RESUMO
Background: Factor H (FH) is a major soluble inhibitor of the complement system and part of a family comprising five related proteins (FHRs 1-5). Deficiency of FHR1 was described to be linked to an elevated risk of systemic lupus erythematosus (SLE). As FHR1 can partially antagonize the functionality of FH, an altered FHR1/FH ratio could not only enhance SLE vulnerability but also affect the disease expression. This study focuses on the analysis of FH and FHR1 at a protein level, and the occurrence of anti-FH autoantibodies (anti-FH) in a large cohort of SLE patients to explore their association with disease activity and/or expression. Methods: We assessed FH and FHR1 levels in plasma from 378 SLE patients compared to 84 healthy controls (normal human plasma, NHP), and sera from another cohort of 84 healthy individuals (normal human serum, NHS), using RayBio® CFH and CFHR1 ELISA kits. Patients were recruited by the Swiss SLE Cohort Study (SSCS). Unmeasurable FHR1 levels were all confirmed by Western blot, and in a subgroup of patients by PCR. Anti-FH were measured in SLE patients with non-detectable FHR1 levels and matched control patients using Abnova's CFH IgG ELISA kit. Results: Overall, FH and FHR1 levels were significantly higher in healthy controls, but there was no significant difference in FHR1/FH ratios between SLE patients and NHPs. However, SLE patients showed a significantly higher prevalence of undetectable FHR1 compared to all healthy controls (35/378 SLE patients versus 6/168 healthy controls; p= 0.0214, OR=2.751, 95% CI = 1.115 - 8.164), with a consistent trend across all ethnic subgroups. Levels of FH and FHR1, FHR1/FH ratios and absence of FHR1 were not consistently associated with disease activity and/or specific disease manifestations, but absence of FHR1 (primarily equivalent to CFHR1 deficiency) was linked to the presence of anti-FH in SLE patients (p=0.039). Conclusions: Deficiency of FHR1 is associated with a markedly elevated risk of developing SLE. A small proportion of FHR1-deficient SLE patients was found to have autoantibodies against FH but did not show clinical signs of microangiopathy.
Assuntos
Autoanticorpos , Fator H do Complemento , Lúpus Eritematoso Sistêmico , Humanos , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/sangue , Feminino , Masculino , Fator H do Complemento/metabolismo , Fator H do Complemento/imunologia , Adulto , Autoanticorpos/sangue , Autoanticorpos/imunologia , Pessoa de Meia-Idade , Proteínas Inativadoras do Complemento C3b/genética , Adulto Jovem , Idoso , Estudos de Casos e Controles , Adolescente , Proteínas SanguíneasRESUMO
Borrelia, spirochetes transmitted by ticks, are the etiological agents of numerous multisystemic diseases, such as Lyme borreliosis (LB) and tick-borne relapsing fever (TBRF). This study focuses on two surface proteins from two Borrelia subspecies involved in these diseases: CspZ, expressed by Borrelia burgdorferi sensu stricto (also named BbCRASP-2 for complement regulator-acquiring surface protein 2), and the factor H binding A (FhbA), expressed by Borrelia hermsii. Numerous subspecies of Borrelia, including these latter, are able to evade the immune defenses of a variety of potential vertebrate hosts in a number of ways. In this context, previous data suggested that both surface proteins play a role in the immune evasion of both Borrelia subspecies by interacting with key regulators of the alternative pathway of the human complement system, factor H (FH) and FH-like protein 1 (FHL-1). The recombinant proteins, CspZ and FhbA, were expressed in Escherichia coli and purified by one-step metal-affinity chromatography, with yields of 15 and 20 mg or pure protein for 1 L of cultured bacteria, respectively. The purity was evaluated by SDS-PAGE and HPLC and is close to about 95%. The mass of CspZ and FhbA was checked by mass spectrometry (MS). Proper folding of CspZ and FhbA was confirmed by circular dichroism (CD), and their biological activity, namely their interaction with purified FH from human serum (recombinant FH15-20 and recombinant FHL-1), was characterized by SPR. Such a study provides the basis for the biochemical characterization of the studied proteins and their biomolecular interactions which is a necessary prerequisite for the development of new approaches to improve the current diagnosis of LB and TBRF. KEY POINTS: ⢠DLS, CD, SEC-MALS, NMR, HPLC, and MS are tools for protein quality assessment ⢠Borrelia spp. possesses immune evasion mechanisms, including human host complement ⢠CspZ and FhbA interact with high affinity (pM to nM) to human FH and rFHL-1.
