Complement Factor H-related Protein Research Articles

Deficiency of complement factor H-related proteins and autoantibody-positive hemolytic uremic syndrome in an infant with combined partial deficiencies and autoantibodies to complement factor H and ADAMTS13.

ABSTRACTA 3-month-old male infant developed an extremely severe episode of atypical hemolytic uremic syndrome (aHUS) associated with partial deficiencies of full-length complement factor H (FH; ∼15% of infant normal) and a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) (39% of normal) and autoantibodies reactive with both proteins. His FH and ADAMTS13 genes were normal, indicating that the partial deficiencies were acquired, probably as the result of autoantibodies against full-length FH and ADAMTS13. The child also had a homozygous deletion of the complement factor H–related (CFHR)3–CFHR1 portion in the complement factor H (CFH) gene cluster. He therefore had deficiency of CFHR proteins and autoantibody-positive hemolytic uremic syndrome (DEAP-HUS) with an unusual early onset associated with a partial deficiency of ADAMTS13 and an anti-ADAMTS13 autoantibody. His clinical episode of aHUS responded to plasma infusion and subsequent treatment with mycophenolate and rituximab. We believe that this is the first report of DEAP-HUS in an infant with partial deficiencies in both ADAMTS13 and full-length FH acquired in association with autoantibodies to both proteins.

Proteome and Metabolome of Subretinal Fluid in Central Serous Chorioretinopathy and Rhegmatogenous Retinal Detachment: A Pilot Case Study.

PurposeTo investigate the molecular composition of subretinal fluid (SRF) in central serous chorioretinopathy (CSCR) and rhegmatogenous retinal detachment (RRD) using proteomics and metabolomics.MethodsSRF was obtained from one patient with severe nonresolving bullous CSCR requiring surgical subretinal fibrin removal, and two patients with long-standing RRD. Proteins were trypsin-digested, labeled with Tandem-Mass-Tag and fractionated according to their isoelectric point for identification and quantification by tandem mass spectrometry. Independently, metabolites were extracted on cold methanol/ethanol, and identified by untargeted ultra-high performance liquid chromatography and high-resolution mass spectrometry. Bioinformatics analyses were conducted.ResultsIn total, 291 proteins and 651 metabolites were identified in SRF samples. Compared with RRD, 128 proteins (77 downregulated; 51 upregulated) and 76 metabolites (43 downregulated; 33 upregulated) differed in the SRF from CSCR. Protein and metabolites notably deregulated in CSCR were related to glycolysis/gluconeogenesis, inflammation (including serum amyloid P component, versican), alternative complement pathway (complement factor H and complement factor H–related protein), cellular adhesion, biliary acid metabolism (farnesoid X receptor/retinoid X receptor), and gluco- and mineralocorticoid systems (aldosterone, angiotensin, and corticosteroid-binding globulin).ConclusionsProteomics and metabolomics can be performed on SRF. A unique SRF sample from CSCR exhibited a distinct molecular profile compared with RRD.Translational RelevanceThis first comparative multiomics analysis of SRF improved the understanding of CSCR and RRD pathophysiology. It identified pathways potentially involved in the better photoreceptor preservation in CSCR, suggesting neuroprotective targets that will require additional confirmation.

Identification of candidate serum biomarkers of childhood-onset growth hormone deficiency using SWATH-MS and feature selection

