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BACKGROUND AND OBJECTIVES: Membranoproliferative GN and C3 glomerulopathy are rare and overlapping disorders associated with dysregulation of the alternative complement pathway. Specific etiologic data for pediatric membranoproliferative GN/C3 glomerulopathy are lacking, and outcome data are based on retrospective studies without etiologic data. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: A total of 80 prevalent pediatric patients with membranoproliferative GN/C3 glomerulopathy underwent detailed phenotyping and long-term follow-up within the National Registry of Rare Kidney Diseases (RaDaR). Risk factors for kidney survival were determined using a Cox proportional hazards model. Kidney and transplant graft survival was determined using the Kaplan–Meier method. RESULTS: Central histology review determined 39 patients with C3 glomerulopathy, 31 with immune-complex membranoproliferative GN, and ten with immune-complex GN. Patients were aged 2–15 (median, 9; interquartile range, 7–11) years. Median complement C3 and C4 levels were 0.31 g/L and 0.14 g/L, respectively; acquired (anticomplement autoantibodies) or genetic alternative pathway abnormalities were detected in 46% and 9% of patients, respectively, across all groups, including those with immune-complex GN. Median follow-up was 5.18 (interquartile range, 2.13–8.08) years. Eleven patients (14%) progressed to kidney failure, with nine transplants performed in eight patients, two of which failed due to recurrent disease. Presence of >50% crescents on the initial biopsy specimen was the sole variable associated with kidney failure in multivariable analysis (hazard ratio, 6.2; 95% confidence interval, 1.05 to 36.6; P50% crescents on the initial biopsy specimen. CONCLUSIONS: Crescentic disease was a key risk factor associated with kidney failure in a national cohort of pediatric patients with membranoproliferative GN/C3 glomerulopathy and immune-complex GN. Presenting eGFR and crescentic disease help define prognostic groups in pediatric C3 glomerulopathy. Acquired abnormalities of the alternative pathway were commonly identified but not a risk factor for kidney failure.

Atypical hemolytic uremic syndrome (aHUS) is caused by dysregulation of the complement system. A humanised anti-C5 monoclonal antibody (eculizumab) is available for the treatment of aHUS. We present the first description of atypical HUS in a child with a coexistent diagnosis of a POL-III leukodystrophy. On standard eculizumab dosing regime, there was evidence of ongoing C5 cleavage and clinical relapses when immunologically challenged. Eculizumab is an effective therapy for aHUS, but the recommended doses may not be adequate for all patients, highlighting the need for ongoing efforts to develop a strategy for monitoring of treatment efficacy and potential individualisation of therapy.

Chromosomal rearrangements affecting the genes encoding complement factor H and the factor H related proteins have been described in aHUS patients. To date such disorders have not been described in other aHUS associated genes. We describe here a heterozygous 875,324bp deletion encompassing the gene (CFI) encoding complement factor I and ten other genes. The index case presented with aHUS and did not recover renal function. No abnormalities were detected on Sanger sequencing of CFI but a low factor I level led to a multiplex ligation-dependent probe amplification assay being undertaken. This showed a complete heterozygous deletion of CFI. The extent of the deletion and the breakpoint was defined. In the Newcastle aHUS cohort we have identified and report here 32 different CFI variants in 56 patients but to date this is the only deletion that we have identified. This finding although rare does suggest that screening for chromosomal rearrangements affecting CFI should be undertaken in all aHUS patients particularly if the factor I level is unexplainably low.

Approximately 50% of aHUS patients have an underlying inherited and/or acquired abnormality of complement which predisposes to excessive activation of the alternative pathway. Use of complement inhibitors such as eculizumab to treat aHUS is therefore logical. Anecdotal reports and subsequent open-label trials demonstrated the efficacy of eculizumab in aHUS leading to approval by both the FDA and EMA. NHS England established in 2013 an interim national service for aHUS including funding for eculizumab for both new patients and those undergoing transplantation. NICE guidance now also recommends eculizumab for funding within the NHS in England under the coordination of an expert centre. The investigation and response to treatment in this cohort provides a unique resource for patient stratification.

Although mitochondrial dysfunction plays a key role in the pathophysiology of acute kidney injury (AKI), the influence of mitochondrial genetic variability in this process remains unclear. We explored the association between the risk of post-cardiac bypass AKI and mitochondrial haplotype – inherited mitochondrial genomic variations of potentially functional significance. Our single-centre study recruited consecutive patients prior to surgery. Exclusions included stage 5 CKD, non-Caucasian race and subsequent off-pump surgery. Haplogroup analysis allowed characterisation of the study population using the common mutations and by phylogenetic supergroup (WXI and HV). Chi-square tests for association allowed the identification of potential predictors of AKI for use in logistic regression analysis. AKI occurred in 12.8% of the study population (n = 881; male 69.6%, non-diabetic 78.5%, median (interquartile range) age 68.0 (61.0–75.0) years). The haplogroup profile comprised H (42.7%), J (12.1%), T (10.9%), U (14.4%) and K (7.6%). Although the regression model was statistically significant (χ2 = 95.483, p

