Contribution of animal models to the mechanistic understanding of Alternative Pathway and Amplification Loop (AP/AL)-driven Complement-mediated Diseases.

This review aimed to capture the key findings that animal models have provided around the role of the alternative pathway and amplification loop (AP/AL) in disease. Animal models, particularly mouse models, have been incredibly useful to define the role of complement and the alternative pathway in health and disease; for instance, the use of cobra venom factor and depletion of C3 provided the initial insight that complement was essential to generate an appropriate adaptive immune response. The development of knockout mice have further underlined the importance of the AP/AL in disease, with the FH knockout mouse paving the way for the first anti-complement drugs. The impact from the development of FB, properdin, and C3 knockout mice closely follows this in terms of mechanistic understanding in disease. Indeed, our current understanding that complement plays a role in most conditions at one level or another is rooted in many of these in vivo studies. That C3, in particular, has roles beyond the obvious in innate and adaptive immunity, normal physiology, and cellular functions, with or without other recognized AP components, we would argue, only extends the reach of this arm of the complement system. Humanized mouse models also continue to play their part. Here, we argue that the animal models developed over the last few decades have truly helped define the role of the AP/AL in disease.

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.

Gain-of-function factor H-related 5 protein impairs glomerular complement regulation resulting in kidney damage.

Genetic variation within the factor H-related (FHR) genes is associated with the complement-mediated kidney disease, C3 glomerulopathy (C3G). There is no definitive treatment for C3G, and a significant proportion of patients develop end-stage renal disease. The prototypical example is CFHR5 nephropathy, through which an internal duplication within a single CFHR5 gene generates a mutant FHR5 protein (FHR5mut) that leads to accumulation of complement C3 within glomeruli. To elucidate how abnormal FHR proteins cause C3G, we modeled CFHR5 nephropathy in mice. Animals lacking the murine factor H (FH) and FHR proteins, but coexpressing human FH and FHR5mut (hFH-FHR5mut), developed glomerular C3 deposition, whereas mice coexpressing human FH with the normal FHR5 protein (hFH-FHR5) did not. Like in patients, the FHR5mut had a dominant gain-of-function effect, and when administered in hFH-FHR5 mice, it triggered C3 deposition. Importantly, adeno-associated virus vector-delivered homodimeric mini-FH, a molecule with superior surface C3 binding compared to FH, reduced glomerular C3 deposition in the presence of the FHR5mut. Our data demonstrate that FHR5mut causes C3G by disrupting the homeostatic regulation of complement within the kidney and is directly pathogenic in C3G. These results support the use of FH-derived molecules with enhanced C3 binding for treating C3G associated with abnormal FHR proteins. They also suggest that targeting FHR5 represents a way to treat complement-mediated kidney injury.

Plasminogen activator-coated nanobubbles targeting cellbound ß2-glycoprotein I as a novel thrombus-specific thrombolytic strategy.

ß2-glycoprotein I (ß2-GPI) is a serum protein widely recognized as the main target of antibodies present in patients with antiphospholipid syndrome (APS). ß2-GPI binds to activated endothelial cells, platelets and leukocytes, key players in thrombus formation. We developed a new targeted thrombolytic agent consisting of nanobubbles (NB) coated with recombinant tissue plasminogen activator (rtPA) and a recombinant antibody specific for cell-bound ß2-GPI. The therapeutic efficacy of targeted NB was evaluated in vitro, using platelet-rich blood clots, and in vivo in three different animal models: i) thrombosis developed in a rat model of APS; ii) ferric chloride-induced mesenteric thrombosis in rats, and iii) thrombotic microangiopathy in a mouse model of atypical hemolytic uremic syndrome (C3-gain-of-function mice). Targeted NB bound preferentially to platelets and leukocytes within thrombi and to endothelial cells through ß2-GPI expressed on activated cells. In vitro, rtPA-targeted NB (rtPA-tNB) induced greater lysis of platelet-rich blood clots than untargeted NB. In a rat model of APS, administration of rtPA-tNB caused rapid dissolution of thrombi and, unlike soluble rtPA that induced transient thrombolysis, prevented new thrombus formation. In a rat model of ferric chloride triggered thrombosis, rtPA-tNB, but not untargeted NB and free rtPA, induced rapid and persistent recanalization of occluded vessels. Finally, treatment of C3-gain-of-function mice with rtPA-tNB, that target ß2-GPI deposited in kidney glomeruli, decreased fibrin deposition, and improved urinalysis data with a greater efficiency than untargeted NB. Our findings suggest that targeting cell-bound ß2-GPI may represent an efficient and thrombus-specific thrombolytic strategy in both APS-related and APS-unrelated thrombotic conditions.

