The complement factor H R1210C mutation is associated with atypical hemolytic uremic syndrome

Mutations in the gene encoding complement factor H (CFH) that alter the C3b/polyanions-binding site in the C-terminal region impair the capacity of factor H to protect host cells. These mutations are also strongly associated with atypical hemolytic uremic syndrome (aHUS). Although most of the aHUS-associated CFH mutations seem "unique" to an individual patient or family, the R1210C mutation has been reported in several unrelated aHUS patients from distinct geographic origins. Five aHUS pedigrees and 7 individual aHUS patients were analyzed to identify potential correlations between the R1210C mutation and clinical phenotype and to characterize the origins of this mutation. The clinical phenotype of aHUS patients carrying the R1210C mutation was heterogeneous. Interestingly, 12 of the 13 affected patients carried at least one additional known genetic risk factor for aHUS. These data are in accord with the 30% penetrance of aHUS in R1210C mutation carriers, as it seems that the presence of other genetic or environmental risk factors significantly contribute to the manifestation and severity of aHUS in these subjects. Genotype analysis of CFH and CFHR3 polymorphisms in the 12 unrelated carriers suggested that the R1210C mutation has a single origin. In conclusion, the R1210C mutation of complement factor H is a prototypical aHUS mutation that is present as a rare polymorphism in geographically separated human populations.     

A Large Family with a Gain-of-Function Mutation of Complement C3 Predisposing to Atypical Hemolytic Uremic Syndrome,Microhematuria, Hypertension and Chronic Renal Failure

Background and objectives: Atypical hemolytic uremic syndrome (aHUS) is associated with mutations in genes encoding complement-regulatory proteins factor H, I and B and membrane cofactor protein. Recently, heterozygous gain-of-function mutations in the complement C3 gene have been found in patients with aHUS.Design, setting, participants, & measurements: A large family with a C3 R570Q mutation is described. Clinical and laboratory findings of carriers of the mutation and unaffected family members are reported.Results: The index patient suffered from recurrent aHUS at age 22 and developed end-stage renal failure. Of 24 family members, nine harbored the C3 R570Q mutation. Carriers showed reduced or borderline C3 levels. Arterial hypertension was found in six family members, microhematuria in five and chronic kidney disease stage 3 in two elderly carrier patients. Despite marked consumption of C3, serum terminal complement complex levels were not elevated in carriers compared with other family members.Conclusions: The penetrance of the C3 R570Q mutation to induce aHUS is incomplete and lower compared with mutations in other genes predisposing to the disease. The mutation is possibly also associated with hypertension, hematuria and chronic kidney disease, all of which may represent consequences of long-term complement activation in the renal vasculature.Hemolytic uremic syndrome (HUS) is a rare disease characterized by microangiopathic hemolytic anemia, thrombocytopenia and acute renal failure. In children, the most frequent form (90% of patients) is the so-called typical or postdiarrheal (D+) HUS, caused by infection with Shiga-toxin (Stx) producing Escherichia coli. Other cases are classified as atypical D-HUS or aHUS and can be either sporadic or familial. A clear link was demonstrated between the disease and genetic abnormalities in complement regulator genes. In about 50% of patients suffering from aHUS, predisposing mutations in genes of complement regulators are identified. These include loss-of-function mutations in soluble complement factors H (CFH) and I (CFI) and membrane-bound membrane co-factor protein (MCP, CD46) (1–9). In about ten percent of cases, loss-of-function is acquired and due to anti-factor H antibodies (10). In addition, gain-of-function mutations in complement factor B (BF) also predispose to aHUS (11). A recent report describes nine heterozygous mutations in the complement C3 gene that are associated with aHUS in 14 patients (12). Whereas two of these caused impaired C3 secretion, five mutated proteins showed reduced binding to MCP and resisted cleavage by CFI. Here we describe in detail a patient carrying one of these mutations (R570Q) and an investigation of the patient''s family. We suggest that this mutation not only predisposes to aHUS, but is probably also associated with hypertension, minor urinary abnormalities and chronic kidney disease (CKD).     