C3 glomerulopathy describes a spectrum of disorders with glomerular pathology associated with C3 cleavage product deposition and with defective complement action and regulation (Fakhouri et al., 2010; Sethi et al., 2012b). Kidney biopsies from these patients show glomerular accumulation or deposition of C3 cleavage fragments, but no or minor deposition of immunoglobulins (Appel et al., 2005; D'Agati and Bomback, 2012; Servais et al., 2007; Sethi and Fervenza, 2011). At present the current situation asks for a better definition of the underlining disease mechanisms, for precise biomarkers, and for a treatment for this disease. The complement system is a self activating and propelling enzymatic cascade type system in which inactive, soluble plasma components are activated spontaneously and lead into an amplification loop (Zipfel and Skerka, 2009). Activation of the alternative pathway is spontaneous, occurs by default, and cascade progression leads to amplification by complement activators. The system however is self-controlled by multiple regulators and inhibitors, like Factor H that control cascade progression in fluid phase and on surfaces. The activated complement system generates a series of potent effector components and activation products, which damage foreign-, as well as modified self cells, recruit innate immune cells to the site of action, coordinate inflammation and the response of the adaptive immune system in form of B cells and T lymphocytes (Kohl, 2006; Medzhitov and Janeway, 2002; Ogden and Elkon, 2006; Carroll, 2004; Kemper and Atkinson, 2007; Morgan, 1999; Muller-Eberhard, 1986; Ricklin et al., 2010). Complement controls homeostasis and multiple reactions in the vertebrate organism including defense against microbial infections (Diaz-Guillen et al., 1999; Mastellos and Lambris, 2002; Nordahl et al., 2004; Ricklin et al., 2010). In consequence defective control of the spontaneous self amplifying cascade or regulation is associated with numerous human disorders (Ricklin and Lambris, 2007; Skerka and Zipfel, 2008; Zipfel et al., 2006). Understanding the exact action and regulation of this sophisticated homeotic cascade system is relevant to understand disease pathology of various complement associated human disorders. Furthermore this knowledge is relevant for a better diagnosis and appropriate therapy. At present diagnosis of C3 glomerulopathy is primarily based on the kidney biopsy, and histological, immmunohistological and electron microscopical evaluation (D'Agati and Bomback, 2012; Fakhouri et al., 2010; Medjeral-Thomas et al., 2014a,b; Sethi et al., 2012b). The challenge is to define the actual cause of the diverse glomerular changes or damages, to define how C3 deposition results in the reported glomerular changes, the location of the cell damage and the formation of deposits.

Atypical hemolytic uremic syndrome is a rare disease associated with genetic or acquired defects in complement regulation which frequently leads to renal failure. Disease often recurs early after kidney transplantation, leading to a rapid irreversible loss of function. Extrarenal features, such as hemolysis and thrombocytopenia, may not always occur, and diagnosis is made by demonstrating the classic features of thrombotic microangiopathy on renal biopsy. Eculizumab, a terminal complement inhibitor, has been used successfully to treat fulminant early recurrent disease after transplantation. We describe a case of disease recurrence presenting in the second year after transplantation with a gradual decline in function and the first report of eculizumab treatment for chronic thrombotic microangiopathy in a transplanted kidney. The resultant diagnostic challenges and successful response to eculizumab in this setting are discussed.

Congenital anomalies of the kidneys and urinary tract (CAKUT) are the most common cause of chronic kidney disease in the first three decades of life. Identification of single-gene mutations that cause CAKUT permits the first insights into related disease mechanisms. However, for most cases the underlying defect remains elusive. We identified a kindred with an autosomal-dominant form of CAKUT with predominant ureteropelvic junction obstruction. By whole exome sequencing, we identified a heterozygous truncating mutation (c.1010delG) of T-Box transcription factor 18 (TBX18) in seven affected members of the large kindred. A screen of additional families with CAKUT identified three families harboring two heterozygous TBX18 mutations (c.1570C>T and c.487A>G). TBX18 is essential for developmental specification of the ureteric mesenchyme and ureteric smooth muscle cells. We found that all three TBX18 altered proteins still dimerized with the wild-type protein but had prolonged protein half life and exhibited reduced transcriptional repression activity compared to wild-type TBX18. The p.Lys163Glu substitution altered an amino acid residue critical for TBX18-DNA interaction, resulting in impaired TBX18-DNA binding. These data indicate that dominant-negative TBX18 mutations cause human CAKUT by interference with TBX18 transcriptional repression, thus implicating ureter smooth muscle cell development in the pathogenesis of human CAKUT.

Atypical hemolytic uremic syndrome (aHUS) is caused by dysregulation of the complement system, leading to complement overactivation. A humanized anti-C5 monoclonal antibody, eculizumab, has been available for the treatment of aHUS since 2011. The long-term safety and efficacy of this novel drug in the pediatric population remain under review. We present a child with a hybrid CFH/CFHR3 gene who, having had multiple disease relapses despite optimal treatment with plasma exchange, commenced eculizumab therapy in August 2010. She remains relapse free in follow-up at 52 months, and treatment has been well tolerated. The risk of meningococcal disease during this treatment is recognized. Despite vaccination against meningococcal disease and appropriate antibiotic prophylaxis, our patient developed meningococcal bacteremia 30 months into treatment. She presented with nonspecific symptoms but recovered without sequelae with appropriate treatment. We recommend that children be vaccinated against invasive meningococcal infection before beginning eculizumab therapy and take appropriate antibiotic prophylaxis during treatment, and we suggest that vaccine responses should be checked and followed annually. Clinicians need to maintain a high index of suspicion for invasive meningococcal disease. Neither vaccination nor antibiotic prophylaxis provides complete protection in patients on eculizumab therapy. The appropriate dosage of eculizumab needed to achieve remission in aHUS in the pediatric population is unknown. Having achieved remission in our patient, we monitor eculizumab and CH50 levels to evaluate ongoing blockade of the terminal complement cascade. Such information may help guide dosing intervals in the future.

The pathogenesis of atypical hemolytic uremic syndrome (aHUS) is strongly linked to dysregulation of the alternative pathway of the complement system. Mutations in complement genes have been identified in about two-thirds of cases, with 5% to 15% being in C3. In this study, 23 aHUS-associated genetic changes in C3 were characterized relative to their interaction with the control proteins factor H (FH), membrane cofactor protein (MCP; CD46), and complement receptor 1 (CR1; CD35). In surface plasmon resonance experiments, 17 mutant recombinant proteins demonstrated a defect in binding to FH and/or MCP, whereas 2 demonstrated reduced binding to CR1. In the majority of cases, decreased binding affinity translated to a decrease in proteolytic inactivation (known as cofactor activity) of C3b via FH and MCP. These results were used to map the putative binding regions of C3b involved in the interaction with MCP and CR1 and interrogated relative to known FH binding sites. Seventy-six percent of patients with C3 mutations had low C3 levels that correlated with disease severity. This study expands our knowledge of the functional consequences of aHUS-associated C3 mutations relative to the interaction of C3 with complement regulatory proteins mediating cofactor activity.

