Atypical hemolytic uremic syndrome (aHUS) is a disease characterized by overactivation of the alternative complement pathway (1). Mutations in the genes encoding complement regulatory proteins complement factor H (CFH) (2–5), complement factor I (CFI) (6–10), and membrane cofactor protein (CD46) (6,11–14) and complement components C3 (C3) (15) and complement factor B (CFB) (16) are associated with aHUS.
As well as inherited defects in complement regulation, acquired defects in the form of autoantibodies to CFH have been described (17–20). These autoantibodies mainly bind to the C-terminal end of CFH, where aHUS-associated mutations cluster (21). This region of the molecule binds to C3b and glycosaminoglycans and is responsible for cell surface complement regulation (22). CFH autoantibodies have been shown to impair cell surface complement regulation, thus mimicking the action of the CFH mutations seen in aHUS (17–20,23).
CFI is a serine protease that cleaves C3b and C4b in the presence of its cofactor proteins, CFH (24), C4 binding protein (25), CD46 (26), and complement receptor 1 (27). By inactivating C3b and C4b through limited proteolytic cleavage and thereby preventing the formation of the C3 and C5 convertases, CFI inhibits the alternative and classic complement pathways. CFI consists of a light chain (which carries the catalytic site) and a heavy chain (of unclear function) linked by a disulphide bond.
Mutations in CFI have been reported in 2–12% of aHUS patients (6–10). Although they are distributed throughout the molecule, they do cluster in the serine protease domain (21). Most aHUS-associated CFI mutations result in decreased secretion, resulting in a quantitative defect in complement regulation. Functional analysis of CFI mutants that are secreted normally has revealed a loss of alternative and classic pathway cofactor activity, both in the fluid phase and on cell surfaces (7,28,29).
Here, we describe the presence of CFI autoantibodies in the Newcastle aHUS cohort, investigate their functional impact, and show that these autoantibodies occur in the presence of additional genetic risk factors.
Materials and Methods
Paired serum and DNA samples were available from 175 patients with aHUS and 100 healthy blood donors (blood donor controls). The study was approved by the Northern and Yorkshire Multi-Center Research Ethics Committee, and informed consent was obtained in accordance with the Declaration of Helsinki.
In individuals with CFI autoantibodies, mutation screening of CFH, CD46, CFI, CFB, C3, and thrombomodulin (THBD) was or had previously been undertaken by direct fluorescent sequencing as described (2,8,12,15,30,31). Variants discovered in these genes were assessed in DNA samples from 300 normal control individuals within the Wellcome Trust Patient Control Consortium (32,33). Genotyping of the following single nucleotide polymorphisms was undertaken by direct sequencing: CD46 −652A>G (rs2796267), CD46 −366A>G (rs2796268), CD46 c.4070T>C (rs7144), CFH −331C>T (rs3753394), CFH c.2016A>G p.Gln672Gln (rs3753396), and CFH c.2808G>T p.Glu936Asp (rs1065489).
CFHR1 and -3 copy number was measured by multiplex ligation-dependent probe amplification with a kit from MRC Holland (SALSA MLPA kit P236-A1 ARMD). CFHR4 copy number was measured by multiplex PCR assay as described (20). Screening for CFH autoantibodies was performed as previously described (20,34).
The anti-CFI ELISA was carried out essentially as previously described for factor H (34), except that 5 µg/ml CFI (purified from pooled serum samples) (35) was substituted for CFH herein and a standard curve was generated using a polyclonal goat anti-CFI (Comptech) followed by rabbit anti-goat horseradish peroxidase (HRP) (Stratech Scientific). The OD450 value for the 1/5000 dilution of goat anti-human CFI was given an arbitrary value of 100,000 relative units (RU). Alternatively, protein A/G column was used to isolated patient and control Ig from sera following manufacturer’s instructions (Pierce, United Kingdom), and the presence of CFI in the samples was detected using 1 µg/ml Medical Research Council of the United Kingdom (MRC) OX21 (gift from Bob Sim, Oxford, United Kingdom) by standard sandwich ELISA of samples.
Purified CFI (35 µg/ml) was diluted in solubilizing buffer, and 20 ml was loaded onto a 10% SDS-PAGE preparative gel and transferred to nitrocellulose, which was then cut into 0.5- to 1-cm-wide strips. After blocking in 5% nonfat milk/PBS, strips were then incubated with individual sera samples (1/25 to 1/100 as appropriate) overnight at 4°C. After extensive washing in PBS/Tween 0.02%, bound autoantibody was detected using goat anti-human IgG-HRP (Stratech Scientific). Alternatively, for CFI immune complex detection, pre- or postcolumn sera (equivalent to 1/20 dilution of fresh serum) or purified Ig (using protein A/G column; Pierce; Thermo Scientific) was concentrated (using 30-kD cutoff spin columns; Sartorius Stedim Biotech) and adjusted to 1 mg/ml after quantification by bicinchoninic acid assay (Pierce; Thermo Scientific) was loaded on SDS-PAGE and blotted. MRC OX21 was used to identify the presence of CFI. Blots were developed using an enhanced chemiluminescence substrate according to the manufacturer’s specifications (Pierce; Thermo Scientific).
