Comprehensive functional characterization of complement factor I rare variant genotypes identified in the SCOPE geographic atrophy cohort.

Rare variants (RVs) in the gene encoding the regulatory enzyme complement factor I (CFI; FI) that reduce protein function or levels increase age-related macular degeneration risk. A total of 3357 subjects underwent screening in the SCOPE natural history study for geographic atrophy secondary to age-related macular degeneration, including CFI sequencing and serum FI measurement. Eleven CFI RV genotypes that were challenging to categorize as type I (low serum level) or type II (normal serum level, reduced enzymatic function) were characterized in the context of pure FI protein in C3b and C4b fluid phase cleavage assays and a novel bead-based functional assay (BBFA) of C3b cleavage. Four variants predicted or previously characterized as benign were analyzed by BBFA for comparison. In all, three variants (W51S, C67R, and I370T) resulted in low expression. Furthermore, four variants (P64L, R339Q, G527V, and P528T) were identified as being highly deleterious with IC50s for C3b breakdown >1 log increased versus the WT protein, while two variants (K476E and R474Q) were ∼1 log reduced in function. Meanwhile, six variants (P50A, T203I, K441R, E548Q, P553S, and S570T) had IC50s similar to WT. Odds ratios and BBFA IC50s were positively correlated (r = 0.76, p < 0.01), while odds ratios versus combined annotation dependent depletion (CADD) scores were not (r = 0.43, p = 0.16). Overall, 15 CFI RVs were functionally characterized which may aid future patient stratification for complement-targeted therapies. Pure protein in vitro analysis remains the gold standard for determining the functional consequence of CFI RVs.

Atypical hemolytic uremic syndrome in the era of terminal complement inhibition: an observational cohort study.

Historically, the majority of patients with complement-mediated atypical hemolytic uremic syndrome (CaHUS) progress to end-stage kidney disease (ESKD). Single-arm trials of eculizumab with a short follow-up suggested efficacy. We prove, for the first time to our knowledge, in a genotype matched CaHUS cohort that the 5-year cumulative estimate of ESKD-free survival improved from 39.5% in a control cohort to 85.5% in the eculizumab-treated cohort (hazard ratio, 4.95; 95% confidence interval [CI], 2.75-8.90; P = .000; number needed to treat, 2.17 [95% CI, 1.81-2.73]). The outcome of eculizumab treatment is associated with the underlying genotype. Lower serum creatinine, lower platelet count, lower blood pressure, and younger age at presentation as well as shorter time between presentation and the first dose of eculizumab were associated with estimated glomerular filtration rate >60 ml/min at 6 months in multivariate analysis. The rate of meningococcal infection in the treated cohort was 550 times greater than the background rate in the general population. The relapse rate upon eculizumab withdrawal was 1 per 9.5 person years for patients with a pathogenic mutation and 1 per 10.8 person years for those with a variant of uncertain significance. No relapses were recorded in 67.3 person years off eculizumab in those with no rare genetic variants. Eculizumab was restarted in 6 individuals with functioning kidneys in whom it had been stopped, with no individual progressing to ESKD. We demonstrated that biallelic pathogenic mutations in RNA-processing genes, including EXOSC3, encoding an essential part of the RNA exosome, cause eculizumab nonresponsive aHUS. Recessive HSD11B2 mutations causing apparent mineralocorticoid excess may also present with thrombotic microangiopathy.

Assessing the Impact of Prophylactic Eculizumab on Renal Graft Survival in Atypical Hemolytic Uremic Syndrome.

BACKGROUND: Atypical hemolytic uremic syndrome (aHUS) is a rare cause of end-stage kidney disease and associated with poor outcomes after kidney transplantation from early disease recurrence. Prophylactic eculizumab treatment at the time of transplantation is used in selected patients with aHUS. We report a retrospective case note review describing transplant outcomes in patients with aHUS transplanted between 1978 and 2017, including those patients treated with eculizumab. METHODS: The National Renal Complement Therapeutics Centre database identified 118 kidney transplants in 86 recipients who had a confirmed diagnosis of aHUS. Thirty-eight kidney transplants were performed in 38 recipients who received prophylactic eculizumab. The cohort not treated with eculizumab comprised 80 transplants in 60 recipients and was refined to produce a comparable cohort of 33 transplants in 32 medium and high-risk recipients implanted since 2002. Complement pathway genetic screening was performed. Graft survival was censored for graft function at last follow-up or patient death. Graft survival without eculizumab treatment is described by complement defect status and by Kidney Disease: Improving Global Outcomes risk stratification. RESULTS: Prophylactic eculizumab treatment improved renal allograft survival ( P = 0.006) in medium and high-risk recipients with 1-y survival of 97% versus 64% in untreated patients. Our data supports the risk stratification advised by Kidney Disease: Improving Global Outcomes. CONCLUSIONS: Prophylactic eculizumab treatment dramatically improves graft survival making transplantation a viable therapeutic option in aHUS.

