with poor quality of life, limited treatment responses and poorly understood aetiology. We performed a genetic association study comparing CD subjects with and without perianal disease and subsequently performed functional follow-up studies for a pCD associated SNP in Complement Factor B (CFB).

Design: Immunochip-based meta-analysis on 4056 pCD and 11 088 patients with CD from three independent cohorts was performed. Serological and clinical variables were analysed by regression analyses. Risk allele of rs4151651 was introduced into human CFB plasmid by site-directed mutagenesis. Binding of recombinant G252 or S252 CFB to C3b and its cleavage was determined in cell-free assays. Macrophage phagocytosis in presence of recombinant CFB or serum from CFB risk, or protective CD or healthy subjects was assessed by flow cytometry.

Results: Perianal complications were associated with colonic involvement, OmpC and ASCA serology, and serology quartile sum score. We identified a genetic association for pCD (rs4151651), a non-synonymous SNP (G252S) in CFB, in all three cohorts. Recombinant S252 CFB had reduced binding to C3b, its cleavage was impaired, and complement-driven phagocytosis and cytokine secretion were reduced compared with G252 CFB. Serine 252 generates a de novo glycosylation site in CFB. Serum from homozygous risk patients displayed significantly decreased macrophage phagocytosis compared with non-risk serum.

Conclusion: pCD-associated rs4151651 in CFB is a loss-of-function mutation that impairs its cleavage, activation of alternative complement pathway, and pathogen phagocytosis thus implicating the alternative complement pathway and CFB in pCD aetiology.

Keywords: IBD - GENETICS; INFLAMMATORY BOWEL DISEASE; META-ANALYSIS.

Figure 1

Serology Quartile Sum Score (QSS) was strongly associated with pCD. Higher perianal CD prevalence was observed in CSMC patients with increasing serology quartile sum score (p=1.39×10–4; OR=1.07 (95% CI 1.04 to 1.11)). Error bars represent 95% CIs centred at the mean. CD, Crohn’s disease; CSMC, Cedars-Sinai Medical Center; pCD, perianal CD.

Figure 2

S252 CFB impairs binding to C3b. (A, B) Plate-bound C3b was incubated with G252 or S252 CFB at the indicated concentration in the presence of C3b (A) or C3b + CFD + properdin (B) for 2 hours at 37°C. Binding of G252 or S252 CFB to C3b was detected using anti-human CFB antibody. (C) Plate-bound G252 or S252 were incubated with C3b or C3b + CFD + properdin for 2 h at 37°C. Binding of C3b to G252 or S252 CFB was detected using anti-human C3 antibody. Data are presented as means±SD. Data are representative of three independent experiments. *p<0.05, **p<0.01, ***p<0.001. CFB, complement factor B;

Figure 3

Serine 252 substitution in CFB impairs C3b-dependent CFB cleavage and results in a de novo glycosylation site. (A) G252 or S252 CFB were incubated with C3b and CFD for 30 min at 37°C. CFB cleavage products were detected with anti-human CFB antibody. (B) G252 or S252 CFB were separated by SDS-PAGE and stained with periodic acid-Schiff (PAS) reagent. (C) Recombinant G252 or S252 CFB (2 μg each), or pooled patient serum (S252/S252, G252/S252, G252/G252) (40 μg/genotype) were incubated with anti-human CFB antibody followed by immunoprecipitation. Samples were separated by SDS-PAGE and stained with PAS reagent. One representative experiment out of three independent experiments is shown (A, B). CFB, complement factor B; SDS-PAGE, sodium dodecyl-sulfate polyacrylamide gel electrophoresis.

Figure 4

Recombinant S252 CFB decreases macrophage phagocytosis and cytokine secretion. (A, B) PBMC-derived macrophages from healthy donors were incubated with fluorescently labelled zymosan particles in the presence of recombinant G252 or S252 CFB. Percentage of phagocytic cells was determined by flow cytometry. (A) Time course of phagocytosis for G252 or S252 CFB (250 ng/mL). (B) Dose response of G252 or S252 CFB (100, 250 and 500 ng/mL) analysed at 15 min. (C, D) PBMC-derived Macrophages were incubated with G252 or S252 CFB (250 ng/mL) in the presence of zymosan (1 ng/mL) for 6 hours. IL-6 (C) and TNF-α (D) concentrations in supernatants were measured by ELISA. Data are presented as means±SD. Data are representative of three (A, B) or five (C, D) independent experiments. *p<0.05, ***p<0.005. CFB, complement factor B; PBMC, peripheral blood mononuclear cells.

Figure 5

Serum from risk subjects (S252/S252, G252/S252) decreases macrophage phagocytosis. PBMC-derived macrophages from healthy donors were incubated with fluorescently labelled zymosan particles for the indicated time points in the presence of 10% serum from patients with CD (S252/S252 n=6, G252/S252 n=15, G252/G252 n=15) (A) or non-IBD controls (G252/S252 n=5, G252/G252 n=5) (B). Phagocytosis was measured by flow cytometry and the percentage of phagocytic cells is shown. Each serum sample was analysed in triplicates. (C) Comparison between G252/S252, G252/G252 non-IBD controls and G252/S252, G252/G252 patients with CD. Data are presented as means±SD. *p<0.05, ***p<0.005. CD, Crohn’s disease; IBD, inflammatory bowel disease; PBMC, peripheral blood mononuclear cells.