Eculizumab, a terminal complement inhibitor, has recently been used successfully to both prevent and treat the recurrence of atypical hemolytic uremic syndrome (aHUS) post renal transplantation. We describe a case that highlights the need to monitor the effects of eculizumab on the complement system and in this case alter the dosage. Despite taking the standard recommended dose of eculizumab for an adult, this aHUS patient developed a low-grade thrombotic microangiopathy on biopsy within months of renal transplantation. Complement assays (trough CH50) showed small amounts of residual terminal pathway activity suggesting inadequate complement blockade on 1,200 mg eculizumab every 2 weeks. Following an increase in the dose of eculizumab to 1,500 mg every 2 weeks, lactate dehydrogenase (LDH), proteinuria, and creatinine decreased, and CH50 assay showed 0%. This case emphasizes the need to monitor clinical parameters and complement activity to ensure that adequate therapeutic blockade is achieved. .

Atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy (C3G) are associated with dysregulation and overactivation of the complement alternative pathway. Typically, gene analysis for aHUS and C3G is undertaken in small patient numbers, yet it is unclear which genes most frequently predispose to aHUS or C3G. Accordingly, we performed a six-center analysis of 610 rare genetic variants in 13 mostly complement genes (CFH, CFI, CD46, C3, CFB, CFHR1, CFHR3, CFHR4, CFHR5, CFP, PLG, DGKE, and THBD) from >3500 patients with aHUS and C3G. We report 371 novel rare variants (RVs) for aHUS and 82 for C3G. Our new interactive Database of Complement Gene Variants was used to extract allele frequency data for these 13 genes using the Exome Aggregation Consortium server as the reference genome. For aHUS, significantly more protein-altering rare variation was found in five genes CFH, CFI, CD46, C3, and DGKE than in the Exome Aggregation Consortium (allele frequency < 0.01%), thus correlating these with aHUS. For C3G, an association was only found for RVs in C3 and the N-terminal C3b-binding or C-terminal nonsurface-associated regions of CFH. In conclusion, the RV analyses showed nonrandom distributions over the affected proteins, and different distributions were observed between aHUS and C3G that clarify their phenotypes.

Autoantibody formation against Factor H (FH) is found in 7–10% of patients who are diagnosed with atypical haemolytic uraemic syndrome (aHUS). These autoantibodies predominately target the C-terminal cell binding recognition domain of FH and are associated with absence of FHR1. Additional autoantibodies have also been identified in association with aHUS, for example autoantibodies to Factor I. Based on this, and that there are genetic mutations in other complement regulators and activators associated with aHUS, we hypothesised that other complement regulator proteins, particularly surface bound regulators in the kidney, might be the target for autoantibody formation in aHUS. Therefore, we assayed serum derived from 89 patients in the Newcastle aHUS cohort for the presence of autoantibodies to CD46 (membrane cofactor protein, MCP), CD55 (decay accelerating factor, DAF), CD35 (complement receptor type 1, CR1; TP10) and CD59. We also assayed 100 healthy blood donors to establish the normal levels of reactivity towards these proteins in the general population. Recombinant proteins CD46 and CD55 (purified from Escherichia coli) as well as soluble CR1 (CD35) and oligomeric C4BP-CD59 (purified from eukaryotic cell media) were used in ELISA to detect high responders. False positive results were established though Western blot and flow cytometric analysis. After excluding false positive responders to bacterial proteins in the CD46 and CD55 preparations, and responses to blood group antigens in CD35, we found no significant level of patient serum IgG reactivity with CD46, CD55, CD35 or CD59 above that detected in the normal population. These results suggest that membrane anchored complement regulators are not a target for autoantibody generation in aHUS.

Contains fulltext : 155154.pdf (Publisher’s version ) (Open Access) Atypical hemolytic uremic syndrome (aHUS) is a rare, possibly life-threatening disease characterized by platelet activation, hemolysis and thrombotic microangiopathy (TMA) leading to renal and other end-organ damage. We originally conducted two phase 2 studies (26 weeks and 1 year) evaluating eculizumab, a terminal complement inhibitor, in patients with progressing TMA (trial 1) and those with long duration of aHUS and chronic kidney disease (trial 2). The current analysis assessed outcomes after 2 years (median eculizumab exposure 100 and 114 weeks, respectively). At all scheduled time points, eculizumab inhibited terminal complement activity. In trial 1 with 17 patients, the platelet count was significantly improved from baseline, and hematologic normalization was achieved in 13 patients at week 26, and in 15 patients at both 1 and 2 years. The estimated glomerular filtration rate (eGFR) was significantly improved compared with baseline and year 1. In trial 2 with 20 patients, TMA event-free status was achieved by 16 patients at week 26, 17 patients at year 1, and 19 patients at year 2. Criteria for hematologic normalization were met by 18 patients at each time point. Improvement of 15 ml/min per 1.73 m(2) or more in eGFR was achieved by 1 patient at week 26, 3 patients at 1 year, and 8 patients at 2 years. The mean change in eGFR was not significant compared with baseline, week 26, or year 1. Eculizumab was well tolerated, with no new safety concerns or meningococcal infections. Thus, a 2-year analysis found that the earlier clinical benefits achieved by eculizumab treatment of aHUS were maintained at 2 years of follow-up.

Key points Finding an inherited complement abnormality in HSCT-associated TMA provides a rationale for the use of a complement inhibitor. Alternative complement inhibitors such as Coversin should be considered in patients who are resistant to eculizumab.

13 p.-3 fig. Goodship, Timothy H.J. et al., In both atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy (C3G) complement plays a primary role in disease pathogenesis. Herein we report the outcome of a 2015 Kidney Disease: Improving Global Outcomes (KDIGO)Controversies Conference where key issues in the management of these 2 diseases were considered by a global panel of experts. Areas addressed included renal pathology, clinical phenotype and assessment, genetic drivers of disease, acquired drivers of disease, and treatment strategies. In order to help guide clinicians who are caring for such patients, recommendations for best treatment strategies were discussed at length, providing the evidence base underpinning current treatment options. Knowledge gaps were identified and a prioritized research agenda was proposed to resolve outstanding controversial issues., The conference was sponsored by Kidney Disease: Improving Global Outcomes (KDIGO) and supported in part by unrestricted educational grants from Achillion Pharmaceuticals, Akari Therapeutics, Alexion Pharmaceuticals, and Omeros.