A novel method for real-time analysis of the complement C3b:FH:FI complex reveals dominant negative CFI variants in age-related macular degeneration.

Age-related macular degeneration (AMD) is linked to 2 main disparate genetic pathways: a chromosome 10 risk locus and the alternative pathway (AP) of complement. Rare genetic variants in complement factor H (CFH; FH) and factor I (CFI; FI) are associated with AMD. FH acts as a soluble cofactor to facilitate FI's cleavage and inactivation of the central molecule of the AP, C3b. For personalised treatment, sensitive assays are required to define the functional significance of individual AP genetic variants. Generation of recombinant FI for functional analysis has thus far been constrained by incomplete processing resulting in a preparation of active and inactive protein. Using an internal ribosomal entry site (IRES)-Furin-CFI expression vector, fully processed FI was generated with activity equivalent to serum purified FI. By generating FI with an inactivated serine protease domain (S525A FI), a real-time surface plasmon resonance assay of C3b:FH:FI complex formation for characterising variants in CFH and CFI was developed and correlated well with standard assays. Using these methods, we further demonstrate that patient-associated rare genetic variants lacking enzymatic activity (e.g. CFI I340T) may competitively inhibit the wild-type FI protein. The dominant negative effect identified in inactive factor I variants could impact on the pharmacological replacement of FI currently being investigated for the treatment of dry AMD.

Long-term outcomes and response to treatment in diacylglycerol kinase epsilon nephropathy.

Recessive mutations in diacylglycerol kinase epsilon (DGKE) display genetic pleiotropy, with pathological features reported as either thrombotic microangiopathy or membranoproliferative glomerulonephritis (MPGN), and clinical features of atypical hemolytic uremic syndrome (aHUS), nephrotic syndrome or both. Pathophysiological mechanisms and optimal management strategies have not yet been defined. In prospective and retrospective studies of aHUS referred to the United Kingdom National aHUS service and prospective studies of MPGN referred to the National Registry of Rare Kidney Diseases for MPGN we defined the incidence of DGKE aHUS as 0.009/million/year and so-called DGKE MPGN as 0.006/million/year, giving a combined incidence of 0.015/million/year. Here, we describe a cohort of sixteen individuals with DGKE nephropathy. One presented with isolated nephrotic syndrome. Analysis of pathological features reveals that DGKE mutations give an MPGN-like appearance to different extents, with but more often without changes in arterioles or arteries. In 15 patients presenting with aHUS, ten had concurrent substantial proteinuria. Identified triggering events were rare but coexistent developmental disorders were seen in six. Nine with aHUS experienced at least one relapse, although in only one did a relapse of aHUS occur after age five years. Persistent proteinuria was seen in the majority of cases. Only two individuals have reached end stage renal disease, 20 years after the initial presentation, and in one, renal transplantation was successfully undertaken without relapse. Six individuals received eculizumab. Relapses on treatment occurred in one individual. In four individuals eculizumab was withdrawn, with one spontaneously resolving aHUS relapse occurring. Thus we suggest that DGKE-mediated aHUS is eculizumab non-responsive and that in individuals who currently receive eculizumab therapy it can be safely withdrawn. This has important patient safety and economic implications.

Functional Characterization of Rare Genetic Variants in the N-Terminus of Complement Factor H in aHUS, C3G, and AMD.

Membranoproliferative glomerulonephritis (MPGN), C3 glomerulopathy (C3G), atypical haemolytic uraemic syndrome (aHUS) and age-related macular degeneration (AMD) have all been strongly linked with dysfunction of the alternative pathway (AP) of complement. A significant proportion of individuals with MPGN, C3G, aHUS and AMD carry rare genetic variants in the CFH gene that cause functional or quantitative deficiencies in the factor H (FH) protein, an important regulator of the AP. In silico analysis of the deleteriousness of rare genetic variants in CFH is not reliable and careful biochemical assessment remains the gold standard. Six N-terminal variants of uncertain significance in CFH were identified in patients with these diseases of the AP and selected for analysis. The variants were produced in Pichia Pastoris in the setting of FH CCPs 1-4, purified by nickel affinity chromatography and size exclusion and characterized by surface plasmon resonance and haemolytic assays as well as by cofactor assays in the fluid phase. A single variant, Q81P demonstrated a profound loss of binding to C3b with consequent loss of cofactor and decay accelerating activity. A further 2 variants, G69E and D130N, demonstrated only subtle defects which could conceivably over time lead to disease progression of more chronic AP diseases such as C3G and AMD. In the variants S159N, A161S, and M162V any functional defect was below the capacity of the experimental assays to reliably detect. This study further underlines the importance of careful biochemical assessment when assigning functional consequences to rare genetic variants that may alter clinical decisions for patients.

