No abstract

Several abnormalities in complement genes reportedly contribute to atypical hemolytic uremic syndrome (aHUS), but incomplete penetrance suggests that additional factors are necessary for the disease to manifest. Here, we sought to describe genotype-phenotype correlations among patients with combined mutations, defined as mutations in more than one complement gene. We screened 795 patients with aHUS and identified single mutations in 41% and combined mutations in 3%. Only 8%-10% of patients with mutations in CFH, C3, or CFB had combined mutations, whereas approximately 25% of patients with mutations in MCP or CFI had combined mutations. The concomitant presence of CFH and MCP risk haplotypes significantly increased disease penetrance in combined mutated carriers, with 73% penetrance among carriers with two risk haplotypes compared with 36% penetrance among carriers with zero or one risk haplotype. Among patients with CFH or CFI mutations, the presence of mutations in other genes did not modify prognosis; in contrast, 50% of patients with combined MCP mutation developed end stage renal failure within 3 years from onset compared with 19% of patients with an isolated MCP mutation. Patients with combined mutations achieved remission with plasma treatment similar to patients with single mutations. Kidney transplant outcomes were worse, however, for patients with combined MCP mutation compared with an isolated MCP mutation. In summary, these data suggest that genotyping for the risk haplotypes in CFH and MCP may help predict the risk of developing aHUS in unaffected carriers of mutations. Furthermore, screening patients with aHUS for all known disease-associated genes may inform decisions about kidney transplantation.

No abstract

C3 glomerulopathies (C3G) are a group of severe renal diseases with distinct patterns of glomerular inflammation and C3 deposition caused by complement dysregulation. Here we report the identification of a familial C3G-associated genomic mutation in the gene complement factor H-related 1 (CFHR1), which encodes FHR1. The mutation resulted in the duplication of the N-terminal short consensus repeats (SCRs) that are conserved in FHR2 and FHR5. We determined that native FHR1, FHR2, and FHR5 circulate in plasma as homo-and hetero-oligomeric complexes, the formation of which is likely mediated by the conserved N-terminal domain. In mutant FHR1, duplication of the N-terminal domain resulted in the formation of unusually large multimeric FHR complexes that exhibited increased avidity for the FHR1 ligands C3b, iC3b, and C3dg and enhanced competition with complement factor H (FH) in surface plasmon resonance (SPR) studies and hemolytic assays. These data revealed that FHR1, FHR2, and FHR5 organize a combinatorial repertoire of oligomeric complexes and demonstrated that changes in FHR oligomerization influence the regulation of complement activation. In summary, our identification and characterization of a unique CFHR1 mutation provides insights into the biology of the FHRs and contributes to our understanding of the pathogenic mechanisms underlying C3G.

The factor H-related protein family (CFHR) is a group of minor plasma proteins genetically and structurally related to complement factor H (fH). Notably, deficiency of CFHR1/CFHR3 associates with protection against age-related macular degeneration and with the presence of anti-fH autoantibodies in atypical hemolytic uremic syndrome (aHUS). We have developed a proteomics strategy to analyze the CFHR proteins in plasma samples from controls, patients with aHUS, and patients with type II membranoproliferative glomerulonephritis. Here, we report on the identification of persons carrying novel deficiencies of CFHR1, CFHR3, and CFHR1/CFHR4A, resulting from point mutations in CFHR1 and CFHR3 or from a rearrangement involving CFHR1 and CFHR4. Remarkably, patients with aHUS lacking CFHR1, but not those lacking CFHR3, present anti-fH autoantibodies, suggesting that generation of these antibodies is specifically related to CFHR1 deficiency. We also report the characterization of a novel CFHR1 polymorphism, resulting from a gene conversion event between CFH and CFHR1, which strongly associates with aHUS. The risk allotype CFHR1*B, with greater sequence similarity to fH, may compete with fH, decreasing protection of cellular surfaces against complement damage. In summary, our comprehensive analyses of the CFHR proteins have improved our understanding of these proteins and provided further insights into aHUS pathogenesis. (Blood. 2009;114:4261-4271)