Large-Scale Whole-Genome Sequencing Reveals the Genetic Architecture of Primary Membranoproliferative GN and C3 Glomerulopathy.

BACKGROUND: Primary membranoproliferative GN, including complement 3 (C3) glomerulopathy, is a rare, untreatable kidney disease characterized by glomerular complement deposition. Complement gene mutations can cause familial C3 glomerulopathy, and studies have reported rare variants in complement genes in nonfamilial primary membranoproliferative GN. METHODS: We analyzed whole-genome sequence data from 165 primary membranoproliferative GN cases and 10,250 individuals without the condition (controls) as part of the National Institutes of Health Research BioResource-Rare Diseases Study. We examined copy number, rare, and common variants. RESULTS: Our analysis included 146 primary membranoproliferative GN cases and 6442 controls who were unrelated and of European ancestry. We observed no significant enrichment of rare variants in candidate genes (genes encoding components of the complement alternative pathway and other genes associated with the related disease atypical hemolytic uremic syndrome; 6.8% in cases versus 5.9% in controls) or exome-wide. However, a significant common variant locus was identified at 6p21.32 (rs35406322) (P=3.29×10-8; odds ratio [OR], 1.93; 95% confidence interval [95% CI], 1.53 to 2.44), overlapping the HLA locus. Imputation of HLA types mapped this signal to a haplotype incorporating DQA1*05:01, DQB1*02:01, and DRB1*03:01 (P=1.21×10-8; OR, 2.19; 95% CI, 1.66 to 2.89). This finding was replicated by analysis of HLA serotypes in 338 individuals with membranoproliferative GN and 15,614 individuals with nonimmune renal failure. CONCLUSIONS: We found that HLA type, but not rare complement gene variation, is associated with primary membranoproliferative GN. These findings challenge the paradigm of complement gene mutations typically causing primary membranoproliferative GN and implicate an underlying autoimmune mechanism in most cases.

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.

Successful simultaneous liver-kidney transplantation for renal failure associated with hereditary complement C3 deficiency.

Hereditary complement C3 deficiency is associated with recurrent bacterial infections and proliferative glomerulonephritis. We describe a case of an adult with complete deficiency of complement C3 due to homozygous mutations in C3 gene: c.1811delT (Val604Glyfs*2), recurrent bacterial infections, crescentic glomerulonephritis, and end-stage renal failure. Following isolated kidney transplantation he would remain C3 deficient with a similar, or increased, risk of infections and glomerulonephritis. As C3 is predominantly synthesized in the liver, with a small proportion of C3 monocyte derived and kidney derived, he proceeded to simultaneous liver-kidney transplantation. The procedure has been successful with restoration of his circulating C3 levels, normal liver and kidney function at 26 months of follow-up. Simultaneous liver-kidney transplant is a viable option to be considered in this rare setting.

CD19 is essential for B cell activation by promoting B cell receptor-antigen microcluster formation in response to membrane-bound ligand.

