C3 Glomerulopathy and Related Disorders in Children: Etiology-Phenotype Correlation and Outcomes.

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; P<0.05). Three distinct C3 glomerulopathy prognostic groups were identified according to presenting eGFR and >50% 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.

Mitochondrial Haplogroup and the Risk of Acute Kidney Injury Following Cardiac Bypass Surgery.

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 < 0.0005), neither the phylogenetic supergroups nor any individual haplogroup was a significant contributor. We found no significant association between common European haplogroups and the risk of post-cardiac bypass AKI. However, given the major role of mitochondrial dysfunction in AKI, there is a need to replicate our findings in other cohorts and with other aetiologies of AKI.

Clinical Relapses of Atypical HUS on Eculizumab: Clinical Gap for Monitoring and Individualised Therapy.

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.

Statistical Validation of Rare Complement Variants Provides Insights into the Molecular Basis of Atypical Hemolytic Uremic Syndrome and C3 Glomerulopathy.

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.

Publisher Correction: Genome-wide linkage and association study implicates the 10q26 region as a major genetic contributor to primary nonsyndromic vesicoureteric reflux.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Genome-wide linkage and association study implicates the 10q26 region as a major genetic contributor to primary nonsyndromic vesicoureteric reflux.

Vesicoureteric reflux (VUR) is the commonest urological anomaly in children. Despite treatment improvements, associated renal lesions - congenital dysplasia, acquired scarring or both - are a common cause of childhood hypertension and renal failure. Primary VUR is familial, with transmission rate and sibling risk both approaching 50%, and appears highly genetically heterogeneous. It is often associated with other developmental anomalies of the urinary tract, emphasising its etiology as a disorder of urogenital tract development. We conducted a genome-wide linkage and association study in three European populations to search for loci predisposing to VUR. Family-based association analysis of 1098 parent-affected-child trios and case/control association analysis of 1147 cases and 3789 controls did not reveal any compelling associations, but parametric linkage analysis of 460 families (1062 affected individuals) under a dominant model identified a single region, on 10q26, that showed strong linkage (HLOD = 4.90; ZLRLOD = 4.39) to VUR. The ~9Mb region contains 69 genes, including some good biological candidates. Resequencing this region in selected individuals did not clearly implicate any gene but FOXI2, FANK1 and GLRX3 remain candidates for further investigation. This, the largest genetic study of VUR to date, highlights the 10q26 region as a major genetic contributor to VUR in European populations.

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.

Use of the complement inhibitor Coversin to treat HSCT-associated TMA.

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.

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.

Atypical hemolytic uremic syndrome and C3 glomerulopathy: conclusions from a "Kidney Disease: Improving Global Outcomes" (KDIGO) Controversies Conference.

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.

Chromosomal rearrangement-A rare cause of complement factor I associated atypical haemolytic uraemic syndrome.

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.

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.

Patient stratification and therapy in atypical haemolytic uraemic syndrome (aHUS).

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.

Use of eculizumab in crescentic IgA nephropathy: proof of principle and conundrum?

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.

Mutations in TBX18 Cause Dominant Urinary Tract Malformations via Transcriptional Dysregulation of Ureter Development.

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.

Case report: Benefits and challenges of long-term eculizumab in atypical hemolytic uremic syndrome.

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.

Autoantibodies to CD59, CD55, CD46 or CD35 are not associated with atypical haemolytic uraemic syndrome (aHUS).

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.

Urinary tract effects of HPSE2 mutations.

Urofacial syndrome (UFS) is an autosomal recessive congenital disease featuring grimacing and incomplete bladder emptying. Mutations of HPSE2, encoding heparanase 2, a heparanase 1 inhibitor, occur in UFS, but knowledge about the HPSE2 mutation spectrum is limited. Here, seven UFS kindreds with HPSE2 mutations are presented, including one with deleted asparagine 254, suggesting a role for this amino acid, which is conserved in vertebrate orthologs. HPSE2 mutations were absent in 23 non-neurogenic neurogenic bladder probands and, of 439 families with nonsyndromic vesicoureteric reflux, only one carried a putative pathogenic HPSE2 variant. Homozygous Hpse2 mutant mouse bladders contained urine more often than did wild-type organs, phenocopying human UFS. Pelvic ganglia neural cell bodies contained heparanase 1, heparanase 2, and leucine-rich repeats and immunoglobulin-like domains-2 (LRIG2), which is mutated in certain UFS families. In conclusion, heparanase 2 is an autonomic neural protein implicated in bladder emptying, but HPSE2 variants are uncommon in urinary diseases resembling UFS.

Factors determining penetrance in familial atypical haemolytic uraemic syndrome.

BACKGROUND: Inherited abnormalities of complement are found in ∼60% of patients with atypical haemolytic uraemic syndrome (aHUS). Such abnormalities are not fully penetrant. In this study, we have estimated the penetrance of the disease in three families with a CFH mutation (c.3643C>G; p. Arg1215Gly) in whom a common lineage is probable. 25 individuals have been affected with aHUS with three peaks of incidence-early childhood (n=6), early adulthood (n=11) and late adulthood (n=8). Eighteen individuals who have not developed aHUS carry the mutation. METHODS: We estimated penetrance at the ages of 4, 27, 60 and 70 years as both a binary and a survival trait using MLINK and Mendel. We genotyped susceptibility factors in CFH, CD46 and CFHR1 in affected and unaffected carriers. RESULTS AND CONCLUSIONS: We found that the estimates of penetrance at the age of 4 years ranged from <0.01 to 0.10, at the age of 27 years from 0.16 to 0.29, at the age of 60 years from 0.39 to 0.51 and at the age of 70 years from 0.44 to 0.64. We found that the CFH haplotype on the allele not carrying the CFH mutation had a significant effect on disease penetrance. In this family, we did not find that the CD46 haplotypes had a significant effect on penetrance.

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

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