Assuntos
Proteínas de Bactérias , Proteínas Recombinantes , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Humanos , Borrelia burgdorferi/genética , Borrelia burgdorferi/metabolismo , Borrelia burgdorferi/imunologia , Cromatografia de Afinidade , Escherichia coli/genética , Escherichia coli/metabolismo , Borrelia/genética , Borrelia/metabolismo , Borrelia/imunologia , Fator H do Complemento/metabolismo , Fator H do Complemento/genética , Doença de Lyme/microbiologia , Proteínas Inativadoras do Complemento C3b/genética , Proteínas Inativadoras do Complemento C3b/metabolismo , Expressão GênicaRESUMO
Purpose: Complement dysregulation is a key component in the pathogenesis of age-related macular degeneration (AMD) and related diseases such as early-onset macular drusen (EOMD). Although genetic variants of complement factor H (CFH) are associated with AMD risk, the impact of CFH and factor H-like protein 1 (FHL-1) expression on local complement activity in human retinal pigment epithelium (RPE) remains unclear. Methods: We identified a novel CFH variant in a family with EOMD and generated patient induced pluripotent stem cell (iPSC)-derived RPE cells. We assessed CFH and FHL-1 co-factor activity through C3b breakdown assays and measured complement activation by immunostaining for membrane attack complex (MAC) formation. Expression of CFH, FHL-1, local alternative pathway (AP) components, and regulators of complement activation (RCA) in EOMD RPE cells was determined by quantitative PCR, western blot, and immunostaining. Isogenic EOMD (cEOMD) RPE was generated using CRISPR/Cas9 gene editing. Results: The CFH variant (c.351-2A>G) resulted in loss of CFH and FHL-1 expression and significantly reduced CFH and FHL-1 protein expression (â¼50%) in EOMD iPSC RPE cells. These cells exhibited increased MAC deposition upon exposure to normal human serum. Under inflammatory or oxidative stress conditions, CFH and FHL-1 expression in EOMD RPE cells paralleled that of controls, whereas RCA expression, including MAC formation inhibitors, was elevated. CRISPR/Cas9 correction restored CFH/FHL-1 expression and mitigated alternative pathway complement activity in cEOMD RPE cells. Conclusions: Identification of a novel CFH variant in patients with EOMD resulting in reduced CFH and FHL-1 and increased local complement activity in EOMD iPSC RPE supports the involvement of CFH haploinsufficiency in EOMD pathogenesis.
Assuntos
Fator H do Complemento , Haploinsuficiência , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas com Domínio LIM , Degeneração Macular , Proteínas Musculares , Epitélio Pigmentado da Retina , Humanos , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Degeneração Macular/genética , Degeneração Macular/metabolismo , Masculino , Feminino , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas Inativadoras do Complemento C3b/genética , Proteínas Inativadoras do Complemento C3b/metabolismo , Ativação do Complemento/genética , Linhagem , Western Blotting , Proteínas do Sistema Complemento/metabolismo , Proteínas do Sistema Complemento/genética , Drusas Retinianas/genética , Drusas Retinianas/metabolismo , Pessoa de Meia-IdadeRESUMO
Mutations in the complement factor H (CFH) gene are associated with complement dysregulation and the development of atypical hemolytic uremic syndrome (aHUS). Several fusion genes that result from genomic structural variation in the CFH and complement factor H-related (CFHR) gene regions have been identified in aHUS. However, one allele has both CFHR gene duplication and CFH::CFHR1 fusion gene have not been reported. An 8-month-old girl (proband) presented with aHUS and was treated with ravulizumab. Her paternal grandfather developed aHUS previously and her paternal great grandmother presented with anti-neutrophil cytoplasmic antibody-associated vasculitis and thrombotic microangiopathy (TMA). However, the proband's parents have no history of TMA. A genetic analysis revealed the presence of CFH::CFHR1 fusion gene and a CFHR3-1-4-2 gene duplication in the patient, her father, and her paternal grandfather. Although several fusion genes resulting from structural variations of the CFH-CFHR genes region have been identified, this is the first report of the combination of a CFH::CFHR1 fusion gene with CFHR gene duplication. Because the CFH-CFHR region is highly homologous, we hypothesized that CFHR gene duplication occurred. These findings indicate a novel pathogenic genomic structural variation associated with the development of aHUS.
