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NDT Advance Access originally published online on May 17, 2007
Nephrology Dialysis Transplantation 2007 22(9):2452-2454; doi:10.1093/ndt/gfm193
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© The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Complement hyperactivation may cause atypical haemolytic uraemic syndrome— gain-of-function mutations in factor B*

Jessica Caprioli1 and Giuseppe Remuzzi2

1Mario Negri Institute for Pharmacological Research, Clinical Research Center for Rare Diseases, Aldo e Cele Daccò, Villa Camozzi, Ranica, Bergamo and 2Division of Nephrology and Dialysis, Azienda Ospedaliera, Ospedali Riuniti di Bergamo, Italy

Correspondence and offprint requests to: Jessica Caprioli, Transplant Research Center, ‘Chiara Cucchi de Alessandri e Gilberto Crespi’, Villa Camozzi, 3 – 24020 Ranica (BG), Italy. Email: caprioli{at}marionegri.it

Keywords: Complement factor B; HUS; mutation



   Introduction
Top
 Introduction
Results and conclusions
 Clinical implications
 Acknowledgements
 References
 
Non-Shiga toxin-associated haemolytic uraemic syndrome (non-Stx-HUS) is a rare disease with manifestations of haemolytic anaemia, thrombocytopenia and renal failure. The clinical outcome is unfavourable, with up to 50% of patients progressing to end-stage renal failure and 25% dying during the acute phase [1]. Genetic abnormalities in complement regulatory proteins, including complement factor H (CFH), membrane cofactor protein (MCP) and complement factor I (CFI), have been reported in about 30, 10 and 5% of patients with non-Stx-HUS, respectively [2–4], thus demonstrating that defective control of the alternative pathway of complement [5] has a pathogenetic role in at least half of the patients. The complement system consists of several plasma proteins that collectively serve to destroy pathogens Figure 1. Its activation is tightly regulated by circulating and membrane-bound inhibitors so as to prevent non-specific damage to host cells and to limit deposition of C3b to the surface of pathogens (Figure 1). Thus, in non-Stx-HUS associated with mutations in CFH, MCP or CFI, upon exposure to an agent that activates complement, C3b is formed in higher than normal amounts, and its deposition on vascular endothelial cells cannot be prevented adequately because of ‘impaired’ function of complement regulatory proteins [5]. On the other hand, abnormally ‘increased’ activity of the alternative pathway enzymes may also result in uncontrolled activation of the complement system, consistent with the pathogenetic mechanism of non-Stx-HUS proposed earlier. Genetic variations in complement-activating proteins have the potential to disrupt the balance between activation and regulation, as it occurs with complement regulators. This dysregulation of the complement system predisposes to tissue damage when the complement system undergoes activation. In line with this hypothesis, Dr. Goicoechea and co-workers [6] describe gain-of-function mutations in CFB and demonstrate their role in non-Stx-HUS.


Figure 1
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  		Fig. 1 The alternative pathway of complement activation and regulation. The first product of the cleavage of C3 by a C3 convertase is C3b that can bind covalently to hydroxyl groups on nearby carbohydrates and protein-acceptor groups. If the acceptor molecule is on a host cell surface (non-activating surface), then protective regulatory mechanisms come into play. This is illustrated by the binding of CFH and MCP to C3b, which act as cofactors for CFI. CFI cleaves C3 into an inactive product, iC3b, releasing a small peptide, C3f. If C3b binds covalently to a bacterium (activating surface), then CFB binds to C3b and it is cleaved by CFD. This leads to the formation of the C3 convertase enzyme C3bBb, which is stabilized by properdin. This enzyme cleaves more C3, leading to the deposition of additional C3b on the bacterium. The subsequent steps of the complement cascade are the cleavage of C5 in C5a and C5b by C3bBb and the formation of the membrane attack complex (MAC) that determines the osmotic lysis of the bacterium.

 
The role of factor B in complement cascade activation
CFB is a zymogen that carries the catalytic site of the complement alternative pathway convertase C3bBb. Upon interaction with C3b, CFB is cleaved by factor D (CFD) into two fragments, Ba and Bb. Ba is released, whereas Bb remains bound to C3b, forming the C3bBb alternative pathway convertase, an active serine protease that cleaves additional C3 into C3b Figure 1. The Bb fragment of CFB comprises two protein domains: a von Willebrand factor type A (vWFA) domain and a serine protease (SP) domain [7]. From structural studies, the CFB vWFA domain is globular, composed of several parallel sheets surrounded by seven helices [8,9]. Site-directed mutagenesis of CFB residues at the C3b–Bb interface in the CFB vWFA domain identified several residues that are critical for the interaction between C3b and CFB and influence the normal dissociation of Bb from C3b, whether it is spontaneous or promoted by the complement regulatory proteins CFH, DAF, or complement receptor 1 (CR1) [10].



