Heterozygous mutations in factor H aggravate pathological damage in a stable IgA deposition model induced by Lactobacillus casei cell wall extract.

Introduction: Activation of complement through the alternative pathway (AP) has a key role in the pathogenesis of IgA nephropathy (IgAN). We previously showed, by intraperitoneal injection of Lactobacillus casei cell wall extract (LCWE), C57BL/6 mice develop mild kidney damage in association with glomerular IgA deposition. To further address complement activity in causing glomerular histological alterations as suggested in the pathogenesis of IgAN, here we used mice with factor H mutation (FHW/R) to render AP overactivation in conjunction with LCWE injection to stimulate intestinal production of IgA. Methods: Dose response to LCWE were examined between two groups of FHW/R mice. Wild type (FHW/W) mice stimulated with LCWE were used as model control. Results: The FHW/R mice primed with high dose LCWE showed elevated IgA and IgA-IgG complex levels in serum. In addition to 100% positive rate of IgA and C3, they display elevated biomarkers of kidney dysfunction, coincided with severe pathological lesions, resembling those of IgAN. As compared to wild type controls stimulated by the same high dose LCWE, these FHW/R mice exhibited stronger complement activation in the kidney and in circulation. Discussion: The new mouse model shares many disease features with IgAN. The severity of glomerular lesions and the decline of kidney functions are further aggravated through complement overactivation. The model may be a useful tool for preclinical evaluation of treatment response to complement-inhibitors.

Case report: A family of atypical hemolytic uremic syndrome involving a CFH::CFHR1 fusion gene and CFHR3-1-4-2 gene duplication.

Mutations in the complement factor H (CFH) gene are associated with complement dysregulation and the development of atypical hemolytic uremic syndrome (aHUS). Several fusion genes that result from genomic structural variation in the CFH and complement factor H-related (CFHR) gene regions have been identified in aHUS. However, one allele has both CFHR gene duplication and CFH::CFHR1 fusion gene have not been reported. An 8-month-old girl (proband) presented with aHUS and was treated with ravulizumab. Her paternal grandfather developed aHUS previously and her paternal great grandmother presented with anti-neutrophil cytoplasmic antibody-associated vasculitis and thrombotic microangiopathy (TMA). However, the proband's parents have no history of TMA. A genetic analysis revealed the presence of CFH::CFHR1 fusion gene and a CFHR3-1-4-2 gene duplication in the patient, her father, and her paternal grandfather. Although several fusion genes resulting from structural variations of the CFH-CFHR genes region have been identified, this is the first report of the combination of a CFH::CFHR1 fusion gene with CFHR gene duplication. Because the CFH-CFHR region is highly homologous, we hypothesized that CFHR gene duplication occurred. These findings indicate a novel pathogenic genomic structural variation associated with the development of aHUS.

A case-based narrative review of pregnancy-associated atypical hemolytic uremic syndrome/complement-mediated thrombotic microangiopathy.

Atypical hemolytic uremic syndrome is a complement-mediated thrombotic microangiopathy caused by uncontrolled activation of the alternative complement pathway in the setting of autoantibodies to or rare pathogenic genetic variants in complement proteins. Pregnancy may serve as a trigger and unmask atypical hemolytic uremic syndrome/complement-mediated thrombotic microangiopathy (aHUS/CM-TMA), which has severe, life-threatening consequences. It can be difficult to diagnose aHUS/CM-TMA in pregnancy due to overlapping clinical features with other thrombotic microangiopathy syndromes including hypertensive disorders of pregnancy. However, the distinction among thrombotic microangiopathy etiologies in pregnancy is important because each syndrome has specific disease management and treatment. In this narrative review, we discuss 2 cases to illustrate the diagnostic challenges and evolving approach in the management of pregnancy-associated aHUS/CM-TMA. The first case involves a 30-year-old woman presenting in the first trimester who was diagnosed with aHUS/CM-TMA and treated with eculizumab from 19 weeks' gestation. Genetic testing revealed a likely pathogenic variant in CFI. She successfully delivered a healthy infant at 30 weeks' gestation. In the second case, a 22-year-old woman developed severe postpartum HELLP syndrome, requiring hemodialysis. Her condition improved with supportive management, yet investigations assessing for aHUS/CM-TMA remained abnormal 6 months postpartum consistent with persistent complement activation but negative genetic testing. Through detailed case discussion describing tests assessing for placental health, fetal anatomy, complement activation, autoantibodies to complement regulatory proteins, and genetic testing for aHUS/CM-TMA, we describe how these results aided in the clinical diagnosis of pregnancy-associated aHUS/CM-TMA and assisted in guiding patient management, including the use of anticomplement therapy.