Acute presentation and persistent glomerulonephritis following streptococcal infection in a patient with heterozygous complement factor H-related protein 5 deficiency

Acute poststreptococcal glomerulonephritis is a common cause of acute nephritis in children. Transient hypocomplementemia and complete recovery are typical, with only a minority developing chronic disease. We describe a young girl who developed persistent kidney disease and hypocomplementemia after a streptococcal throat infection. Kidney biopsy 1 year after presentation showed isolated glomerular complement C3 deposition, membranoproliferative changes, and subendothelial, intramembranous and occasional subepithelial electron-dense deposits consistent with C3 glomerulopathy. Complement gene screening revealed a heterozygous single nucleotide insertion in exon 4 of the complement factor H-related protein 5 gene (CFHR5), resulting in a premature stop codon. This variant was not detected in 198 controls. Serum CFHR5 levels were reduced. The mother and sister of the index patient were heterozygous for the sequence variant, with no overt evidence of kidney disease. We speculate that this heterozygous CFHR5 sequence variant is a risk factor for the development of chronic kidney disease after streptococcal infection.     

Combined Complement Gene Mutations in Atypical Hemolytic Uremic Syndrome Influence Clinical Phenotype

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.Hemolytic uremic syndrome (HUS) is a rare disease of microangiopathic hemolysis, thrombocytopenia, and renal failure.^1,2 The most common form in children is associated with infection by certain strains of Escherichia coli, which produce Shiga-like toxins.³ This form has a good prognosis.¹ There are rarer atypical forms (aHUS), not associated with Shiga-like toxins-producing bacteria, that have a worse outcome, with up to 50% of cases progressing to end stage renal failure (ESRF) and 10%–15% dying during the acute phase.^1,4Inherited defects that determine uncontrolled activation of the alternative complement pathway have been well documented in aHUS patients.^2,5,6 Research in recent years has identified more than 120 different mutations, accounting for around 40%–60% of cases, in the genes encoding complement factor H (CFH),^7–9 membrane cofactor protein (MCP),^10–13 complement factor I (CFI),^14–16 C3,¹⁷ complement factor B (CFB),^18,19 CFH-related 5 (CFHR5),²⁰ and thrombomodulin (THBD).^20,21 In addition, anti-CFH autoantibodies have been described mostly in children that lack CFHR1 and CFHR3 because of a deletion of the corresponding genes.^22–26 Novel genetic abnormalities of CFHR1, CFHR3, and CFHR4 and genomic rearrangement between CFH and CFHR1 have recently been described.^27,28Incomplete penetrance of aHUS has been reported in mutation carriers,^12,29–31 indicating that complement gene mutations confer predisposition to develop aHUS, with additional genetic and/or environmental hits necessary for disease manifestation.^7,32,33 In keeping with this hypothesis, patients with mutations in more than one complement gene (combined gene mutations) have been described.^20,29,34,35 This study was designed to (1) determine the frequency of combined complement gene mutations among four cohorts of aHUS patients; (2) compare short- and long-term outcomes, response to plasma treatment, and outcome of kidney transplantation among patients carrying mutations in different gene combinations; and (3) compare clinical parameters in patients carrying combined mutations versus patients with mutations in a single complement gene. Thanks to a joint effort by the European Working Party on Complement Genetics in Renal Diseases, we genotyped almost 800 aHUS patients for aHUS-associated genes, identifying 27 patients with combined gene mutations.     

A mutation in factor I that is associated with atypical hemolytic uremic syndrome does not affect the function of factor I in complement regulation

Factor I (FI) is the major complement inhibitor that degrades C3b and C4b in the presence of cofactors such as factor H (FH) and membrane cofactor protein (MCP). Recently, mutations and polymorphisms in complement regulator molecules FH and MCP but also in FI have been associated with atypical hemolytic uremic syndrome (aHUS). HUS is a disorder characterized by hemolytic anemia, thrombocytopenia and acute renal failure. In this study, we report three unrelated patients with an identical heterozygous mutation, G261D, in the FI heavy chain who developed severe aHUS at different time points in their lives. Two of the patients also have polymorphisms in FH previously associated with risk of developing aHUS. Testing in particular one patient and control serum samples we did not observe major differences in complement hemolytic activity, FI plasma levels or the capability to degrade C4b or C3b. A recombinant protein was produced in order to analyze the functional consequences of the mutation. Mutant FI had a slightly different migration pattern during electrophoresis under reducing conditions. An alteration due to alternative splicing or glycosylation was ruled out, thus the altered migration may be due to proximity of the mutation to a cysteine residue. The recombinant mutant FI degraded C3b and C4b in a manner comparable to wild-type protein. In conclusion, despite the association between the heterozygous mutation in FI and aHUS we did not observe any abnormalities in the function of FI regarding complement regulation.     

