Functional Study of Rare Genetic Variants in Complement Factor Haidsinterpretation Of

Functional study of Rare Genetic Variants in Complement Factor HAidsInterpretation of TheirSignificance in Complement-mediated Renal Disease.

Introduction: Membranoproliferative glomerulonephritis (MPGN), C3 glomerulopathy (C3G) and atypical haemolytic uraemic syndrome (aHUS) are rare renal diseases associated with dysregulation of the alternative pathway (AP) of complement. Mutation screening in candidate genes in AP, including complement factor H (CFH) is routine in aHUS and is increasing in cases of MPGN and C3G. Correct interpretation of the pathological significance of rare genetic variants is critical to appropriate clinical management.

CFH is the major fluid phase regulator of AP. The regulatory functions are mediated via the N-terminal domains. Mutations in the N-terminal region of CFH(R53C, R53H, R78G, R83S and DelK224) have been reported in cases of MPGN, C3G and aHUS. Functional studies demonstrated loss of regulatory function and confirm the significance of these mutations. We now examine the functional significance of additional novel variants identified in cases of MPGN, C3G and aHUS.

Methods: Novel genetic variants in the N-terminal region ofCFHwere identified in patients with MPGN, C3G or aHUS.Structural modelling was used to predict the functional effects of the mutated protein. Recombinant wild type and mutant sequence variants were generated in Pichia pastoris in the setting of the N-terminal domain of CFH. We determined affinity of CFH to C3b using surface plasmon resonance. We investigated decay activity using surface plasmon resonance and sheep erythrocyte haemolytic assays and co-factor activity using fluid phase and sheep erythrocyte haemolytic assays.

Results: We studied 5 variants identified in cases of MPGN, C3G or aHUS. Structural modelling predicted that Q81P was at the CFH/C3b interface. The variantsS159N, A161S and M162V were in close proximity to each other and at the interface between FH/C3b but distant from Q81P. D130N was at the surface of CFH and predicted to interface with complement factor I. Q81P showed complete loss of binding, decay and co-factor activity. The other variants showed little or no differences in functional assays.

Discussion: Mutation screening reveals rare genetic variants in the N-terminal region of CFH in cases of MPGN, C3G and aHUS. Q81P results in a loss of regulatory function. These effects are consistent with the mutants R78G and R83S that are in close proximity to Q81P. This suggests a critical interface between CFH and C3b. Other mutants in this study cause minimal disruption to function suggesting that other interfaces between CFH and C3b are less critical. It is likely that these different variants are not of the same pathological significance. This worksuggests the ongoing importance of functional studies to interpret the significance of future novel sequence variants identified in the N-terminal region of CFHto guide appropriate clinical management.