Severe type I interferonopathy and unrestrained interferon signaling due to a homozygous germline mutation in STAT2.

Excessive type I interferon (IFNα/ß) activity is implicated in a spectrum of human disease, yet its direct role remains to be conclusively proven. We investigated two siblings with severe early-onset autoinflammatory disease and an elevated IFN signature. Whole-exome sequencing revealed a shared homozygous missense Arg148Trp variant in STAT2, a transcription factor that functions exclusively downstream of innate IFNs. Cells bearing STAT2R148W in homozygosity (but not heterozygosity) were hypersensitive to IFNα/ß, which manifest as prolonged Janus kinase-signal transducers and activators of transcription (STAT) signaling and transcriptional activation. We show that this gain of IFN activity results from the failure of mutant STAT2R148W to interact with ubiquitin-specific protease 18, a key STAT2-dependent negative regulator of IFNα/ß signaling. These observations reveal an essential in vivo function of STAT2 in the regulation of human IFNα/ß signaling, providing concrete evidence of the serious pathological consequences of unrestrained IFNα/ß activity and supporting efforts to target this pathway therapeutically in IFN-associated disease.

Glucose-6-Phosphate Dehydrogenase Deficiency Mimicking Atypical Hemolytic Uremic Syndrome.

A 4-year-old boy presented with nonimmune hemolysis, thrombocytopenia, and acute kidney injury. Investigations for an underlying cause failed to identify a definitive cause and a putative diagnosis of complement-mediated atypical hemolytic uremic syndrome (aHUS) was made. The patient was started initially on plasma exchange and subsequently eculizumab therapy, after which his kidney function rapidly improved. While on eculizumab therapy, despite adequate complement blockade, he presented 2 more times with hemolytic anemia and thrombocytopenia, but without renal involvement. Genetic analysis did not uncover a mutation in any known aHUS gene (CFH, CFI, CFB, C3, CD46, THBD, INF2, and DGKE) and anti-factor H antibodies were undetectable. Whole-exome sequencing was undertaken to identify a cause for the eculizumab resistance. This revealed a pathogenic variant in G6PD (glucose-6-phosphate dehydrogenase), which was confirmed by functional analysis demonstrating decreased erythrocyte G6PD activity. Eculizumab therapy was withdrawn. Complement-mediated aHUS is a diagnosis of exclusion and this case highlights the diagnostic difficulty that remains without an immediately available biomarker for confirmation. This case of G6PD deficiency presented with a phenotype clinically indistinguishable from complement-mediated aHUS. We recommend that G6PD deficiency be included in the differential diagnosis of patients presenting with aHUS and suggest measuring erythrocyte G6PD concentrations in these patients.

Thrombotic Microangiopathy and the Kidney.

Thrombotic microangiopathy can manifest in a diverse range of diseases and is characterized by thrombocytopenia, microangiopathic hemolytic anemia, and organ injury, including AKI. It can be associated with significant morbidity and mortality, but a systematic approach to investigation and prompt initiation of supportive management and, in some cases, effective specific treatment can result in good outcomes. This review considers the classification, pathology, epidemiology, characteristics, and pathogenesis of the thrombotic microangiopathies, and outlines a pragmatic approach to diagnosis and management.

Complement C5-inhibiting therapy for the thrombotic microangiopathies: accumulating evidence, but not a panacea.

Thrombotic microangiopathy (TMA), characterized by organ injury occurring consequent to severe endothelial damage, can manifest in a diverse range of diseases. In complement-mediated atypical haemolytic uraemic syndrome (aHUS) a primary defect in complement, such as a mutation or autoantibody leading to over activation of the alternative pathway, predisposes to the development of disease, usually following exposure to an environmental trigger. The elucidation of the pathogenesis of aHUS resulted in the successful introduction of the complement inhibitor eculizumab into clinical practice. In other TMAs, although complement activation may be seen, its role in the pathogenesis remains to be confirmed by an interventional trial. Although many case reports in TMAs other than complement-mediated aHUS hint at efficacy, publication bias, concurrent therapies and in some cases the self-limiting nature of disease make broader interpretation difficult. In this article, we will review the evidence for the role of complement inhibition in complement-mediated aHUS and other TMAs.