Here we describe the spatiotemporal architecture, at high molecular resolution, of receptors and signaling molecules during the early events of mouse B cell activation. In response to membrane-bound ligand stimulation, antigen aggregation occurs in B cell antigen receptor (BCR) microclusters containing immunoglobulin (Ig) M and IgD that recruit the kinase Syk and transiently associate with the coreceptor CD19. Unexpectedly, CD19-deficient B cells were significantly defective in initiation of BCR-dependent signaling, accumulation of downstream effectors and cell spreading, defects that culminated in reduced microcluster formation. Hence, we have defined the dynamics of assembly of the main constituents of the BCR 'signalosome' and revealed an essential role for CD19, independent of the costimulatory molecule CD21, in amplifying early B cell activation events in response to membrane-bound ligand stimulation.

Modulation of the Alternative Pathway of Complement by Murine Factor H-Related Proteins.

Factor H (FH) is a key alternative pathway regulator that controls complement activation both in the fluid phase and on specific cell surfaces, thus allowing the innate immune response to discriminate between self and foreign pathogens. However, the interrelationships between FH and a group of closely related molecules, designated the FH-related (FHR) proteins, are currently not well understood. Whereas some studies have suggested that human FHR proteins possess complement regulatory abilities, recent studies have shown that FHR proteins are potent deregulators. Furthermore, the roles of the FHR proteins have not been explored in any in vivo models of inflammatory disease. In this study, we report the cloning and expression of recombinant mouse FH and three FHR proteins (FHR proteins A-C). Results from functional assays show that FHR-A and FHR-B proteins antagonize the protective function of FH in sheep erythrocyte hemolytic assays and increase cell-surface C3b deposition on a mouse kidney proximal tubular cell line (TEC) and a human retinal pigment epithelial cell line (ARPE-19). We also report apparent KD values for the binding interaction of mouse C3d with mouse FH (3.85 µM), FHR-A (136 nM), FHR-B (546 nM), and FHR-C (1.04 µM), which directly correlate with results from functional assays. Collectively, our work suggests that similar to their human counterparts, a subset of mouse FHR proteins have an important modulatory role in complement activation. Further work is warranted to define the in vivo context-dependent roles of these proteins and determine whether FHR proteins are suitable therapeutic targets for the treatment of complement-driven diseases.

An Engineered Complement Factor H Construct for Treatment of C3 Glomerulopathy.

Background C3 glomerulopathy (C3G) is associated with dysregulation of the alternative pathway of complement activation, and treatment options for C3G remain limited. Complement factor H (FH) is a potent regulator of the alternative pathway and might offer a solution, but the mass and complexity of FH makes generation of full-length FH far from trivial. We previously generated a mini-FH construct, with FH short consensus repeats 1-5 linked to repeats 18-20 (FH1-5^18-20), that was effective in experimental C3G. However, the serum t1/2 of FH1-5^18-20 was significantly shorter than that of serum-purified FH.Methods We introduced the oligomerization domain of human FH-related protein 1 (denoted by R1-2) at the carboxy or amino terminus of human FH1-5^18-20 to generate two homodimeric mini-FH constructs (FHR1-2^1-5^18-20 and FH1-5^18-20^R1-2, respectively) in Chinese hamster ovary cells and tested these constructs using binding, fluid-phase, and erythrocyte lysis assays, followed by experiments in FH-deficient Cfh-/- mice.Results FHR1-2^1-5^18-20 and FH1-5^18-20^R1-2 homodimerized in solution and displayed avid binding profiles on clustered C3b surfaces, particularly FHR1-2^1-5^18-20 Each construct was >10-fold more effective than FH at inhibiting cell surface complement activity in vitro and restricted glomerular basement membrane C3 deposition in vivo significantly better than FH or FH1-5^18-20 FH1-5^18-20^R1-2 had a C3 breakdown fragment binding profile similar to that of FH, a >5-fold increase in serum t1/2 compared with that of FH1-5^18-20, and significantly better retention in the kidney than FH or FH1-5^18-20Conclusions FH1-5^18-20^R1-2 may have utility as a treatment option for C3G or other complement-mediated diseases.

Disease-linked mutations in factor H reveal pivotal role of cofactor activity in self-surface-selective regulation of complement activation.