C3 and C4 levels were measured by rate nephelometry (Beckman Array 360). CFH and CFI levels were measured by radioimmunodiffusion (Binding Site). Cell surface expression of CD46 was measured by flow cytometry as previously described (12,36).
Alternative Pathway Assays
Cell-bound complement activity was carried out essentially as previously described with minor modifications (37). Briefly, rabbit red blood cells (TCS Biologic) were washed several times in PBS and subsequently transferred to alternative pathway buffer (APB; 3.12 mM Barbital, 0.9 mM Na Barbital, 145 mM NaCl2, 7.83 mM MgCl2, 0.25 mM CaCl2, 10 mM EGTA, and 0.1% w/v gelatin, final pH 7.2). Cells were resuspended at 0.1% v/v, and 100 µl were plated out on round-bottomed 96-well plates containing 100 µl triplicate serial dilutions of normal human serum or patient sera in APB. Wells were supplemented with purified patient or normal control Ig (100 µg/well), CFI (0.7 µg/well), or CFH (5 µg/well) before adding rabbit red blood cells. Plates were incubated at 37°C for 30 minutes before red cells were pelleted at 500 g for 5 min. Absorbance of supernatant was measured at OD410.
Fluid Phase C3b Breakdown
Fluid phase complement activity was established as previously described (38). Briefly, purified C3b (4 µg), CFH (0.5 µg), and CFI (titration from 1 µg; obtained from Comptech, TX) were mixed with APB. Patient and control Ig (25 µg) in APB were mixed with the C3b- and CFH-containing solution before addition of CFI. Samples were then taken to 37°C for 3 min before being heated to 95°C for 5 min; 10% SDS-PAGE gels were stained with Coomassie blue or alternatively subjected to Western blotting as appropriate. C3b breakdown was visualized using a sheep anti-C3 at 1/500 (gift from B. P. Morgan, Cardiff, United Kingdom) followed by 1/2000 donkey anti-sheep HRP (Stratech Scientific). Western blots were visualized for 30 seconds. Image analysis was carried out on scanned gels and autorad films as follows. A set grid was used to compare pixel intensity (grayscale gradient) for each band within a lane. Results were standardized for loading based on the β-chain of C3.
In both the blood donor controls (BDCs) and aHUS patients, CFI autoantibody titer (in RU) was not normally distributed (Figure 1A) (determined by the Kolmogorov–Smirnov test). The median (range; mean) antibody titer in BDC and aHUS patients was 64 RU (32–504; 97) and 56 RU (22–34,921; 310), respectively. Compared with the BDC group, aHUS patients had increased levels of CFI autoantibody (P<0.02). We used the 0.975 fractile of the BDC group to determine autoantibody positivity as recommended by the International Federation of Clinical Chemistry for data with non-normal distribution (39). This use equated to 423 RU, and six patients were above this threshold (Figure 1A). However, our experience with anti-CFH autoantibodies has suggested that it is prudent to confirm presence of autoantibodies using a second technique (34). Thus, Western blotting was used to confirm the presence of autoantibodies in samples with a titer ≥423. Only the three patients with the highest titer of CFI autoantibodies were confirmed to have CFI autoantibodies after Western blotting analysis. Detection of autoantibodies in patient 1’s serum required a shorter film exposure (Figure 1B) and was readily detectable when less serum was used than for patients 2 and 3 (Figure 1C), consistent with the ELISA results. Additional Western analysis indicated the CFI autoantibody detected in patient 1 bound to the heavy chain of CFI (Figure 1D). Using specific HRP-conjugated secondaries (mouse monoclonal antibodies: MH17–15, -22, -32, -42; Invitrogen, United Kingdom) in our ELISA, P1 and P3 autoantibodies were established as being predominately IgG1 subclass, whereas P2 autoantibodies were IgG3 subclass (data not shown). Thus, of the 175 aHUS patients that we screened, 3 patients were confirmed to possess significant levels of CFI autoantibodies.
