Abstract
Purpose :
Complement Factor H (CFH) functions as a negative regulator of the complement system alternative pathway (AP) through a number of activities. Of these, CFH competes with Factor B for C3b binding, preventing AP C3 convertase (C3bBb) formation. Once formed, CFH can dissociate C3bBb, defined as decay acceleration activity (DAA). CFH acts as a cofactor for Complement Factor I (CFI) to irreversibly inactivate C3b to iC3b, thereby regulating C3bBb activity as well as enabling opsonin mediated debris clearance mechanisms. Additionally, CFH binds to mammalian cell surfaces to protect self-cells from complement activation. There are CFH coding variants known to be associated with age-related macular degeneration (AMD) and it is of interest to understand which functions, if any, get impacted in these variants. The functional implications of a subset of the AMD-associated CFH variants were evaluated in comparison to plasma derived human CFH in a panel of in vitro activity assays.
Methods :
Full-length human CFH coding variants, R53H, S58A, R175Q and R1210C, were tested for activity in the following five assays compared to plasma purified human CFH; SPR-based human C3b binding and DAA of C3bBb, cofactor activity in the CFI-mediated cleavage of C3b, protection of sheep erythrocytes from hemolysis in CFH-deficient human serum and terminal complement pathway membrane attack complex formation in Weislab® assay. Functional impairment in each of the assays was defined by the variant having a response more than one standard deviation outside of the observed average for the plasma derived human CFH control among multiple (≥2) runs of the assay.
Results :
The AMD-associated CFH variants, were found to be functionally impaired in one or more of the assays. R53H was functionally impaired in the DAA, cofactor, hemolysis and Weislab® assays, S58A in the cofactor assay and R175Q in the C3b binding, DAA, cofactor and Weislab® assays. R1210C function was marginally impacted in all tested assays.
Conclusions :
This study confirmed impairment of functionality of CFH in AMD-associated CFH coding variants R53H, S58A, R175Q and R1210C. The molecule’s functionality is affected in one or more of five tested assays. Prediction of AMD associated risk with newly-discovered CFH variants of unknown significance may need to factor in functional impairment in a panel of assays rather than a single select assay.
This is a 2020 ARVO Annual Meeting abstract.