A typical clinical manifestation of growth hormone deficiency (GHD) is a short stature resulting from delayed growth, but GHD affects bone health, cardiovascular function and metabolic profile and therefore quality of life. Although early GH treatment during childhood has been shown to improve outcomes, no single biochemical parameter is currently available for the accurate diagnosis of GHD in children. There is hence a need for non-invasive biomarkers. In this study, the relative abundance of serum proteins from GHD children and healthy controls was measured by next-generation proteomics SWATH-MS technology. The data generated was analysed by machine-learning feature-selection algorithms in order to discover the minimum number of protein biomarkers that best discriminate between both groups. The analysis of serum proteins by a SWATH-MS approach yielded a useful method for discovering potential biomarkers of GHD in children. A total of 263 proteins were confidently detected and quantified in each sample. Pathway analysis indicated an effect on tissue/organ structure and morphogenesis. The top ten serum protein biomarker candidates were identified after applying feature-selection data analysis. The combination of three proteins – apolipoprotein A-IV, complement factor H-related protein 4 and platelet basic protein – demonstrated the best classification performance for our data. In addition, the apolipoprotein group resulted in strong over-representation, thus highlighting these proteins as an additional promising biomarker panel. SignificanceCurrently there is no single biochemical parameter available for the accurate diagnosis of growth hormone (GH) deficiency (GHD) in children. Simple GH measurements are not an option: because GH is released in a pulsatile action, its blood levels fluctuate throughout the day and remain nearly undetectable for most of that time. This makes measurements of GH in a single blood sample useless for assessing GH deficiency. Actually, the diagnosis of GHD includes a combination of direct and indirect non-accurate measurements, such as taking several body measurements, testing GH levels in multiple blood samples after provocative tests (GH peak <7.3ng/mL, using radioimmunoassay), and conducting magnetic resonance imaging (MRI), among others. Therefore, there is a need for simple, non-invasive, accurate and cost-effective biomarkers. Here we report a case-control study, where relative abundance of serum proteins were measured by next-generation proteomics SWATH-MS technology in 15 GHD children and 15healthy controls matched by age, sex, and not receiving any treatment. Data generated was analysed by machine learning feature selection algorithms. 263 proteins could be confidently detected and quantified on each sample. The top 10 serum protein biomarker candidates could be identified after applying a feature selection data analysis. The combination of three proteins, apolipoprotein A-IV, complement factor H-related protein 4 and platelet basic protein, showed the best classification performance for our data. In addition, the fact that the pathway and GO analysis we performed pointed to the apolipoproteins as over-represented highlights this protein group as an additional promising biomarker panel for the diagnosis of GHD and for treatment evaluation.

Factor H-Related (FHR)-1 and FHR-2 Form Homo- and Heterodimers, while FHR-5 Circulates Only As Homodimer in Human Plasma.

The complement factor H-related (FHR) proteins are hypothesized to fine-tune the regulatory role of complement factor H (FH) in the alternative pathway of the complement system. Moreover, FHR-1, FHR-2, and FHR-5 have been proposed to be dimers, which further complicates accurate analysis. As FHRs are highly similar among themselves and toward FH, obtaining specific reagents for quantification of serum levels and functional analysis is challenging. In this study, we generated antibodies and developed ELISAs to measure FHR-1, FHR-2, and FHR-5 in serum. We used both recombinant and serum-derived proteins to show that four dimers occur in human circulation: homodimers of FHR-1, FHR-2, and FHR-5, as well as FHR-1/FHR-2 heterodimers. Heterodimers containing FHR-5 were not found. In individuals with homozygous CFHR1 deletions or compound heterozygous CFHR2 missense/nonsense mutations identified in this study, the respective FHR-1 and FHR-2 homo- and heterodimers were absent. Using FRET, we found that recombinant FHR dimers exchange monomers rapidly. This was confirmed ex vivo, using FHR-1- and FHR-2-deficient sera. Of all FHR dimers, FHR-5/5 homodimers demonstrated strong binding affinity toward heparin. Specific ELISAs demonstrated that serum levels of FHR-1/1, FHR-1/2, FHR-2/2, and FHR-5/5 dimers were low compared to FH, which circulates at a 10- to 200-fold molar excess. In summary, FHR-1, FHR-2, and FHR-5 homodimerize, with FHR-1 and FHR-2 forming heterodimers as well, and equilibrate quickly in plasma.

Complement factor H-related (CFHR) proteins 2 and 5 as well as iC3b and properdin are deposited in different glomerular diseases

Complement factor H-related (CFHR) proteins 2 and 5 as well as iC3b and properdin are deposited in different glomerular diseases

Circulating complement factor H–related proteins 1 and 5 correlate with disease activity in IgA nephropathy

