The MFHR1 Fusion Protein Is a Novel Synthetic Multitarget Complement Inhibitor with Therapeutic Potential.

The complement system is essential for host defense, but uncontrolled complement system activation leads to severe, mostly renal pathologies, such as atypical hemolytic uremic syndrome or C3 glomerulopathy. Here, we investigated a novel combinational approach to modulate complement activation by targeting C3 and the terminal pathway simultaneously. The synthetic fusion protein MFHR1 links the regulatory domains of complement factor H (FH) with the C5 convertase/C5b-9 inhibitory fragment of the FH-related protein 1. In vitro, MFHR1 showed cofactor and decay acceleration activity and inhibited C5 convertase activation and C5b-9 assembly, which prevented C3b deposition and reduced C3a/C5a and C5b-9 generation. Furthermore, this fusion protein showed the ability to escape deregulation by FH-related proteins and form multimeric complexes with increased inhibitory activity. In addition to substantially inhibiting alternative and classic pathway activation, MFHR1 blocked hemolysis mediated by serum from a patient with aHUS expressing truncated FH. In FH-/- mice, MFHR1 administration augmented serum C3 levels, reduced abnormal glomerular C3 deposition, and ameliorated C3 glomerulopathy. Taking the unique design of MFHR1 into account, we suggest that the combination of proximal and terminal cascade inhibition together with the ability to form multimeric complexes explain the strong inhibitory capacity of MFHR1, which offers a novel basis for complement therapeutics.

Atypical hemolytic uremic syndrome due to factor H autoantibody.

Atypical hemolytic uremic syndrome (aHUS) is a disease caused by pathologies in the alternative complement system. The prevalence of aHUS is 10% of all aHUS cases. The subgroup of aHUS designated as DEAP (DEficiency of CFHR Proteins and CFH Autoantibody Positive)-HUS because of autoantibody to complement factor H (CFH) and CFH-related protein deficiency is seen very rarely, and the prevalence is 6% of all aHUS cases in the literature. We present here a female patient with DEAP-HUS. A 7.5-year-old girl with recurrent attacks of HUS had low C3 level. We initiated plasmapheresis treatment. After further analysis of the complement system, the result was compatible with DEAP-HUS, so we initiated immunosuppressive treatment. There were also family members with deficiency of CFHR-1 and CFHR-3, but they had no CFH autoantibody and no symptoms of HUS. In atypical cases of HUS, we should investigate complement status, especially for factor H autoantibody, for which treatment options differ from those of the other types of aHUS.

Complement factor H-related protein 1 deficiency and factor H antibodies in pediatric patients with atypical hemolytic uremic syndrome.

BACKGROUND AND OBJECTIVES: This study evaluated the relevance of complement factor H (CFH)-related protein (CFHR) 1 deficiency in pediatric patients with atypical hemolytic uremic syndrome (aHUS) by evaluating both the frequency of deletions in CFHR1 and the presence of complement factor H (CFH) antibodies. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: A total of 116 patients (mainly from central Europe) and 118 healthy blood donors were included from 2001 to 2012. The presence of CFHR1 gene deletions was determined in 90 pediatric patients with aHUS and 118 controls by an easy, fast, and cheap PCR assay; 100 patients with aHUS and 42 controls were tested for CFH antibodies by ELISA. Questionnaires were administered to evaluate the clinical and laboratory data. RESULTS: Homozygous deletion in CFHR1 was detected in 32% of the patients with aHUS tested, compared with 2.5% of controls (P<0.001). CFH antibodies were present in 25% of the patients and none of the controls. CFH antibodies were detected in 82% of patients with homozygous CFHR1 gene deletion and in 6% of patients without. CFH antibody-positive patients with aHUS showed a significantly lower platelet nadir at disease onset and significantly less frequent involvement of the central nervous system than did antibody-negative patients. Antibody-positive patients also received plasma therapy more often. CONCLUSION: Homozygous deletion in CFHR1 is strongly associated with occurrence of CFH antibodies in pediatric patients with aHUS. However, despite this apparent genetic disease predisposition, it cannot be considered an exclusive cause for aHUS. Initial presentation of Shiga toxin-negative HUS with severe thrombocytopenia and no central nervous system complications in pediatric patients is especially suspicious for CFH antibody aHUS.

DEAP-HUS: deficiency of CFHR plasma proteins and autoantibody-positive form of hemolytic uremic syndrome.

