Variation in factor B (BF) and complement component 2 (C2) genes is associated with age-related macular degeneration

Abstract

Age-related macular degeneration (AMD) is the most common form of irreversible blindness in developed countries. Variants in the factor H gene (CFH, also known as HF1), which encodes a major inhibitor of the alternative complement pathway, are associated with the risk for developing AMD. Here we test the hypothesis that variation in genes encoding other regulatory proteins of the same pathway is associated with AMD. We screened factor B (BF) and complement component 2 (C2) genes, located in the major histocompatibility complex class III region, for genetic variation in two independent cohorts comprising approximately 900 individuals with AMD and approximately 400 matched controls. Haplotype analyses identify a statistically significant common risk haplotype (H1) and two protective haplotypes. The L9H variant of BF and the E318D variant of C2 (H10), as well as a variant in intron 10 of C2 and the R32Q variant of BF (H7), confer a significantly reduced risk of AMD (odds ratio = 0.45 and 0.36, respectively). Combined analysis of the C2 and BF haplotypes and CFH variants shows that variation in the two loci can predict the clinical outcome in 74% of the affected individuals and 56% of the controls. These data expand and refine our understanding of the genetic risk for AMD.

Figure 1

Diagram and haplotype analysis of SNPs in BF and C2. The SNPs used in the study are shown along with the predicted haplotypes, odds ratios (OR), P values and frequencies in the combined analysis of affected individuals (CAS) and controls (CON). The 95% c.i. for H7 is 0.33–0.61 and for H10 is 0.23–0.56. The ancestral (chimpanzee) haplotype is designated as Anc. Examples of haplotype H2 (AL662849), H5 (AL645922, NG_004658) and H7 (NG_000013) have been sequenced, and no additional nonsynonymous variants in either the C2 or BF genes are present.

Figure 2

Combined complement gene analyses. Individual SNP analyses suggested several possible combinations of SNPs that protect an individual from developing AMD. To test these, an empirical model was applied first. (a,b) Fraction accounted for by hypothesized model. The model gives four possible combinations of genotypes (a) that would protect from AMD: (i) rs547154 (R32Q) is G/A AND rs1061170 (Y402H) is C/T, (ii) rs547154 is G/A AND rs1061170 is C/C, (iii) rs4151667 (L9H) is T/A AND rs1061170 is C/T, and (iv) rs4151667 is T/A AND rs1061170 is C/C. Application of this model resulted in the distributions shown in b for the Iowa, Columbia and combined cohorts. The case percentage is the percentage of cases for which the model was false; that is, they did not have protection as described by the model. The control percentage is the percentage of controls that did have the protective factors. (c,d) Fraction accounted for by the Exemplar machine-built model. c shows the best-performing model, which describes four possible individual or combinations of genotypes that protect from AMD (that is, combinations resulting in the model being ‘true’): (i) rs1048709 (R150R) is G/G AND rs1061170 is C/C, OR (ii) rs547154 is G/A, OR (iii) rs4151667 is T/A OR (iv) CFH intron 1 variant is delTT. The model performance is shown in d for the Iowa, Columbia, and combined cohorts.

Figure 3

Immunolocalization of proteins along the retinal pigment epithelium (RPE)-choroid (Ch) complex in sections from an unfixed eye of a 72-year-old donor with early-stage AMD. (a) BF. (b) Ba (a fragment of the full-length factor B). (c) C3. Anti-BF (red) labels drusen (D), particularly along their rims, Bruch’s membrane and the choroidal stroma. Anti-Ba (purple) labels Bruch’s membrane and RPE-associated patches. The distribution of BF is similar to that of C3. Brown coloration in the RPE cytoplasm and choroid is due to melanin.