Rare Variants in the Functional Domains of Complement Factor H Are Associated With Age-Related Macular Degeneration

Abstract

Purpose: Age-related macular degeneration (AMD) has a substantial genetic risk component, as evidenced by the risk from common genetic variants uncovered in the first genome-wide association studies. More recently, it has become apparent that rare genetic variants also play an independent role in AMD risk. We sought to determine if rare variants in complement factor H (CFH) played a role in AMD risk.

Methods: We had previously collected DNA from a large population of patients with advanced age-related macular degeneration (A-AMD) and controls for targeted deep sequencing of candidate AMD risk genes. In this analysis, we tested for an increased burden of rare variants in CFH in 1665 cases and 752 controls from this cohort.

Results: We identified 65 missense, nonsense, or splice-site mutations with a minor allele frequency ≤ 1%. Rare variants with minor allele frequency ≤ 1% (odds ratio [OR] = 1.5, P = 4.4 × 10⁻²), 0.5% (OR = 1.6, P = 2.6 × 10⁻²), and all singletons (OR = 2.3, P = 3.3 × 10⁻²) were enriched in A-AMD cases. Moreover, we observed loss-of-function rare variants (nonsense, splice-site, and loss of a conserved cysteine) in 10 cases and serum levels of FH were decreased in all 5 with an available sample (haploinsufficiency). Further, rare variants in the major functional domains of CFH were increased in cases (OR = 3.2; P = 1.4 × 10⁻³) and the magnitude of the effect correlated with the disruptive nature of the variant, location in an active site, and inversely with minor allele frequency.

Conclusions: In this large A-AMD cohort, rare variants in the CFH gene were enriched and tended to be located in functional sites or led to low serum levels. These data, combined with those indicating a similar, but even more striking, increase in rare variants found in CFI, strongly implicate complement activation in A-AMD etiopathogenesis as CFH and CFI interact to inhibit the alternative pathway.

Figure 1

Rare CFH variants within CCP1-4 repeats cluster at the C3b:FH interface. Variants present only in A-AMD cases are shown in purple or red. Variants that are nonsense or remove a conserved cysteine are shown in red (these are only seen in cases). Variants seen only in controls are shown in green. One variant was observed in both cases and controls (magenta). The variants in repeats CCP3 contact the CUB domain and variants in repeats CCP4 contact the TED of C3b. Complement component 3 α-chain is in teal (foreground) and the β-chain is in red (background). The structure of FH and C3b (PDB ID 2wII) was displayed in Pymol (Delano Scientific). C345C (NTR – Netrin C-terminal domain); CUB, complement C1r/C1s, Uegf, Bmp1; TED, thioester domain.

Figure 2

Complement Factor H rare variants in CCP20 cluster near R1210C. Factor H CCP19-20 is in orange; C3d is in teal. Variant R1210C is shown in black (arrow). The crystal structure of CCP19-20 contains two C3d fragments (PDB ID 2XQW) and was displayed in Pymol (Delano Scientific). Mutations seen only in A-AMD cases are shown in dark blue. The CCP19 variant (Q1143E, in one control) is not present in the crystal structure. The rare variants cluster on the same face as R1210C, known to affect heparin/GAG binding.