IgA nephropathy (IgAN) is a common cause of chronic kidney disease and end-stage renal failure, especially in young people. Due to a wide range of clinical outcomes and difficulty in predicting response to immunosuppression, we need to understand why and identify which patients with IgAN will develop progressive renal impairment. A deletion polymorphism affecting the genes encoding the complement factor H-related protein (FHR)-1 and FHR-3 is robustly associated with protection against IgAN. Some FHR proteins, including FHR-1 and FHR-5, antagonize the ability of complement factor H (fH), the major negative regulator of the complement alternative pathway, to inhibit complement activation on surfaces, a process termed fH deregulation. From a large cohort of patients, we demonstrated that plasma FHR-1 and the FHR-1/fH ratio were elevated in IgAN and associated with progressive disease. Plasma FHR-1 negatively correlated with eGFR but remained elevated in patients with IgAN with normal eGFR. Serum FHR5 was slightly elevated in IgAN but did not correlate with eGFR. Neither FHR5 levels nor the FHR-5/fH ratio was associated with progressive disease. However, higher serum FHR-5 levels were associated with a lack of response to immunosuppression, the presence of endocapillary hypercellularity, and histology scores of disease severity (the Oxford Classification MEST score). Thus, FHR-1 and FHR-5 have a role in IgAN disease progression.

Indications of underdiagnosis of atypical haemolytic uraemic syndrome in a cohort referred to the Coagulation Unit in Malmo, Sweden, for analysis of ADAMTS13 2007-2012.

Complement-mediated atypical haemolytic uraemic syndrome (aHUS) is a rare disease with high mortality and morbidity if left untreated. The diagnostic work-up is complicated and the manifestations overlap with other conditions. Therefore, we hypothesize that complement-mediated aHUS is an under diagnosed disease. A cohort of 768 referrals referred to the Coagulation Unit in Malmo, Sweden, for analysis of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), 2007-2012, were retrospectively reviewed. Subjects were included on the basis of presence of haemolytic anaemia, thrombocytopaenia, renal failure and ADAMTS13 > 0.05. They were excluded if tested positive for Escherichia coli. Included subjects were categorized as "suspected HUS" with and without potential causes and triggers. Levels of C3 and C4, presence of complement factor H (CFH)-specific antibodies and associated deficiency in complement factor H related protein 1 (CFHR1) were analyzed on frozen samples. In total, 134/316 (42%) unique subjects fulfilled inclusion criteria; 103 were categorized as "suspected HUS associated with potential causes/triggers" and 31 subjects categorized as "suspected HUS" without such association. One case of complement-mediated aHUS had been confirmed during the treatment period. Laboratory analyses performed showed that in total 78 cases had findings consistent with complement-mediated aHUS: 24 cases indicated presence of CFH-specific antibodies whereof five cases had isolated low C3 titres and six cases had deficiency of CFHR1. Additionally 54 cases indicated isolated alternative pathway consumption. The results suggest that the presence of complement-mediated aHUS was under diagnosed in this cohort calling for improvement of diagnostic availability.

Changes in complement factor H-related protein 2 in children with pulmonary arterial hypertension associated with congenital heart disease and its potential clinical significance

Objective To detect the changes and the clinical significance in plasma protein-complement factor H-related protein 2 (CFHR2) in pulmonary arterial hypertension (PAH) associated with congenital heart disease (CHD) children. Methods Various types of 66 CHD patients with or without PAH and 16 healthy children(healthy control group) were studied, including 11 ventricular septal defects(VSD) with PAH (VSD-PAH), 11 isolated VSD, 11 atrial septal defects with PAH (ASD-PAH), 11 isolated ASD, 11 mixed type of heart defects [two or more defects of VSD, ASD and patent ductus arteriosus (PDA)] with PAH (Mix-PAH) and 11 cases without PAH (Mix). CFHR2 was validated by enzyme linked immunosorbent assay in the sample plasma. Results Compared with the healthy control group, the CFHR2 concentration in VSD-PAH patients [(189.10±24.01) μg/L vs.(42.99±4.53) μg/L, t=4.975, P 0.05). The plasma CFHR2 level in Mix-PAH patients [(189.10±24.01) μg/L vs.(83.23±15.96) μg/L, t=3.314, P 0.05] had no difference compared with the healthy control group, but had statistical significance with Mix-PAH group (t=2.370, P<0.05). Conclusions The decrease of CFHR2 protein may demonstrate the deficiency of the immune system and coagulation mechanism in these patients and can be consi-dered as biomarker of CHD-PAH disease. Key words: Pulmonary arterial hypertension; Congenital heart disease; Complement factor H-related protein 2; Biomarker

Complement activation in pauci-immune necrotizing and crescentic glomerulonephritis: results of a proteomic analysis.

