Clinicopathologic Implications of Complement Genetic Variants in Kidney Transplantation
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Genetic testing has uncovered rare variants in complement proteins associated with thrombotic microangiopathy (TMA) and C3 glomerulopathy (C3G). Approximately 50% are classified as variants of uncertain significance (VUS). Clinical risk assessment of patients carrying a VUS remains challenging primarily due to a lack of functional information, especially in the context of multiple confounding factors in the setting of kidney transplantation. Our objective was to evaluate the clinicopathologic significance of genetic variants in TMA and C3G in a kidney transplant cohort. We used whole exome next-generation sequencing to analyze complement genes in 76 patients, comprising 60 patients with a TMA and 16 with C3G. Ten variants in complement factor H ( CFH ) were identified; of these, four were known to be pathogenic, one was likely benign and five were classified as a VUS (I372V, I453L, G918E, T956M, L1207I). Each VUS was subjected to a structural analysis and was recombinantly produced; if expressed, its function was then characterized relative to the wild-type (WT) protein. Our data indicate that I372V, I453L, and G918E were deleterious while T956M and L1207I demonstrated normal functional activity. Four common polymorphisms in CFH (E936D, N1050Y, I1059T, Q1143E) were also characterized. We also assessed a family with a pathogenic variant in membrane cofactor protein ( MCP ) in addition to CFH with a unique clinical presentation featuring valvular dysfunction. Our analyses helped to determine disease etiology and defined the recurrence risk after kidney transplant, thereby facilitating clinical decision making for our patients. This work further illustrates the limitations of the prediction models and highlights the importance of conducting functional analysis of genetic variants particularly in a complex clinicopathologic scenario such as kidney transplantation.Keywords:
Thrombotic microangiopathy
As an important tool of the innate immune system, the complement system rapidly recognizes and clears invading microbes and host debris. Furthermore, it stimulates B- and T-cell development through cross talks with the adaptive immune system. Complement is activated through specific danger pattern recognition by the classical and lectin pathways, unspecific initiation and amplification by the alternative pathway, resulting in cell lysis by terminal pathway. The classical pathway, lectin and alternative pathways induce opsonization of C3b on targeted cells, which ultimately results in the clearance of targets. The complement system is tightly controlled to avoid unwanted damage of healthy host cells. Over-activation of complement due to mutations in complement proteins or regulators, or the presence of autoantibodies against the regulators, has been linked to diseases like atypical hemolytic uremic syndrome (aHUS), age-related macular degeneration (AMD) and paroxysmal nocturnal hemoglobinuria (PNH). ‘Regulators of complement activity’ (RCA) proteins form a group of complement regulators. They down-regulate the activity of the central enzyme complexes of the complement system, i.e. C3 convertases, through two mechanisms: (1) they assist serine protease Factor I to induce degradation of C3b and C4b (cofactor activity) and (2) they accelerate the irreversible dissociation of C3 convertases, C3bBb and C4b2a (decay acceleration activity). Many viruses functionally mimic the host complement-regulator proteins to avoid complement attacks. Investigations on complement regulation elucidated the mechanisms of how regulators bind to C3b. The recent published structures of C3b in complex with factor H (FH), complement receptor 1 (CR1), membrane cofactor protein (MCP), smallpox inhibitor of complement enzymes (SPICE) and decay accelerating factor (DAF) imply that all the regulators utilize a common platform in C3b for binding. However, the molecular mechanisms of complement regulation, especially the activity control of C3 convertases, are still largely unexplored. In this thesis, we investigated the molecular basis of cofactor activity and decay accelerating activity in the alternative pathway, as well as the regulatory mechanisms in the classical pathway, through determining a series crystal structures and performing systematic biochemical and biophysical experiments.
