Factor H Deficiency Research Articles

Proof of concept of a new plasma complement Factor H from waste plasma fraction.

Complement factor H (FH) is a major regulator of the complement alternative pathway, its mutations predispose to an uncontrolled activation in the kidney and on blood cells and to secondary C3 deficiency. Plasma exchange has been used to correct for FH deficiency and although the therapeutic potential of purified FH has been suggested by in vivo experiments in animal models, a clinical approved FH concentrate is not yet available. We aimed to develop a purification process of FH from a waste fraction rather than whole plasma allowing a more efficient and ethical use of blood and plasma donations. Waste fractions from industrial plasma fractionation (pooled human plasma) were analyzed for FH content by ELISA. FH was purified from unused fraction III and its decay acceleration, cofactor, and C3 binding capacity were characterized in vitro. Biodistribution was assessed by high-resolution dynamic PET imaging. Finally, the efficacy of the purified FH preparation was tested in the mouse model of C3 glomerulopathy (Cfh-/- mice). Our purification method resulted in a high yield of highly purified (92,07%), pathogen-safe FH. FH concentrate is intact and fully functional as demonstrated by in vitro functional assays. The biodistribution revealed lower renal and liver clearance of human FH in Cfh-/- mice than in wt mice. Treatment of Cfh-/- mice documented its efficacy in limiting C3 activation and promoting the clearance of C3 glomerular deposits. We developed an efficient and economical system for purifying intact and functional FH, starting from waste material of industrial plasma fractionation. The FH concentrate could therefore constitute possible treatments options of patients with C3 glomerulopathy, particularly for those with FH deficiency, but also for patients with other diseases associated with alternative pathway activation.

A rare association between factor H deficiency and lupus: Case report and experimental treatment with curcumin

Factor H (FH) is one of the most important regulatory proteins of the alternative pathway of the complement system. FH deficiency is a rare condition that causes unregulated C3 consumption, leading to an increased susceptibility to infections and glomerulopathies. Our previous studies have demonstrated a FH deficient patient carrying a c.452G > A, p.R127H FH mutation which leads to a misfolded protein and its retention in the endoplasmic reticulum. In his cultured fibroblasts, FH-delayed secretion was partially rescued when treated with curcumin, and once secreted, exhibited normal regulatory function. Here, we report a childhood-onset systemic lupus erythematosus (cSLE) in this FH deficient patient and the results of experimental treatment with curcumin aiming to rescue FH secretion and regulatory activity.

Local complement factor H protects kidney endothelial cell structure and function

Factor H (FH) is a critical regulator of the alternative complement pathway and its deficiency or mutation underlie kidney diseases such as dense deposit disease. Since vascular dysfunction is an important facet of kidney disease, maintaining optimal function of the lining endothelial cells is important for vascular health. To investigate the molecular mechanisms that are regulated by FH in endothelial cells, FH deficient and sufficient mouse kidney endothelial cell cultures were established. Endothelial FH deficiency resulted in cytoskeletal remodeling, increased angiogenic potential, loss of cellular layer integrity and increased cell proliferation. FH reconstitution prevented these FH-dependent proliferative changes. Respiratory flux analysis showed reduced basal mitochondrial respiration, ATP production and maximal respiratory capacity in FH deficient endothelial cells, while proton leak remained unaltered. Similar changes were observed in FH deficient human glomerular endothelial cells indicating the translational potential of these studies. Gene expression analysis revealed that the FH-dependent gene changes in mouse kidney endothelial cells include significant upregulation of genes involved in inflammation and the complement system. The transcription factor nuclear factor-kB, that regulates many biological processes, was translocated from the cytoplasm to the nucleus in the absence of FH. Thus, our studies show the functional relevance of intrinsic FH in kidney endothelial cells in man and mouse.

Absence of complement factor H reduces physical performance in C57BL6 mice

Complement (C) system is a double edge sword acting as the first line of defense on the one hand and causing aggravation of disease on the other. C activation when unregulated affects different organs including muscle regeneration. However, the effect of factor H (FH), a critical regulator of the alternative C pathway in muscle remains to be studied. FH deficiency results in excessive C activation and generates proinflammatory fragments C5a and C3a as byproducts. C3a and C5a signal through their respective receptors, C5aR and C3aR. In this study, we investigated the role of FH and downstream C5a/C5aR signaling in muscle architecture and function. Using the FH knockout (fh-/-) and fh-/-/C5aR-/double knockout mice we explored the role of C, specifically the alternative C pathway in muscle dysfunction. Substantial C3 and C9 deposits occur along the walls of the fh-/- muscle fibers indicative of unrestricted C activation. Physical performance assessments of the fh-/- mice show reduced grip endurance (76 %), grip strength (14 %) and rotarod balance (36 %) compared to controls. Histological analysis revealed a shift in muscle fiber populations indicated by an increase in glycolytic MHC IIB fibers and reduction in oxidative MHC IIA fibers. Consistent with this finding, mitochondrial DNA (mtDNA) and citrate synthase (CS) expression were both reduced indicating possible reduction in mitochondrial biomass. In addition, our results showed a significant increase in TGFβ expression and altered TGFβ localization in this setting. The architecture of cytoskeletal proteins actin and vimentin in the fh-/- muscle was changed that could lead to contractile weakness and loss of skeletal muscle elasticity. The muscle pathology in fh-/- mice was reduced in fh-/-/C5aR-/- double knockout (DKO) mice, highlighting partial C5aR dependence. Our results for the first time demonstrate an important role of FH in physical performance and skeletal muscle health.