Clinical Details: Genotyping and Background Analysis of the Three Patients with CFI Autoantibodies
A summary of the clinical details of the three patients with CFI autoantibodies is shown in Table 1, with a full summary available in Supplemental Material. Briefly, all required renal replacement therapy at initial presentation and plasma therapy was instituted. However, only patient 3 recovered renal function. In the remaining two patients, a total of three renal transplants were undertaken in the absence of any specific therapy to remove autoantibodies. Patient 1 showed recurrent aHUS in the allograft. Table 1 also shows the values for serum levels of C3, C4, CFH, and CFI, the results of screening for CFH autoantibodies, and the measurement of CD46 expression on the original samples. In all individuals, systemic alternative pathway complement activation was shown with low levels of C3, but in only one individual was the C4 level low. Mutation screening showed that patient 1 had a heterozygous mutation in CFH (c.3468dupA) and a sequence variant in CFI (c.1657 C>T; p.Pro553Ser) that was present in normal controls at a frequency of 5/574 chromosomes (Table 2). Patient 2 had a heterozygous mutation in CFH (c.2018G>A; p.Cys673Tyr). No mutations were detected in genes previously associated with aHUS in patient 3, and all three patients had two copies of CFHR1, -3, and -4. Genotyping for CFH and CD46 susceptibility factors revealed that both patients 1 and 2 are heterozygous for the at-risk haplotype CFHTGTGGT (H3) haplotype (20), whereas patient 2 is homozygous and patient 1 is heterozygous for the at-risk CD46GGAAC haplotype (40) (Table 3).
Time Course of Ig Class Switching and Titer
Patient 1 showed a high titer of IgM CFI antibodies at the time of initial presentation with aHUS (Figure 2), but by the time of the first renal transplant (2 years after presenting with aHUS), the titer of IgM CFI autoantibodies was at background levels, where they have remained. Notably, the first renal transplant was lost to recurrent aHUS, and at that time, there were no detectable IgM or IgG CFI autoantibodies. By 42 months after the initial presentation and 24 months after the post-transplant recurrent episode of aHUS, IgG CFI autoantibodies are present in high titers. This finding coincides with the second renal transplant, which was not associated with recurrent aHUS. The sample used in our cohort analysis was 6 months after the peak IgG response (Figure 2), and levels had dropped to 50% maximal detected response by this time. The titer of the IgG CFI autoantibodies then declined slowly and currently rests at the ELISA positive cutoff.
CFI and CFI Autoantibodies Form a Circulating Immune Complex
Despite the apparent lack of correlation between CFI autoantibody titer and the clinical course of the disease in patient 1, we wished to establish if the presence of CFI autoantibodies might be disease-modifying. We hypothesized that immune complexes of CFI and CFI autoantibodies could both lead to the generation of additional proinflammatory stimuli (41) and elude detection by standard ELISA. Western blot analysis of a column-purified IgG sample from freshly obtained patient 1 serum (i.e., anti-CFI at 650 RU) showed that CFI had remained associated with IgG (Figure 3A). The post-IgG affinity column sample from patient 1 had less CFI than the normal control sample (despite similar precolumn levels established by anti-CFI sandwich ELISA), suggesting that it was bound out of the serum with IgG and then eluted from the IgG during the wash. In a second approach, we determined that Ig was associated with captured CFI extracted from archived serum samples from patient 1 (Figure 3B). Therefore, we consistently detect immune complexes of IgG autoantibody and CFI in patient 1, which may contribute to disease.
Purified Patient Ig Interferes with CFI Function in a Fluid Phase C3b Breakdown Assay
Detection of immune complexes suggests that CFI autoantibodies readily associate with CFI in the fluid phase. Therefore, we next assessed whether purified total Ig (containing CFI autoantibodies) isolated from patient 1 could alter CFI function in a fluid phase C3b breakdown assay. Purified C3b, CFH, and CFI were incubated with control or patient total Ig. The presence of Ig from patient 1 slowed the breakdown of C3b in the fluid phase (Figure 4). A 50% reduction in C3b breakdown over the 3-minute time period was found when limiting concentrations of CFI were mixed with patient Ig before the addition of the C3b to the reaction mixture (Figure 4C).
Alternative Pathway Hemolytic Activity Significantly Increased in Patient 1 Largely Because of Lower CFH Levels
From the fluid phase assays, there was evidence that CFI autoantibody did interfere with CFI function. However, cell surface complement regulation is critical in aHUS (42). Using standard alternative pathway hemolysis assays (37), freshly isolated patient 1 serum had similar hemolytic activity compared with normal human serum (NHS) (Figure 5A). This finding was counterintuitive considering that the patient serum had both lower CFH levels and CFI autoantibody, albeit currently at low levels. We surmised that the low serum C3 levels in this patient could be undermining this assay. Therefore, we mixed patient and NHS serum 1:1 to replenish C3 levels. In this analysis, the 1:1 mix gave significantly greater lysis than NHS only, suggesting failure to control complement activation in serum from patient 1. Addition of purified CFI (Figure 5B) or CFH (Figure 5C) into this mixed sample indicated that the majority of the defect was caused by the loss of CFH. To test whether the CFI autoantibody had any effect on complement regulation, we supplemented NHS with 100 µg purified patient or control Ig. Addition of patient Ig had no impact on hemolysis compared with control Ig (Figure 5D). These findings were also replicated in the sheep red blood cell alternative pathway assay (43), and addition of purified CFI autoantibody to a standard classic pathway hemolytic assay (37) again showed no clear increase in lysis as a result of interference with factor I function (data not shown).
