Functional Assessment of Rare Genetic Variants of Complement Factor I in Age Related Macular Degeneration

Abstract

Age-related macular degeneration (AMD) is the leading cause of blindness in the developed world. There is no cure for the disease, and treatment options remain limited to anti vascular endothelial growth factor (VEGF) therapeutics and dietary supplementation. Several pivotal studies have associated both rare and common genetic variants in complement factor I (CFI) with an increased AMD susceptibility, with rare variants carrying a particularly increased burden (Seddon et al., 2013; Fritsche et al., 2016). Three rare genetic variants of CFI nominally associated with AMD were identified for further functional analysis (R406H, K441R and P553S). Their selection was based upon occurrence in the literature, in silico analysis and structural modelling within the alternative pathway (AP) regulatory trimolecular complex (TMC). Each of the three variants are secreted and are reported within the normal range for serum FI levels. A simplified method was developed for the purification of both recombinant and plasma FI without the need for a polyhistidine tag. Recombinant protein production led to the generation of a mixture of proteins, Pro-I and FI. Pro-I is proposed to be functionally inactive, however this has not been proven. To facilitate the removal, and subsequent analysis of Pro I, the generation of an antibody was attempted, however this was unsuccessful. Using ion exchange chromatography, Pro-I could be purified from both plasma and supernatant and has been confirmed as completely inactive and incapable of forming the AP regulatory TMC. Additionally, a novel method for generating completely processed, fully functional recombinant FI has been developed. Through the modelling of K441R, R406H and P553S on both a WT and inactive (S525A) CFI backbone, this has enabled the most extensive analysis of these variants recorded in the literature. K441R, R406H, P553S all demonstrate proteolytic activity, however P553S demonstrates a significantly reduced activity in a variety of fluid-phase cofactor assays. When modelled on the AP regulatory TMC, K441R performed similarly to WT, R406H demonstrated a slightly reduced response, and P553S exhibited noticeably decreased binding. These results led to the hypothesis that patients carrying the variants P553S and R406H may benefit from complement inhibition or FI supplementation, due to a defect in AP regulation which may exacerbate their disease.