Assuntos
Síndrome Hemolítico-Urêmica Atípica , Fator H do Complemento , Humanos , Feminino , Lactente , Fator H do Complemento/genética , Síndrome Hemolítico-Urêmica Atípica/diagnóstico , Síndrome Hemolítico-Urêmica Atípica/genética , Duplicação Gênica , Proteínas do Sistema Complemento/genética , Mutação , Proteínas Sanguíneas/genética , Proteínas Inativadoras do Complemento C3b/genéticaRESUMO
The aim of this study is to investigate the role of CFHR1 in bile duct carcinoma (BDC) and its mechanism of action, and we hope that our analysis and research will contribute to a better understanding of cholangiocarcinoma (BDC) disease genesis, progression and the development of new therapeutic strategies. The prognostic receiver operating characteristic curve of CFHR1 was generated using survival ROC. The ROC curve for CFHR1 showed that there is a correlation between CFHR1 expression and clinicopathological parameters and has an impact on poor prognosis. STRING was used to predict the protein-protein interaction network of the identified genes, and the Microenvironment Cell Populations counter algorithm was used to analyze immune cell infiltration within the BDC. The combined analysis showed that CFHR1 was found to be upregulated in BDC tissues, along with a total of 20 related differentially expressed genes (DEGs) (8 downregulated and 12 upregulated genes). Also, the results showed that the expression of CFHR1 is correlated with immune cell infiltration in tumor and immune cell markers in BDC (P < 0.05). In addition, we have verified experimentally the biological function of CFHR1. These findings suggest that CFHR1 may be a prognostic marker and a potential therapeutic target for BDC. Information regarding the detailed roles of CFHR1 in BDC could be valuable for improving the diagnosis and treatment of this rare cancer.
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Neoplasias dos Ductos Biliares , Colangiocarcinoma , Humanos , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/diagnóstico , Neoplasias dos Ductos Biliares/patologia , Colangiocarcinoma/genética , Biomarcadores , Prognóstico , Ductos Biliares Intra-Hepáticos/patologia , Microambiente Tumoral , Proteínas Inativadoras do Complemento C3bRESUMO
BACKGROUND: We present this challenging case report of Atypical Haemolytic Uremic Syndrome (aHUS) presenting with multi-organ involvement in a patient and heterozygous CFHR1/CFHR3 gene variant, which was refractory to initial eculizumab therapy. CASE PRESENTATION: A forty-three year old female presented with aHUS and had heterozygous disease-associated deletions in the complement genes CFHR1/CFHR3. She had progressive kidney failure and severe extra-renal manifestations including cardiomyopathy and haemorrhagic cystitis; as well as pulmonary, gastrointestinal and neurological involvement. The initial kidney biopsy revealed thrombotic microangiopathy (TMA) changes involving all glomeruli. Clinical improvement was initially seen during eculizumab initiation with suppressed CH50 level, but a new rhinovirus/enterovirus upper respiratory tract infection triggered further severe multi-organ disease activity. The extra-renal manifestations stabilised, then ultimately improved after a period of eculizumab dose intensification. However, the impact on dose intensification on this improvement is unclear. Despite the extra-renal clinical improvement, she ultimately progressed to end-stage kidney disease (ESKD), commencing peritoneal dialysis for three years before undergoing a successful uncomplicated cadaveric kidney transplant without prophylactic eculizumab. Two years after transplant, she has excellent transplant graft function without any further disease recurrence. CONCLUSIONS: This case highlights the concept of extra-renal manifestations in aHUS initially resistant to eculizumab, which potentially responded to dose intensification. Whilst organ injuries are potentially reversible with timely targeted treatment, it appears that the kidneys are most vulnerable to injury.
Assuntos
Síndrome Hemolítico-Urêmica Atípica , Falência Renal Crônica , Transplante de Rim , Feminino , Humanos , Adulto , Deleção de Genes , Síndrome Hemolítico-Urêmica Atípica/complicações , Síndrome Hemolítico-Urêmica Atípica/diagnóstico , Síndrome Hemolítico-Urêmica Atípica/tratamento farmacológico , Rim , Falência Renal Crônica/genética , Proteínas Sanguíneas , Proteínas Inativadoras do Complemento C3b/genéticaRESUMO
Atypical hemolytic uremic syndrome (aHUS) is a rare complement-mediated disease that manifests as the triad of thrombotic microangiopathy. We identified two aHUS patients with neither anti-complement factor H (CFH) antibodies nor causative variants of seven aHUS-related genes (CFH, CFI, CFB, C3, MCP, THBD, and DGKE); however, their plasma showed increased levels of hemolysis by hemolytic assay, which strongly suggests CFH-related abnormalities. Using a copy number variation (CNV) analysis of the CFH/CFHR gene cluster, we identified CFH-CFHR1 hybrid genes in these patients. We verified the absence of aHUS-related abnormal CNVs of the CFH gene in control genomes of 2036 individuals in the general population, which suggests that pathogenicity is related to these hybrid genes. Our study emphasizes that, for patients suspected of having aHUS, it is important to perform an integrated analysis based on a clinical examination, functional analysis, and detailed genetic investigation.