In this study, we report for the first time the presence of CFI autoantibodies in aHUS patients. There was, however, little evidence to correlate the genesis of IgG isotype CFI autoantibodies with the course of aHUS in the patient with the highest recorded titer of CFI autoantibodies. Furthermore, functional analysis of freshly isolated CFI autoantibodies suggests that, currently, their presence results in only a minor modification of complement regulator capacity of CFI. This finding leads us to question whether these autoantibodies are disease-modifying or an epiphenomenon.
At a frequency of ∼2% (3/175) in the Newcastle aHUS cohort, CFI autoantibodies are less frequent than CFH autoantibodies (5–10%) (17–20). In patient 1, we have for the first time seen evidence of the class switch event in a complement protein autoantibody. Intriguingly, there is a large interval between the initial IgM and IgG response (Figure 2). Review of the patient’s history has provided no clear indication of the trigger for either the initial or subsequent events, and critically, the levels of autoantibody do not seem to be associated with the course of the disease. That we show that CFI autoantibodies exist both free in solution and in an immune complex with CFI suggests the autoantibodies may have low to intermediate affinity to native CFI.
A deletion incorporating CFHR1 and -3 is strongly associated with CFH autoantibodies in aHUS (19,44). Here, we found that all three individuals with CFI autoantibodies have two copies of CFHR1 and -3, suggesting that their absence only plays a specific role in the development CFH autoantibodies. That two of three individuals with CFI autoantibodies also carried mutations in CFH supports the hypothesis that chronic increased complement activation may predispose to the generation of autoantibodies against complement components. In patient 1, there was a heterozygous duplication of a single base pair (c.3468dupA), leading to a frame shift and premature stop codon in CFH. This finding is consistent with the low levels of CFH seen in this patient. Furthermore, patients with CFI deficiency also display low CFH levels. Therefore, the low levels of CFH seen in this patient could result from both immune complex removal of CFH associated with excess C3b (45) as well as the effects of the CFH mutation. Patient 2 has a heterozygous nonsynonymous CFH mutation (c.2018G>A; p.Cys673Tyr) that would be predicted to result in failure of secretion from that allele (3), but CFH levels, presumably produced from other allele, were found to be in the normal range (Table 1). In addition to mutations and autoantibodies, several studies have now identified CFH and CD46 risk haplotypes for aHUS. Intriguingly, both patients 1 and 2 do possess at-risk haplotypes on CFH and CD46 (Table 3) (CFHTGTGGT [H3] haplotype  and CD46GGAAC haplotype ), whereas patient 3, who had the best clinical outcome, did not have additional genetic risk factors for aHUS, a phenomenon previously noted in aHUS patients with CFI mutations (29).
Thus, as in patients with CFH autoantibodies, individuals with CFI autoantibodies may have additional genetic risk factors predisposing to aHUS. Furthermore, despite having a functionally significant mutation in CFH, this latent predisposition to disease did not manifest in patient 1 until the age of 26 years when she developed the disease in association with pregnancy (46). Likewise, patient 3 only developed aHUS after a diarrheal illness (not shiga toxin-associated). Thus, these three patients also show that, in addition to inherited and acquired susceptibility factors, a trigger is needed for the disease to be manifest. Recent analysis of cohorts of aHUS patients with complement mutations has identified upper respiratory tract infections (6), viruses (36), pregnancy (6,47), drugs (6), and non-Escherichia coli diarrheal illnesses (48) as potential triggers.
We have found CFI autoantibodies in ∼2% of the Newcastle aHUS cohort. These autoantibodies were associated with other susceptibility factors and support the theory that multiple hits are necessary in most patients before aHUS presents clinically (49). Importantly, CFI autoantibodies do not seem to track with disease in the patient with highest antibody titer, raising the possibility that these autoantibodies could be a marker of disease rather than a direct factor in disease development.
T.H.J.G. is a scientific advisor for Alexion Pharmaceuticals and has received grant support from the same company.
This work was supported by the Medical Research Council (G0701325), Kidney Research UK, the Northern Counties Kidney Research Fund, the Mason Medical Research Foundation, and the Academy of Medical Sciences. D.K. is a Wellcome Intermediate Clinical Fellow. We acknowledge use of DNA from the UK Blood Services Collection of Common Controls funded by Wellcome Trust Grant 076113/C/04/Z, Juvenile Diabetes Research Foundation Grant WT061858, and the National Institutes of Health Research of England. The collection was established as part of the Wellcome Trust Patient Control Consortium.
D.K. and I.Y.P. contributed equally to this work.
Published online ahead of print. Publication date available at www.cjasn.org.