Complement Cascade Proteins Correlate with Fibrosis and Inflammation in Early-Stage Type 1 Diabetic Kidney Disease in the Ins2Akita Mouse Model.

Diabetic kidney disease (DKD) is characterized by histological changes including fibrosis and inflammation. Evidence supports that DKD is mediated by the innate immune system and more specifically by the complement system. Using Ins2Akita T1D diabetic mice, we studied the connection between the complement cascade, inflammation, and fibrosis in early DKD. Data were extracted from a previously published quantitative-mass-spectrometry-based proteomics analysis of kidney glomeruli of 2 (early DKD) and 4 months (moderately advanced DKD)-old Ins2Akita mice and their controls A Spearman rho correlation analysis of complement- versus inflammation- and fibrosis-related protein expression was performed. A cross-omics validation of the correlation analyses' results was performed using public-domain transcriptomics datasets (Nephroseq). Tissue sections from 43 patients with DKD were analyzed using immunofluorescence. Among the differentially expressed proteins, the complement cascade proteins C3, C4B, and IGHM were significantly increased in both early and later stages of DKD. Inflammation-related proteins were mainly upregulated in early DKD, and fibrotic proteins were induced in moderately advanced stages of DKD. The abundance of complement proteins with fibrosis- and inflammation-related proteins was mostly positively correlated in early stages of DKD. This was confirmed in seven additional human and mouse transcriptomics DKD datasets. Moreover, C3 and IGHM mRNA levels were found to be negatively correlated with the estimated glomerular filtration rate (range for C3 rs = -0.58 to -0.842 and range for IGHM rs = -0.6 to -0.74) in these datasets. Immunohistology of human kidney biopsies revealed that C3, C1q, and IGM proteins were induced in patients with DKD and were correlated with fibrosis and inflammation. Our study shows for the first time the potential activation of the complement cascade associated with inflammation-mediated kidney fibrosis in the Ins2Akita T1D mouse model. Our findings could provide new perspectives for the treatment of early DKD as well as support the use of Ins2Akita T1D in pre-clinical studies.

Concurrent Cobalamin C and Plasminogen Deficiencies in a Patient with Chronic Thrombotic Microangiopathy.

BACKGROUND: Although most patients with atypical hemolytic uremic syndrome (aHUS) have variants in genes participating in alternative complement pathways, rare variants in non-complement pathway-related genes, including DGKE, INF2, MMACHC, PLG, and THBD, have also been described. CASE PRESENTATION: We report an 18-year-old male patient with renal biopsy-proven chronic thrombotic microangiopathy that raised suspicion of aHUS. Whole-exome sequencing revealed a novel pathogenic homozygous MMACHC c.484G>T (p.Gly162Trp) variant. Subsequently, clinical and laboratory findings confirmed cobalamin C (Cbl C) deficiency. Also, homozygous missense c.1112C>T PLG (p.Thr371Ile) variant was detected (it had been reported as a variant of unknown significance). However, the low serum plasminogen (PLG) activity proved the pathogenicity of c.1112C>T. Hence, the patient was diagnosed with concurrent Cbl C and PLG deficiencies. Segregation analysis revealed that the mother and father had the same heterozygous PLG and MMACHC variants. PLG variants have generally been described in aHUS patients concomitant with complement gene variants in the literature; therefore, the association between aHUS and PLG variants is controversial. The possible contribution of PLG deficiency to thrombotic microangiopathy was also discussed in this case. CONCLUSION: Non-complement-mediated aHUS is an exceptional disorder. A limited number of genes are involved in this entity. To our knowledge, this is the first aHUS patient diagnosed with both Cbl C and PLG deficiencies in the literature.

Defective C3d caused by C3 p.W1034R in inherited atypical hemolytic uremic syndrome.

