Genetic variability shapes the alternative pathway complement activity and predisposition to complement-related diseases.

The implementation of next-generation sequencing technologies has provided a sharp picture of the genetic variability in the components and regulators of the alternative pathway (AP) of the complement system and has revealed the association of many AP variants with different rare and common diseases. An important finding that has emerged from these analyses is that each of these complement-related diseases associate with genetic variants altering specific aspects of the activation and regulation of the AP. These genotype-phenotype correlations have provided valuable insights into their pathogenic mechanisms with important diagnostic and therapeutic implications. While genetic variants in coding regions and structural variants are reasonably well characterized and occasionally have been instrumental to uncover unknown features of the complement proteins, data about complement expressed quantitative trait loci are still very limited. A crucial task for future studies will be to identify these quantitative variations and to determine their impact in the overall activity of the AP. This is fundamental as it is now clear that the consequences of genetic variants in the AP are additive and that susceptibility or resistance to disease is the result of specific combinations of genetic variants in different complement components and regulators ("complotypes").

Functional characterization of 105 factor H variants associated with aHUS: lessons for variant classification.

Atypical hemolytic uremic syndrome (aHUS) is a life-threatening thrombotic microangiopathy that can progress, when untreated, to end-stage renal disease. Most frequently, aHUS is caused by complement dysregulation due to pathogenic variants in genes that encode complement components and regulators. Among these genes, the factor H (FH) gene, CFH, presents with the highest frequency (15% to 20%) of variants and is associated with the poorest prognosis. Correct classification of CFH variants as pathogenic or benign is essential to clinical care but remains challenging owing to the dearth of functional studies. As a result, significant numbers of variants are reported as variants of uncertain significance. To address this knowledge gap, we expressed and functionally characterized 105 aHUS-associated FH variants. All FH variants were categorized as pathogenic or benign and, for each, we fully documented the nature of the pathogenicity. Twenty-six previously characterized FH variants were used as controls to validate and confirm the robustness of the functional assays used. Of the remaining 79 uncharacterized variants, only 29 (36.7%) alter FH expression or function in vitro and, therefore, are proposed to be pathogenic. We show that rarity in control databases is not informative for variant classification, and we identify important limitations in applying prediction algorithms to FH variants. Based on structural and functional data, we suggest ways to circumvent these difficulties and, thereby, improve variant classification. Our work highlights the need for functional assays to interpret FH variants accurately if clinical care of patients with aHUS is to be individualized and optimized.

Molecular bases for the association of FHR-1 with atypical hemolytic uremic syndrome and other diseases.

Factor H (FH)-related proteins are a group of partly characterized complement proteins thought to promote complement activation by competing with FH in binding to surface-bound C3b. Among them, FH-related protein 1 (FHR-1) is remarkable because of its association with atypical hemolytic uremic syndrome (aHUS) and other important diseases. Using a combination of biochemical, immunological, nuclear magnetic resonance, and computational approaches, we characterized a series of FHR-1 mutants (including 2 associated with aHUS) and unraveled the molecular bases of the so-called deregulation activity of FHR-1. In contrast with FH, FHR-1 lacks the capacity to bind sialic acids, which prevents C3b-binding competition between FH and FHR-1 in host-cell surfaces. aHUS-associated FHR-1 mutants are pathogenic because they have acquired the capacity to bind sialic acids, which increases FHR-1 avidity for surface-bound C3-activated fragments and results in C3b-binding competition with FH. FHR-1 binds to native C3, in addition to C3b, iC3b, and C3dg. This unexpected finding suggests that the mechanism by which surface-bound FHR-1 promotes complement activation is the attraction of native C3 to the cell surface. Although C3b-binding competition with FH is limited to aHUS-associated mutants, all surface-bound FHR-1 promotes complement activation, which is delimited by the FHR-1/FH activity ratio. Our data indicate that FHR-1 deregulation activity is important to sustain complement activation and C3 deposition at complement-activating surfaces. They also support that abnormally elevated FHR-1/FH activity ratios would perpetuate pathological complement dysregulation at complement-activating surfaces, which may explain the association of FHR-1 quantitative variations with diseases.

Thrombotic microangiopathy in patients with malignant hypertension.

BACKGROUND: Thrombotic microangiopathy (TMA) is a complication of malignant hypertension (mHTN) attributed to high blood pressure (BP). However, no studies have investigated in patients with mHTN of different aetiologies whether the presence of TMA is associated with specific causes of mHTN. METHODS: We investigated the presence of TMA (microangiopathic haemolytic anaemia and thrombocytopenia) in a large and well-characterized cohort of 199 patients with mHTN of different aetiologies [primary HTN 44%, glomerular diseases 16.6%, primary atypical haemolytic uraemic syndrome (aHUS) 13.1%, renovascular HTN 9.5%, drug-related HTN 7%, systemic diseases 5.5%, endocrine diseases 4.5%]. Outcomes of the study were kidney recovery and kidney failure. RESULTS: Patients with TMA [40 cases (20.1%)] were younger, were more likely female and had lower BP levels and worse kidney function at presentation. Their underlying diseases were primary aHUS (60%), drug-related mHTN (15%), glomerular diseases [all of them immunoglobulin A nephropathy (IgAN); 10%], systemic diseases (10%) and primary HTN (5%). The presence of TMA was 92.3% in primary aHUS, 42.9% in drug-related HTN, 36.4% in systemic diseases, 12.1% in glomerular diseases and 2.3% in primary HTN. No patient with renovascular HTN or mHTN caused by endocrine diseases developed TMA, despite BP levels as high as patients with TMA. A higher proportion of TMA patients developed kidney failure as compared with patients without TMA (56.4% versus 38.9%, respectively). CONCLUSIONS: The presence of TMA in patients with mHTN should guide the diagnosis towards primary aHUS, drug-related mHTN, some systemic diseases and IgAN, while it is exceptional in other causes of mHTN.