This article contains supplemental material online at http://cjasn.asnjournals.org/lookup/suppl/doi:10.2215/CJN.05750611/-/DCSupplemental.
1. Kavanagh D, Richards A, Atkinson J: Complement regulatory genes and hemolytic uremic syndromes. Annu Rev Med 59: 293–309, 2008
2. Richards A, Buddles MR, Donne RL, Kaplan BS, Kirk E, Venning MC, Tielemans CL, Goodship JA, Goodship TH: Factor H mutations in hemolytic uremic syndrome cluster in exons 18-20, a domain important for host cell recognition. Am J Hum Genet 68: 485–490, 2001
3. Dragon-Durey MA, Frémeaux-Bacchi V, Loirat C, Blouin J, Niaudet P, Deschenes G, Coppo P, Herman Fridman W, Weiss L: Heterozygous and homozygous factor h deficiencies associated with hemolytic uremic syndrome or membranoproliferative glomerulonephritis: Report and genetic analysis of 16 cases. J Am Soc Nephrol 15: 787–795, 2004
4. Caprioli J, Bettinaglio P, Zipfel PF, Amadei B, Daina E, Gamba S, Skerka C, Marziliano N, Remuzzi G, Noris M; Itaslian Registry of Familial and Recurrent HUS/TTP: The molecular basis of familial hemolytic uremic syndrome: Mutation analysis of factor H gene reveals a hot spot in short consensus repeat 20. J Am Soc Nephrol 12: 297–307, 2001
5. Pérez-Caballero D, González-Rubio C, Gallardo ME, Vera M, López-Trascasa M, Rodríguez de Córdoba S, Sánchez-Corral P: Clustering of missense mutations in the C-terminal region of factor H in atypical hemolytic uremic syndrome. Am J Hum Genet 68: 478–484, 2001
6. Caprioli J, Noris M, Brioschi S, Pianetti G, Castelletti F, Bettinaglio P, Mele C, Bresin E, Cassis L, Gamba S, Porrati F, Bucchioni S, Monteferrante G, Fang CJ, Liszewski MK, Kavanagh D, Atkinson JP, Remuzzi G; International Registry of Recurrent and Familial HUS/TTP: Genetics of HUS: The impact of MCP, CFH, and IF mutations on clinical presentation, response to treatment, and outcome. Blood 108: 1267–1279, 2006
7. Kavanagh D, Richards A, Noris M, Hauhart R, Liszewski MK, Karpman D, Goodship JA, Fremeaux-Bacchi V, Remuzzi G, Goodship TH, Atkinson JP: Characterization of mutations in complement factor I (CFI) associated with hemolytic uremic syndrome. Mol Immunol 45: 95–105, 2008
8. Kavanagh D, Kemp EJ, Mayland E, Winney RJ, Duffield JS, Warwick G, Richards A, Ward R, Goodship JA, Goodship TH: Mutations in complement factor I predispose to development of atypical hemolytic uremic syndrome. J Am Soc Nephrol 16: 2150–2155, 2005
9. Sellier-Leclerc AL, Fremeaux-Bacchi V, Dragon-Durey MA, Macher MA, Niaudet P, Guest G, Boudailliez B, Bouissou F, Deschenes G, Gie S, Tsimaratos M, Fischbach M, Morin D, Nivet H, Alberti C, Loirat C; French Society of Pediatric Nephrology: Differential impact of complement mutations on clinical characteristics in atypical hemolytic uremic syndrome. J Am Soc Nephrol 18: 2392–2400, 2007
10. Fremeaux-Bacchi V, Dragon-Durey MA, Blouin J, Vigneau C, Kuypers D, Boudailliez B, Loirat C, Rondeau E, Fridman WH: Complement factor I: A susceptibility gene for atypical haemolytic uraemic syndrome. J Med Genet 41: e84, 2004
11. Fremeaux-Bacchi V, Moulton EA, Kavanagh D, Dragon-Durey MA, Blouin J, Caudy A, Arzouk N, Cleper R, Francois M, Guest G, Pourrat J, Seligman R, Fridman WH, Loirat C, Atkinson JP: Genetic and functional analyses of membrane cofactor protein (CD46) mutations in atypical hemolytic uremic syndrome. J Am Soc Nephrol 17: 2017–2025, 2006
12. Richards A, Kemp EJ, Liszewski MK, Goodship JA, Lampe AK, Decorte R, Müslümanoğlu MH, Kavukcu S, Filler G, Pirson Y, Wen LS, Atkinson JP, Goodship TH: Mutations in human complement regulator, membrane cofactor protein (CD46), predispose to development of familial hemolytic uremic syndrome. Proc Natl Acad Sci USA 100: 12966–12971, 2003