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Síndrome Hemolítico-Urêmica Atípica , Humanos , Síndrome Hemolítico-Urêmica Atípica/diagnóstico , Síndrome Hemolítico-Urêmica Atípica/genética , Variações do Número de Cópias de DNA/genética , Proteínas do Sistema Complemento/genética , Proteínas Inativadoras do Complemento C3b/genéticaRESUMO
The aim of the study was to better understand the interplay between genetic factors and the aging process in the human retina through mapping complement factor H (CFH) and related proteins. Two human eyes, from 92- and 64-year-old donors, were genotyped for the expression of CFH-related 1 (CFHR1) and CFH-related 3 (CFHR3) genes. Deoxyribonucleic acid (DNA) was extracted and analyzed for concentration and purity with a spectrophotometer, at 260 nm. The results showed a DNA concentration of 469.17 ng∕µL in the aged retina and of 399.20 ng∕µL in the younger one. Through polymerase chain reaction (PCR) genotyping, the DNA CFHR1 and CFHR3 were visible as bands of 175 bp and 181 bp. Immunohistochemistry by immunofluorescence method was used with a panel of specific antibodies for CFH, CFHR1, CFHR3 and GFAP, a marker for Müller cells. All the samples were examined, and images captured using confocal microscopy. In the younger retina, CFH was localized in the inner plexiform layer and below the outer nuclear layer, while in the aged retina, it was found in the photoreceptors. CFH was also detected in the choriocapillaris and within the end-feet of the Müller cells. Our controls showed autofluorescence of the retinal pigment epithelium shedding light on a false positive CFH immunostaining of this layer. GFAP immunoreactivity highlighted an increased gliosis within the aged retina. CFHR3 signal was found in the microglia, while CFHR1 was detected in the choriocapillaris. In summary, underpinning the expression of these components can show the potential involvement of these modulators in implementing new treatment strategies.
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Envelhecimento , Proteínas Inativadoras do Complemento C3b , Idoso , Envelhecimento/genética , Biomarcadores , Proteínas Inativadoras do Complemento C3b/genética , Proteínas Inativadoras do Complemento C3b/metabolismo , DNA , Genótipo , Humanos , RetinaRESUMO
Thrombotic microangiopathy (TMA) is a disease that causes organ damage due to microvascular hemolytic anemia, thrombocytopenia, and microvascular platelet thrombosis. Streptococcus pneumoniae-associated TMA (spTMA) is a rare complication of invasive pneumococcal infection. In addition, atypical hemolytic uremic syndrome (aHUS) is TMA associated with congenital or acquired dysregulation of complement activation. We report the case of a nine-month-old boy with refractory nephrotic syndrome complicated by spTMA in the setting of heterozygous complement factor-I (CFI) gene mutation and CFHR3-CFHR1 deletion. He repeatedly developed thrombocytopenia, anemia with schistocytes, hypocomplementemia, and abnormal coagulation triggered by infection, which manifested clinically with convulsions and an intraperitoneal hematoma. Eculizumab (a monoclonal humanized anti-C5 antibody) provided transient symptomatic benefit including improvement in thrombocytopenia; however, he developed unexplained cardiac arrest and was declared brain dead a few days later. In this report, we highlight the diagnostic challenges of this case and the causal relationship between spTMA and complement abnormalities and consider the contribution of heterozygous mutation of CFI and CFHR3-CFHR1 deletion.
