Margarita López-Trascasa

Gain-of-function mutations in complement factor B are associated with atypical hemolytic uremic syndrome

Hemolytic uremic syndrome (HUS) is an important cause of acute renal failure in children. Mutations in one or more genes encoding complement-regulatory proteins have been reported in approximately one-third of nondiarrheal, atypical HUS (aHUS) patients, suggesting a defect in the protection of cell surfaces against complement activation in susceptible individuals. Here, we identified a subgroup of aHUS patients showing persistent activation of the complement alternative pathway and found within this subgroup two families with mutations in the gene encoding factor B (BF), a zymogen that carries the catalytic site of the complement alternative pathway convertase (C3bBb). Functional analyses demonstrated that F286L and K323E aHUS-associated BF mutations are gain-of-function mutations that result in enhanced formation of the C3bBb convertase or increased resistance to inactivation by complement regulators. These data expand our understanding of the genetic factors conferring predisposition to aHUS, demonstrate the critical role of the alternative complement pathway in the pathogenesis of aHUS, and provide support for the use of complement-inhibition therapies to prevent or reduce tissue damage caused by dysregulated complement activation.

Clustering of Missense Mutations in the C-Terminal Region of Factor H in Atypical Hemolytic Uremic Syndrome

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.

Structural and functional characterization of factor H mutations associated with atypical hemolytic uremic syndrome.

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.

Human C3 mutation reveals a mechanism of dense deposit disease pathogenesis and provides insights into complement activation and regulation

Dense deposit disease (DDD) is a severe renal disease characterized by accumulation of electron-dense material in the mesangium and glomerular basement membrane. Previously, DDD has been associated with deficiency of factor H (fH), a plasma regulator of the alternative pathway (AP) of complement activation, and studies in animal models have linked pathogenesis to the massive complement factor 3 (C3) activation caused by this deficiency. Here, we identified a unique DDD pedigree that associates disease with a mutation in the C3 gene. Mutant C(3923ΔDG), which lacks 2 amino acids, could not be cleaved to C3b by the AP C3-convertase and was therefore the predominant circulating C3 protein in the patients. However, upon activation to C3b by proteases, or to C3(H₂O) by spontaneous thioester hydrolysis, C(3923ΔDG) generated an active AP C3-convertase that was regulated normally by decay accelerating factor (DAF) but was resistant to decay by fH. Moreover, activated C(3b923ΔDG) and C3(H₂O)(923ΔDG) were resistant to proteolysis by factor I (fI) in the presence of fH, but were efficiently inactivated in the presence of membrane cofactor protein (MCP). These characteristics cause a fluid phase-restricted AP dysregulation in the patients that continuously activated and consumed C3 produced by the normal C3 allele. These findings expose structural requirements in C3 that are critical for recognition of the substrate C3 by the AP C3-convertase and for the regulatory activities of fH, DAF, and MCP, all of which have implications for therapeutic developments.

Hereditary angioedema due to C1 inhibitor deficiency : patient registry and approach to the prevalence in Spain

BACKGROUND Hereditary angioedema (HAE) is a rare disease caused by C1 inhibitor mutations. Although more than 100 mutations have been described, epidemiologic data are lacking; therefore, we developed a Spanish HAE patient registry. OBJECTIVE To study the prevalence of HAE and the current state of diagnosis and treatment of this disease in Spain. METHODS Epidemiologic data were obtained by direct contact with physicians who treat patients with HAE and with patients themselves. Diagnosis was evaluated by measuring C1 inhibitor levels and function, and most families also underwent genetic studies. RESULTS We registered 444 patients (minimal prevalence, 1.09 per 100,000 inhabitants), many of whom are asymptomatic (never having symptoms) (n = 61, 13.7%). Most symptomatic patients (62.9%) receive long-term prophylaxis with attenuated androgens (80.9%) and antifibrinolytic agents (22.8%), alone or in combination, but no patients are receiving long-term prophylaxis with C1 inhibitor. There is a long delay in diagnosis (mean, 13.1 years). Nine patients underwent a tracheotomy as a consequence of a laryngeal attack, and 30 families recalled a total of 38 relatives who died of HAE, which underlines the severity of the illness. CONCLUSIONS The detected minimal prevalence of HAE in Spain is 1.09 per 100,000 inhabitants. Because this is a rare disease and some patients may be misdiagnosed, this prevalence could be higher.