IgA nephropathy (IgAN) is characterized by a variable clinical course and multifaceted pathophysiology. There is substantial evidence to suggest that complement activation plays a pivotal role in the pathogenesis of the disease. Therefore, complement inhibition using the humanized anti-C5 monoclonal antibody eculizumab could be a rational treatment. We report here a 16-year-old male with the vasculitic form of IgAN who failed to respond to aggressive conventional therapy including high-dose steroids, cyclophosphamide and plasma exchange and who was treated with four weekly doses of 900 mg eculizumab followed by a single dose of 1200 mg. He responded rapidly to this treatment and has had a stable creatinine around 150 µmol/L (1.67 mg/dL) for >6 months. However, proteinuria was unabated on maximal conventional anti-proteinuric treatment, and a repeat renal biopsy 11 months after presentation revealed severe chronic changes. We believe this case provides proof of principle that complement inhibition may be beneficial in IgAN but also that development of chronicity may be independent of complement.

Summary Thrombotic thrombocytopenic purpura (TTP) and atypical haemolytic uraemic syndrome (aHUS) are acute, rare life‐threatening thrombotic microangiopathies that require rapid diagnosis and treatment. They are defined by microangiopathic haemolytic anaemia and thrombocytopenia, with renal involvement primarily in aHUS and neurological and cardiological sequelae in TTP. Prompt treatment for most cases of both conditions is with plasma exchange initially and monoclonal therapy (rituximab in TTP and eculizumab in aHUS) as the mainstay of therapy. Here we discuss the diagnosis and therapy for both disorders.

The renal disorder C3 glomerulopathy with dense deposit disease (C3G-DDD) pattern results from complement dysfunction and primarily affects children and young adults. There is no effective treatment, and patients often progress to end-stage renal failure. A small fraction of C3G-DDD cases linked to factor H or C3 gene mutations as well as autoantibodies have been reported. Here, we examined an index family with 2 patients with C3G-DDD and identified a chromosomal deletion in the complement factor H-related (CFHR) gene cluster. This deletion resulted in expression of a hybrid CFHR2-CFHR5 plasma protein. The recombinant hybrid protein stabilized the C3 convertase and reduced factor H-mediated convertase decay. One patient was refractory to plasma replacement and exchange therapy, as evidenced by the hybrid protein quickly returning to pretreatment plasma levels. Subsequently, complement inhibitors were tested on serum from the patient for their ability to block activity of CFHR2-CFHR5. Soluble CR1 restored defective C3 convertase regulation; however, neither eculizumab nor tagged compstatin had any effect. Our findings provide insight into the importance of CFHR proteins for C3 convertase regulation and identify a genetic variation in the CFHR gene cluster that promotes C3G-DDD. Monitoring copy number and sequence variations in the CFHR gene cluster in C3G-DDD and kidney patients with C3G-DDD variations will help guide treatment strategies.

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.

Background/Aims: Twenty-five members of a family from the county of Devon in England have been affected by atypical haemolytic uraemic syndrome (aHUS) associated with a CFH mutation (c.3643C>G; p.Arg1215Gly). A 65-year-old male was diagnosed with aHUS after losing a renal transplant to a thrombotic microangiopathy. Subsequent mutation screening revealed the same CFH mutation without him being knowingly related to the local kindred. We designed a study to investigate the prevalence of this mutation in the local area. In addition, we examined the diagnoses of pre-existing haemodialysis patients to determine whether other patients might unknowingly be at risk of carrying the same CFH mutation. Methods: The Exeter Ten Thousand (EXTEND) study aims to recruit 10,000 healthy volunteers over the age of 18 years living within 25 miles of Exeter in Devon. We genotyped DNA from 4,000 EXTEND subjects for CFH c.3643C>G; p.Arg1215Gly. We reviewed the diagnoses of 294 haemodialysis patients in the Devon area and genotyped 7 patients with either end-stage renal disease of unknown aetiology, malignant hypertension or renovascular disease. Results: CFH c.3643C>G; p.Arg1215Gly was not detected in any of the 7 haemodialysis patients or the 4,000 individuals within the EXTEND study. Conclusions: We conclude that CFH c.3643C>G; p.Arg1215Gly is not endemic in Devon. This reinforces our existing practice of genotyping only patients with kidney disease and evidence of a thrombotic microangiopathy for this mutation. This is the first study looking at the prevalence of CFH mutations in the general population.

A 2-year-old patient with a neuroblastoma developed haemolytic uraemic syndrome (HUS) following treatment with cisplatin and carboplatin. Following treatment with eculizumab, there was a substantial improvement in renal function with the recovery of the platelet count and the cessation of haemolysis. Subsequent investigations showed a novel, heterozygous CD46 splice site mutation with reduced peripheral blood neutrophil CD46 expression. Withdrawal of eculizumab was followed by the recurrence of disease activity, which resolved with re-introduction of therapy. Abnormal regulation of complement may be associated with other cases of cisplatin-induced HUS and treatment with eculizumab may be appropriate for other affected individuals.

Background Research on complement factor H (fH) in human disease is hampered by lack of an assay suitable for use in large-scale epidemiological studies. We describe the development and validation of a high throughput nephelometric assay for fH. Methods Reagents from a commercial radial immunodiffusion (RID) assay (The Binding Site) were adapted for use on the Siemens BNII high throughput nephelometric instrument. The assay was calibrated with a highly purified human fH preparation with rigorously determined concentration, and assay performance was comprehensively evaluated using samples from healthy human volunteers, with the commercial RID assay as a comparator. The distribution and determinants of circulating fH concentration in humans were then investigated in a large representative population sample. Results The nephelometric assay had recovery close to 100%, was reproducible with intra- and inter-assay CV's of 11% and 5–15% respectively, and had a wider operating range than the RID assay. fH values were unaffected after multiple freeze-thaw cycles demonstrating that it is evidently a stable analyte for immunoassay. fH concentration was unaltered by an acute inflammatory stimulus. The population study showed that plasma fH concentration is associated with circulating lipids and indices of body fat. Conclusion We present the first high throughput assay for circulating fH; the assay is accurate and reliable with reproducible measures from stored samples. It has established the distribution of fH values at a population level and demonstrated important associations with circulating lipids and indices of body fat, thus providing an important reference for future clinical and epidemiological investigations.