INTRODUCTION: Atypical hemolytic uremic syndrome (aHUS) is a rare form of thrombotic microangiopathy. Personal genome analyses have revealed numerous aHUS-causing variants, mainly complement-related genes. However, not all aHUS-causing variants have been functionally validated. METHODS: An exome sequence analysis of a Japanese multiplex family composed of three patients diagnosed with aHUS in infancy and showing frequent relapses clustered in a dominant transmission mode was performed. Protein interaction between the C3d and C-terminal domains of factor H was analyzed using a quartz crystal microbalance. RESULTS: Following filtering by heterozygous variants, amino acid substitutions, and allele frequency, the analysis revealed eight rare variants shared by the affected individuals. Variant prioritization listed C3 p.W1034R as the most likely candidate gene mutation in the affected individuals, despite being classified as a variant of uncertain significance. Binding of recombinant C3d harboring 1034R to recombinant short consensus repeats 15 to 20 of factor H was significantly suppressed compared with that of C3 with 1034W. CONCLUSION: C3 p.W1034R results in an inherited form of aHUS that often presents with recurrent episodes, possibly because of impaired interactions between the C3d and C-terminal domains of factor H. Following comprehensive genomic analysis, functional validation of C3 p.W1034R strengthens the molecular basis for aHUS pathophysiology.

A Transcriptome Array-Based Approach to Link SGLT-2 and Intrarenal Complement C5 Synthesis in Diabetic Nephropathy.

Diabetic nephropathy is a common microvascular complication of diabetes mellitus. It is characterized by progressive chronic kidney disease (CKD) with decline of kidney function by hyperfiltration. On a mechanistic level, activation of the complement system has been implicated in the pathogenesis of diabetic nephropathy. Therefore, here we pursued a transcriptome array-based approach to link intrarenal SGLT-2 and the synthesis of distinct complement components in diabetic nephropathy. Publicly available datasets for SLC5A2 (encoding SGLT-2) and complement system components were extracted specifically from microdissected tubulointerstitial (healthy controls: n = 31, diabetic nephropathy: n = 17) and glomerular compartments (healthy controls: n = 21, diabetic nephropathy: n = 12). First, we compared tubulointerstitial and glomerular log2SLC5A2 mRNA expression levels and confirmed a predominant synthesis within the tubulointerstitial compartment. Among various complement components and receptors, the only significant finding was a positive association between SLC5A2 and the tubulointerstitial synthesis of the complement component C5 in diabetic nephropathy (p = 0.0109). Finally, intrarenal expression of SLC5A2 was associated predominantly with pathways involved in metabolic processes. Interestingly, intrarenal complement C5 synthesis was also associated with enrichment of metabolic signaling pathways, overlapping with SLC5A2 for "metabolism" and "biological oxidations". These observations could be of relevance in the pathogenesis of diabetic nephropathy and implicate a mechanistic link between SGLT-2 and intrarenal complement synthesis.

Clinical research advances of CFHR5 nephropathy: a recent review.

CFHR5 nephropathy is a type of clinical C3 glomerulopathy, which is a monogenic genetic disease caused by the internal replication of CFHR5 gene, a protein related to the complement regulatory factor H family. The disease seems to be prevalent only in people of Greek Cypriot descent. Because of the special variation of the internal replication of exon 2 and exon 3 of CFHR5 protein in the occurrence of disease, it has had a serious impact on local residents. At present, the mechanism of glomerular damage caused by CFHR5 protein mutations is still unclear. The purpose of this article is to review the clinical research advances of this disease in the past 10 years, including the study of mutant genes, the analysis of mutant proteins and the role of alternative pathways in glomerular injury. It covers the progress in diagnosis and clinical treatment of the disease and looks forward to the future development prospects of its treatment. It is hoped that the recent results will be summarized for the follow-up in-depth study of CFHR5 nephropathy.

Gene Therapy for Immunoglobulin E, Complement-Mediated, and Eosinophilic Disorders.

Immunoglobulin E, complement, and eosinophils play an important role in host defense, but dysfunction of each of these components can lead to a variety of human disorders. In this review, we summarize how investigators have adapted gene therapy and antisense technology to modulate immunoglobulin E, complement, and/or eosinophil levels to treat these disorders.

Genetic investigation of Nordic patients with complement-mediated kidney diseases.