Rare genetic variants in Shiga toxin-associated haemolytic uraemic syndrome: genetic analysis prior to transplantation is essential.

We present a case of haemolytic uraemic syndrome (HUS) in a 16-year-old female with serological evidence of acute Escherichia coli O157:H7 infection. She progressed to established renal failure and received a deceased donor kidney transplant. Shiga toxin-associated HUS (STEC-HUS) does not recur following renal transplantation, but unexpectedly this patient did experience rapid and severe HUS recurrence. She responded to treatment with the terminal complement inhibitor eculizumab and subsequent genetic analysis revealed a rare variant in a complement gene. This highlights the importance of genetic analysis in patients with STEC-HUS prior to renal transplantation so that management can be individualized.

Factor H autoantibody is associated with atypical hemolytic uremic syndrome in children in the United Kingdom and Ireland.

Factor H autoantibodies can impair complement regulation, resulting in atypical hemolytic uremic syndrome, predominantly in childhood. There are no trials investigating treatment, and clinical practice is only informed by retrospective cohort analysis. Here we examined 175 children presenting with atypical hemolytic uremic syndrome in the United Kingdom and Ireland for factor H autoantibodies that included 17 children with titers above the international standard. Of the 17, seven had a concomitant rare genetic variant in a gene encoding a complement pathway component or regulator. Two children received supportive treatment; both developed established renal failure. Plasma exchange was associated with a poor rate of renal recovery in seven of 11 treated. Six patients treated with eculizumab recovered renal function. Contrary to global practice, immunosuppressive therapy to prevent relapse in plasma exchange-treated patients was not adopted due to concerns over treatment-associated complications. Without immunosuppression, the relapse rate was high (five of seven). However, reintroduction of treatment resulted in recovery of renal function. All patients treated with eculizumab achieved sustained remission. Five patients received renal transplants without specific factor H autoantibody-targeted treatment with recurrence in one who also had a functionally significant CFI mutation. Thus, our current practice is to initiate eculizumab therapy for treatment of factor H autoantibody-mediated atypical hemolytic uremic syndrome rather than plasma exchange with or without immunosuppression. Based on this retrospective analysis we see no suggestion of inferior treatment, albeit the strength of our conclusions is limited by the small sample size.

Hemolytic Uremic Syndrome in Pregnancy and Postpartum.

BACKGROUND: Pregnancy is associated with various forms of thrombotic microangiopathy, including hemolytic uremic syndrome. A previous small French study suggested that pregnancy-associated hemolytic uremic syndrome was to be included in the spectrum of atypical hemolytic uremic syndrome linked to complement alternative pathway dysregulation. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We sought to retrospectively analyze the presentation, outcome, and frequency of complement alternative pathway gene variants in a larger international (France, United Kingdom, Italy) cohort of patients with pregnancy-associated hemolytic uremic syndrome. RESULTS: Eighty-seven patients with pregnancy-associated hemolytic uremic syndrome were included. Hemolytic uremic syndrome occurred mainly during the first pregnancy (58%) and in the postpartum period (76%). At diagnosis, 56 (71%) patients required dialysis. Fifty-six (78%) patients underwent plasma exchanges, 21 (41%) received plasma infusions, and four (5%) received eculizumab. During follow-up (mean duration of 7.2 years), 41 (53%) patients reached ESRD, 15 (19%) had CKD, and 18 (28%) patients experienced hemolytic uremic syndrome relapse. Twenty-four patients (27%) received a kidney transplant and a recurrence of hemolytic uremic syndrome occurred in 13 (54%) patients. Variants in complement genes were detected in 49 (56%) patients, mainly in the CFH (30%) and CFI genes (9%). CONCLUSIONS: Pregnancy-associated hemolytic uremic syndrome and atypical hemolytic uremic syndrome nonrelated to pregnancy have the same severity at onset and during follow-up and the same frequency of complement gene variants.

Thrombotic Microangiopathy in Inverted Formin 2-Mediated Renal Disease.