Several abnormalities in complement genes reportedly contribute to atypical hemolytic uremic syndrome (aHUS), but incomplete penetrance suggests that additional factors are necessary for the disease to manifest. Here, we sought to describe genotype–phenotype correlations among patients with combined mutations, defined as mutations in more than one complement gene. We screened 795 patients with aHUS and identified single mutations in 41% and combined mutations in 3%. Only 8%–10% of patients with mutations in CFH , C3 , or CFB had combined mutations, whereas approximately 25% of patients with mutations in MCP or CFI had combined mutations. The concomitant presence of CFH and MCP risk haplotypes significantly increased disease penetrance in combined mutated carriers, with 73% penetrance among carriers with two risk haplotypes compared with 36% penetrance among carriers with zero or one risk haplotype. Among patients with CFH or CFI mutations, the presence of mutations in other genes did not modify prognosis; in contrast, 50% of patients with combined MCP mutation developed end stage renal failure within 3 years from onset compared with 19% of patients with an isolated MCP mutation. Patients with combined mutations achieved remission with plasma treatment similar to patients with single mutations. Kidney transplant outcomes were worse, however, for patients with combined MCP mutation compared with an isolated MCP mutation. In summary, these data suggest that genotyping for the risk haplotypes in CFH and MCP may help predict the risk of developing aHUS in unaffected carriers of mutations. Furthermore, screening patients with aHUS for all known disease-associated genes may inform decisions about kidney transplantation.

Rare kidney diseases encompass at least 150 different conditions, most of which are inherited. Although individual rare kidney diseases raise specific issues, as a group these rare diseases can have overlapping challenges in diagnosis and treatment. These challenges include small numbers of affected patients, unidentified causes of disease, lack of biomarkers for monitoring disease progression, and need for complex care. To address common clinical and patient issues among rare kidney diseases, the KDIGO Controversies Conference entitled, Common Elements in Rare Kidney Diseases, brought together a panel of multidisciplinary clinical providers and patient advocates to address five central issues for rare kidney diseases. These issues encompassed diagnostic challenges, management of kidney functional decline and progression of chronic kidney disease, challenges in clinical study design, translation of advances in research to clinical care, and provision of practical and integrated patient support. Thus, by a process of consensus, guidance for addressing these challenges was developed and is presented here.

We describe a case of invasive meningococcal disease due to a vaccine-preventable and penicillin-resistant strain in a fully immunized young adult on long-term complement inhibitor therapy and daily penicillin chemoprophylaxis. Eculizumab is a humanized monoclonal antibody that binds human complement C5 protein and inhibits the terminal complement pathway. It is currently recommended for the treatment of complement-mediated thrombotic microangiopathies. An unwanted complication of inhibiting complement, however, is an increased risk of invasive meningococcal disease. Here, we report the first case of meningococcal group B vaccine failure in a young adult receiving eculizumab for atypical hemolytic uremic syndrome. She developed invasive meningococcal disease due to a vaccine-preventable and penicillin-resistant meningococcal group B strain 4 months after receiving 2 doses of meningococcal group B vaccine while on oral penicillin prophylaxis against meningococcal infection.

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.

1 p. SI: XXVI ICW Kanazawa 2016

C3 nephritic factors are autoantibodies that prolong the half-life or prevent regulation of the alternative pathway C3 convertase, resulting in uncontrolled complement activation. They are strongly associated with renal disease but their role in pathogenesis remains controversial. Here we optimized and compared a panel of assays to identify and interrogate nephritic factor activities. Of 101 patients with histologic or clinically evident disease, 48 were positive in some or all assays. In the presence of properdin, binding of autoantibody was detected in 39 samples and convertase stabilization was detected in 36. Forty-two of 48 nephritic factors tested prevented convertase decay by factor H, and most of these by decay accelerating factor (28) and complement receptor 1 (34). Representative properdin-independent nephritic factors had no effect on C5 cleavage and terminal pathway activity, while properdin-dependent nephritic factors enhanced activity. Biacore analysis of four purified IgG samples confirmed resistance to decay and showed that properdin-independent nephritic factors increased convertase half-life over 50-fold, whereas properdin-dependent nephritic factors increased the half-life 10- to 20-fold and also increased activity of the C3 convertase up to 10-fold. Thus, our study provides a rational approach to detect and characterize nephritic factors in patients.

Factor H autoantibodies are found in ~10% of aHUS patients. Most are associated with complete deficiency of factor H related proteins 1/3 and bind to the C terminal recognition domain. MPGN, like aHUS, is characterised by complement activation. In this study we, therefore, examined the hypothesis that factor H autoantibodies are associated with MPGN. We screened sera from 16 MPGN patients and 100 normal controls using ELISA and detected strongly positive IgG factor H autoantibodies in 2 patients. One patient had type II (DDD) MPGN (male aged 24 yrs) with C3NeF and the other type I (female aged 26 yrs) with no detectable C3NeF. We identified the binding site of the autoantibodies using small SCR domain fragments in the ELISA and showed that the autoantibodies in both patients bound predominately to the N terminal complement regulatory domain of factor H. We measured CFHR 1/3 copy number using MLPA and showed that both patients had 2 copies of CFHR1 and 3. Finally, we examined the functionality of detected factor H autoantibodies using purified patient IgG and observed increased haemolysis when purified IgG from both patients was added to normal human sera prior to incubation with rabbit red blood cells. Thus, in a cohort of MPGN patients we have found a high titre of functionally significant factor H autoantibodies in two patients with MPGN. Antibody depleting therapy may have a role in such patients and we suggest that screening for factor H autoantibodies should be undertaken in all patients with MPGN.

We report here a young female who underwent a successful deceased donor liver transplant for hepatic vein thrombosis. Five years after transplantation she developed postpartum atypical hemolytic uremic syndrome (aHUS). She did not recover renal function. Mutation screening of complement genes in her DNA did not show any abnormality. Mutation screening of DNA available from the donor showed a nonsense CFH mutation leading to factor H deficiency. Genotyping of the patient showed that she was homozygous for an aHUS CD46 at-risk haplotype. In this individual, the development of aHUS has been facilitated by the combination of a trigger (pregnancy), an acquired rare genetic variant (CFH mutation) and a common susceptibility factor (CD46 haplotype).