Hemolytic-uremic syndrome (HUS) is a microvasculature disorder leading to microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. Most cases of HUS are associated with epidemics of diarrhea caused by verocytotoxin-producing bacteria, but atypical cases of HUS not associated with diarrhea (aHUS) also occur. Early studies describing the association of aHUS with deficiencies of factor H suggested a role for this complement regulator in aHUS. Molecular evidence of factor H involvement in aHUS was first provided by Warwicker et al., who demonstrated that aHUS segregated with the chromosome 1q region containing the factor H gene (HF1) and who identified a mutation in HF1 in a case of familial aHUS with normal levels of factor H. We have performed the mutational screening of the HF1 gene in a novel series of 13 Spanish patients with aHUS who present normal complement profiles and whose plasma levels of factor H are, with one exception, within the normal range. These studies have resulted in the identification of five novel HF1 mutations in four of the patients. Allele HF1 Delta exon2, a genomic deletion of exon 2, produces a null HF1 allele and results in plasma levels of factor H that are 50% of normal. T956M, W1183L, L1189R, and V1197A are missense mutations that alter amino acid residues in the C-terminal portion of factor H, within a region--SCR16-SCR20--that is involved in the binding to solid-phase C3b and to negatively charged cellular structures. This remarkable clustering of mutations in HF1 suggests that a specific dysfunction in the protection of cellular surfaces by factor H is a major pathogenic condition underlying aHUS.

The efficiency of the complement system as an innate immune defense mechanism depends on a fine control that restricts its action to pathogens and prevents non-specific damage to host tissues. Genetic and functional analyses have shown that this critical control of complement activation may be impaired in atypical hemolytic uremic syndrome (aHUS) patients. Mutations in HF1, MCP or FI have been found in aHUS patients, but incomplete penetrance of the disease in individuals carrying these mutations is relatively frequent and no genetic defect has yet been found in a majority of aHUS patients. We report here the identification of a specific SNP haplotype block, spanning the MCP gene in the regulators of complement activation gene cluster, which is over-represented in aHUS patients and strongly associates with the severity of the disease. Linkage disequilibrium analyses suggest that this SNP haplotype also includes the CR1, DAF and C4BP genes. Initial studies identified two SNPs in the haplotype that influence the transcription activity of the MCP promoter in transient transfection experiments. Notably, the SNP haplotype block was found to be particularly frequent among patients who carry mutations in HF1, MCP or FI. These findings and the identification of aHUS patients carrying mutations in two complement regulatory genes provide an important insight into the etiology of aHUS. Together, they suggest that complement regulatory molecules act as a protein network and that multiple hits, involving plasma- and membrane-associated complement regulatory proteins, are necessary to impair protection to host tissues and to confer significant predisposition to aHUS.

Genetic studies have demonstrated the involvement of the complement regulator factor H in nondiarrheal, nonverocytotoxin (i.e., atypical) cases of hemolytic uremic syndrome. Different factor H mutations have been identified in 10%-30% of patients with atypical hemolytic uremic syndrome (aHUS), and most of these mutations alter single amino acids in the C-terminal region of factor H. Although these mutations are considered to be responsible for the disease, the precise role that factor H plays in the pathogenesis of aHUS is unknown. We report here the structural and functional characterization of three different factor H proteins purified from the plasma of patients with aHUS who carry the factor H mutations W1183L, V1197A, or R1210C. Structural anomalies in factor H were found only in R1210C carriers; these individuals show, in their plasma, a characteristic high-molecular-weight factor H protein that results from the covalent interaction between factor H and human serum albumin. Most important, all three aHUS-associated factor H proteins have a normal cofactor activity in the proteolysis of fluid-phase C3b by factor I but show very low binding to surface-bound C3b. This functional impairment was also demonstrated in recombinant mutant factor H proteins expressed in COS7 cells. These data support the hypothesis that patients with aHUS carry a specific dysfunction in the protection of cellular surfaces from complement activation, offering new possibilities to improve diagnosis and develop appropriate therapies.