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Activation of the alternative pathway of complement is implicated in common neurodegenerative diseases including age-related macular degeneration (AMD). We explored the impact of common variation in genes encoding proteins of the alternative pathway on complement activation in human blood and in AMD. Genetic variation across the genes encoding complement factor H (CFH), factor B (CFB) and component 3 (C3) was determined. The influence of common haplotypes defining transcriptional and translational units on complement activation in blood was determined in a quantitative genomic association study. Individual haplotypes in CFH and CFB were associated with distinct and novel effects on plasma levels of precursors, regulators and activation products of the alternative pathway of complement in human blood. Further, genetic variation in CFH thought to influence cell surface regulation of complement did not alter plasma complement levels in human blood. Plasma markers of chronic activation (split-products Ba and C3d) and an activating enzyme (factor D) were elevated in AMD subjects. Most of the elevation in AMD was accounted for by the genetic variation controlling complement activation in human blood. Activation of the alternative pathway of complement in blood is under genetic control and increases with age. The genetic variation associated with increased activation of complement in human blood also increased the risk of AMD. Our data are consistent with a disease model in which genetic variation in the complement system increases the risk of AMD by a combination of systemic complement activation and abnormal regulation of complement activation in local tissues.
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The pathophysiology of atypical haemolytic-uraemic syndrome (aHUS) occurring de novo after renal transplantation may include genetic mutations of regulators of complement activation, but they are still rarely determined. A 41-year-old female renal transplant recipient presented two very different episodes of thrombotic microangiopathy. The first episode was associated with antibody-mediated rejection and the second was an isolated, acute aHUS, successfully treated with eculizumab. The diagnosis included a genetic analysis and we found a synonymous variant in the Complement Factor H (CFH) gene, c2634C>T (p.His878=) and low factor H (FH) activity during both events. In conclusion, the diagnosis of aHUS should be considered when TMA is associated with an AMR episode. In this setting, a silent polymorphism of factor H may be responsible for these rare cases of "de novo" aHUS after transplantation.
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Atypical haemolytic–uraemic syndrome (aHUS) is characterized by endothelial damage leading to renal failure. aHUS has a proven genetic background [1]. To date, underlying genetic factors have been identified in complement components and complement regulators among ∼50% of aHUS patients [2, 3]. The disease-associated genes include C3 and factor B, which form the complement amplification convertase C3bBb and the regulators factor H (CFH, a plasma gycoprotein), membrane cofactor protein (CD46, MCP) and factor I (a plasma serine protease) which concur to controlling C3 convertase activity (Figure 1). In addition, autoantibodies recognizing factor H have been identified as being associated with haemolytic–uraemic syndrome (HUS) [4]. Over the last decade, a clear link has been demonstrated between this disorder and a defective regulation of the complement system components [5]. Thus, it is generally accepted that aHUS is a disease of excessive complement activation in the kidney glomerular and arteriolar cells. Following yet not well-identified triggering events, endothelial cells lose their integrity and become a target for an uncontrolled complement attack. The release of C5a and the inactive form—in terms of cytolytic effect—of the membrane attack complex (MAC) C5b-9 induces a procoagulant phenotype. This deleterious triggering of the complement and the coagulation cascades leads to the development of the thrombotic microangiopathy (TMA) that characterizes aHUS [6].
Fig. 1.
The complement system is a major innate immune defence mechanism. Complement may be activated by the classical, lectin or alternative pathways, all leading to the cleavage of the inactive central component C3 to biologically active C3b. C3b binds covalently ...
aHUS is a severe and frequently relapsing disorder comprising a typical triad of thrombocytopaenia, haemolysis and acute renal failure, in the absence of Shiga toxin-producing Escherichia coli infection, and detectable ADAMST13 in the serum. It portends a dismal prognosis. More than 50% of patients with aHUS progress to chronic renal failure and 10% die from complications of the disease. The disease can affect patients of all ages and its prognosis correlates to a certain extent with the identified genetic defects. Patients with CFH mutations carry the worst prognosis [7]. At the end of the first year following the onset of aHUS, 60% of patients with CFH mutations die or progress to dialysis dependency. In addition, when renal transplantation is undertaken, loss of the graft occurs in over 50% of renal transplants, due to HUS recurrence [8]. Despite the fact that plasma exchanges are the treatment of choice in severe thrombotic thrombocytopaenic purpura [9], their efficacy in aHUS is not guaranteed. Therefore, other therapeutic procedures, in particular complement-blocking agents, may be of benefit, and considering the severity of this condition are fully justified. As the activation of C5 plays a pivotal role in the pathophysiology of aHUS, the newly elaborated complement antagonist eculizumab represents a reasonable hope as a promising treatment for the disease [10].