13. Richards A, Kathryn Liszewski M, Kavanagh D, Fang CJ, Moulton E, Fremeaux-Bacchi V, Remuzzi G, Noris M, Goodship TH, Atkinson JP: Implications of the initial mutations in membrane cofactor protein (MCP; CD46) leading to atypical hemolytic uremic syndrome. Mol Immunol 44: 111–122, 2007
14. Sullivan M, Erlic Z, Hoffmann MM, Arbeiter K, Patzer L, Budde K, Hoppe B, Zeier M, Lhotta K, Rybicki LA, Bock A, Berisha G, Neumann HP: Epidemiological approach to identifying genetic predispositions for atypical hemolytic uremic syndrome. Ann Hum Genet 74: 17–26, 2010
15. Frémeaux-Bacchi V, Miller EC, Liszewski MK, Strain L, Blouin J, Brown AL, Moghal N, Kaplan BS, Weiss RA, Lhotta K, Kapur G, Mattoo T, Nivet H, Wong W, Gie S, Hurault de Ligny B, Fischbach M, Gupta R, Hauhart R, Meunier V, Loirat C, Dragon-Durey MA, Fridman WH, Janssen BJ, Goodship TH, Atkinson JP: Mutations in complement C3 predispose to development of atypical hemolytic uremic syndrome. Blood 112: 4948–4952, 2008
16. Goicoechea de Jorge E, Harris CL, Esparza-Gordillo J, Carreras L, Arranz EA, Garrido CA, López-Trascasa M, Sánchez-Corral P, Morgan BP, Rodríguez de Córdoba S: Gain-of-function mutations in complement factor B are associated with atypical hemolytic uremic syndrome. Proc Natl Acad Sci USA 104: 240–245, 2007
17. Dragon-Durey MA, Loirat C, Cloarec S, Macher MA, Blouin J, Nivet H, Weiss L, Fridman WH, Frémeaux-Bacchi V: Anti-factor H autoantibodies associated with atypical hemolytic uremic syndrome. J Am Soc Nephrol 16: 555–563, 2005
18. Józsi M, Strobel S, Dahse HM, Liu WS, Hoyer PF, Oppermann M, Skerka C, Zipfel PF: Anti factor H autoantibodies block C-terminal recognition function of factor H in hemolytic uremic syndrome. Blood 110: 1516–1518, 2007
19. Józsi M, Licht C, Strobel S, Zipfel SL, Richter H, Heinen S, Zipfel PF, Skerka C: Factor H autoantibodies in atypical hemolytic uremic syndrome correlate with CFHR1/CFHR3 deficiency. Blood 111: 1512–1514, 2008
20. Moore I, Strain L, Pappworth I, Kavanagh D, Barlow PN, Herbert AP, Schmidt CQ, Staniforth SJ, Holmes LV, Ward R, Morgan L, Goodship TH, Marchbank KJ: Association of factor H autoantibodies with deletions of CFHR1, CFHR3, CFHR4, and with mutations in CFH, CFI, CD46, and C3 in patients with atypical hemolytic uremic syndrome. Blood 115: 379–387, 2010
21. Kavanagh D, Goodship T: Genetics and complement in atypical HUS. Pediatr Nephrol 25: 2431–2442, 2010
22. Schmidt CQ, Herbert AP, Kavanagh D, Gandy C, Fenton CJ, Blaum BS, Lyon M, Uhrín D, Barlow PN: A new map of glycosaminoglycan and C3b binding sites on factor H. J Immunol 181: 2610–2619, 2008
23. Ferreira VP, Herbert AP, Cortés C, McKee KA, Blaum BS, Esswein ST, Uhrín D, Barlow PN, Pangburn MK, Kavanagh D: The binding of factor H to a complex of physiological polyanions and C3b on cells is impaired in atypical hemolytic uremic syndrome. J Immunol 182: 7009–7018, 2009
24. Whaley K, Ruddy S: Modulation of C3b hemolytic activity by a plasma protein distinct from C3b inactivator. Science 193: 1011–1013, 1976
25. Nagasawa S, Stroud RM: Mechanism of action of the C3b inactivator: Requirement for a high molecular weight cofactor (C3b-C4bINA cofactor) and production of a new C3b derivative (C3b’). Immunochemistry 14: 749–756, 1977
26. Liszewski MK, Post TW, Atkinson JP: Membrane cofactor protein (MCP or CD46): Newest member of the regulators of complement activation gene cluster. Annu Rev Immunol 9: 431–455, 1991
27. Medof ME, Iida K, Mold C, Nussenzweig V: Unique role of the complement receptor CR1 in the degradation of C3b associated with immune complexes. J Exp Med 156: 1739–1754, 1982
28. Nilsson SC, Kalchishkova N, Trouw LA, Fremeaux-Bacchi V, Villoutreix BO, Blom AM: Mutations in complement factor I as found in atypical hemolytic uremic syndrome lead to either altered secretion or altered function of factor I. Eur J Immunol 40: 172–185, 2010