Summary Background and objectives Atypical hemolytic uremic syndrome is a disease associated with mutations in the genes encoding the complement regulators factors H and I. In addition, factor H autoantibodies have been reported in ;10% of patients with atypical hemolytic uremic syndrome. This study searched for the presence of factor I autoantibodies in atypical hemolytic uremic syndrome. Design, setting, participants, & measurements This study screened 175 atypical hemolytic uremic syndrome patients for factor I autoantibodies using ELISA with confirmatory Western blotting. Functional studies using purified immunoglobulin from one patient were subsequently undertaken. ResultsFactor Iautoantibodieswere detectedinthree patients.Inone patient withah ightiterofautoantibody, the titer was tracked over time and was found to have no association with disease activity. This study found evidence of animmune complex of antibody and factor Ii nthis patient,but purifiedIgG,isolated from current serum samples, had only a minor effect on fluid phase and cell surface complement regulation. Genetic analysis of the three patients with factor I autoantibodies revealed that they had two copies of the genes encoding factor H–related proteins 1 and 3 and therefore, did no th ave ad eletion commonly associated with factor H autoantibodies in atypical hemolytic uremic syndrome. Two patients, however, had functionally significant mutations in complement factor H. Conclusions These findings reinforce the concept of multiple concurrent risk factors being associated with atypical hemolytic uremic syndrome but question whether autoantibodies per se predispose to atypical hemolytic uremic syndrome. Clin J Am Soc Nephrol 7: 417–426, 2012. doi: 10.2215/CJN.05750611

The evolution of man has been characterised by recurrent episodes of migration and settlement with infectious disease a constant threat. This long history of demographic change, together with the action of evolutionary forces such as natural selection and genetic drift, has shaped human genetic diversity. In particular, the interaction between humans, pathogens and the environment has played a crucial role in generating patterns of human genetic variation. The complement system plays a crucial role in the early protective immune response after exposure to a pathogen. Pathogens, over time, have developed mechanisms to circumvent the effects of complement which in turn has led to development of a more complex complement system. During the evolution of the complement system genes coding complement proteins have evolved polymorphisms, some of which have a functional effect, and this may reflect human-pathogen interaction and geographical origin. An example is the polymorphism Ile62Val (rs800292 (A>G)) in the complement regulator Factor H gene which alters the susceptibility to age-related macular degeneration (AMD), with the Ile62 polymorphism protecting against AMD. When sub-Saharan African and European populations are compared, the frequency of this polymorphism shows a very marked geographical distribution. Polymorphisms in other complement genes such as complement factor B show similar trends. This paper describes the geographical variation present in complement genes and discusses the implications of these observations. The analysis of genetic variation in complement genes is a promising tool to unravel mechanisms of host-pathogen interaction and can provide new insights into the evolution of the human immune system.

Atypical hemolytic uremic syndrome (aHUS) is now well recognized to be a disease characterized by excessive complement activation in the microvasculature. In both the familial and sporadic forms, inherited and acquired abnormalities affecting components of the alternative complement pathway are found in 60% of patients. These include mutations in the genes encoding both complement regulators (factor H, factor I, membrane cofactor protein, and thrombomodulin) and activators (factors B and C3) and autoantibodies against factor H. Multiple hits are necessary for the disease to manifest, including a trigger, mutations, and at-risk haplotypes in complement genes. The prognosis for aHUS is poor, with most patients developing end-stage renal failure. Renal transplantation in most patients also has a poor prognosis, with frequent loss of the allograft to recurrent disease. However, improving results with combined liver-kidney transplantation and the advent of complement inhibitors such as eculizumab offer hope that the prognosis for aHUS will improve in future years. Definition of aHUS The term hemolytic uremic syndrome (HUS) was first used by the Swiss hematologist Conrad Von Gasser in a paper published in 1955. 1 In this paper, he described 5 patients with renal failure, thrombocytopenia, and acquired hemolytic anemia. Since then, the term HUS has mainly been used in association with a thrombotic microangiopathy occurring after infection with verocytotoxin (Shigalike toxin)‐producing bacteria, particularly enterohemorrhagicEscherichia coli. Most individuals with this condition (Stx-HUS) recover renal function. Less often, patients present with the features of HUS without any evidence of infection with veryocytotoxinproducing bacteria. In this group of patients, the prognosis is worse, with a significant number developing end-stage renal failure. Over time, the prefix “atypical” has been added to define this group. The nomenclature used to describe those diseases characterized by a thrombotic microangiopathy has been a source of debate for decades. Defining the molecular mechanisms underlying these conditions has in the past decade enabled the development of a mechanistic classification. 2 We review herein the genetic basis underlying aHUS and discuss how this has changed patient management. Complement and aHUS aHUS can be both familial and sporadic. 3 In both the familial and sporadic forms of aHUS, a series of studies undertaken since the late 1990s have established that dysregulation and/or excessive activation of the alternative pathway of complement plays a pivotal role in the pathogenesis of the disease. Both inactivating mutations in genes encoding complement regulators (factor H, factor I, and membrane cofactor protein) and gain-of-function mutations in genes encoding the complement activators (C3 and factor B) have been described. 4 Mutations in the gene encoding thrombomodulin, a membrane-bound glycoprotein with anticoagulant properties that modulates complement activation on cell surfaces, have also been described in aHUS. 5 The following sections contain descriptions of the genes that may be mutated in aHUS. The protein products of these genes are involved in regulation of the activity of the alternative pathway of complement amplification, C3 convertase. For an overview of the function of each protein within the alternative pathway, see the accompanying article in this section by Atkinson and Liszewski entitled “Too much of a good thing at the site of tissue injury: The instructive example of the complement system predisposing to thrombotic microangiopathy.”

Primary vesicoureteric reflux (VUR) can coexist with reflux nephropathy (RN) and impaired renal function. VUR appears to be an inherited condition and is reported in approximately one third of siblings of index cases. The objective was to establish a DNA collection and clinical database from U.K. families containing affected sibling pairs for future VUR genetics studies. The cohort's clinical characteristics have been described.Most patients were identified from tertiary pediatric nephrology centers; each family had an index case with cystography-proven primary, nonsyndromic VUR. Affected siblings had radiologically proven VUR and/or radiographically proven RN.One hundred eighty-nine index cases identified families with an additional 218 affected siblings. More than 90% were20 years at the study's end. Blood was collected and leukocyte DNA extracted from all 407 patients and from 189 mothers and 183 fathers. Clinical presentation was established in 122; 92 had urinary tract infections and 16 had abnormal antenatal renal scans. RN was radiologically proven in 223 patients. Four patients had been transplanted; none were on dialysis. In 174 others aged1 year, estimated GFR (eGFR) was calculated. Five had eGFR 15 to 59 and 48 had eGFR 60 to 89 ml/min per 1.73 m(2). Values were lower in bilateral RN patients than in those with either unilateral or absent RN.The large DNA collection from families with VUR and associated RN constitutes a resource for researchers exploring the most likely complex, genetic components predisposing to VUR and RN.