Eculizumab (Soliris; Alexion) is a humanized monoclonal antibody indicated for the treatment of paroxysmal nocturnal haemoglobinuria [11]. It binds specifically and with high affinity to the complement protein C5, thereby preventing the release of the anaphylatoxin C5a and the assembly of the terminal complement complex C5b-9. To this day, >50 cases of patients who received eculizumab for aHUS have been published or included in an international multicentre prospective Phase II trial [8, 12–24, 25]. Eculizumab was used in patients who had experienced aHUS in their native kidney, in order to rescue or prevent post-transplant recurrence. There are still few reports indicating that the C5 mAb antagonist eculizumab may be an effective form of treatment for aHUS. The results of clinical trials have now confirmed that the drug arrests the TMA process and improves renal function or stabilizes its decline [26, 27]. Kim et al. [28] and Garjou et al. [29], in two articles that appear in this issue of the Clinical Kidney Journal, describe the outcome of aHUS in two additional cases of patients who received eculizumab for this syndrome occurring in their native kidneys. In both cases, a mutation in complement genes was identified and intensive plasmaphaereses were of no avail to abate the HUS. Plasma exchanges were inefficient to arrest the haematological and renal symptoms in a little girl with CFH mutation who presented with the disease at the age of 7 months. It comes as no surprise that plasma exchanges were not beneficial in the patient with MCP mutation. MCP is classically associated with a childhood onset, a spontaneous abortion of the TMA and recurrent episodes. Interestingly, this observation highlights the poor outcome of patients with an MCP mutation who presented with an onset of the disease at adult age. After failure of plasma exchanges, eculizumab induced an increase in platelet counts and haemolysis was stopped. The patient with MCP mutation who was treated later than 3 months after the onset did not recover his kidney function. Conversely, in the paediatric case with CFH mutation, eculizumab was started 4 months after the onset. In this patient, a slow improvement in the kidney function was observed after a period of peritoneal dialysis. Duran et al. [30], in this issue of the Clinical Kidney Journal, report a significant recovery of renal function in a patient with a CFH mutation who received eculizumab 3 months after being started on dialysis following recurrence of HUS after kidney transplantation.
By blocking the terminal pathway of the complement cascade, eculizumab entails an increased risk of Neisseria meningitides infection. As recommended, the patients were vaccinated before they were commenced on eculizumab. In these cases, the tolerability was good. It appears that eculizumab, the first targeted terminal complement inhibitor, is able to provide an effective and generally well-tolerated treatment for patients suffering from aHUS. Eculizumab should therefore be considered for treating all patients with childhood or adult onset of atypical HUS. Unfortunately, renal function recovery cannot be expected when irreversible histologic injury to the kidney has already been established before the treatment is undertaken. In other cases, this new targeted monoclonal antibody represents a real hope as a promising treatment to rescue the kidney function following the onset of TMA.
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Abstract Vaccinia virus complement control protein (VCP) is a virulence determinant of vaccinia virus that helps protect the virus from the complement attack of the host. To characterize the interaction of VCP with C3 and C4 and understand the mechanism by which VCP inactivates complement, we have expressed VCP in a yeast expression system and compared the biologic activity of the purified protein to that of human factor H and complement receptor 1 (CR1). Recombinant VCP bound to C3 and the proteolytically cleaved form of C3 (C3b), but not to the 135,300-m.w. fragment of C3 generated using elastase (C3c) and the 35,000-m.w. fragment of C3 generated using elastase (C3d) and inhibited both the classical and alternative pathways of complement activation. Although rVCP was less effective at inhibiting the alternative pathway than factor H or CR1, it was more effective than factor H at inhibiting the classical pathway. Unlike factor H, rVCP was unable discriminate between alternative pathway-mediated lysis of rabbit and sheep E. A comparison of the cofactor activity in factor I-mediated cleavage of C3b suggested that in contrast to factor H and CR1, which displayed cofactor activity for the three sites, rVCP displayed cofactor activity primarily for the first site, leading to generation of C3b cleaved by factor I between Arg1281-Ser1282 (iC3b1). Its cofactor activity for C4b cleavages was similar to that of soluble complement receptor type 1. Purification and functional analysis of iC3b1 showed that it was unable to interact with factor B to form the alternative pathway C3 convertase, C3b,Bb. These results suggest that the interaction of VCP with C3 is different from that of factor H and CR1 and that VCP-supported first cleavage of C3b by factor I is sufficient to render C3b nonfunctional.