29. Bienaime F, Dragon-Durey MA, Regnier CH, Nilsson SC, Kwan WH, Blouin J, Jablonski M, Renault N, Rameix-Welti MA, Loirat C, Sautés-Fridman C, Villoutreix BO, Blom AM, Fremeaux-Bacchi V: Mutations in components of complement influence the outcome of Factor I-associated atypical hemolytic uremic syndrome. Kidney Int 77: 339–349, 2010
30. Kavanagh D, Burgess R, Spitzer D, Richards A, Diaz-Torres ML, Goodship JA, Hourcade DE, Atkinson JP, Goodship TH: The decay accelerating factor mutation I197V found in hemolytic uraemic syndrome does not impair complement regulation. Mol Immunol 44: 3162–3167, 2007
31. Kavanagh D, Goodship TH: Membrane cofactor protein and factor I: Mutations and transplantation. Semin Thromb Hemost 32: 155–159, 2006
32. Wellcome Trust Case Control Consortiumperson-group>: Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls. Nature 447: 661–678, 2007
33. Burton PR, Clayton DG, Cardon LR, Craddock N, Deloukas P, Duncanson A, Kwiatkowski DP, McCarthy MI, Ouwehand WH, Samani NJ, Todd JA, Donnelly P, Barrett JC, Davison D, Easton D, Evans DM, Leung HT, Marchini JL, Morris AP, Spencer CC, Tobin MD, Attwood AP, Boorman JP, Cant B, Everson U, Hussey JM, Jolley JD, Knight AS, Koch K, Meech E, Nutland S, Prowse CV, Stevens HE, Taylor NC, Walters GR, Walker NM, Watkins NA, Winzer T, Jones RW, McArdle WL, Ring SM, Strachan DP, Pembrey M, Breen G, St Clair D, Caesar S, Gordon-Smith K, Jones L, Fraser C, Green EK, Grozeva D, Hamshere ML, Holmans PA, Jones IR, Kirov G, Moskivina V, Nikolov I, O’Donovan MC, Owen MJ, Collier DA, Elkin A, Farmer A, Williamson R, McGuffin P, Young AH, Ferrier IN, Ball SG, Balmforth AJ, Barrett JH, Bishop TD, Iles MM, Maqbool A, Yuldasheva N, Hall AS, Braund PS, Dixon RJ, Mangino M, Stevens S, Thompson JR, Bredin F, Tremelling M, Parkes M, Drummond H, Lees CW, Nimmo ER, Satsangi J, Fisher SA, Forbes A, Lewis CM, Onnie CM, Prescott NJ, Sanderson J, Matthew CG, Barbour J, Mohiuddin MK, Todhunter CE, Mansfield JC, Ahmad T, Cummings FR, Jewell DP, Webster J, Brown MJ, Lathrop MG, Connell J, Dominiczak A, Marcano CA, Burke B, Dobson R, Gungadoo J, Lee KL, Munroe PB, Newhouse SJ, Onipinla A, Wallace C, Xue M, Caulfield M, Farrall M, Barton A, Bruce IN, Donovan H, Eyre S, Gilbert PD, Hilder SL, Hinks AM, John SL, Potter C, Silman AJ, Symmons DP, Thomson W, Worthington J, Dunger DB, Widmer B, Frayling TM, Freathy RM, Lango H, Perry JR, Shields BM, Weedon MN, Hattersley AT, Hitman GA, Walker M, Elliott KS, Groves CJ, Lindgren CM, Rayner NW, Timpson NJ, Zeggini E, Newport M, Sirugo G, Lyons E, Vannberg F, Hill AV, Bradbury LA, Farrar C, Pointon JJ, Wordsworth P, Brown MA, Franklyn JA, Heward JM, Simmonds MJ, Gough SC, Seal S, Stratton MR, Rahman N, Ban M, Goris A, Sawcer SJ, Compston A, Conway D, Jallow M, Newport M, Sirugo G, Rockett KA, Bumpstead SJ, Chaney A, Downes K, Ghori MJ, Gwilliam R, Hunt SE, Inouye M, Keniry A, King E, McGinnis R, Potter S, Ravindrarajah R, Whittaker P, Widden C, Withers D, Cardin NJ, Davison D, Ferreira T, Pereira-Gale J, Hallgrimsdo’ttir IB, Howie BN, Su Z, Teo YY, Vukcevic D, Bentley D, Brown MA, Compston A, Farrall M, Hall AS, Hattersley AT, Hill AV, Parkes M, Pembrey M, Stratton MR, Mitchell SL, Newby PR, Brand OJ, Carr-Smith J, Pearce SH, McGinnis R, Keniry A, Deloukas P, Reveille JD, Zhou X, Sims AM, Dowling A, Taylor J, Doan T, Davis JC, Savage L, Ward MM, Learch TL, Weisman MH, Brown M; Wellcome Trust Case Control ConsortiumAustralo-Anglo-American Spondylitis Consortium (TASC)Biologics in RA Genetics and Genomics Study Syndicate (BRAGGS) Steering CommitteeBreast Cancer Susceptibility Collaboration (UK): Association scan of 14,500 nonsynonymous SNPs in four diseases identifies autoimmunity variants. Nat Genet 39: 1329–1337, 2007