Characterization of mutations in complement factor I (CFI) associated with hemolytic uremic syndrome

Recent studies have identified mutations in the complement regulatory gene factor I (CFI) that predispose to atypical hemolytic uremic syndrome (aHUS). CFI is a two-chain serine protease in which the light chain carries the catalytic domain while the heavy chain's function is unclear. It downregulates the alternative and classical complement pathways by cleaving the alpha' chains of C3b and C4b in the presence of cofactor proteins (known as cofactor activity). Many CFI mutations in aHUS result in low CFI levels with a consequent quantitative defect in complement regulation. In others, the mutant protein is present in normal amounts but the presumed functional deficiency has not yet been defined. In this report we examine the nature of the functional defect in aHUS-associated CFI mutations. The I322T, D501N and D506V mutations reside in the serine protease domain of CFI and result in secreted proteins that lack C3b and C4b cofactor activity. The delTTCAC (1446-1450) mutant leads to a protein that is not secreted. The R299W mutant lies in a region of the CFI heavy chain of no known function. Our assessments demonstrate decreased C3b and C4b cofactor activity, providing evidence that this region is important for cofactor activity. In two other heavy chain mutants and one probable polymorphic variant, no functional deficiency was identified. These defective mutant proteins will result in an inability to appropriately control the complement cascade at sites of endothelial cell injury. The excessive complement activation for a given degree of damage may result in generation of a procoagulant state and aHUS.     

Selective disappearance of C3NeF IgG autoantibody in the plasma of a patient with membranoproliferative glomerulonephritis following renal fransplantation

The serum of patients with membranopro-liferative glomerulonephritis(MPGN) often exhibits C3 nephritic factor activity (C3NeF) associatedwith autoantibodies directed against the C3bBb alternative complementpathway C3 convertase. In the present study, we have sequentiallyassessed C3NeF activity in the purified IgG fraction of theserum of a patient with MPGN type II and end-stage renal diseaseevery month for 1 year following renal transplantation and bilateralnephrectomy. C3NeF activity in the patient's purified serumIgG decreased with time from the first month after transplantationand became undetectable after forty-five weeks. At that time,plasma levels of C3 and factor B had returned to normal values.The disappearance of C3NeF activity in patient's IgG was selectivein that no change in activity of two other autoantibodies andof one antibody against a non-self antigen was observed withtime, indicating that the decrease in C3NeF activity was notrelated to a non specilic effect of immunosuppressive therapy.Loss of C3NeF activity was not related to the generation ofanti-idiotypic antibodies against C3NeF. This study documentsfor the first time the selective disappearance of C3NeF activityfollowing renal transplantation and bilateral nephrectomy. Theresults suggest that the autoantibody represents an antigen-drivenexpansion of self reactive B cell clones in response to a specificdisease process in the kidney of patients with MPGN.     

Membrane cofactor protein mutations in atypical hemolytic uremic syndrome (aHUS), fatal Stx-HUS, C3 glomerulonephritis, and the HELLP syndrome

The hemolytic uremic syndrome (HUS) is a triad of microangiopathic hemolytic anemia, thrombocytopenia, and renal impairment. Genetic studies demonstrate that heterozygous mutations of membrane cofactor protein (MCP;CD46) predispose to atypical HUS (aHUS), which is not associated with exposure to Shiga toxin (Stx). Among the initial 25 MCP mutations in patients with aHUS were 2, R69W and A304V, that were expressed normally and for which no dysfunction was found. The R69W mutation is in complement control protein module 2, while A304V is in the hydrophobic transmembrane domain. In addition to 3 patients with aHUS, the A304V mutation was identified in 1 patient each with fatal Stx-HUS, the HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome, and glomerulonephritis with C3 deposits. A major goal was to assess if these putative mutations lead to defective complement regulation. Permanent cell lines expressing the mutated proteins were complement "challenged," and membrane control of C3 fragment deposition was monitored. Both the R69W and A304V MCP mutations were deficient in their ability to control the alternative pathway of complement activation on a cell surface, illustrating the importance of modeling transmembrane proteins in situ.