Mutations in complement C3 predispose to development of atypical hemolytic uremic syndrome

Abstract

Atypical hemolytic uremic syndrome (aHUS) is a disease of complement dysregulation. In approximately 50% of patients, mutations have been described in the genes encoding the complement regulators factor H, MCP, and factor I or the activator factor B. We report here mutations in the central component of the complement cascade, C3, in association with aHUS. We describe 9 novel C3 mutations in 14 aHUS patients with a persistently low serum C3 level. We have demonstrated that 5 of these mutations are gain-of-function and 2 are inactivating. This establishes C3 as a susceptibility factor for aHUS.

Figure 1

Location of C3 mutations associated with aHUS. (A) Gene structure of C3, domains of the encoded protein, and position of the mutations. Note that the genomic structure numbering begins with the start site ATG, while the protein structures begin with the first amino acid of the mature protein. (B) Structures of complement component C3 and C3b showing the locations of mutations identified in atypical hemolytic uremic syndrome (aHUS) patients. Ribbon representation of C3b and C3 (2 views) with the domains containing mutations (labeled spheres).

Figure 2

Ligand binding and cofactor activity of the C3 proteins. Proteins were transiently expressed in 293T cells, concentrated and quantified before analysis (see Document S1). (A) Binding to MCP, factor H, factor B, and soluble CR1 (sCR1) in ELISA. For MCP and factor H, C3 protein was incubated at 15 ng/mL and for factor B, 200 ng/mL. Detection was made with chicken anti–human C3 and HRP-linked donkey anti–chicken IgY. * indicates a significant reduction in binding (P < .05). (B) Kinetic analysis of cofactor activity. C3 preparations were incubated for 0 to 30 minutes at 37°C with factor I and a cofactor protein (MCP, factor H, or sCR1). The zero control is before the addition of factor I. The last lane is an iC3b control. Samples were reduced and analyzed by Western blotting using either chicken anti–human C3 or goat anti–human C3 (insets) followed by an HRP-linked antiglobulin (see Document S1). Cofactor activity is assessed by the loss of the α chain and appearance of the α₁ and α₄₁ kDa major cleavage fragments. The minor α₄₃ kDa cleavage fragment is more variable but no pattern was observed that was specific for a mutant. Data are representative of 5 similar experiments.