Dengue virus and the complement alternative pathway.

Dengue disease is an inflammatory-driven pathology, and complement overactivation is linked to disease severity and vascular leakage. Additionally, dysregulation of complement alternative pathway (AP) components has been described, such as upregulation of complement factor D and downregulation of complement factor H (FH), which activate and inhibit the AP, respectively. Thus, the pathology of severe dengue could in part result from AP dysfunction, even though complement and AP activation usually provide protection against viral infections. In dengue virus-infected macrophages and endothelial cells (ECs), the site of replication and target for vascular pathology, respectively, the AP is activated. The AP activation, reduced FH and vascular leakage seen in dengue disease in part parallels other complement AP pathologies associated with FH deficiency, such as atypical haemolytic uraemic syndrome (aHUS). aHUS can be therapeutically targeted with inhibitors of complement terminal activity, raising the idea that strategies such as inhibition of complement or delivery of FH or other complement regulatory components to EC may be beneficial to combat the vascular leakage seen in severe dengue.

Molecular Basis of Factor H R1210C Association with Ocular and Renal Diseases.

The complement factor H (FH) mutation R1210C, which was described in association with atypical hemolytic uremic syndrome (aHUS), also confers high risk of age-related macular degeneration (AMD) and associates with C3 glomerulopathy (C3G). To reveal the molecular basis of these associations and to provide insight into what determines the disease phenotype in FH-R1210C carriers, we identified FH-R1210C carriers in our aHUS, C3G, and AMD cohorts. Disease status, determined in patients and relatives, revealed an absence of AMD phenotypes in the aHUS cohort and, vice versa, a lack of renal disease in the AMD cohort. These findings were consistent with differences in the R1210C-independent overall risk for aHUS and AMD between mutation carriers developing one pathology or the other. R1210C is an unusual mutation that generates covalent complexes between FH and HSA. Using purified FH proteins and surface plasmon resonance analyses, we demonstrated that formation of these FH-HSA complexes impairs accessibility to all FH functional domains. These data suggest that R1210C is a unique C-terminal FH mutation that behaves as a partial FH deficiency, predisposing individuals to diverse pathologies with distinct underlying pathogenic mechanisms; the final disease outcome is then determined by R1210C-independent genetic risk factors.

Partial Complement Factor H Deficiency Associates with C3 Glomerulopathy and Thrombotic Microangiopathy.

The complement-mediated renal diseases C3 glomerulopathy (C3G) and atypical hemolytic uremic syndrome (aHUS) strongly associate with inherited and acquired abnormalities in the regulation of the complement alternative pathway (AP). The major negative regulator of the AP is the plasma protein complement factor H (FH). Abnormalities in FH result in uncontrolled activation of C3 through the AP and associate with susceptibility to both C3G and aHUS. Although previously developed FH-deficient animal models have provided important insights into the mechanisms underlying susceptibility to these unique phenotypes, these models do not entirely reproduce the clinical observations. FH is predominantly synthesized in the liver. We generated mice with hepatocyte-specific FH deficiency and showed that these animals have reduced plasma FH levels with secondary reduction in plasma C3. Unlike mice with complete FH deficiency, hepatocyte-specific FH-deficient animals developed neither plasma C5 depletion nor accumulation of C3 along the glomerular basement membrane. In contrast, subtotal FH deficiency associated with mesangial C3 accumulation consistent with C3G. Although there was no evidence of spontaneous thrombotic microangiopathy, the hepatocyte-specific FH-deficient animals developed severe C5-dependent thrombotic microangiopathy after induction of complement activation within the kidney by accelerated serum nephrotoxic nephritis. Taken together, our data indicate that subtotal FH deficiency can give rise to either spontaneous C3G or aHUS after a complement-activating trigger within the kidney and that the latter is C5 dependent.