   Results and conclusions
Top
 Introduction
 Results and conclusions
Clinical implications
 Acknowledgements
 References
 
Factor B mutations in HUS
The authors found two heterozygous mutations in affected members of two Spanish pedigrees, determining aminoacidic changes in the vWFA domain of CFB (F286L and K323E). Both mutations alter residues at the C3b–Bb interface: the mutation K323E changes a fully exposed residue with a positive charge to one with a negative charge. The mutation F286L influences Y319, another fully exposed residue, modifying the edge-to-face stacking interaction between F286 and Y319. Plasmon resonance experiments demonstrated that each of the mutant CFB proteins cause increased alternative pathway activation. Formation of the C3bB proenzyme by F286L mutant CFB is much enhanced, which produces more active enzyme in vivo, whereas K323E mutant CFB forms a C3bBb enzyme more resistant to decay by DAF and CFH, also causing increased enzyme activity in vivo.

This report of gain-of-function mutations in the CFB gene and their association with non-Stx-HUS illustrates the important contribution of the alternative pathway C3 convertase to the pathogenesis of the disease. These data offer a complementary view to the previously described loss-of-function mutations in complement regulators and definitively establish the critical role of the complement alternative pathway in the pathogenesis of non-Stx-HUS.

The observation in the article derives from a series of 74 non-Stx-HUS patients. Five of them were selected for having very low C3 levels and normal or elevated levels of C4 over multiple determinations. The selected patients do not carry mutations in CFH, MCP and CFI genes. Mutations in CFB were found in two of these patients (40%). In another report [11], 20 non-Stx-HUS patients were screened, but no mutation was found in CFB gene. However, the authors may have failed to find mutations in CFB gene due to the small number of patients screened (the mutation rate in a general cohort of non-Stx patients is 2–3%). Moreover, the 20 patients were not selected for having the C3 and normal or high C4 levels, the criteria used by Dr. Goicoechea.

Incomplete penetrance of the disease phenotype is reported. A large pedigree was analysed for the F286L mutation and 11 carriers were found (seven of them are affected). The authors assess that the ‘healthy carriers are old enough to assume that they are not at risk’. We suspect that this may not be the case, as in face of a strong trigger, unaffected mutation carriers may still develop the disease at older ages. In our cohort of HUS patients, we found many CFH or MCP unaffected mutation carriers [2,12] who developed the disease only at older ages, in concomitance with pregnancies or infections [2]. The most striking of our cases is a CFH mutation carrier who developed HUS at the age of 82 years, due to acute pneumonia. On the other hand, all affected members of the pedigree described by Dr. Goicoechea and co-workers [13] carry the MCPggaac risk allele, which may contribute to the full manifestation of the disease. The same kind of situation has been reported in articles showing the association of CFH and MCP polymorphisms with the disease. Non-Stx-HUS appears to be a multigenic and multifactorial disease, and mutations in genes encoding for complement regulatory and activating proteins are susceptibility factors that confer a predisposition to developing HUS, rather than directly causing the disease. Other risk factors (polymorphisms, environmental triggers, drugs, systemic diseases) may contribute to the full manifestation of the phenotype.



   Clinical implications
Top
 Introduction
 Results and conclusions
 Clinical implications
Acknowledgements
 References
 
As observed for CFH, MCP and CFI genes [2,14], screening for CFB mutations may provide some information on clinical outcome. In fact, though other studies are needed, the clinical course of the disease in CFB mutated subjects appears to be serious, with end-stage renal disease (ESRD) being the final outcome in all but one patient.

Moreover, clinicians should now be aware that a new gene has been added to the list of those to be screened before proper genetic counselling can be offered to a patient, in particular in view of a kidney transplant. CFH and CFI are plasma proteins mainly produced by the liver. Thus a kidney transplant in CFH or CFI mutation carriers should be discouraged, as it will not correct the genetic defect in those patients. At variance, MCP is a transmembrane protein: transplantation of a kidney that expresses normal MCP should therefore correct the defect in patients with an MCP mutation [2,14]. What do we expect in case of a CFB mutation? CFB is a circulating protein, so one could speculate that HUS recurrence may take place on the transplanted kidney, and patients may experience graft failure. The only data available are in line with this hypothesis, as one of the Spanish patients reported by Dr. Goicoechea received a kidney transplant and experienced a recurrence of HUS.