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Síndrome Hemolítico-Urêmica Atípica , Microangiopatias Trombóticas , Humanos , Lactente , Masculino , Anticorpos Monoclonais , Síndrome Hemolítico-Urêmica Atípica/complicações , Síndrome Hemolítico-Urêmica Atípica/genética , Proteínas Sanguíneas/genética , Proteínas Inativadoras do Complemento C3b/genética , Fator I do Complemento/genética , Mutação/genética , Streptococcus pneumoniae , Microangiopatias Trombóticas/genéticaRESUMO
Atypical hemolytic uremic syndrome (aHUS) an important form of a thrombotic microangiopathy (TMA) that can frequently lead to acute kidney injury (AKI). An important subset of aHUS is the anti-factor H associated aHUS. This variant of aHUS can occur due to deletion of the complement factor H genes, CFHR1 and CFHR3, along with the presence of anti-factor H antibodies. However, it is a point of interest to note that not all patients with anti-factor H associated aHUS have a CFHR1/R3 deletion. Factor-H has a vital role in the regulation of the complement system, specifically the alternate pathway. Therefore, dysregulation of the complement system can lead to inflammatory or autoimmune diseases. Patients with this disease respond well to treatment with plasma exchange therapy along with Eculizumab and immunosuppressant therapy. Anti-factor H antibody associated aHUS has a certain genetic predilection therefore there is focus on further advancements in the diagnosis and management of this disease. In this article we discuss the baseline characteristics of patients with anti-factor H associated aHUS, their triggers, various treatment modalities and future perspectives.
Assuntos
Injúria Renal Aguda , Síndrome Hemolítico-Urêmica Atípica , Proteínas do Sistema Complemento , Injúria Renal Aguda/genética , Injúria Renal Aguda/imunologia , Injúria Renal Aguda/terapia , Anticorpos/genética , Anticorpos/imunologia , Síndrome Hemolítico-Urêmica Atípica/complicações , Síndrome Hemolítico-Urêmica Atípica/genética , Síndrome Hemolítico-Urêmica Atípica/imunologia , Síndrome Hemolítico-Urêmica Atípica/terapia , Proteínas Sanguíneas/genética , Proteínas Inativadoras do Complemento C3b/genética , Fator H do Complemento/antagonistas & inibidores , Fator H do Complemento/genética , Fator H do Complemento/imunologia , Proteínas do Sistema Complemento/genética , Proteínas do Sistema Complemento/imunologia , Humanos , Troca PlasmáticaRESUMO
De novo systemic atypical hemolytic uremic syndrome (aHUS) post-kidney transplant is an uncommon entity associated with unfavorable outcome. We herein report a case of systemic and fulminant de novo aHUS accompanied by heart and respiratory failure in a 48-year-old male receiving ABO-incompatible living-related kidney transplant who was successfully treated with the anti-C5 monoclonal antibody eculizumab with complete recovery of allograft function. Genetic testing demonstrated a novel pathogenic heterozygous complement factor H-related 1 gene mutation in both the donor and the recipient. Our study highlights the high risks of post-transplant aHUS due to the complement gene mutations in both donor and recipient in living-related transplantation. Early intervention with eculizumab may be effective for reversing systemic aHUS in kidney transplant recipients.
Assuntos
Síndrome Hemolítico-Urêmica Atípica , Transplante de Rim , Anticorpos Monoclonais Humanizados/uso terapêutico , Síndrome Hemolítico-Urêmica Atípica/diagnóstico , Síndrome Hemolítico-Urêmica Atípica/tratamento farmacológico , Síndrome Hemolítico-Urêmica Atípica/genética , Proteínas Inativadoras do Complemento C3b/genética , Humanos , Transplante de Rim/efeitos adversos , Masculino , Pessoa de Meia-Idade , MutaçãoRESUMO
OBJECTIVES: Atypical hemolytic uremic syndrome (aHUS) is characterized by a triad of thrombocytopenia, microangiopathic hemolytic anemia, and acute renal failure resulting from platelet thrombi in the microcirculation of the kidney and other organs, in the absence of a preceding diarrheal illness. This report describes a case in which copy number variation (CNV) analysis using next-generation sequencing (NGS) identified the CFHR3/CFHR1 deletion in a patient with aHUS. METHODS: A 49-year-old Korean female was diagnosed with aHUS based on clinical findings, including schistocytes in peripheral blood and marked thrombocytopenia, suggesting the presence of thrombotic microangiopathy, elevated serum lactate dehydrogenase, and acute kidney injury. Sequence variants and CNV generated from NGS data were estimated to determine if there was a potential genetic cause. Multiplex ligation-dependent probe amplification (MLPA) was conducted to confirm the CFHR3/CFHR1 deletion identified by NGS with CNV analysis. RESULTS: No known or novel pathogenic single nucleotide variant or small insertion/deletion that would be predicted to have damaging effects that could lead to aHUS were identified. However, CNV analysis of NGS data identified the heterozygous CFHR3/CFHR1 deletion. MLPA confirmed this loss of one copy number between the CFHR3 and the CFHR1 genes on chromosome 1q31.3. CONCLUSION: We genetically diagnosed a Korean woman harboring a heterozygous CFHR3/CFHR1 deletion of a known causative gene for aHUS. Our report emphasizes the need for CNV analysis of NGS data and gene dosage assays, such as MLPA, to evaluate large-scale deletions or duplications and generate hybrid CFH genes in patients with suspected aHUS.