Complement activation plays an important role in the pathophysiology of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), although it remains unclear which pathway is activated. Whether pauci-immune necrotizing crescentic glomerulonephritis (pauci-immune GN) with negative ANCA serology is part of the spectrum of AAV or a different disease entity is essentially unknown. We used proteomic analysis to delineate the complement profile in a series of 13 kidney biopsies of patients with pauci-immune GN, with either proteinase 3 (PR3) (five patients) or myeloperoxidase (MPO) antibodies (four patients) or with consistently negative ANCA serology (four patients). Immunofluorescence staining of glomeruli was essentially negative in the PR3-ANCA and MPO-ANCA groups, while a mild staining for C3 was seen in the ANCA-negative cases. No electron-dense deposits were found in the PR3-ANCA and MPO-ANCA groups, but mesangial and few subepithelial deposits were clearly present in the ANCA-negative specimens. Mass spectrometry revealed low spectra numbers for C3 and immunoglobulins in both PR3-positive and MPO-positive patients with minimal or no C4 and C9. In contrast, larger spectra numbers for C3, moderate spectra numbers for C9, complement factor H-related protein-1 and low spectra numbers for C4, C5 and immunoglobulins were found in the ANCA-negative cases. While complement activation is noted in AAV, the complement activation appears to be more prominent in the ANCA-negative glomerulonephritis. The larger amount of C3 and moderate amount of C9 in the ANCA-negative glomerulonephritis implies activation of the alternate and terminal pathway of complement, suggesting that this entity may be caused or promoted by a genetic or acquired defect in the alternative pathway.

Eculizumab and Belatacept for De Novo Atypical Hemolytic Uremic Syndrome Associated With CFHR3-CFHR1 Deletion in a Kidney Transplant Recipient: A Case Report

BackgroundAtypical hemolytic uremic syndrome (aHUS) is associated with significant morbidity and mortality and occurs due to genetic or acquired abnormalities that result in the dysregulation of the alternative complement pathway. Case ReportWe report a case of post-living kidney transplantation de novo aHUS in a setting of heterozygous deletion in the complement factor H-related protein (CFHR)3-CFHR1 gene. The aHUS episode was possibly triggered by antibody-mediated rejection or tacrolimus. The patient responded well to eculizumab and substituting belatacept for tacrolimus. Her serum creatinine level was stable at 1.5 mg/dL after 2.5 years of follow-up. ConclusionThis case highlights the success of using a strategy that combines eculizumab and belatacept, as an alternative to calcineurin inhibitors, in treating aHUS in a patient with heterozygous deletion in the CFHR3-CFHR1 gene.

Does complement Factor H-Related protein 5 Nephropathy (Troodos Nephropathy) protect from rickettsial infections?

Complement Factor H-Related protein 5 Nephropathy (CFHR5N) is an endemic hereditary renal disease in the island of Cyprus. Although only very recently recognized, it has provided insight into previously unknown genetic aspects of complement-mediated renal diseases and in fact it has contributed to the introduction of the new disease classification, ‘C3 Glomerulopathy’. Herein, based on evidence from epidemiological, genetic, clinical and basic research studies, the hypothesis that CFHR5N could be protective from rickettsial infections is proposed. Confirming this hypothesis, could have significant implications for the study of Complement Factor- H Related Proteins (CFHRs) in renal diseases and rickettsial molecular microbiology.

Plasma Proteomic Study in Pulmonary Arterial Hypertension Associated with Congenital Heart Diseases

Pulmonary arterial hypertension associated with congenital heart disease (CHD-PAH) has serious consequence and plasma protein profiles in CHD-PAH are unknown. We aimed to reveal the differential plasma proteins in 272 CHD patients with or without PAH. Various types of CHD-PAH were studied. Differential plasma proteins were first detected by iTRAQ proteomic technology and those with significant clinical relevance were selected for further ELISA validation in new cohort of patients. Among the 190 differential plasma proteins detected by iTRAQ, carbamoyl-phosphate synthetase I (CPSI, related to urea cycle and endogenous nitric oxide production) and complement factor H-related protein 2 (CFHR2, related to complement system and coagulant mechanism) were selected for further ELISA validation in new cohort of 152 patients. Both CPSI and CFHR2 were down-regulated with decreased plasma levels (p < 0.01). Thus, we for the first time in CHD-PAH patients identified a large number of differential plasma proteins. The decreased CPSI expression in CHD-PAH patients may reveal a mechanism related to endogenous nitric oxide and the decrease of CFHR2 protein may demonstrate the deficiency of the immune system and coagulation mechanism. The findings may open a new direction for translational medicine in CHD-PAH with regard to the diagnosis and progress of the disease.