Anti-IgA antibodies in selective IgA deficiency and in primary immunodeficient patients treated with γ-globulin

Sera from 106 blood donors, 40 patients with primary immunodeficiencies (ID) treated with gamma-globulin, and 46 patients with selective IgA deficiency were analyzed by an enzyme-linked immunosorbent assay for anti-IgA antibodies. Increased levels of antibodies to IgA were found in 5.6% of the blood donors, 17.5% of the ID patients, and 36.8% of the isolated IgA deficiencies. The percentage was higher in patients with IgA and IgG2 deficiencies (50%). The percentage of patients having increased levels of anti-IgA antibodies was similar to the total prevalence of the 10 other autoantibodies studied. These anti-IgA antibodies were mainly of the IgG class, except from one blood donor with IgM antibodies, and two patients, one with isolated IgA deficiency and the other with common variable immunodeficiency who had anti-IgA antibodies of the IgE class. The latter patient developed a near fatal anaphylactic reaction when intravenous gamma-globulin was administered. Most of the patients with severe adverse reactions to gamma-globulin did not present anti-IgA antibodies. Our data suggest that at least in some immunodeficient patients the elevated amounts of anti-IgA antibodies are not related to the administration of exogenous IgA. The importance of measuring anti-IgA antibodies of the IgG and IgE isotypes in IgA-deficient patients as well as in patients in treatment with gamma-globulin is emphasized.

Sensitive and specific assays for C3 nephritic factors clarify mechanisms underlying complement dysregulation

C3 nephritic factors are autoantibodies that prolong the half-life or prevent regulation of the alternative pathway C3 convertase, resulting in uncontrolled complement activation. They are strongly associated with renal disease but their role in pathogenesis remains controversial. Here we optimized and compared a panel of assays to identify and interrogate nephritic factor activities. Of 101 patients with histologic or clinically evident disease, 48 were positive in some or all assays. In the presence of properdin, binding of autoantibody was detected in 39 samples and convertase stabilization was detected in 36. Forty-two of 48 nephritic factors tested prevented convertase decay by factor H, and most of these by decay accelerating factor (28) and complement receptor 1 (34). Representative properdin-independent nephritic factors had no effect on C5 cleavage and terminal pathway activity, while properdin-dependent nephritic factors enhanced activity. Biacore analysis of four purified IgG samples confirmed resistance to decay and showed that properdin-independent nephritic factors increased convertase half-life over 50-fold, whereas properdin-dependent nephritic factors increased the half-life 10- to 20-fold and also increased activity of the C3 convertase up to 10-fold. Thus, our study provides a rational approach to detect and characterize nephritic factors in patients.