Background: Complement activation in atypical hemolytic uremic syndrome (aHUS), C3 glomerulonephropathy (C3G) and immune complex-mediated membranoproliferative glomerulonephritis (IC-MPGN) may be associated with rare genetic variants. Here we describe gene variants in the Swedish and Norwegian populations. Methods: Patients with these diagnoses (N=141) were referred for genetic screening. Sanger or next-generation sequencing were performed to identify genetic variants in 16 genes associated with these conditions. Nonsynonymous genetic variants are described when they have a minor allele frequency of <1% or were previously reported as being disease-associated. Results: In patients with aHUS (n=94, one also had IC-MPGN) 68 different genetic variants or deletions were identified in 60 patients, of which 18 were novel. Thirty-two patients had more than one genetic variant. In patients with C3G (n=40) 29 genetic variants, deletions or duplications were identified in 15 patients, of which 9 were novel. Eight patients had more than one variant. In patients with IC-MPGN (n=7) five genetic variants were identified in five patients. Factor H variants were the most frequent in aHUS and C3 variants in C3G. Seventeen variants occurred in more than one condition. Conclusion: Genetic screening of patients with aHUS, C3G and IC-MPGN is of paramount importance for diagnostics and treatment. In this study, we describe genetic assessment of Nordic patients in which 26 novel variants were found.

Immunological risk and complement genetic evaluations in early onset de novo thrombotic microangiopathy after living donor kidney transplantation: A Japanese multicenter registry.

BACKGROUND: Thrombotic microangiopathy (TMA) after kidney transplantation (KTx), particularly early onset de novo (dn) TMA, requires immediate interventions to prevent irreversible organ damage. This multicenter study was performed to investigate the allogeneic clinical factors and complement genetic background of dnTMA after KTx. METHODS: Perioperative dnTMA after KTx within 1 week after KTx were diagnosed based on pathological or/and hematological criteria at each center, and their immunological backgrounds were researched. Twelve aHUS-related gene variants were examined in dnTMA cases. RESULTS: Seventeen recipients (15 donors) were enrolled, and all dnTMA cases were onset within 72-h of KTx, and 16 of 17 cases were ABO incompatible. The implementation rate of pre-transplant plasmaphereses therapies were low, including cases with high titers of anti-A/anti-B antibodies. Examination of aHUS-related gene variants revealed some deletions and variants with minor allele frequency (MAF) in Japan or East Asian genome databases in genes encoding alternative pathways and complement regulatory factors. These variants was positive in 8 cases, 6 of which were positive in both recipient and donor, but only in one graft loss case. CONCLUSIONS: Although some immunological risks were found for dnTMA after KTx, only a few cases developed into TMA. The characteristic variations revealed in the present study may be novel candidates related to dnTMA in Japanese or Asian patients, but not pathogenic variants of aHUS. Future studies on genetic and antigenic factors are needed to identify factors contributing to dnTMA after KTx.

Alzheimer's disease-associated complement gene variants influence plasma complement protein levels.

BACKGROUND: Alzheimer's disease (AD) has been associated with immune dysregulation in biomarker and genome-wide association studies (GWAS). GWAS hits include the genes encoding complement regulators clusterin (CLU) and complement receptor 1 (CR1), recognised as key players in AD pathology, and complement proteins have been proposed as biomarkers. MAIN BODY: To address whether changes in plasma complement protein levels in AD relate to AD-associated complement gene variants we first measured relevant plasma complement proteins (clusterin, C1q, C1s, CR1, factor H) in a large cohort comprising early onset AD (EOAD; n = 912), late onset AD (LOAD; n = 492) and control (n = 504) donors. Clusterin and C1q were significantly increased (p < 0.001) and sCR1 and factor H reduced (p < 0.01) in AD plasma versus controls. ROC analyses were performed to assess utility of the measured complement biomarkers, alone or in combination with amyloid beta, in predicting AD. C1q was the most predictive single complement biomarker (AUC 0.655 LOAD, 0.601 EOAD); combining C1q with other complement or neurodegeneration makers through stepAIC-informed models improved predictive values slightly. Effects of GWS SNPs (rs6656401, rs6691117 in CR1; rs11136000, rs9331888 in CLU; rs3919533 in C1S) on protein concentrations were assessed by comparing protein levels in carriers of the minor vs major allele. To identify new associations between SNPs and changes in plasma protein levels, we performed a GWAS combining genotyping data in the cohort with complement protein levels as endophenotype. SNPs in CR1 (rs6656401), C1S (rs3919533) and CFH (rs6664877) reached significance and influenced plasma levels of the corresponding protein, whereas SNPs in CLU did not influence clusterin levels. CONCLUSION: Complement dysregulation is evident in AD and may contribute to pathology. AD-associated SNPs in CR1, C1S and CFH impact plasma levels of the encoded proteins, suggesting a mechanism for impact on disease risk.