DEAP-HUS [Deficiency of CFHR (complement factor H-related) plasma proteins and Autoantibody Positive form of Hemolytic Uremic Syndrome] represents a novel subtype of hemolytic uremic syndrome (HUS) with unique characteristics. It affects children and requires special clinical attention in terms of diagnosis and therapy. DEAP-HUS and other atypical forms of HUS share common features, such as microangiopathic hemolytic anemia, acute renal failure, and thrombocytopenia. However, DEAP-HUS has the unique combination of an acquired factor in the form of autoantibodies to the complement inhibitor Factor H and a genetic factor which, in most cases, is the chromosomal deletion of a 84-kbp fragment within human chromosome 1 that results in the absence of the CFHR1 and CFHR3 proteins in plasma. Special attention is required to diagnose and treat DEAP-HUS patients. Most patients show a favorable response to the reduction of autoantibody titers by either plasma therapy, steroid treatment, and/or immunosuppression. In addition, in those DEAP-HUS patients with end-stage renal disease, the reduction of autoantibody titers prior to transplantation is expected to prevent post-transplant disease recurrence by aiming for full complement control at the endothelial cell surface in order to minimize adverse complement and immune reactions.

Successful renal transplantation in factor H autoantibody associated HUS with CFHR1 and 3 deficiency and CFH variant G2850T.

Factor H (CFH) autoantibodies are associated with atypical hemolytic uremic syndrome (aHUS). Peritransplantation plasma exchange therapy and intensification of immunosuppression, with adjuvant use of anti-CD20 monoclonal antibodies has recently been advocated for cases of CFH-autoantibody associated aHUS. In this report, we describe successful deceased donor renal transplantation in a case of CFH-autoantibody associated aHUS with combined CFHR1 and 3 deficiency in addition to the CFH sequence variant, (cG2850T, pGln950His). CFH-autoantibodies were detected 2 weeks prior to transplantation. Disease recurrence was not observed using basiliximab, an IL2-receptor antagonist and high-dose corticosteroids with mycophenolate mofetil. Adjuvant therapies such as Rituximab nor intensification of plasma therapy were employed. Consequently, careful consideration needs to be given to the use of additional immunosuppression in certain cases of CFH-autoantibody associated aHUS. Serial measurement of CFH-autoantibodies is required in the immediate pre- and posttransplantation period to further clarify their role as a factor in the recurrence of aHUS posttransplantation. Furthermore, delineation of the functional significance of CFH-autoantibodies is warranted in individual cases.

Characterization of complement factor H-related (CFHR) proteins in plasma reveals novel genetic variations of CFHR1 associated with atypical hemolytic uremic syndrome.

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.

Atypical hemolytic uremic syndrome associated with complement factor H autoantibodies and CFHR1/CFHR3 deficiency.

Although genetic defect of complement factor H (CFH) is a common cause of atypical hemolytic uremic syndrome (aHUS), development of autoantibodies to CFH (CFH-Ab) is also known to be an acquired cause of aHUS. Recently, a correlation between the development of CFH-Ab and the deficiency of the CFH-related proteins, CFHR1 and CFHR3, was identified. In this study, plasma complement profiles were measured and genetic analysis of the CFH, CFI, MCP, CFHR1, and CFHR3 genes were performed in three female patients diagnosed with aHUS with positive CFH-Ab. Acute stage plasmas of all the three patients revealed low C3, low or low-normal CFH antigenic levels, and high titers of CFH-Ab. All the patients also showed complete plasma CFHR1 deficiency and homozygous genomic deletion of CFHR1/CFHR3, but none had CFH, CFI, or MCP mutations. All the patients were treated with plasmapheresis, and two patients required additional immunosuppressive therapy. These patients had a novel subgroup of aHUS characterized by a combination of genetic (a homozygous deletion of CFHR1/CFHR3) and acquired (development of CFH-Ab) factors. Patients with this disease may need intensive immunosuppressive therapy in addition to plasmapheresis. Screening for CFH-Ab and the CFHR1/CFHR3 deficiency should be included in the diagnostic tests for patients with aHUS.

Autoantibodies in haemolytic uraemic syndrome (HUS).

