AMD: The Biology of the Genetics

Abstract

Purpose: : The goal of our studies is to understand how the polymorphisms in the coding regions of complement proteins associated with AMD risk affect complement activity. Age-related macular degeneration (AMD) is the most common cause of vision impairment and blindness in the developed world. Genetic studies have identified mutations/deletions in the genes encoding for proteins in the complement pathway, including C3, factor B, factor H, and factor H related proteins CFHR1 and CFHR3, to be strongly associated with AMD. Although the prevailing hypothesis is that mutations in the complement pathway components lead to chronic over-activation of the pathway, a correlation between the biophysical and biochemical properties of mutant complement proteins with disease-causing biology has yet to be established.

Methods: : The variant proteins encoded by the risk and protective alleles for C3, factor B, and factor H were expressed transiently in HEK 293T cells. All proteins were purified to >99% homogeneous monomers. Functional activities of the proteins were measured by concentration titration in three assays: 1) C3b deposition on zymosan-coated plates; 2) MAC deposition on zymosan-coated plates; 3) hemolysis of rabbit red blood cells.

Results: : For the factor B variants, in each assay the R32 risk variant was more active than the Q32 protective variant. Further analysis of the factor B variants using an alpha-screen proximity assay to measure association of factor B with C3b indicated that the binding affinity of the R32 variant to C3b was almost four times stronger than the Q32 variant, suggesting that the R32 variant would generate more of the stable C3 convertase (C3Bb) than that of the Q32 variant. For factor H variants, the risk variants V62 and H402 were less efficient than the protective variants I62 and Y402 in inactivating C3 convertase in hemolytic assays using factor H depleted human serum.

Conclusions: : Variants in complement pathway proteins C3 and factor B that raise risk for AMD over-activate the pathway, while the inhibitory factor H risk variants associated with AMD result in attenuated inactivation of the pathway. The data support the hypothesis that over-activation of the complement pathway has a role in the pathogenesis of AMD.

Keywords: age-related macular degeneration • genetics • inflammation