34. Dhillon B, Wright AF, Tufail A, Pappworth I, Hayward C, Moore I, Strain L, Kavanagh D, Barlow PN, Herbert AP, Schmidt CQ, Armbrecht AM, Laude A, Deary IJ, Staniforth SJ, Holmes LV, Goodship TH, Marchbank KJ: Complement factor h autoantibodies and age-related macular degeneration. Invest Ophthalmol Vis Sci 51: 5858–5863, 2010
35. Roversi P, Johnson S, Caesar JJ, McLean F, Leath KJ, Tsiftsoglou SA, Morgan BP, Harris CL, Sim RB, Lea SM: Structural basis for complement factor I control and its disease-associated sequence polymorphisms. Proc Natl Acad Sci USA 108: 12839–12844, 2011
36. Bento D, Mapril J, Rocha C, Marchbank KJ, Kavanagh D, Barge D, Strain L, Goodship TH, Meneses-Oliveira C: Triggering of atypical hemolytic uremic syndrome by influenza A (H1N1). Ren Fail 32: 753–756, 2010
37. Morgan BP: Measurement of Complement Hemolytic Activity, Generation of Complement-Depleted Sera, Production of Hemolytic Intermediates. In: Complement Methods and Protocols, edited by Morgan BP, Totowa, NJ, Humana Press, 2000, pp 61–72
38. Sahu A, Isaacs SN, Soulika AM, Lambris JD: Interaction of vaccinia virus complement control protein with human complement proteins: factor I-mediated degradation of C3b to iC3b1 inactivates the alternative complement pathway. J Immunol 160: 5596–5604, 1998
39. Solberg HE: International Federation of Clinical Chemistry. Scientific committee, Clinical Section. Expert Panel on Theory of Reference Values and International Committee for Standardization in Haematology Standing Committee on Reference Values. Approved recommendation (1986) on the theory of reference values. Part 1. The concept of reference values. Clin Chim Acta 165: 111–118, 1987
40. Esparza-Gordillo J, Goicoechea de Jorge E, Buil A, Carreras Berges L, López-Trascasa M, Sánchez-Corral P, Rodríguez de Córdoba S: Predisposition to atypical hemolytic uremic syndrome involves the concurrence of different susceptibility alleles in the regulators of complement activation gene cluster in 1q32. Hum Mol Genet 14: 703–712, 2005
41. Holers VM: The spectrum of complement alternative pathway-mediated diseases. Immunol Rev 223: 300–316, 2008
42. Roumenina LT, Loirat C, Dragon-Durey MA, Halbwachs-Mecarelli L, Sautes-Fridman C, Fremeaux-Bacchi V: Alternative complement pathway assessment in patients with atypical HUS. J Immunol Methods 365: 8–26, 2011
43. Sánchez-Corral P, González-Rubio C, Rodríguez de Córdoba S, López-Trascasa M: Functional analysis in serum from atypical Hemolytic Uremic Syndrome patients reveals impaired protection of host cells associated with mutations in factor H. Mol Immunol 41: 81–84, 2004
44. Zipfel PF, Edey M, Heinen S, Józsi M, Richter H, Misselwitz J, Hoppe B, Routledge D, Strain L, Hughes AE, Goodship JA, Licht C, Goodship TH, Skerka C: Deletion of complement factor H-related genes CFHR1 and CFHR3 is associated with atypical hemolytic uremic syndrome. PLoS Genet 3: e41, 2007
45. Naked GM, Florido MP, Ferreira de Paula P, Vinet AM, Inostroza JS, Isaac L: Deficiency of human complement factor I associated with lowered factor H. Clin Immunol 96: 162–167, 2000
46. George JN: The association of pregnancy with thrombotic thrombocytopenic purpura-hemolytic uremic syndrome. Curr Opin Hematol 10: 339–344, 2003
47. Goodship TH, Kavanagh D: Pulling the trigger in atypical hemolytic uremic syndrome: The role of pregnancy. J Am Soc Nephrol 21: 731–732, 2010
48. Fakhouri F, Roumenina L, Provot F, Sallée M, Caillard S, Couzi L, Essig M, Ribes D, Dragon-Durey MA, Bridoux F, Rondeau E, Frémeaux-Bacchi V: Pregnancy-associated hemolytic uremic syndrome revisited in the era of complement gene mutations. J Am Soc Nephrol 21: 859–867, 2010
49. Rodríguez de Córdoba S: aHUS: A disorder with many risk factors. Blood 115: 158–160, 2010