Haemolytic uraemic syndrome (HUS) is a severe disease with renal failure, microangiopathic anemia and thrombocytopenia. Several mechanisms leading to HUS have been identified, like infections with enterohaemorrhagic Escherichia coli, as well as genetic mutations of complement genes, which result in defective complement control on the surface of host cells. The complement system forms the first defense line of innate immunity and mediates the attack against foreign microorganisms. Defective regulation of this cascade results in attack of self cells and in autoimmune disease. Apparently, the alternative pathway convertase C3bBb is central for the pathophysiology of HUS as gene mutations of the components (C3 and Factor B) or of regulators (Factor H, Factor I and MCP/CD46) are observed in the genetic form of HUS. Recently, a novel mechanism leading to atypical HUS (aHUS) was identified, in form of autoantibodies that bind the complement inhibitor Factor H. Here we summarize the current concept of HUS and focus in particular on the novel subgroup of aHUS patients with IgG autoantibodies to Factor H which develop on the genetic background of CFHR1/CFHR3 deficiency, and which define a new subform termed DEAP-HUS (deficient for CFHR proteins and Factor H autoantibody positive).

Factor H autoantibodies in atypical hemolytic uremic syndrome correlate with CFHR1/CFHR3 deficiency.

Atypical hemolytic uremic syndrome (aHUS) is a severe renal disease that is associated with defective complement regulation caused by multiple factors. We previously described the deficiency of factor H-related proteins CFHR1 and CFHR3 as predisposing factor for aHUS. Here we identify in an extended cohort of 147 aHUS patients that 16 juvenile individuals (ie, 11%) who either lacked the CFHR1/CFHR3 completely (n = 14) or showed extremely low CFHR1/CFHR3 plasma levels (n = 2) are positive for factor H (CFH) autoantibodies. The binding epitopes of all 16 analyzed autoantibodies were localized to the C-terminal recognition region of factor H, which represents a hot spot for aHUS mutations. Thus we define a novel subgroup of aHUS, termed DEAP HUS (deficiency of CFHR proteins and CFH autoantibody positive) that is characterized by a combination of genetic and acquired factors. Screening for both factors is obviously relevant for HUS patients as reduction of CFH autoantibody levels represents a therapeutic option.

Deletion of complement factor H-related genes CFHR1 and CFHR3 is associated with atypical hemolytic uremic syndrome.

Atypical hemolytic uremic syndrome (aHUS) is associated with defective complement regulation. Disease-associated mutations have been described in the genes encoding the complement regulators complement factor H, membrane cofactor protein, factor B, and factor I. In this study, we show in two independent cohorts of aHUS patients that deletion of two closely related genes, complement factor H-related 1 (CFHR1) and complement factor H-related 3 (CFHR3), increases the risk of aHUS. Amplification analysis and sequencing of genomic DNA of three affected individuals revealed a chromosomal deletion of approximately 84 kb in the RCA gene cluster, resulting in loss of the genes coding for CFHR1 and CFHR3, but leaving the genomic structure of factor H intact. The CFHR1 and CFHR3 genes are flanked by long homologous repeats with long interspersed nuclear elements (retrotransposons) and we suggest that nonallelic homologous recombination between these repeats results in the loss of the two genes. Impaired protection of erythrocytes from complement activation is observed in the serum of aHUS patients deficient in CFHR1 and CFHR3, thus suggesting a regulatory role for CFHR1 and CFHR3 in complement activation. The identification of CFHR1/CFHR3 deficiency in aHUS patients may lead to the design of new diagnostic approaches, such as enhanced testing for these genes.

Role for specific complement phenotypes and deficiencies in the clinical expression of IgA nephropathy.

IgA nephropathy, the most commonly occurring type of chronic glomerulonephritis in individuals of European and Asian descent, exhibits marked heterogeneity of clinical signs and ultimate prognosis. Based upon their studies of regional clustering of the ancestors of related patients in eastern Kentucky, the authors have postulated the existence of an inherited disease susceptibility for IgA nephropathy. They examined serum concentrations of individual complement proteins and phenotypes for C3, C4A, C4B, and factor B (Bf) for related and unrelated patients with IgA nephropathy from Kentucky and for patients from the Mid-South region of Tennessee, Mississippi, and Alabama. In these populations, they have described partial complement deficiencies or specific phenotypes which may be associated with the disease. Their findings include the following: (1) partial deficiencies for C2, beta 1H (H), properdin (P), or C4 binding protein (C4BP) in four patients with end-stage renal disease, (2) an association between the C3*F allele with IgA nephropathy in the combined group of unrelated patients from Kentucky and the Mid-South, (3) the occurrence of C4B deficiency in two siblings with IgA nephropathy, and (4) an association between C4A deficiency and poor outcome in patients with IgA nephropathy diagnosed as adults. In addition, the related patients differ from the unrelated patients from Kentucky with respect to frequencies of Bf*F and the BfF (FF + FS + F1F + F1S) phenotype, suggestive of immunogenetic difference between these groups. Important functional differences exist between C4A and C4B isotypes and functional differences are also possible based upon C3 or Bf phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)

Inherited complement deficiency states: implications for immunity and immunological disease.