Assuntos
Síndrome Hemolítico-Urêmica Atípica , Síndrome Hemolítico-Urêmica Atípica/genética , Proteínas Sanguíneas , Proteínas Inativadoras do Complemento C3b/genética , Fator H do Complemento/genética , Variações do Número de Cópias de DNA , Feminino , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Pessoa de Meia-IdadeRESUMO
BACKGROUND: C3 glomerulopathy (C3G) is a heterogeneous group of chronic renal diseases characterized predominantly by glomerular C3 deposition and complement dysregulation. Mutations in factor H-related (FHR) proteins resulting in duplicated dimerization domains are prototypical of C3G, although the underlying pathogenic mechanism is unclear. METHODS: Using in vitro and in vivo assays, we performed extensive characterization of an FHR-1 mutant with a duplicated dimerization domain. To assess the FHR-1 mutant's association with disease susceptibility and renal prognosis, we also analyzed CFHR1 copy number variations and FHR-1 plasma levels in two Spanish C3G cohorts and in a control population. RESULTS: Duplication of the dimerization domain conferred FHR-1 with an increased capacity to interact with C3-opsonized surfaces, which resulted in an excessive activation of the alternative pathway. This activation does not involve C3b binding competition with factor H. These findings support a scenario in which mutant FHR-1 binds to C3-activated fragments and recruits native C3 and C3b; this leads to formation of alternative pathway C3 convertases, which increases deposition of C3b molecules, overcoming FH regulation. This suggests that a balanced FHR-1/FH ratio is crucial to control complement amplification on opsonized surfaces. Consistent with this conceptual framework, we show that the genetic deficiency of FHR-1 or decreased FHR-1 in plasma confers protection against developing C3G and associates with better renal outcome. CONCLUSIONS: Our findings explain how FHR-1 mutants with duplicated dimerization domains result in predisposition to C3G. They also provide a pathogenic mechanism that may be shared by other diseases, such as IgA nephropathy or age-related macular degeneration, and identify FHR-1 as a potential novel therapeutic target in C3G.
Assuntos
Proteínas Inativadoras do Complemento C3b , Glomerulonefrite por IGA , Proteínas Sanguíneas , Complemento C3/genética , Complemento C3/metabolismo , Proteínas Inativadoras do Complemento C3b/genética , Proteínas Inativadoras do Complemento C3b/metabolismo , Fator H do Complemento/genética , Variações do Número de Cópias de DNA , Suscetibilidade a Doenças , Glomerulonefrite por IGA/genética , Glomerulonefrite por IGA/metabolismo , Humanos , PrognósticoRESUMO
Components of the extracellular matrix (ECM), when exposed to body fluids may promote local complement activation and inflammation. Pathologic complement activation at the glomerular basement membrane and at the Bruch's membrane is implicated in renal and eye diseases, respectively. Binding of soluble complement inhibitors to the ECM, including factor H (FH), is important to prevent excessive complement activation. Since the FH-related (FHR) proteins FHR1 and FHR5 are also implicated in these diseases, our aim was to study whether these FHRs can also bind to ECM components and affect local FH activity and complement activation. Both FH and the FHRs showed variable binding to ECM components. We identified laminin, fibromodulin, osteoadherin and PRELP as ligands of FHR1 and FHR5, and found that FHR1 bound to these ECM components through its C-terminal complement control protein (CCP) domains 4-5, whereas FHR5 bound via its middle region, CCPs 3-7. Aggrecan, biglycan and decorin did not bind FH, FHR1 and FHR5. FHR5 also bound to immobilized C3b, a model of surface-deposited C3b, via CCPs 3-7. By contrast, soluble C3, C3(H2O), and the C3 fragments C3b, iC3b and C3d bound to CCPs 8-9 of FHR5. Properdin, which was previously described to bind via CCPs 1-2 to FHR5, did not bind in its physiologically occurring serum forms in our assays. FHR1 and FHR5 inhibited the binding of FH to the identified ECM proteins in a dose-dependent manner, which resulted in reduced FH cofactor activity. Moreover, both FHR1 and FHR5 enhanced alternative complement pathway activation on immobilized ECM proteins when exposed to human serum, resulting in the increased deposition of C3-fragments, factor B and C5b-9. Thus, our results identify novel ECM ligands of FH family proteins and indicate that FHR1 and FHR5 are competitive inhibitors of FH on ECM and, when bound to these ligands, they may enhance local complement activation and promote inflammation under pathological conditions.
