Abstract
Purpose :
Factor I (FI) is a serine protease inhibitor of the alternative pathway of the complement system. This regulation of the complement system is achieved by cleavage of C3b (known as cofactor activity). Heterozygous rare genetic variants in CFI are associated with AAMD. The clinical impact of these variants is unknown since a majority have not been functionally characterized and are classified based on predictive models as ‘variants of uncertain significance’ (VUS). This study assessed the functional significance of VUS in CFI.
Methods :
Our previous cross-sectional study demonstrated that heterozygous CFI variants in AAMD can be categorized into three major types [Java et al. Trans Vis Sci Tech 2020; 9(9):37]. Type 1 variants caused a quantitative deficiency of FI (decreased FI level with a proportional decrease in function). Type 2 variants demonstrated a qualitative deficiency (normal FI level but decreased function). However, Type 3 variants consisted of VUS that were less dysfunctional than Types 1 and 2 but were not as biologically active as wild-type. In this study we present an in-depth functional assessment of the Type 3 variants. We utilized serum FI antigenic levels, site-directed mutagenesis followed by expression of the recombinant variant and a comprehensive set of functional assays to characterize nine Type 3 variants identified in 34 individuals (R202I, Q217H, S221Y, G263V, G362A, K441R, Q462H, I492L and N536K).
Results :
Our study established that the expression of the recombinant protein compared to wild-type by 293T cells was reduced for Q217H, S221Y and G263V. Further, R202I and G362A had significantly reduced cofactor activity. These results led to recategorization of Q217H, S221Y and G263V as Type 1 variants and to reclassification of R202I and G362A as Type 2. The remaining Type 3 variants (K441R, Q462H, I492L and N536K) are best classified as being benign at this time. This study highlights the utility of an in-depth functional analysis in defining the pathologic and clinical implications of complement variants in AAMD and provides further insights relative to the pathogenic mechanisms underlying AMD.
Conclusions :
This approach will be a useful model system for guiding more appropriate therapeutic decisions relative to an individualized treatment plan for patients and thereby pave the way for precision medicine in AMD.
This is a 2021 ARVO Annual Meeting abstract.