Mapping the Complement Factor H-Related Protein 1 (CFHR1):C3b/C3d Interactions.

Complement factor H-related protein 1 (CFHR1) is a complement regulator which has been reported to regulate complement by blocking C5 convertase activity and interfering with C5b surface association. CFHR1 also competes with complement factor H (CFH) for binding to C3b, and may act as an antagonist of CFH-directed regulation on cell surfaces. We have employed site-directed mutagenesis in conjunction with ELISA-based and functional assays to isolate the binding interaction that CFHR1 undertakes with complement components C3b and C3d to a single shared interface. The C3b/C3d:CFHR1 interface is identical to that which occurs between the two C-terminal domains (SCR19-20) of CFH and C3b. Moreover, we have been able to corroborate that dimerization of CFHR1 is necessary for this molecule to bind effectively to C3b and C3d, or compete with CFH. Finally, we have established that CFHR1 competes with complement factor H-like protein 1 (CFHL-1) for binding to C3b. CFHL-1 is a CFH gene splice variant, which is almost identical to the N-terminal 7 domains of CFH (SCR1-7). CFHR1, therefore, not only competes with the C-terminus of CFH for binding to C3b, but also sterically blocks the interaction that the N-terminus of CFH undertakes with C3b, and which is required for CFH-regulation.

The complement factor H-related proteins.

The role of the complement factor H-related (FHR) proteins in homeostasis, pathogen defense, and autoimmune disease has recently attracted considerable interest. We highlight the exciting research that has contributed to our understanding of the FHR protein family. Unlike factor H, a potent negative regulator of complement C3 activation, the FHR proteins appear to promote C3 activation. These data have important implications for understanding complement-mediated diseases because, depending on the context, the balance between the actions of factor H and the FHR proteins determines the degree of complement activation.

Generation of mice with deficiency of complement factor H and the complement factor H-related proteins

Generation of mice with deficiency of complement factor H and the complement factor H-related proteins

A novel, multiplexed targeted mass spectrometry assay for quantification of complement factor H (CFH) variants and CFH-related proteins 1–5 in human plasma

A novel, multiplexed targeted mass spectrometry assay for quantification of complement factor H (CFH) variants and CFH-related proteins 1–5 in human plasma

Dimerization of complement factor H-related (FHR) proteins: FHR-5 forms homodimers whereas FHR-1 and FHR-2 both homodimerize and heterodimerize with each other

Dimerization of complement factor H-related (FHR) proteins: FHR-5 forms homodimers whereas FHR-1 and FHR-2 both homodimerize and heterodimerize with each other

CFHR2-rs2986127 as a genetic protective marker for acute anterior uveitis in Chinese patients.

Complement factor H (CFH) related proteins (CFHRs) play important roles in complement activation pathways, whereas previous studies have only shown that CFH can affect the development of uveitis. In the present study, we investigated the potential associations between one of single-nucleotide polymorphisms in the CFHR2 gene with acute anterior uveitis (AAU). A total of 571 subjects, 283 patients diagnosed with AAU and 288 healthy adult controls, were recruited for this case-control study. CFHR2-rs2986127 was detected using Sequenom MassARRAY technology (Sequenom, San Diego, CA, USA). The stratified analyses for AAU patients with ankylosing spondylitis (AS) revealed a reduced frequency of the A allele in CFHR2-rs2986127 compared to controls (p = 0.033, odds ratio = 0.563, 95% confidence interval = 330-0.960). Further stratified analyses revealed a similar significantly reduced frequency in male AAU patients with AS compared to male controls (p = 0.036, odds ratio = 0.514, 95% confidence interval = 0.274-0.965) and in AAU patients without posterior segment involvement compared to controls (p = 0.048). The present study reveals an association between CFHR2-rs2986127 and AAU diagnosis, especially with respect to gender, AS status and other clinical signs, such as posterior segment involvement. Our results may further enrich the growing understanding of uveitis genetics, and raise the clinical diagnostic accuracy of this disease. Copyright © 2016 John Wiley & Sons, Ltd.

