A prevalent C3 mutation in aHUS patients causes a direct C3 convertase gain of function

Abstract

Atypical hemolytic uremic syndrome (aHUS) is a rare renal thrombotic microangiopathy commonly associated with rare genetic variants in complement system genes, unique to each patient/family. Here, we report 14 sporadic aHUS patients carrying the same mutation, R139W, in the complement C3 gene. The clinical presentation was with a rapid progression to end-stage renal disease (6 of 14) and an unusually high frequency of cardiac (8 of 14) and/or neurologic (5 of 14) events. Although resting glomerular endothelial cells (GEnCs) remained unaffected by R139W-C3 sera, the incubation of those sera with GEnC preactivated with pro-inflammatory stimuli led to increased C3 deposition, C5a release, and procoagulant tissue-factor expression. This functional consequence of R139W-C3 resulted from the formation of a hyperactive C3 convertase. Mutant C3 showed an increased affinity for factor B and a reduced binding to membrane cofactor protein (MCP; CD46), but a normal regulation by factor H (FH). In addition, the frequency of at-risk FH and MCP haplotypes was significantly higher in the R139W-aHUS patients, compared with normal donors or to healthy carriers. These genetic background differences could explain the R139W-aHUS incomplete penetrance. These results demonstrate that this C3 mutation, especially when associated with an at-risk FH and/or MCP haplotypes, becomes pathogenic following an inflammatory endothelium-damaging event.

Figure 1

Localization of R139W C3 mutation. (A) Position within the C3 gene and the protein primary structure. (B) Representative histogram for the sequencing of a patient, carrier of R139W. (C) Mapping of R139W on the surface of C3 using Pymol software.

Figure 2

Complement activation on glomerular endothelial cells, incubated with sera from R139W-aHUS patients and their healthy relatives. (A) C3 deposition on resting or TNFα/IFNγ activated GEnCs in the presence of sera from 50 individual normal donors, FHdpl (4 different lots), R139W-aHUS patients (P2, P5, and P14), healthy relatives of patient P5 bearing the mutation (5.F indicates father; and 5.S1 and 5.S2, sisters) or mutation-free relatives of patient P5, indicated as family (5.M indicates mother; and 5.B, brother). C3 depositions (RFI) obtained with each patient or healthy donor were normalized by the C3 deposition from one normal human serum on resting cells, considered as a standard and run in each experiment to obtain the fold increase. Each point is a mean of 3-5 independent experiments. Statistical significance (***P < .001) was calculated by ANOVA. (B) Levels of C3a, C5a, and soluble C5b-9, released after incubation of the TNFα/IFNγ activated GEnCs with serum from normal donors (n = 6) or R139W sera (n = 3), were measured by ELISA. The level of C3a, C5a, or sC5b-9 in the supernatant (one-third diluted serum) from resting cells was subtracted from the corresponding levels on activated cells, to obtain the specific amount of C5a and sC5b-9 released because of complement activation. Results are expressed as fold increase, compared with a standard normal serum as in panel A. The statistical analysis was a Mann-Whitney test. (C) Tissue-factor expression on TNFα/IFNγ activated or resting GEnCs after overnight incubation with sera from normal donors (n = 6) or R139W-positive sera (n = 4). The percentage of TF-positive cells was measured by flow cytometry. The statistical analysis was the Mann-Whitney test.

Figure 3

Effects of blocking FH and MCP on the regulation of C3 deposition from WT or R139W sera. (A) GEnCs preactivated with TNFα/IFNγ were incubated for 30 minutes with the standard normal human serum, FHdpl, or R139W sera, in the presence or absence of blocking mAbs against FH (Ox24) or MCP (GB24). The C3 deposition was evaluated by flow cytometry as in Figure 2A. ***P < .0001, unpaired t test. Unless specifically mentioned, the comparison was made with the NHS (the first bar with a black and white pattern). (B) GEnCs activated with TNFα/IFNγ were incubated for 30 minutes with normal human sera, FHdpl, or R139W sera in the presence of increasing doses of purified FH. C3 deposition in the absence of FH for the FHdpl or R139W-positive sera (from aHUS patient and healthy carriers) was taken as 100% and each C3 level percentage, in the presence of different doses of FH, was calculated. The starting FH concentration was in the normal range for P5, 5.F, and 5.S1. The level of C3 deposition from a normal serum is given as a straight line.

Figure 4

Interaction of R139W with MCP and FH. R139 position (A) on the structure of C3b in a complex with FH CCP1-4 or (B) in a model complex with MCP. R139 is close to CCP3 binding site of both FH and MCP. Direct binding of recombinant WT or mutant C3 to (C) FH or (D) MCP, studied by ELISA. FCS-free supernatant, containing recombinant WT or mutant C3 produced by stably transfected CHO cells, was used as a source of C3. SN0 is the supernatant of cells transfected with a plasmid not containing the C3 gene. The real-time binding of recombinant WT or mutant C3 to (E) FH or (F) MCP was studied by SPR, using recombinant C3 proteins purified from serum-free culture supernatants by DEAE-Sepharose column.

Figure 5

Interaction of R139W with factor B. (A) R139 position on the structures of C3b with FB in closed (refractory to cleavage by FD) and open (prone to cleavage by FD) conformations and on the structure of the C3bBb complex. The α and β chains of C3b are depicted in blue and green and FB is colored in magenta. (B) Binding of FB to WT or mutant recombinant C3, bound to an anti-C3d mAb, coated to the ELISA plate. Serum-free supernatant was used as a source of recombinant C3 molecules. (C) The binding of FB to recombinant WT or R139W C3, bound to an anti-C3d mAb on the CM5 biosensor chip, was studied by SPR using Biacore. Recombinant C3 proteins, purified from serum-free culture supernatants by DEAE-Sepharose column, were used. (D) Formation of a C3 convertase on the biosensor chip by subsequent injection of C3+FB+FD, followed by an injection of FB+FD. Purified WT or R139W recombinant C3 were mixed with native C3, purified from plasma. Proteins deposition on the chip was followed over time. For panels B through D, 1 representative experiment of 3, performed with 3 independent productions of C3, is presented.