The demonstration of impaired C regulation in the thrombotic microangiopathy (TMA) atypical hemolytic uremic syndrome (aHUS) resulted in the successful introduction of the C inhibitor eculizumab into clinical practice. C abnormalities account for approximately 50% of aHUS cases; however, mutations in the non-C gene diacylglycerol kinase-ε have been described recently in individuals not responsive to eculizumab. We report here a family in which the proposita presented with aHUS but did not respond to eculizumab. Her mother had previously presented with a post-renal transplant TMA. Both the proposita and her mother also had Charcot-Marie-Tooth disease. Using whole-exome sequencing, we identified a mutation in the inverted formin 2 gene (INF2) in the mutational hotspot for FSGS. Subsequent analysis of the Newcastle aHUS cohort identified another family with a functionally-significant mutation in INF2 In this family, renal transplantation was associated with post-transplant TMA. All individuals with INF2 mutations presenting with a TMA also had aHUS risk haplotypes, potentially accounting for the genetic pleiotropy. Identifying individuals with TMAs who may not respond to eculizumab will avoid prolonged exposure of such individuals to the infectious complications of terminal pathway C blockade.

A De Novo Deletion in the Regulators of Complement Activation Cluster Producing a Hybrid Complement Factor H/Complement Factor H-Related 3 Gene in Atypical Hemolytic Uremic Syndrome.

The regulators of complement activation cluster at chromosome 1q32 contains the complement factor H (CFH) and five complement factor H-related (CFHR) genes. This area of the genome arose from several large genomic duplications, and these low-copy repeats can cause genome instability in this region. Genomic disorders affecting these genes have been described in atypical hemolytic uremic syndrome, arising commonly through nonallelic homologous recombination. We describe a novel CFH/CFHR3 hybrid gene secondary to a de novo 6.3-kb deletion that arose through microhomology-mediated end joining rather than nonallelic homologous recombination. We confirmed a transcript from this hybrid gene and showed a secreted protein product that lacks the recognition domain of factor H and exhibits impaired cell surface complement regulation. The fact that the formation of this hybrid gene arose as a de novo event suggests that this cluster is a dynamic area of the genome in which additional genomic disorders may arise.

Plasma resistant atypical hemolytic uremic syndrome associated with a CFH mutation treated with eculizumab: a case report.

INTRODUCTION: Thrombotic microangiopathies are a group of diseases presenting as microangiopathic hemolytic anemia, thrombocytopenia and end-organ dysfunction. As the role of the complement system was elucidated in atypical hemolytic uremic syndrome pathogenesis, eculizumab was successfully introduced into clinical practice. We present a large pedigree with multiple individuals carrying a functionally significant novel factor H mutation. We describe the proband's presentation following a presumed infectious trigger requiring plasma exchange and hemodialysis. CASE PRESENTATION: A 32-year-old Caucasian woman presented with pyrexia and headache lasting one week to our Emergency Department. She gave no history of diarrhea or other symptoms to account for her high temperature. She was not taking any medication. She was pyrexial (38°C), tachycardic (110 bpm) and hypertensive (160/110 mmHg). Her fundoscopy revealed grade IV hypertensive retinopathy. She had mild pretibial and periorbital edema, with oliguria (450 mL/day). She had a pregnancy one year previously, during which she had hypertension, proteinuria and edema, with successful delivery at term. Her mother had died in her early 30s with a clinical picture consistent with thrombotic microangiopathy. Her laboratory evaluation showed microangiopathic hemolytic anemia. After 22 sessions of plasma exchange, her lactate dehydrogenase levels started to climb. As a result, she was classified as plasma resistant and eculizumab therapy was instituted. Her lactate dehydrogenase level and platelet count normalized, and her renal function recovered after three months of dialysis. CONCLUSIONS: We demonstrate that, even in patients with atypical hemolytic uremic syndrome and prolonged dialysis dependence, recovery of renal function can be seen with eculizumab treatment. We suggest a treatment regime of at least three months prior to evaluation of efficacy.

Protecting the kidney from complement: atypical haemolytic uraemic syndrome.

Atypical haemolytic uraemic syndrome is a thrombotic microangiopathy with a predominant renal phenotype. Research developments in the last 15 years have led to the elucidation of the role of complement over activation in the pathogenesis of the disease. This was to lead to the successful introduction of targeted pharmacological therapy, in the form of the complement inhibitor, eculizumab.

Atypical haemolytic uraemic syndrome associated with a CD46 mutation triggered by Shigella flexneri.

We present a case of haemolytic uraemic syndrome (HUS) triggered by Shigella flexneri. Of the Shigella species, only S. dysenteriae type 1 is said to produce Shiga toxin and consequently cause HUS. Investigation of the complement system in this patient revealed a CD46 mutation. In individuals with mutations in complement genes incomplete penetrance of atypical HUS (aHUS) is seen, suggesting that a trigger, such as infection, is required for disease to manifest. In an era of complement modulatory therapy for aHUS it is important to be alert to unusual presentations of diarrhoeal-associated disease.