Structural evolution of an immune evasion determinant shapes pathogen host tropism.

Modern infectious disease outbreaks often involve changes in host tropism, the preferential adaptation of pathogens to specific hosts. The Lyme disease-causing bacterium Borrelia burgdorferi (Bb) is an ideal model to investigate the molecular mechanisms of host tropism, because different variants of these tick-transmitted bacteria are distinctly maintained in rodents or bird reservoir hosts. To survive in hosts and escape complement-mediated immune clearance, Bb produces the outer surface protein CspZ that binds the complement inhibitor factor H (FH) to facilitate bacterial dissemination in vertebrates. Despite high sequence conservation, CspZ variants differ in human FH-binding ability. Together with the FH polymorphisms between vertebrate hosts, these findings suggest that minor sequence variation in this bacterial outer surface protein may confer dramatic differences in host-specific, FH-binding-mediated infectivity. We tested this hypothesis by determining the crystal structure of the CspZ-human FH complex, and identifying minor variation localized in the FH-binding interface yielding bird and rodent FH-specific binding activity that impacts infectivity. Swapping the divergent region in the FH-binding interface between rodent- and bird-associated CspZ variants alters the ability to promote rodent- and bird-specific early-onset dissemination. We further linked these loops and respective host-specific, complement-dependent phenotypes with distinct CspZ phylogenetic lineages, elucidating evolutionary mechanisms driving host tropism emergence. Our multidisciplinary work provides a novel molecular basis for how a single, short protein motif could greatly modulate pathogen host tropism.

Balancing selection on the complement system of a wild rodent.

BACKGROUND: Selection pressure exerted by pathogens can influence patterns of genetic diversity in the host. In the immune system especially, numerous genes encode proteins involved in antagonistic interactions with pathogens, paving the way for coevolution that results in increased genetic diversity as a consequence of balancing selection. The complement system is a key component of innate immunity. Many complement proteins interact directly with pathogens, either by recognising pathogen molecules for complement activation, or by serving as targets of pathogen immune evasion mechanisms. Complement genes can therefore be expected to be important targets of pathogen-mediated balancing selection, but analyses of such selection on this part of the immune system have been limited. RESULTS: Using a population sample of whole-genome resequencing data from wild bank voles (n = 31), we estimated the extent of genetic diversity and tested for signatures of balancing selection in multiple complement genes (n = 44). Complement genes showed higher values of standardised ß (a statistic expected to be high under balancing selection) than the genome-wide average of protein coding genes. One complement gene, FCNA, a pattern recognition molecule that interacts directly with pathogens, was found to have a signature of balancing selection, as indicated by the Hudson-Kreitman-Aguadé test (HKA) test. Scans for localised signatures of balancing selection in this gene indicated that the target of balancing selection was found in exonic regions involved in ligand binding. CONCLUSION: The present study adds to the growing evidence that balancing selection may be an important evolutionary force on components of the innate immune system. The identified target in the complement system typifies the expectation that balancing selection acts on genes encoding proteins involved in direct interactions with pathogens.

Complement Activation by Adeno-Associated Virus-Neutralizing Antibody Complexes.

Treatment of monogenetic disorders using vectors based on adeno-associated virus (AAV) is an area of intense interest. AAV is non-pathogenic human virus, and preexisting capsid antibodies are prevalent in the population posing a challenge to the safety and efficacy of AAV-mediated gene therapies. In this study, we investigated the risk of AAV-mediated complement activation when sera from a cohort of human donors were exposed to AAV9 capsid. Seropositive donor sera carrying neutralizing antibodies from a previous environmental exposure activated complement when admixed with AAV9 capsids and complement activation was associated with donors who had higher levels of anti-AAV IgG1 antibodies. These findings were consistent with mass spectrometry analysis that identified increased binding of immunoglobulins and complement factors when AAV9 capsids were admixed with seropositive sera. Finally, complement activation was abrogated after IgG-depletion using affinity columns or serum pretreatment with an IgG degrading enzyme. Overall, these results demonstrate an important role of preexisting neutralizing antibodies in activating complement; a risk that can be mitigated by using adequate immunosuppression strategies when dosing seropositive patients with vector.

Combined Heterozygous Genetic Variations in Complement C2 and C8B: An Explanation for Multidimensional Immune Imbalance?