The study of complement deficiency states and their influence on immune function has generated new insights and still provides a challenge to continued investigation. The association of classical pathway deficiencies (C1, C4, C2 or C3) with immunological diseases such as SLE and glomerulonephritis has contributed to current knowledge concerning complement-dependent immune complex handling and elimination. Susceptibility to systemic infection with encapsulated bacteria is encountered in most forms of inherited complement deficiency. Recurrent neisserial infection is the only clinical manifestation clearly associated with defects of the membranolytic sequence C5-C9, while deficiency of properdin, a component of the alternative activation pathway, appears to predispose to nonrecurrent meningococcal disease. Inherited complement deficiency is rare, but the perspective is widened by the more common occurence of acquired defects in immunological diseases, and the apparent requirement for efficient complement recruitment in host defense. Another aspect is the possibility that complement deficiency might alleviate or prevent inflammatory symptoms. Notably, complement deficiency has not been reported in classical rheumatoid arthritis. Considerations of this kind would be refuted or modified by findings of complement deficiency in single patients.

Metabolism of C3 and factor B in patients with congenital factor I deficiency.

The metabolism of complement factor B and C3 was analyzed in three previously described patients with congenital deficiency of factor I (C3b/C4b inactivator). Samples taken at steady state contained elevated levels of Ba and Bb, whereas native factor B was not detectable. Following plasma infusion in two of the patients Ba was rapidly cleared (within 8-12 hr) from the circulation and native factor B increased transiently, reaching normal levels within 22-24 hr. In parallel with the increase in Ba, factor B decreased during the following days, reaching preinfusion level after seven days. This was in contrast to a continued increase in C3 concentration, which was still within the normal range 15 days after infusion, despite the presence of only trace amounts of native B. In vitro complement activation experiments, employing purified C3Nef IgG as alternative pathway activator and aggregated IgG as classical pathway activator, were performed on selected serum samples (base-line, 12 hr and 15 days postinfusion samples) from the plasma infusion series. It was demonstrated that (a) C3Nef could not induce alternative pathway C3-conversion in factor B depleted samples and (b) C3-degradation by CP-activation could still occur in B-depleted samples, although at a much slower rate compared to normal human serum. These results may partly explain the difference in kinetics of factor B and C3 metabolism seen after plasma infusion in patients with factor I deficiency.(ABSTRACT TRUNCATED AT 250 WORDS)

Hereditary, complete deficiency of complement factor H associated with recurrent meningococcal disease.

Complement factor H (beta-1H globulin) is an important regulatory protein which inhibits the spontaneous complement activation via the alternative pathway. We describe a 15-year-old girl without any detectable factor H in plasma. She has had two episodes of meningococcal disease, but is otherwise completely healthy. Secondary to the factor-H deficiency, the levels of factor B, properdin, C3, and C5-C9 were strongly reduced due to spontaneous in vivo activation of the alternative complement pathway. Plasma C3dg was strongly elevated in spite of the factor-H deficiency; apparently erythrocyte CR1 substitutes for factor H in C3 degradation. Neither C3 nor complement lesions were demonstrable on her erythrocytes which did, however, show increased, spontaneous haemolysis in vitro in citrate plasma, but not in serum. The patient is a single child and her parents, who are unrelated and healthy, had half-normal levels of factor H. This reduction of factor H is sufficient to cause increased, spontaneous activation of the alternative pathway.

Combined homozygous factor H and heterozygous C2 deficiency in an Italian family.

Three of four children in a family have homozygous (less than 1% of normal) deficiency of factor H of the complement system and both parents, who are first cousins, are heterozygous for the same defect. The father and two of the H-deficient siblings also have a partial C2 deficiency. One of the children with combined deficiencies is affected by systemic lupus erythematosus with nephritis. No increased susceptibility to infections has been observed in the family. H deficiency is inherited in an autosomal codominant manner and is independently transmitted from C2 deficiency and HLA haplotypes. In the homozygous state it is associated with very low serum concentrations of B and C3, barely demonstrable as activated molecules. C5 is greatly reduced (less than 5%). Also, properdin and C6-9 are decreased. The findings in this family demonstrate that the occurrence of systemic lupus erythematosus in one of the children affected by a combined deficiency of factor H and C2 raises the question whether this pathology is related to the complete factor H or to the heterozygous C2 deficiency. Complete H deficiency is not necessarily accompanied by overt illness.