Atypical hemolytic uremic syndrome (aHUS) is a disease characterized by overactivation of complement. Recurrence following renal transplantation is determined by a genetic predisposition. Genetic screening of all individuals with aHUS should be performed prior to listing for transplantation. Individuals with isolated mutations in MCP have a low risk of recurrence and may be considered for kidney transplantation alone. In individuals with CFH and CFI mutations, the risk of recurrence following renal transplantation is high. Combined liver/kidney transplantation has been used successfully in individuals with CFH mutations following the introduction of perioperative plasma exchange; however, such a procedure is not without its risks. Liver/kidney transplantation has yet to be performed on individuals with CFI and C3 mutations but may be predicted to be successful. In individuals with CFH autoantibodies, a reduction in titer through plasma exchange and rituximab has been successful. Clinical trials of the complement C5 inhibitor eculizumab may improve prospects for isolated renal transplantation in individuals with complement protein mutations.

Purpose of review The last few years revealed a molecular distinction between thrombotic thrombocytopenic purpura, a disease characterized by a lack of ADAMTS13 activity, and atypical hemolytic uremic syndrome (aHUS), a disease of complement overactivation. Many different predisposing genetic factors resulting in complement overactivation have been described in aHUS. Additionally, autoantibodies against complement regulatory proteins have been reported. Recent findings The last year has seen the description of a new risk factor for aHUS in the form of mutations in thrombomodulin. As with other genetic risk factors seen in aHUS, these mutations result in impaired regulation of complement. It is increasingly recognized that a confluence of risk factors resulting in complement overactivation may be required for the disease to manifest. In the last year the complement inhibitor eculizumab has been used successfully to treat patients with aHUS. Summary The characterization of the molecular defect in aHUS has allowed targeted therapy to be used. Although early reports of the efficacy of the complement inhibitor eculizumab are promising, the outcome of a recent clinical trial is awaited.

We report a case of atypical hemolytic uremic syndrome (aHUS) triggered by influenza A (H1N1) in a 17-year-old boy with a mutation in the gene (CD46) encoding the transmembrane complement regulator membrane cofactor protein. The patient recovered completely following treatment with oseltamivir, plasma exchange, and hemodialysis. We describe the case and discuss this unusual association of diseases.

Central to the pathogenesis of atypical hemolytic uremic syndrome (aHUS) is over-activation of the alternative pathway of complement. Following the initial discovery of mutations in the complement regulatory protein, factor H, mutations have been described in factor I, membrane cofactor protein and thrombomodulin, which also result in decreased complement regulation. Autoantibodies to factor H have also been reported to impair complement regulation in aHUS. More recently, gain of function mutations in the complement components C3 and Factor B have been seen. This review focuses on the genetic causes of aHUS, their functional consequences, and clinical effect.

Atypical hemolytic uremic syndrome (aHUS) is associated with mutations in the gene CFH encoding the complement regulator factor H (CFH). We previously reported a family, in which three individuals had partial CFH deficiency but only one was affected by aHUS. We have investigated this family further to show that the partial CFH deficiency is associated with a heterozygous CFH mutation (c.2768T>G, p.Tyr899Asp). We used the polymorphic CFH variant p.His402Tyr to track expression of p.Tyr899Asp, and found that this mutant was expressed in minimal quantities in serum. In the one affected individual we found a second CFH mutation (c.3581G>A, p.Gly1194Asp) on the other allele which was expressed normally. We showed that this mutant, which has been described previously in aHUS, has impaired regulation of cell surface complement activation. The affected individual in this family is therefore a compound heterozygote for two functionally significant CFH mutations. Two individuals (mother and male sib) in the pedigree carried only c.2768T>G, p.Tyr899Asp and one (father) carried only c.3581G>A, p.Gly1194Asp, and all three were asymptomatic. Thus, further investigation of this family has enabled us to clarify the genotype-phenotype correlation.

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.

Atypical haemolytic uraemic syndrome (aHUS) is associated with a poor prognosis with regard to survival at presentation, recovery of renal function and transplantation. It is now established that aHUS is a disease of complement dysregulation with mutations in the genes encoding both complement regulators and activators, and autoantibodies against the complement regulator factor H. Identification of the underlying molecular abnormality in an individual patient can now help to guide their future management. In these guidelines we make recommendations for the investigation and management of aHUS patients both at presentation and in the long-term. We particularly address the role of renal transplantation alone and combined liver-kidney transplantation.

Factor H (CFH) autoantibodies are associated with atypical hemolytic uremic syndrome (aHUS). Peritransplantation plasma exchange therapy and intensification of immunosuppression, with adjuvant use of anti-CD20 monoclonal antibodies has recently been advocated for cases of CFH-autoantibody associated aHUS. In this report, we describe successful deceased donor renal transplantation in a case of CFH-autoantibody associated aHUS with combined CFHR1 and 3 deficiency in addition to the CFH sequence variant, (cG2850T, pGln950His). CFH-autoantibodies were detected 2 weeks prior to transplantation. Disease recurrence was not observed using basiliximab, an IL2-receptor antagonist and high-dose corticosteroids with mycophenolate mofetil. Adjuvant therapies such as Rituximab nor intensification of plasma therapy were employed. Consequently, careful consideration needs to be given to the use of additional immunosuppression in certain cases of CFH-autoantibody associated aHUS. Serial measurement of CFH-autoantibodies is required in the immediate pre- and posttransplantation period to further clarify their role as a factor in the recurrence of aHUS posttransplantation. Furthermore, delineation of the functional significance of CFH-autoantibodies is warranted in individual cases.