Spontaneous activation enables the complement system to respond very rapidly to diverse threats. This activation is efficiently suppressed by complement factor H (CFH) on self-surfaces but not on foreign surfaces. The surface selectivity of CFH, a soluble protein containing 20 complement-control protein modules (CCPs 1-20), may be compromised by disease-linked mutations. However, which of the several functions of CFH drives this self-surface selectivity remains unknown. To address this, we expressed human CFH mutants in Pichia pastoris We found that recombinant I62-CFH (protective against age-related macular degeneration) and V62-CFH functioned equivalently, matching or outperforming plasma-derived CFH, whereas R53H-CFH, linked to atypical hemolytic uremic syndrome (aHUS), was defective in C3bBb decay-accelerating activity (DAA) and factor I cofactor activity (CA). The aHUS-linked CCP 19 mutant D1119G-CFH had virtually no CA on (self-like) sheep erythrocytes (ES) but retained DAA. The aHUS-linked CCP 20 mutant S1191L/V1197A-CFH (LA-CFH) had dramatically reduced CA on ES but was less compromised in DAA. D1119G-CFH and LA-CFH both performed poorly at preventing complement-mediated hemolysis of ES PspCN, a CFH-binding Streptococcus pneumoniae protein domain, binds CFH tightly and increases accessibility of CCPs 19 and 20. PspCN did not improve the DAA of any CFH variant on ES Conversely, PspCN boosted the CA, on ES, of I62-CFH, R53H-CFH, and LA-CFH and also enhanced hemolysis protection by I62-CFH and LA-CFH. We conclude that CCPs 19 and 20 are critical for efficient CA on self-surfaces but less important for DAA. Exposing CCPs 19 and 20 with PspCN and thus enhancing CA on self-surfaces may reverse deficiencies of some CFH 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.

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.

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.

An extended mini-complement factor H molecule ameliorates experimental C3 glomerulopathy.

Abnormal regulation of the complement alternative pathway is associated with C3 glomerulopathy. Complement factor H is the main plasma regulator of the alternative pathway and consists of 20 short consensus repeat (SCR) domains. Although recombinant full-length factor H represents a logical treatment for C3 glomerulopathy, its production has proved challenging. We and others have designed recombinant mini-factor H proteins in which 'non-essential' SCR domains have been removed. Here, we report the in vitro and in vivo effects of a mini-complement factor H protein, FH1-5^18-20, using the unique factor H-deficient (Cfh-/-) mouse model of C3 glomerulopathy. FH1-5^18-20 is comprised of the key complement regulatory domains (SCRs 1-5) linked to the surface recognition domains (SCRs 18-20). Intraperitoneal injection of FH1-5^18-20 in Cfh-/- mice reduced abnormal glomerular C3 deposition, similar to full-length factor H. Systemic effects on plasma alternative pathway control were comparatively modest, in association with a short half-life. Thus, FH1-5^18-20 is a potential therapeutic agent for C3 glomerulopathy and other renal conditions with alternative pathway-mediated tissue injury.

Characterization of a factor H mutation that perturbs the alternative pathway of complement in a family with membranoproliferative GN.

Complement C3 activation is a characteristic finding in membranoproliferative GN (MPGN). This activation can be caused by immune complex deposition or an acquired or inherited defect in complement regulation. Deficiency of complement factor H has long been associated with MPGN. More recently, heterozygous genetic variants have been reported in sporadic cases of MPGN, although their functional significance has not been assessed. We describe a family with MPGN and acquired partial lipodystrophy. Although C3 nephritic factor was shown in family members with acquired partial lipodystrophy, it did not segregate with the renal phenotype. Genetic analysis revealed a novel heterozygous mutation in complement factor H (R83S) in addition to known risk polymorphisms carried by individuals with MPGN. Patients with MPGN had normal levels of factor H, and structural analysis of the mutant revealed only subtle alterations. However, functional analysis revealed profoundly reduced C3b binding, cofactor activity, and decay accelerating activity leading to loss of regulation of the alternative pathway. In summary, this family showed a confluence of common and rare functionally significant genetic risk factors causing disease. Data from our analysis of these factors highlight the role of the alternative pathway of complement in MPGN.