C3 dysregulation due to factor H deficiency is mannan-binding lectin-associated serine proteases (MASP)-1 and MASP-3 independentin vivo

Uncontrolled activation of the complement alternative pathway is associated with complement-mediated renal disease. Factor B and factor D are essential components of this pathway, while factor H (FH) is its major regulator. In complete FH deficiency, uncontrolled C3 activation through the alternative pathway results in plasma C3 depletion and complement-mediated renal disease. These are dependent on factor B. Mannan-binding lectin-associated serine proteases 1 and 3 (MASP-1, MASP-3) have been shown recently to contribute to alternative pathway activation by cleaving pro-factor D to its active form, factor D. We studied the contribution of MASP-1 and MASP-3 to uncontrolled alternative pathway activation in experimental complete FH deficiency. Co-deficiency of FH and MASP-1/MASP-3 did not ameliorate either the plasma C3 activation or glomerular C3 accumulation in FH-deficient mice. Our data indicate that MASP-1 and MASP-3 are not essential for alternative pathway activation in complete FH deficiency.

Atypical hemolytic uremic syndrome and mutation analysis of factor H gene in two Tunisian families

We carried out a protein and genetic investigation of the factor H gene mutations within two families presenting with a diagnostic suspicion of atypical hemolytic uremic syndrome (aHUS). The results within the patients of the first family revealed a factor H-deficiency. Direct sequencing allowed the detection of a 4-nucleotide deletion in the factor H gene. This deletion was found as the homozygote form in the proband and as the heterozygote form in the parents. Protein and functional analyses of the complement system were normal in all members of the second family. However, the molecular investigation for the father showed the presence of an amino acid substitution in the FH gene. Unfortunately, his two affected children died without being investigated for mutations. The functional consequences of these abnormal proteins are still to be demonstrated.

Complete factor H deficiency-associated atypical hemolytic uremic syndrome in a neonate

Recent advances have shown that atypical hemolytic uremic syndrome (aHUS) is a disease of complement dysregulation. Almost 50% of cases are associated with mutations in the three complement regulatory genes, factor H (HF1), membrane co-factor protein (MCP) and factor I (IF). The corresponding gene products act in concert and affect the same enzyme, alternative pathway convertase C3bBb, which initiates the alternative pathway and amplification of the complement system. Factor H (FH) deficiency-associated aHUS usually occurs in infants to middle-aged adults and only rarely in neonates. Moreover, the vast majority of patients are heterozygous for the HF1 gene mutations. We report on a case of neonatal-onset aHUS associated with complete FH deficiency due to novel compound heterozygous mutations in the HF1 gene. A 22-day-old baby girl developed acute renal failure and a remarkably low serum complement C3 level, which was rapidly followed by the development of micro-angiopathic hemolytic anemia. Western blot analysis revealed nearly zero plasma FH levels, and an HF1 gene study showed compound heterozygous mutations, C1077W/Q1139X. Renal pathology findings were compatible with glomerular involvement in HUS. The baby recovered completely after the repetitive infusion of fresh frozen plasma. During follow-up (until she was 20 months old) after the initial plasma therapy, the disease recurred three times; twice after the tapering off of plasma therapy, and once during a weekly plasma infusion. All recurrence episodes were preceded by an upper respiratory tract infection, and were successfully managed by restarting or increasing the frequency of plasma therapy.

The Factor H Variant Associated with Age-related Macular Degeneration (His-384) and the Non-disease-associated Form Bind Differentially to C-reactive Protein, Fibromodulin, DNA, and Necrotic Cells

Recently, a polymorphism in the complement regulator factor H (FH) gene has been associated with age-related macular degeneration. When histidine instead of tyrosine is present at position 384 in the seventh complement control protein (CCP) domain of FH, the risk for age-related macular degeneration is increased. It was recently shown that these allotypic variants of FH, in the context of a recombinant construct corresponding to CCPs 6-8, recognize polyanionic structures differently, which may lead to altered regulation of the alternative pathway of complement. We show now that His-384, corresponding to the risk allele, binds C-reactive protein (CRP) poorly compared with the Tyr-384 form. We also found that C1q and phosphorylcholine do not compete with FH for binding to C-reactive protein. The interaction with extracellular matrix protein fibromodulin, which we now show to be mediated, at least in part, by CCP6-8 of FH, occurs via the polypeptide of fibromodulin and not through its glycosaminoglycan modifications. The Tyr-384 variant of FH bound fibromodulin better than the His-384 form. Furthermore, we find that CCP6-8 is able to interact with DNA and necrotic cells, but in contrast the His-384 allotype binds these ligands more strongly than the Tyr-384 variant. The variations in binding affinity of the two alleles indicate that complement activation and local inflammation in response to different targets will differ between His/His and Tyr/Tyr homozygotes.