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Ativação do Complemento , Proteínas Inativadoras do Complemento C3b , Fator H do Complemento , Proteínas do Sistema Complemento , Proteínas Inativadoras do Complemento C3b/metabolismo , Proteínas do Sistema Complemento/metabolismo , Matriz Extracelular , Humanos , Inflamação , LigantesRESUMO
Complement factor H (CFH) and its related proteins have an essential role in regulating the alternative pathway of the complement system. Mutations and structural variants (SVs) of the CFH gene cluster, consisting of CFH and its five related genes (CFHR1-5), have been reported in renal pathologies as well as in complex immune diseases like age-related macular degeneration and systemic lupus erythematosus. SV analysis of this cluster is challenging because of its high degree of sequence homology. Following first-line next-generation sequencing gene panel sequencing, we applied Genomic Vision's Molecular Combing Technology to detect and visualize SVs within the CFH gene cluster and resolve its structural haplotypes completely. This approach was tested in three patients with atypical hemolytic uremic syndrome and known SVs and 18 patients with atypical hemolytic uremic syndrome or complement factor 3 glomerulopathy with unknown CFH gene cluster haplotypes. Three SVs, a CFH/CFHR1 hybrid gene in two patients and a rare heterozygous CFHR4/CFHR1 deletion in trans with the common CFHR3/CFHR1 deletion in a third patient, were newly identified. For the latter, the breakpoints were determined using a targeted enrichment approach for long DNA fragments (Samplix Xdrop) in combination with Oxford Nanopore sequencing. Molecular combing in addition to next-generation sequencing was able to improve the molecular genetic yield in this pilot study. This (cost-)effective approach warrants validation in larger cohorts with CFH/CFHR-associated disease.
Assuntos
Síndrome Hemolítico-Urêmica Atípica , Fator H do Complemento , Família Multigênica , Síndrome Hemolítico-Urêmica Atípica/diagnóstico , Síndrome Hemolítico-Urêmica Atípica/genética , Proteínas Inativadoras do Complemento C3b/genética , Proteínas Inativadoras do Complemento C3b/metabolismo , Fator H do Complemento/genética , Haplótipos , Humanos , Projetos PilotoRESUMO
Human complement Factor H-related protein 1 (FHR-1) is one of complement Factor H-related proteins (FHRs) and plays an important role in innate immunity. In particular, FHR-1 promotes complement activation by competing with Factor H (FH) for ligands on different surfaces or directly binding to C3b and native C3. Paradoxically, FHR-1 restrains complement activation by inhibiting C5 convertase and terminal complement complex (TCC) formation, and in vitro assays showed that the physiological concentration of FHR-1 had no obvious C3 and C5 regulatory activity. FHR-1 also plays a role in the inflammatory process. MDA-bound FHR-1 promotes inflammatory cytokine release from monocytes in a complement-independent manner. However, its deficiency increases TNFα, IL1ß, IL6, and IL10 secretion from monocytes stimulated with LPS and R484. These contradictory effects of FHR-1 in innate immunity indicate that FHR-1 may function differently in different scenarios. Dysregulation of innate immunity due to frequent CFHR1 variations is associated with various immune inflammatory disorders. Mutations in the C-terminus of FHR-1 that increase its similarity with FH are associated with atypical haemolytic uraemia syndrome (aHUS). In contrast, mutations in the N-terminus that increase the multimerization of FHRs are associated with C3 glomerulopathy (C3G). Changes in FHR-1 concentration have been observed in other diseases. The different functions of the C-terminus and N-terminus of FHR-1 and the distinct function of FHR-1 under various conditions may explain the association of CFHR1 variations with different diseases. Here, we summarized the recent progress on FHR-1 and dissected its role in various immune inflammatory disorders, helping to comprehend and further explore the disease pathogenesis.