A novel hybrid CFH/CFHR3 gene generated by a microhomology-mediated deletion in familial atypical hemolytic uremic syndrome.

Genomic disorders affecting the genes encoding factor H (fH) and the 5 factor H related proteins have been described in association with atypical hemolytic uremic syndrome. These include deletions of CFHR3, CFHR1, and CFHR4 in association with fH autoantibodies and the formation of a hybrid CFH/CFHR1 gene. These occur through nonallelic homologous recombination secondary to the presence of large segmental duplications (macrohomology) in this region. Using multiplex ligation-dependent probe amplification to screen for such genomic disorders, we have identified a large atypical hemolytic uremic syndrome family where a deletion has occurred through microhomology-mediated end joining rather than nonallelic homologous recombination. In the 3 affected persons of this family, we have shown that the deletion results in formation of a CFH/CFHR3 gene. We have shown that the protein product of this is a 24 SCR protein that is secreted with normal fluid-phase activity but marked loss of complement regulation at cell surfaces despite increased heparin binding. In this study, we have therefore shown that microhomology in this area of chromosome 1 predisposes to disease associated genomic disorders and that the complement regulatory function of fH at the cell surface is critically dependent on the structural integrity of the whole molecule.

Genotype/phenotype correlations in complement factor H deficiency arising from uniparental isodisomy.

We report a male infant who presented at 8 months of age with atypical hemolytic uremic syndrome (aHUS) responsive to plasma therapy. Investigation showed him to have complement factor H (CFH) deficiency associated with a homozygous CFH mutation (c.2880delT [p.Phe960fs]). Mutation screening of the child's parents revealed that the father was heterozygous for this change but that it was not present in his mother. Chromosome 1 uniparental isodisomy of paternal origin was confirmed by genotyping chromosome 1 SNPs. CD46 SNP genotyping was undertaken in this individual and another patient with CFH deficiency associated with chromosome 1 uniparental isodisomy. This showed a homozygous aHUS risk haplotype (CD46GGAAC) in the patient with aHUS and a homozygous glomerulonephritis risk haplotype (CD46AAGGT) in the patient with endocapillary glomerulonephritis. We also showed that FHL-1 (factor H-like protein 1) was present in the patient with aHUS and absent in the patient with glomerulonephritis. This study emphasizes that modifiers such as CD46 and FHL-1 may determine the kidney phenotype of patients who present with homozygous CFH deficiency.

Association of factor H autoantibodies with deletions of CFHR1, CFHR3, CFHR4, and with mutations in CFH, CFI, CD46, and C3 in patients with atypical hemolytic uremic syndrome.

Factor H autoantibodies have been reported in approximately 10% of patients with atypical hemolytic uremic syndrome (aHUS) and are associated with deficiency of factor H-related proteins 1 and 3. In this study we examined the prevalence of factor H autoantibodies in the Newcastle cohort of aHUS patients, determined whether the presence of such autoantibodies is always associated with deficiency of factor H-related proteins 1 and 3, and examined whether such patients have additional susceptibility factors and/or mutations in the genes encoding complement regulator/activators. We screened 142 patients with aHUS and found factor H autoantibodies in 13 individuals (age 1-11 years). The presence of the autoantibodies was confirmed by Western blotting. By using multiplex ligation-dependent probe amplification we measured complement factor H-related (CFHR)1 and CFHR3 copy number. In 10 of the 13 patients there were 0 copies of CFHR1, and in 3 patients there were 2. In 3 of the patients with 0 copies of CFHR1 there was 1 copy of CFHR3, and these individuals exhibited a novel deletion incorporating CFHR1 and CFHR4. In 5 patients mutations were identified: 1 in CFH, 1 in CFI, 1 in CD46, and 2 in C3. The latter observation emphasizes that multiple concurrent factors may be necessary in individual patients for disease manifestation.