Background. A child, who presented atypical haemolytic uraemic syndrome (aHUS) at the age of 1 month, developed cerebral ischaemic events at the age of 10 years. Results. Stenoses of both carotid arteries, left subclavian and vertebral arteries, several intracranial, right humeral, several coronary, and all pulmonary arteries were demonstrated. At the age of 13 years, left subclavian and right cervical carotid arteries were occluded. Right carotid recanalization induced intracranial dissection and death. The child had a Lys350Asp factor B mutation. Conclusion. Arterial steno-occlusive lesions appear as potential complications of dysregulated complement activation in aHUS. Endovascular treatment should be considered cautiously in this setting.

Kidney transplant in patients with atypical hemolytic uremic syndrome (aHUS) is associated with a poor outcome because of recurrent disease, especially in patients known to have a factor H mutation. Long-term prophylactic plasma exchange and combined liver-kidney transplant have prevented graft loss caused by recurrence. However, the mortality associated with liver transplant is not negligible, and prophylactic plasma exchange requires permanent vascular access and regular hospitalization and exposes the patient to potential allergic reactions to plasma. Eculizumab is a high-affinity humanized monoclonal antibody that binds to C5 and thus prevents generation of C5a and the membrane attack complex. We report the case of a 17-year-old girl with aHUS associated with a mutation in the gene for complement factor H (CFH; c.3572C > T, Ser1191Leu) who was highly dependent on plasma exchange. Because of severe allergic reactions to plasma after the third renal graft, eculizumab was introduced in place of plasma exchange without problems. This and other reports suggest that the promise of complement inhibitors in the management of aHUS is going to be fulfilled. Am J Kidney Dis 55:708-711. (C) 2010 by the National Kidney Foundation, Inc

Familial juvenile hyperuricaemic nephropathy (FJHN), an autosomal dominant disorder, is caused by mutations in the UMOD gene, which encodes Uromodulin, a glycosylphosphatidylinositol-anchored protein that is expressed in the thick ascending limb of the loop of Henle and excreted in the urine. Uromodulin contains three epidermal growth factor (EGF)-like domains, a cysteine-rich region which includes a domain of eight cysteines and a zona pellucida (ZP) domain. Over 90% of UMOD mutations are missense, and 62% alter a cysteine residue, implicating a role for protein misfolding in the disease. We investigated 20 northern European FJHN probands for UMOD mutations. Wild-type and mutant Uromodulins were functionally studied by expression in HeLa cells and by the use of western blot analysis and confocal microscopy. Six different UMOD missense mutations (Cys32Trp, Arg185Gly, Asp196Asn, Cys217Trp, Cys223Arg and Gly488Arg) were identified. Patients with UMOD mutations were phenotypically similar to those without UMOD mutations. The mutant Uromodulins had significantly delayed maturation, retention in the endoplasmic reticulum (ER) and reduced expression at the plasma membrane. However, Gly488Arg, which is the only mutation we identified in the ZP domain, was found to be associated with milder in vitro abnormalities and to be the only mutant Uromodulin detected in conditioned medium from transfected cells, indicating that the severity of the mutant phenotypes may depend on their location within the protein. Thus, FJHN-causing Uromodulin mutants are retained in the ER, with impaired intracellular maturation and trafficking, thereby indicating mechanisms whereby Uromodulin mutants may cause the phenotype of FJHN.

There is substantial evidence to suggest that complement activation plays a pivotal role in the pathogenesis of IgA nephropathy. Mesangial C3 deposition is seen in approximately 90% of patients and polymeric IgA has been shown to activate the alternative and lectin pathways. In addition there have been reports of deficiency and mutations in the serum complement regulator factor H (CFH) in association with IgA nephropathy. In this study we have examined the hypothesis that CFH is a susceptibility factor for IgA nephropathy. In 46 IgA nephropathy patients we undertook genotyping of three CFH SNPS (rs3753394, rs3753396 and rs1065489). There was no significant difference in the allele frequency of these 3 SNPs between the patients and normal controls. In the same group of patients we undertook mutation screening of CFH exons 18-23 using direct sequencing and found no abnormalities. All the patients had a normal serum factor H concentration. In this small cohort of IgA nephropathy patients we have not found evidence to support the hypothesis that factor H is a major susceptibility factor for the disease.

Atypical hemolytic uremic syndrome (aHUS) is a disorder characterized by hemolytic anemia, thrombocytopenia, and acute renal failure. Mutations, polymorphisms, and copy number variation in complement factors and inhibitors are associated with aHUS. In this study, we report the first functional non-synonymous polymorphism in the complement inhibitor C4b-binding protein (C4BP) α-chain (c.719G>A; p.Arg240His), which is associated with aHUS. This heterozygous change was found in 6/166 aHUS patients compared with 5/542 normal (χ2 = 6.021; p = 0.014), which was replicated in a second cohort of aHUS patients in which we found 5/170 carriers. The polymorphism does not decrease expression efficiency of C4BP. p.Arg240His is equally efficient as the wild type in binding and supporting degradation of C4BP but its ability to bind C3b and act as cofactor to its degradation both in fluid phase and on surfaces is impaired. This observation supports the hypothesis that dysregulation of the alternative pathway of complement is pivotal for aHUS. Three of the patients carry also mutations in membrane cofactor protein and factor H strengthening the hypothesis that individuals may carry multiple susceptibility factors with an additive effect on the risk of developing aHUS.

The hemolytic uremic syndrome (HUS) is a triad of microangiopathic hemolytic anemia, thrombocytopenia, and renal impairment. Genetic studies demonstrate that heterozygous mutations of membrane cofactor protein (MCP;CD46) predispose to atypical HUS (aHUS), which is not associated with exposure to Shiga toxin (Stx). Among the initial 25 MCP mutations in patients with aHUS were 2, R69W and A304V, that were expressed normally and for which no dysfunction was found. The R69W mutation is in complement control protein module 2, while A304V is in the hydrophobic transmembrane domain. In addition to 3 patients with aHUS, the A304V mutation was identified in 1 patient each with fatal Stx-HUS, the HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome, and glomerulonephritis with C3 deposits. A major goal was to assess if these putative mutations lead to defective complement regulation. Permanent cell lines expressing the mutated proteins were complement “challenged,” and membrane control of C3 fragment deposition was monitored. Both the R69W and A304V MCP mutations were deficient in their ability to control the alternative pathway of complement activation on a cell surface, illustrating the importance of modeling transmembrane proteins in situ.