The complement system plays a crucial role in host defense, homeostasis, and tissue regeneration and bridges the innate and the adaptive immune systems. Although the genetic variants in complement C2 (c.839_849+17del; p.(Met280Asnfs*5)) and C8B (c.1625C>T; p.(Thr542Ile)) are known individually, here, we report on a patient carrying their combination in a heterozygous form. The patient presented with a reduced general condition and suffers from a wide variety of autoimmune diseases. While no autoimmune disease-specific autoantibodies could be detected, genetic analysis revealed abnormalities in the two complement genes C2 and C8B. Therefore, we performed a comprehensive investigation of the innate immune system on a cellular and humoral level to define the functional consequences. We found slightly impaired functionality of neutrophils and monocytes regarding phagocytosis and reactive oxygen species generation and a diminished expression of the C5aR1. An extensive complement analysis revealed a declined activation potential for the alternative and classical pathway. Reconstitution with purified C2 and C8 into patient serum failed to normalize the dysfunction, whereas the addition of C3 improved the hemolytic activity. In clinical transfer, in vitro supplementation of the patient's plasma with FFP as a complement source could fully restore full complement functionality. This study describes for the first time a combined heterozygous genetic variation in complement C2 and C8B which, however, cannot fully explain the overall dysfunctions and calls for further complement deficiency research and corresponding therapies.

CFH-CFHR1 hybrid genes in two cases of atypical hemolytic uremic syndrome.

Atypical hemolytic uremic syndrome (aHUS) is a rare complement-mediated disease that manifests as the triad of thrombotic microangiopathy. We identified two aHUS patients with neither anti-complement factor H (CFH) antibodies nor causative variants of seven aHUS-related genes (CFH, CFI, CFB, C3, MCP, THBD, and DGKE); however, their plasma showed increased levels of hemolysis by hemolytic assay, which strongly suggests CFH-related abnormalities. Using a copy number variation (CNV) analysis of the CFH/CFHR gene cluster, we identified CFH-CFHR1 hybrid genes in these patients. We verified the absence of aHUS-related abnormal CNVs of the CFH gene in control genomes of 2036 individuals in the general population, which suggests that pathogenicity is related to these hybrid genes. Our study emphasizes that, for patients suspected of having aHUS, it is important to perform an integrated analysis based on a clinical examination, functional analysis, and detailed genetic investigation.

An ex vivo test to investigate genetic factors conferring susceptibility to atypical haemolytic uremic syndrome.

Introduction: Comprehensive genetic analysis is essential to clinical care of patients with atypical haemolytic uremic syndrome (aHUS) to reinforce diagnosis, and to guide treatment. However, the characterization of complement gene variants remains challenging owing to the complexity of functional studies with mutant proteins. This study was designed: 1) To identify a tool for rapid functional determination of complement gene variants; 2) To uncover inherited complement dysregulation in aHUS patients who do not carry identified gene variants. Methods: To address the above goals, we employed an ex-vivo assay of serum-induced C5b-9 formation on ADP-activated endothelial cells in 223 subjects from 60 aHUS pedigrees (66 patients and 157 unaffected relatives). Results: Sera taken from all aHUS patients in remission induced more C5b-9 deposition than control sera, independently from the presence of complement gene abnormalities. To avoid the possible confounding effects of chronic complement dysregulation related to aHUS status, and considering the incomplete penetrance for all aHUS-associated genes, we used serum from unaffected relatives. In control studies, 92.7% of unaffected relatives with known pathogenic variants exhibited positive serum-induced C5b-9 formation test, documenting a high sensitivity of the assay to identify functional variants. The test was also specific, indeed it was negative in all non-carrier relatives and in relatives with variants non-segregating with aHUS. All but one variants in aHUS-associated genes predicted in-silico as likely pathogenic or of uncertain significance (VUS) or likely benign resulted as pathogenic in the C5b-9 assay. At variance, variants in putative candidate genes did not exhibit a functional effect, with the exception of a CFHR5 variant. The C5b-9 assay in relatives was helpful in defining the relative functional effect of rare variants in 6 pedigrees in which the proband carried more than one genetic abnormality. Finally, for 12 patients without identified rare variants, the C5b-9 test in parents unmasked a genetic liability inherited from an unaffected parent. Discussion: In conclusion, the serum-induced C5b-9 formation test in unaffected relatives of aHUS patients may be a tool for rapid functional evaluation of rare complement gene variants. When combined with exome sequencing the assay might be of help in variant selection, to identify new aHUS-associated genetic factors.