Two generations with familial thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) is a rare multi-system disease characterised by the pentad of microangiopathic haemolytic anaemia, thrombocytopenia, renal dysfunction, fever and neurologic changes. A hereditary form of recurrent familial TTP has been described, which usually presents in adolescence or early adulthood and can lead to recurrent or chronic relapsing TTP. Genetic analyses of patients with familial TTP have linked the disease to chromosome 9q34, and an increased incidence is seen in people with HLA-B40 group antigens. We describe here an 11-year-old Egyptian girl with no significant past medical history who presented with new onset of bruising, petechial rash, fatigue and fevers and was diagnosed with familial TTP. Further testing revealed that both the patient and her father had the HLA-B40 group antigen and also had ADAMTS-13 von Willebrand factor-cleaving protease deficiency as well as factor-H deficiency.

Comparison of surface recognition and C3b binding properties of mouse and human complement factor H

Factor H (FH) is a central complement regulator both in plasma and on certain cellular and acellular surfaces that are in contact with plasma. Although FH deficiency has been shown to lead to similar diseases in man and mice (membranoproliferative glomerulonephritis or dense deposit disease) little is known about the similarity between the human and murine FH functions. We here characterize the interactions of murine FH (mFH) with C3b, glycosaminoglycans, and endothelial cells and compare these interactions with those of human FH (hFH). To achieve this we purified mFH and murine C3 from plasma, prepared murine C3b, and expressed recombinant mFH constructs containing domains 1–5 and 18–20 (mFH1–5 and mFH18–20). For comparisons, hFH, human C3b, and recombinant hFH1–5 and hFH18–20 were used. We demonstrate that mFH and mFH1–5 do act as cofactors for factor I-mediated cleavage of human C3b. Surface plasmon resonance analysis showed binding of mFH18–20 to murine C3b and weak binding to human C3b. The mFH18–20 construct bound to heparin in a manner comparable to hFH18–20. It was demonstrated by flow cytometry that mFH and mFH18–20 bind to human endothelial cells in a similar manner to hFH and hFH18–20. Taken together, locations of the key functions of mFH, i.e. complement regulation and surface recognition, are comparable to hFH. Recently, mutations in the carboxy-terminal end of hFH have been found to be associated with atypical hemolytic uremic syndrome (aHUS). Based on the results in this report it is conceptually attractive to establish a murine model for aHUS.

Anti–Factor H Autoantibodies Associated with Atypical Hemolytic Uremic Syndrome

Several studies have demonstrated genetic predisposition in non-shigatoxin-associated hemolytic uremic syndrome (HUS), involving regulatory proteins of the complement alternative pathway: Factor H (FH) and membrane co-factor protein (CD46). Regarding the observations of thrombotic thrombocytopenic purpura patients, in whom a von Willebrand factor protease (ADAMST-13) deficiency may be inherited or acquired secondary to IgG antibodies, it was speculated that HUS might occur in a context of an autoimmune disease with the development of anti-FH antibodies leading to an acquired FH deficiency. The presence of FH autoantibodies was investigated by an ELISA method using coated purified human FH in a series of 48 children who presented with atypical HUS and were recruited from French university hospitals. Anti-FH IgG antibodies were detected in the plasma of three children who presented with recurrent HUS. The anti-FH specificity was conserved by the Fab'2 fraction. The plasma FH activity was found to be decreased, whereas plasma FH antigenic levels and FH gene analysis were normal, indicating that the presence of anti-FH antibodies led to an acquired functional FH deficiency. This report supports for the first time that HUS may occur in a context of an autoimmune disease with the development of anti-FH-specific antibody leading to an acquired FH deficiency. This new mechanism of functional FH deficiency may lead to the design of new approaches of diagnosis and treatment with a particular interest in plasma exchanges or immunosuppressive therapies.

Familial hemolytic uremic syndrome associated with complement factor H deficiency

Atypical hemolytic uremic syndrome (HUS) associated with factor H deficiency (FHD) carries a poor prognosis. A 3-year-old girl with FHD-HUS reached end-stage renal disease at age 6 months after experiencing numerous relapses; she underwent a cadaveric renal transplant at age 46 months. One month after transplantation, she experienced an extensive non-hemorrhagic cerebral infarction. Later, hematologic and renal manifestations of HUS developed, followed by another massive cerebral infarction and death in spite of multiple plasma transfusions. A 14-month-old boy with FHD-HUS experienced numerous HUS episodes starting at the age of 2 weeks. Daily plasma transfusions during relapses brought about only a temporary state of remission. However, prophylactic twice-weekly plasma therapy has been successful in preventing relapses and preserving renal function. With this regimen, serum factor H was increased from 6 mg/dL to subnormal values of 12 to 25 mg/dL (normal >60 mg/dL). We conclude that FHD-HUS recurs because FHD is not corrected by renal transplantation. A hypertransfusion protocol may prevent FHD-HUS. (J Pediatr 2001;138:412-7)

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.