Characterization of mutations in complement factor I (CFI) associated with hemolytic uremic syndrome
Introduction
The hemolytic uremic syndrome (HUS) comprises the clinical triad of thrombocytopenia, microangiopathic hemolytic anaemia, and acute renal failure (Richards et al., 2002). The most common form of HUS is associated with a preceding diarrheal illness caused by a verocytotoxin-producing bacteria, most typically Escherichia coli O157:H7. Those not preceded by a diarrheal illness are classified as atypical HUS (aHUS) (Kavanagh and Goodship, 2006). aHUS is rare, may be familial, and has a poorer prognosis, with death rates of up to 25% in the acute phase and 50% requiring ongoing renal replacement therapy (Noris and Remuzzi, 2005).
Recent advances have shown that aHUS is a disease of complement dysregulation (reviewed in Atkinson et al., 2005), with 50% of cases involving the complement regulatory genes, factor H (CFH) (Buddles et al., 2000, Caprioli et al., 2001, Dragon-Durey et al., 2004, Neumann et al., 2003, Perez-Caballero et al., 2001, Richards et al., 2001, Warwicker et al., 1998), membrane cofactor protein (MCP; CD46) (Caprioli et al., 2006, Esparza-Gordillo et al., 2005, Fremeaux-Bacchi et al., 2006, Noris et al., 2003, Richards et al., 2003, Richards et al., 2006) and factor I (CFI) (Caprioli et al., 2006, Fremeaux-Bacchi et al., 2004, Geelen et al., 2007, Kavanagh et al., 2005, Nilsson et al., 2007). Goicoechea de Jorge et al. have also reported gain of function mutations in complement factor B associated with aHUS reinforcing the importance of the alternative complement pathway in the pathogenesis of aHUS (Goicoechea de Jorge et al., 2007).
CFI is an 88 kDa serum glycoprotein predominantly synthesised by the liver (Morris et al., 1982). It is a serine protease that cleaves the α′ chains of C3b and C4b but only in the presence of its cofactor proteins (Morgan and Harris, 1999). These cofactors are CFH for C3b (Pangburn et al., 1977, Whaley and Ruddy, 1976), C4 binding protein (C4BP) for C4b (Nagasawa and Stroud, 1977, Shiraishi and Stroud, 1975) and MCP and complement receptor 1 (CR1; CD35) for both (Liszewski et al., 1991, Medof et al., 1982, Ross et al., 1982). 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 the classical complement pathways. Since the alternative pathway comprises a feedback loop mechanism triggered by C3b deposition on a target, inhibition of C3b's ability to participate in this process by proteolytic cleavage is a key regulatory outcome that requires CFI.
CFI is a heterodimer, consisting of a heavy chain disulfide bonded to a catalytic light chain (Fearon, 1977, Pangburn et al., 1977). The heavy chain contains two low-density lipoprotein receptor (LDLr) domains, a CD5 domain, and a module found only in CFI and complement proteins C6 and C7 (factor I module). Additional 24- and 32-residue sequences are present at the N-terminus and C-terminus of the heavy chain (Fig. 1) (Kunnath-Muglia et al., 1993, Minta et al., 1996). The light chain contains the catalytic serine protease domain (Catterall et al., 1987, Goldberger et al., 1987, Kunnath-Muglia et al., 1993).
Over 30 families with complete CFI deficiency have now been described (Reis et al., 2006). The clinical manifestations of increased susceptibility to recurrent infection with encapsulated micro-organisms are present from early childhood. In complete CFI deficiency there is uncontrolled activation of the amplification loop of the alternative pathway resulting in a consumptive loss of C3. This secondary deficiency of C3 leads to a defect in opsonization, immune adherence and phagocytosis (Lambris, 1988).
Two patients with complete CFI deficiency have been reported to have renal disease. One patient had serological evidence of SLE and diffuse proliferative glomerulonephritis on renal biopsy (Amadei et al., 2001). The other patient presented with a multi-system inflammatory disorder characterized by hepatitis, pneumonitis, myositis, and histological evidence of a microangiopathic vasculitis. This patient subsequently developed focal segmental glomerulosclerosis (Sadallah et al., 1999).
All CFI mutations described so far in aHUS have been heterozygous (Caprioli et al., 2006, Esparza-Gordillo et al., 2005, Fremeaux-Bacchi et al., 2004, Geelen et al., 2007, Kavanagh et al., 2005, Nilsson et al., 2007). Mutations in CFI appear to be an uncommon cause of aHUS with several studies reporting a frequency of less than 5% (Caprioli et al., 2006, Esparza-Gordillo et al., 2005, Kavanagh et al., 2005), although the study by Fremeaux-Bacchi et al. reported a frequency of 12% (Fremeaux-Bacchi et al., 2004). Approximately 40% of the CFI mutants associated with aHUS result in low levels of CFI (Kavanagh et al., 2006). The remaining mutations produce a mutant protein that is secreted but may not be functionally active. However, in none of the studies reporting these mutations has a functional defect in the protein been demonstrated. We chose a representative subset of these aHUS-associated mutants (Caprioli et al., 2006, Geelen et al., 2007, Kavanagh et al., 2005, Kavanagh et al., 2006, Nilsson et al., 2007) to assess consequent functional defects. We analyzed the functional consequences of multiple missense mutations and one deletion mutation using recombinant CFI produced in human embryonic kidney 293T cells. Activity of the purified CFI was assessed by C3b and C4b cofactor assays. We provide experimental evidence for secreted but non-functional CFI mutants in patients with aHUS.
Section snippets
Factor I mutants
The CFI mutations studied include those identified in the cohorts of aHUS patients at the Institute of Human Genetics, Newcastle upon Tyne, UK (Kavanagh et al., 2005), and the Mario Negri Institute for Pharmacological Research, Bergamo, Italy (Caprioli et al., 2006) and also those reported elsewhere in the literature (Fremeaux-Bacchi et al., 2004, Geelen et al., 2007). Residue numbering is from the N-terminus of the mature protein and the 18 amino acid signal sequence is omitted corresponding
Clinical histories
Only clinical histories of patients not previously reported are described here.
Mutation M120I: Patient Nw#77 presented at age 26 with malignant hypertension and end stage renal failure. Renal biopsy was consistent with HUS. After 1 year he received a cadaveric renal transplant with good renal function for a decade. After 11 years there was deterioration in transplant function accompanied by a microangiopathy on blood film. Renal biopsy confirmed recurrent HUS. Despite plasma exchange the
Discussion
CFI gene mutations have been reported in patients with aHUS (Caprioli et al., 2006, Fremeaux-Bacchi et al., 2004, Geelen et al., 2007, Kavanagh et al., 2005, Kavanagh et al., 2006, Nilsson et al., 2007). These mutations, like those previously reported in CFH and MCP associated with aHUS, are of two main types. In Type 1, the mutant protein is absent or present in lower amounts in the serum resulting in a quantitative (haploinsufficient) defect in complement regulation. In Type 2, the mutant
Acknowledgments
D. Kavanagh is supported by Kidney Research UK and the Peel Medical Research Trust. AR is supported by the Fulbright US/UK exchange programme and the Peel Medical Research Trust. THJG is supported by the Foundation for Children with Atypical HUS and the Robin Davies Trust. MKL and JPA are supported by NIH RO1 AI37618. MN and GR are supported by grants from Telethon project GGP02162, from Associazione Ricerca Trapianto (ART), from NIH R21 DK071221, and from the Foundation for Children with
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