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Síndrome Hemolítico-Urêmica Atípica , Doenças do Sistema Imunitário , Ativação do Complemento , Convertases de Complemento C3-C5/metabolismo , Complemento C3b/metabolismo , Proteínas Inativadoras do Complemento C3b/genética , Fator H do Complemento , Proteínas do Sistema Complemento/metabolismo , Humanos , Ligação ProteicaRESUMO
AIM: Pregnancy-associated hemolytic uremic syndrome (P-aHUS) is an important cause of peripartum acute kidney injury. Studies from Europe have described mutations in complement regulator genes, and data in Indian patients is scarce. Hence this study used multiplex ligation-dependent probe amplification (MLPA) to identify variants in complement genes in P-aHUS patients. METHODS: We present 17 patients of P-aHUS who were investigated for complement protein levels and genetic analysis with MLPA for complement genes. Plasma exchange therapy was offered to all patients presenting in acute phase. RESULTS: Mean age 26.74 (3.36) years with 15/17 delivered by caesarean section. Eleven patients received early (within 7 days) plasma exchange, three were dialysis-dependent at 3 months and seven were dialysis-free. Only one of the three patients receiving late (after 7 days) plasma exchange was dialysis-free. MLPA showed that 11 patients had heterozygous deletions of exons 3, 5, 6 of CFHR1 and upstream region of exons 1, 2, 3, 6 and intron 4 of CFHR3 gene while four patients had homozygous deletions at the same loci. Two patients had no MLPA-detectable variations. CONCLUSION: This study reports a high proportion of deletions of exons of CFHR1 & CFHR3 genes in Indian P-aHUS patients detectable by MLPA by copy number variations. This needs confirmation in large multicentre studies. Plasma exchange can be an effective therapy in the non-availability of Eculizumab.
Assuntos
Proteínas Sanguíneas/genética , Proteínas Inativadoras do Complemento C3b/genética , Deleção de Genes , Síndrome Hemolítico-Urêmica/genética , Complicações na Gravidez/genética , Adulto , Feminino , Humanos , Índia , Gravidez , Estudos Prospectivos , Adulto JovemRESUMO
Hemolytic uremic syndrome (HUS) is a thrombotic microangiopathy (TMA) affecting the kidneys. Compared with typical HUS due to an infection from shiga toxin-producing Escherichia coli, atypical HUS involves a genetic or acquired dysregulation of the complement alternative pathway. In the presence of a mutation in a complement gene, a second trigger is often necessary for the development of the disease. We report a case of a 54-year-old female, with a past medical history of pulmonary tuberculosis, who was admitted to the emergency service with general malaise and reduction in urine output, 5 days after vaccination with ChAdOx1 nCoV-19. Laboratory results revealed microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury. Given the clinical picture of TMA, plasma exchange (PEX) was immediately started, along with hemodialysis. Complementary laboratory workup for TMA excluded thrombotic thrombocytopenic purpura and secondary causes. Complement study revealed normal levels of factors H, B, and I, normal activity of the alternate pathway, and absence of anti-factor H antibodies. Genetic study of complement did not show pathogenic variants in the 12 genes analyzed, but revealed a deletion in gene CFHR3/CFHR1 in homozygosity. Our patient completed 10 sessions of PEX, followed by eculizumab, with both clinical and laboratorial improvement. Actually, given the short time lapse between vaccination with ChAdOx1 nCoV-19 and the clinical manifestations, we believe that vaccine was the trigger for the presentation of aHUS in this particular case.
Assuntos
Síndrome Hemolítico-Urêmica Atípica/etiologia , Proteínas Sanguíneas/genética , ChAdOx1 nCoV-19/efeitos adversos , Proteínas Inativadoras do Complemento C3b/genética , Deleção de Genes , Homozigoto , Feminino , Humanos , Pessoa de Meia-IdadeRESUMO
Atherosclerotic cardiovascular disease (ACVD) is a lipid-driven inflammatory disease and one of the leading causes of death worldwide. Lipid deposits in the arterial wall lead to the formation of plaques that involve lipid oxidation, cellular necrosis, and complement activation, resulting in inflammation and thrombosis. The present study found that homozygous deletion of the CFHR1 gene, which encodes the plasma complement protein factor H-related protein 1 (FHR-1), was protective in two cohorts of patients with ACVD, suggesting that FHR-1 accelerates inflammation and exacerbates the disease. To test this hypothesis, FHR-1 was isolated from human plasma and was found to circulate on extracellular vesicles and to be deposited in atherosclerotic plaques. Surface-bound FHR-1 induced the expression of pro-inflammatory cytokines and tissue factor in both monocytes and neutrophils. Notably, plasma concentrations of FHR-1, but not of factor H, were significantly (p < 0.001) elevated in patients with ACVD, and correlated with the expression of the inflammation markers C-reactive protein, apolipoprotein serum amyloid protein A, and neopterin. FHR-1 expression also significantly correlated with plasma concentrations of low-density lipoprotein (LDL) (p < 0.0001) but not high-density lipoprotein (HDL). Taken together, these findings suggest that FHR-1 is associated with ACVD.