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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.
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Dysregulation of the alternative pathway (AP) of complement cascade has been implicated in the pathogenesis of age-related macular degeneration (AMD), the leading cause of blindness in the elderly. To further test the hypothesis that defective control of complement activation underlies AMD, parameters of complement activation in blood plasma were determined together with disease-associated genetic markers in AMD patients. Plasma concentrations of activation products C3d, Ba, C3a, C5a, SC5b-9, substrate proteins C3, C4, factor B and regulators factor H and factor D were quantified in patients (n = 112) and controls (n = 67). Subjects were analyzed for single nucleotide polymorphisms in factor H (CFH), factor B-C2 (BF-C2) and complement C3 (C3) genes which were previously found to be associated with AMD. All activation products, especially markers of chronic complement activation Ba and C3d (p<0.001), were significantly elevated in AMD patients compared to controls. Similar alterations were observed in factor D, but not in C3, C4 or factor H. Logistic regression analysis revealed better discriminative accuracy of a model that is based only on complement activation markers Ba, C3d and factor D compared to a model based on genetic markers of the complement system within our study population. In both the controls' and AMD patients' group, the protein markers of complement activation were correlated with CFH haplotypes.This study is the first to show systemic complement activation in AMD patients. This suggests that AMD is a systemic disease with local disease manifestation at the ageing macula. Furthermore, the data provide evidence for an association of systemic activation of the alternative complement pathway with genetic variants of CFH that were previously linked to AMD susceptibility.
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Atypical haemolytic-uraemic syndrome (aHUS) is characterised by microangiopathic haemolytic anaemia, thrombocytopenia and renal failure in the absence of shigatoxin1. aHUS is rare, accounting for approximately 10% of all cases of HUS, and has a poor prognosis2. aHUS can occur at any age and may be sporadic or familial. It is now known that aHUS is due to the loss of complement regulation resulting from mutations in the complement regulatory proteins complement factor H (CFH), factor I, complement factor H related (CFHR)1–3, CFHR-5, CD46 membrane cofactor protein (MCP), thrombomodulin (THBD) or complement activators such as factor B, and complement 3 (C3)2,3. The presence of a mutation, or variant (single nucleotide polymorphisms and haplotype blocks), and a triggering event increasing complement activation may be necessary for manifestation of the disease4,5. In this paper we report the onset of aHUS in association with heterozygous deletion of CFHR1 and a mutation in CFH, c.497G>T (p.Arg166Leu) that has not previously been reported with aHUS. We also report this patient’s response to complement inhibition with eculizumab and the effect of the patient’s subsequent pregnancy.
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A typical haemolytic uraemic syndrome (aHUS) is a rare disease involving haemolytic anaemia, thrombocytopenia, and renal failure. There is growing evidence that the disease is associated with defective control of the alternative pathway of complement [1].
Genetic abnormalities in complement regulatory proteins, including complement factor H (CFH), membrane cofactor protein, and complement factor I, have been reported in 30%, 10%, and 5% of patients with aHUS, respectively [2–4]. CFH is a plasma protein produced by the liver that acts as a central regulator in the alternative pathway of complement activation. CFH acts as a cofactor for complement factor I in the inactivation of the central complement protein C3b to form iC3b. CFH also accelerates the decay of the C3 convertase C3bBb of the alternative pathway, and competes with factor B for binding to C3b [5].
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