Eculizumab in the Treatment of Atypical Hemolytic Uremic Syndrome in Infants

A 28-day-old male newborn weighing 3.6 kg was given a diagnosis of atypical hemolytic-uremic syndrome, new-onset thrombotic microangiopathy (TMA; hemoglobin, 7.7 g/dL; schistocytes, 9%), thrombocytopenia (platelets, 49 × 10(3)/μL [49 × 10(9)/L]), and acute kidney failure (serum creatinine, 1.13 mg/dL [99.8 μmol/L], corresponding to estimated glomerular filtration rate [eGFR] of 15 mL/min/1.73 m(2) [0.25 mL/s/1.73 m(2)]). Repeated high-volume plasma infusions were ineffective. Plasma exchange was attempted, but not tolerated. The patient required mechanical ventilation and continuous renal replacement therapy. He developed multiple intestinal perforations and leg skin necrosis due to systemic TMA. A low C3 level (36 mg/dL) suggested complement activation. Eculizumab, 300 mg, was administered, and within 48 hours the patient recovered from acute kidney failure, with complete hematologic remission 2 weeks later. The infant, 14 months old at the time of writing, continues to receive eculizumab, 300 mg, every 3 weeks; he is free of disease activity and has a normal creatinine level of 0.2 mg/dL (17.68 μmol/L; corresponding to eGFR of 110 mL/min/1.73 m(2) [1.83 mL/s/1.73 m(2)]), but mild proteinuria (urinary protein-creatine ratio, 1 mg/g). Results of additional studies, including probing for cobalamin anomalies and measuring levels of ADAMTS13, complement factor H (CFH), factor I (CFI), and membrane cofactor protein (MCP), were unremarkable. Antibodies to CFH were undetectable, and mutation testing of the genes for CFH, CFI, and MCP gave negative results. Treatment with eculizumab was life saving, and with continued treatment, the patient showed sustained freedom from clinical TMA complications.

Functional C1-inhibitor diagnostics in hereditary angioedema: assay evaluation and recommendations.

Hereditary angioedema (HAE) is an autosomal dominant disease characterized by recurrent episodes of potentially life-threatening angioedema. The most widespread underlying genetic deficiency is a heterozygous deficiency of the serine protease inhibitor C1 esterase inhibitor (C1-Inh). In addition to low C4 levels, the most important laboratory parameter for correct diagnosis of HAE or angioedema due to acquired C1-Inh deficiency is reduced C1-Inh function (fC1-Inh). No direct recommendations about the assays for fC1-Inh or sample handling conditions are available, although this would prove especially useful when a laboratory first starts to offer assays on fC1-Inh for HAE diagnosis. In the present study we evaluated the performance of fC1-Inh assays in the 15 different laboratories that are specialised in HAE diagnostics and assessed inter-laboratory variation with each laboratory using their own assays and standards. A double-blind survey was conducted using plasma/serum samples from healthy donors and HAE patients and the uniformity of HAE diagnosis was evaluated. It can be concluded that the diagnosis of fC1-Inh deficiency was made correctly in most cases in this survey. We can recommend the chromogenic assay for the determination of fC1-Inh, while the complex ELISA needs further investigation.

Atypical Hemolytic Uremic Syndrome-Associated Variants and Autoantibodies Impair Binding of Factor H and Factor H-Related Protein 1 to Pentraxin 3

Atypical hemolytic uremic syndrome (aHUS) is a renal disease associated with complement alternative pathway dysregulation and is characterized by endothelial injury. Pentraxin 3 (PTX3) is a soluble pattern recognition molecule expressed by endothelial cells and upregulated under inflammatory conditions. PTX3 activates complement, but it also binds the complement inhibitor factor H. In this study, we show that native factor H, factor H-like protein 1, and factor H-related protein 1 (CFHR1) bind to PTX3 and that PTX3-bound factor H and factor H-like protein 1 maintain their complement regulatory activities. PTX3, when bound to extracellular matrix, recruited functionally active factor H. Residues within short consensus repeat 20 of factor H that are relevant for PTX3 binding were identified using a peptide array. aHUS-associated factor H mutations within this binding site caused a reduced factor H binding to PTX3. Similarly, seven of nine analyzed anti-factor H autoantibodies isolated from aHUS patients inhibited the interaction between factor H and PTX3, and five autoantibodies also inhibited PTX3 binding to CFHR1. Moreover, the aHUS-associated CFHR1*B variant showed reduced binding to PTX3 in comparison with CFHR1*A. Thus, the interactions of PTX3 with complement regulators are impaired by certain mutations and autoantibodies affecting factor H and CFHR1, which could result in an enhanced local complement-mediated inflammation, endothelial cell activation, and damage in aHUS.