Functional Analysis of the Complement Inhibitor Factor I in Sera of Patients with Advanced Age-Related Macular Degeneration

Abstract

Purpose : Rare genetic variants in the complement alternative pathway regulator Factor I (FI) are associated with increased risk for developing advanced age-related macular degeneration (AAMD). Genetic screening frequently identifies variants of uncertain significance. To provide more insight into the function of these variants, we used an optimized biochemical assay of serum to identify both quantitative and qualitative FI deficiency in individuals with and without AAMD with and without rare FI variants.

Methods : FI activity was assessed in serum (0.2%, final conc) with 1.9 mM C3b and 290 nM factor-H followed by determinations of iC3b level using an ELISA with further confirmation by Western blot. Validation of the assay specificity was conducted by comparing results with samples in the presence and absence of exogenous C3b or serum. Each sample was assessed in at least two separate determinations.

Results : FI activity was determined in the serum of 119 individuals across 40 distinct rare FI variants and compared to the activity of FI in 94 wildtype controls. Presence of AAMD was 71% (n=84) within the population containing rare variants while 52% (n=49) had AAMD in the non-carrier (no FI rare variant) population. Analysis of individual variants identified two main categories of functional defect. Type 1 variants had a reduced FI serum level and reduced iC3b production in our assay which normalized when expressed per unit of FI. These variants (e.g. G119R, G287R) cause a quantitative deficiency of FI. Type 2 variants did not have low levels of FI but had reduced iC3b production which did not normalize when expressed per unit of FI. This second group is in keeping with a FI variant that is secreted but has decreased function (e.g. P553S).

Conclusions : We present a high throughput functional screening assay that detects impaired FI activity as a consequence of type 1 (impaired secretion) and type 2 (impaired functional activity) mutations. This assay may allow rapid stratification of patient populations in clinical trials ultimately leading to personalized patient care.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.