In conclusion, the article by Dr. Goicoechea et al. adds a plug in the comprehension of the genetic causes of non-Stx-HUS and drives attention towards those genes whose alterations determine an abnormally increased activity of the alternative pathway of complement. Proteins such as properdin and C3 may be candidate genes contributing to non-Stx-HUS susceptibility, at least in those patients (around 50%) in which the genetic cause of the disease is still unknown.



   Acknowledgements
Top
 Introduction
 Results and conclusions
 Clinical implications
 Acknowledgements
References
 
This work was partially supported by grants from Telethon (Project GGP02162), National Institute of Health (Project NIH-NIDDKD 1R21DK071221) and Associazione ALICE.

Take home message. Gain-of-function mutations in CFB result in uncontrolled activation of the complement system, consistent with the pathogenetic mechanism of non-Stx-HUS.

Conflict of interest statement. None declared.



   Notes
 
*Comment on Goicoechea de Jorge E, Harris CL, Esparza-Gordillo J et al. Gain-of-function mutations in complement factor B are associated with atypical haemolytic uraemic syndrome. Proc Natl Acad Sci USA 2007; 104: 240–245. Back



   References
Top
 Introduction
 Results and conclusions
 Clinical implications
 Acknowledgements
 References
 

  1. Ruggenenti P, Noris M, Remuzzi G. Thrombotic microangiopathy, hemolytic uremic syndrome, and thrombotic thrombocytopenic purpura. Kidney Int (2001) 60:831–846.[CrossRef][ISI][Medline]
  2. Caprioli J, Noris M, Brioschi S, et al. Genetics of HUS: the impact of MCP, CFH, and IF mutations on clinical presentation, response to treatment, and outcome. Blood (2006) 108:1267–1279.[Abstract/Free Full Text]
  3. Richards A, Buddles MR, Donne RL, et al. Factor H mutations in hemolytic uremic syndrome cluster in exons 18-20, a domain important for host cell recognition. Am J Hum Genet (2001) 68:485–490.[CrossRef][ISI][Medline]
  4. Fremeaux-Bacchi V, Moulton EA, Kavanagh D, et al. Genetic and functional analyses of membrane cofactor protein (CD46) mutations in atypical hemolytic uremic syndrome. J Am Soc Nephrol (2006) 17:2017–2025.[Abstract/Free Full Text]
  5. Noris M, Remuzzi G. Hemolytic uremic syndrome. J Am Soc Nephrol (2005) 16:1035–1050.[Free Full Text]
  6. Goicoechea de Jorge E, Harris CL, Esparza-Gordillo J, et al. Gain-of-function mutations in complement factor B are associated with atypical hemolytic uremic syndrome. Proc Natl Acad Sci USA (2007) 104:240–245.[Abstract/Free Full Text]
  7. Smith CA, Vogel CW, Muller-Eberhard HJ. MHC Class III products: an electron microscopic study of the C3 convertases of human complement. J Exp Med (1984) 159:324–329.[Abstract/Free Full Text]
  8. Bhattacharya AA, Lupher ML Jr, Staunton DE, Liddington RC. Crystal structure of the A domain from complement factor B reveals an integrin-like open conformation. Structure (2004) 12:371–378.[Medline]
  9. Ponnuraj K, Xu Y, Macon K, Moore D, Volanakis JE, Narayana SV. Structural analysis of engineered Bb fragment of complement factor B: insights into the activation mechanism of the alternative pathway C3-convertase. Mol Cell (2004) 14:17–28.[CrossRef][ISI][Medline]
  10. Harris CL, Abbott RJ, Smith RA, Morgan BP, Lea SM. Molecular dissection of interactions between components of the alternative pathway of complement and decay accelerating factor (CD55). J Biol Chem (2005) 280:2569–2578.[Abstract/Free Full Text]
  11. Kavanagh D, Kemp EJ, Richards A, et al. Does complement factor B have a role in the pathogenesis of atypical HUS? Mol Immunol (2006) 43:856–859.[CrossRef][ISI][Medline]
  12. Caprioli J, Bettinaglio P, Zipfel PF, et al. 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 (2001) 12:297–307.[Abstract/Free Full Text]
  13. Esparza-Gordillo J, Goicoechea de Jorge E, Buil A, et al. 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 (2005) 14:703–712.[Abstract/Free Full Text]
  14. Bresin E, Daina E, Noris M, et al. Outcome of renal transplantation in patients with non-Shiga-toxin-associated hemolytic-uremic-syndrome: prognostic significance of genetic backgound. Clin J Am Soc Nephrol (2006) 1:88–99.[Abstract/Free Full Text]
Received for publication: 28. 2.07
Accepted in revised form: 13. 3.07


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