Proteomic profile of circulating immune complexes in chronic Chagas disease

Immune complexes (ICs) are the direct and real-time products of humoral immune responses. The identification of constituent foreign or autoantigens within ICs might bring new insights into the pathology of infectious diseases. We applied immune complexome analysis of plasma to the study of Chagas disease caused by Trypanosoma cruzi. Twenty seropositive plasma samples including cardiac and/or megacolon determinate patients (n = 11) and indeterminate (n = 9) were analysed along with 10 seronegative individuals to characterize the antigens bound to circulating ICs. We identified 39 T. cruzi antigens and 114 human autoantigens specific to patients with Chagas. Among those antigens, two T. cruzi antigens (surface protease GP63, glucose-6-isomerase) and six human autoantigens (CD180 antigen, ceruloplasmin, fibrinogen beta chain, fibrinogen beta chain isoform 2 preprotein, isoform gamma-A of fibrinogen γ-chain, serum paraoxonase) were detected in more than 50% of the patients tested. Human isoform short of complement factor H-related protein 2 and trans-sialidase of T. cruzi were more frequently found in the indeterminate (5/9 for both) compared with in the determinate Chagas (0/11, P = 0·046 for human, 1/11, P = 0·0498 for T. cruzi). The immune complexome could illustrate the difference of immune status between clinical forms of chronic Chagas disease.

Characterisation of a novel hybrid CFH/CFHR3 protein with impaired cell surface complement regulation in atypical haemolytic uraemic syndrome

Background60% of patients with atypical haemolytic uraemic syndrome have a rare genetic variant in C3, complement factor B (CFB), complement factor H (CFH), complement factor I (CFI), or CD46. Many of these variants are in the 3' cell recognition domain of CFH. The genes encoding CFH and five CFH-related proteins are found adjacently in the regulators of complement activation (RCA) cluster on chromosome 1q32. This cluster is a region of genomic instability that can lead to genomic disorders causing atypical haemolytic uraemic syndrome. MethodsUsing multiplex-ligation dependent probe amplification, we screened patients with atypical haemolytic uraemic syndrome who did not have a disease-causing genetic variant in C3, CFB, CFH, CFI, or CD46 for a genomic disorder. We used Sanger sequencing to define the breakpoint and identify a transcript for a hybrid CFH/CFHR3 gene. We purified the hybrid CFH/CFHR3 protein using allele specific monoclonal antibodies to the Y402H polymorphism of CFH. We tested its regulatory function using cell surface sheep erythrocyte lytic assays. FindingsWe found one patient who had evidence of a de-novo deletion in CFH. We identified a 6·3 kb deletion extending from exon 20 of CFH to the CFH 3' intergenetic region incorporating exons 21, 22, and 23. Directly flanking the breakpoint was a 7 base-pair region of microhomology. A putative hybrid gene was predicted to include exons 1 to 20 of CFH and exons 2 to 6 of CFHR3. We confirmed mRNA for a CFH/CFHR3 hybrid gene. Western blot analysis showed a hybrid CFH/CFHR3 protein at ∼160 kDa that lacked the cell recognition domain of CFH compared with 150 kDa for CFH. Peptide fragments from the larger band confirmed the CFH/CFHR3 hybrid protein. The purified CFH/CFHR3 hybrid protein showed less cell surface decay acceleration and cofactor activity compared with control. InterpretationScreening for genomic disorders in this patient identified a hybrid CFH/CFHR3 protein that lacked cell surface regulation of complement. This valuable method has led to insight into the disease mechanism of atypical haemolytic uraemic syndrome, thus aiding in patient stratification for future management and prognosis. That the deletion was a de-novo event suggests that it is a dynamic region of the genome and that we can expect to discover further genomic disorders in atypical haemolytic uraemic syndrome. FundingMedical Research Council, Wellcome Trust, EURenOmics.