Variants in Complement Factor H and Complement Factor H-Related Protein Genes, CFHR3 and CFHR1, Affect Complement Activation in IgA Nephropathy

Abstract

Complement activation is common in patients with IgA nephropathy (IgAN) and associated with disease severity. Our recent genome-wide association study of IgAN identified susceptibility loci on 1q32 containing the complement regulatory protein-encoding genes CFH and CFHR1-5, with rs6677604 in CFH as the top single-nucleotide polymorphism and CFHR3-1 deletion (CFHR3-1∆) as the top signal for copy number variation. In this study, to explore the clinical effects of variation in CFH, CFHR3, and CFHR1 on IgAN susceptibility and progression, we enrolled two populations. Group 1 included 1178 subjects with IgAN and available genome-wide association study data. Group 2 included 365 subjects with IgAN and available clinical follow-up data. In group 1, rs6677604 was associated with mesangial C3 deposition by genotype-phenotype correlation analysis. In group 2, we detected a linkage between the rs6677604-A allele and CFHR3-1∆ and found that the rs6677604-A allele was associated with higher serum levels of CFH and lower levels of the complement activation split product C3a. Furthermore, CFH levels were positively associated with circulating C3 levels and negatively associated with mesangial C3 deposition. Moreover, serum levels of the pathogenic galactose-deficient glycoform of IgA1 were also associated with the degree of mesangial C3 deposition in patients with IgAN. Our findings suggest that genetic variants in CFH, CFHR3, and CFHR1 affect complement activation and thereby, predispose patients to develop IgAN.

Keywords: IgA nephropathy; complement activation; complement factor H; complement factor H receptor 3-1Δ deletion.

Figure 1.

Contributors associated with mesangial C3 deposits in IgAN. (A) The rs6677604-A allele was associated with lower intensity of mesangial C3 deposits. Patients with IgAN with the AA/AG genotype of rs6677604 (n=95) had high intensity of mesangial C3 deposits compared with those with the rs6677604-GG genotype (n=1083) (0, 1+, 2+, and 3+–4+: 13.7%, 28.4%, 31.6%, and 26.3%, respectively, in rs6677604-AA/AG versus 10.1%, 16.4%, 39.2%, and 34.3%, respectively, in rs6677604-GG; P=0.01). (B) Lower circulating C3 levels were associated with greater mesangial C3 deposition. Circulating C3 levels decreased significantly from 0 to 3+–4+ mesangial C3 deposition in patients with IgAN (0, 1+, 2+, and 3+–4+: 1.09±0.26, 1.08±0.20, 1.03±0.23, and 0.96±0.21 g/L, respectively; P<0.001; n=106, n=169, n=382, and n=351, respectively). (C and D) Higher circulating IgA and Gd-IgA1 levels were associated with greater mesangial C3 deposition. From 0 to 3+–4+ mesangial C3 deposition, significantly increased levels of circulating IgA (0, 1+, 2+, and 3+–4+: 2.95±1.19, 3.08±1.22, 3.19±1.10, and 3.39±1.23 g/L, respectively; P=0.001; n=109, n=173, n=390, and n=357, respectively) and Gd-IgA1 levels (0, 1+, 2+, and 3+–4+: 279.61±144.68, 306.50±128.64, 313.29±128.89, and 350.15±165.54 units/ml, respectively; P<0.009; n=38, n=82, n=177, and n=183, respectively) were observed in patients with IgAN.

Figure 2.

Minor allele A of rs6677604 highly tagged CFHR3–1 deletion (CFHR3–1∆). Pie charts show the proportion of different copy numbers of CFHR3 and CFHR1 in patients with IgAN and healthy controls with rs6677604-AA, -AG, and -GG genotypes. In our population, 100% of individuals (41 of 41 patients with IgAN and 28 of 28 healthy controls) with rs6677604-GG had two copies of CFHR3 and CFHR1. All individuals (42 of 42 patients with IgAN and 55 of 55 healthy controls) with rs6677604-AG had one copy of CFHR3, whereas all individuals (5 of 5 healthy controls) with rs6677604-AA had no copies of CFHR3. In patients with IgAN with rs6677604-AG, 93% (39 of 42) had one copy of CFHR1, and the remaining 7% (3 of 42) had two copies of CFHR1. In healthy controls with rs6677604-AG, 89% (49 of 55) had one copy of CFHR1, 7% (4 of 55) had two copies of CFHR1, and 4% (2 of 55) had no copies of CFHR1. Of five controls with rs6677604-AA, four (80%) had no copies of CFHR1, and one (20%) had one copy of CFHR1.

Figure 3.

Comparison of CFH levels according to rs6677604 genotype in patients with IgAN and healthy controls. Individuals with rs6677604-A alleles showed significantly higher plasma CFH levels than those with rs6677604-G alleles in both patients with IgAN (AG versus GG: 431.29±131.71 versus 376.27±147.06 µg/ml; P=0.03; n=39 and n=326, respectively) and healthy controls (AA versus AG versus GG: 426.26±71.73 versus 373.37±97.71 versus 328.81±80.03 µg/ml; P<0.01; n=5, n=56, and n=56, respectively). P values for each comparison between two groups are shown.

Figure 4.

Comparison of C3 activation split product C3a serum levels according to the rs6677604 genotype in patients with IgAN. Patients with IgAN with the rs6677604-AG genotype showed significantly lower C3a levels than those without the genotype (464.0 [275.3–687.6] versus 2715.1 [1044.8–4405.3] ng/ml; P<0.001; n=37 and n=31, respectively). Each symbol corresponds to a value from one individual.

Figure 5.

Correlation of CFH levels with circulating C3 and mesangial C3 deposition in patients with IgAN. (A) The circulating CFH level was positively correlated with the circulating C3 level (correlation coefficient=0.360; P<0.001). (B) Lower CFH levels were associated with greater mesangial C3 deposition. Circulating CFH levels decreased significantly from 0 to 3+–4+ mesangial C3 deposition in patients with IgAN (0, 1+, 2+, and 3+–4+: 445.30±186.41, 401.25±150.82, 363.97±147.14, and 378.78±135.09 µg/ml, respectively; 0 versus 1+, P=0.21; 0 versus 2+, P=0.008; 0 versus 3+–4+, P=0.03; n=28, n=47, n=114, and n=176, respectively).

Figure 6.

A hypothesis for a potential mechanism by which variants of CFH, CFHR3, and CFHR1 genes affect complement activation in IgAN. Previous studies revealed the pathogenesis of IgAN, in which Gd-IgA1 molecules bound by antiglycan autoantibodies form circulating IgA1-containing immune complexes that ultimately deposit in the glomerular mesangium, inducing glomerular injury. Meanwhile, complement C3 was observed in both circulating and deposited immune complexes. In this study, we found that variants in CFH, CFHR3, and CFHR1 (rs6677604-A allele or CFHR3–1Δ) were associated with higher CFH levels. Because genetic deletion of CFHR3 and CFHR1 was reported to lead to absence of these proteins and because CFHR1 was a competitive antagonist of CFH to modulate complement activation, higher CFH levels together with the absence of antagonist (CFHR1 protein) resulted in the robust complement inhibition represented by higher circulating C3 and lower C3a. Furthermore, the associations between mesangial C3 deposition and circulating CFH levels as well as genetic variants suggested that variants in CFH, CFHR3, and CFHR1 influenced the formation and deposition of pathogenic immune complexes in IgAN.