C4BP(ß-)-mediated immunomodulation attenuates inflammation in DSS-induced murine colitis and in myeloid cells from IBD patients.

The most recent and promising therapeutic strategies for inflammatory bowel disease (IBD) have engaged biologics targeting single effector components involved in major steps of the immune-inflammatory processes, such as tumor necrosis factor, interleukins or integrins. Nevertheless, these molecules have not yet met expectations regarding efficacy and safety, resulting in a significant percentage of refractory or relapsing patients. Thus, novel treatment options are urgently needed. The minor isoform of the complement inhibitor C4b-binding protein, C4BP(ß-), has been shown to confer a robust anti-inflammatory and immunomodulatory phenotype over inflammatory myeloid cells. Here we show that C4BP(ß-)-mediated immunomodulation can significantly attenuate the histopathological traits and preserve the intestinal epithelial integrity in dextran sulfate sodium (DSS)-induced murine colitis. C4BP(ß-) downregulated inflammatory transcripts, notably those related to neutrophil activity, mitigated circulating inflammatory effector cytokines and chemokines such as CXCL13, key in generating ectopic lymphoid structures, and, overall, prevented inflammatory immune cell infiltration in the colon of colitic mice. PRP6-HO7, a recombinant curtailed analogue with only immunomodulatory activity, achieved a similar outcome as C4BP(ß-), indicating that the therapeutic effect is not due to the complement inhibitory activity. Furthermore, both C4BP(ß-) and PRP6-HO7 significantly reduced, with comparable efficacy, the intrinsic and TLR-induced inflammatory markers in myeloid cells from both ulcerative colitis and Crohn's disease patients, regardless of their medication. Thus, the pleiotropic anti-inflammatory and immunomodulatory activity of PRP6-HO7, able to "reprogram" myeloid cells from the complex inflammatory bowel environment and to restore immune homeostasis, might constitute a promising therapeutic option for IBD.

Factor H-Related Protein 1 Drives Disease Susceptibility and Prognosis in C3 Glomerulopathy.

BACKGROUND: C3 glomerulopathy (C3G) is a heterogeneous group of chronic renal diseases characterized predominantly by glomerular C3 deposition and complement dysregulation. Mutations in factor H-related (FHR) proteins resulting in duplicated dimerization domains are prototypical of C3G, although the underlying pathogenic mechanism is unclear. METHODS: Using in vitro and in vivo assays, we performed extensive characterization of an FHR-1 mutant with a duplicated dimerization domain. To assess the FHR-1 mutant's association with disease susceptibility and renal prognosis, we also analyzed CFHR1 copy number variations and FHR-1 plasma levels in two Spanish C3G cohorts and in a control population. RESULTS: Duplication of the dimerization domain conferred FHR-1 with an increased capacity to interact with C3-opsonized surfaces, which resulted in an excessive activation of the alternative pathway. This activation does not involve C3b binding competition with factor H. These findings support a scenario in which mutant FHR-1 binds to C3-activated fragments and recruits native C3 and C3b; this leads to formation of alternative pathway C3 convertases, which increases deposition of C3b molecules, overcoming FH regulation. This suggests that a balanced FHR-1/FH ratio is crucial to control complement amplification on opsonized surfaces. Consistent with this conceptual framework, we show that the genetic deficiency of FHR-1 or decreased FHR-1 in plasma confers protection against developing C3G and associates with better renal outcome. CONCLUSIONS: Our findings explain how FHR-1 mutants with duplicated dimerization domains result in predisposition to C3G. They also provide a pathogenic mechanism that may be shared by other diseases, such as IgA nephropathy or age-related macular degeneration, and identify FHR-1 as a potential novel therapeutic target in C3G.

Intracellular complement activation sustains T cell homeostasis and mediates effector differentiation.

Complement is viewed as a critical serum-operative component of innate immunity, with processing of its key component, C3, into activation fragments C3a and C3b confined to the extracellular space. We report here that C3 activation also occurred intracellularly. We found that the T cell-expressed protease cathepsin L (CTSL) processed C3 into biologically active C3a and C3b. Resting T cells contained stores of endosomal and lysosomal C3 and CTSL and substantial amounts of CTSL-generated C3a. While "tonic" intracellular C3a generation was required for homeostatic T cell survival, shuttling of this intracellular C3-activation-system to the cell surface upon T cell stimulation induced autocrine proinflammatory cytokine production. Furthermore, T cells from patients with autoimmune arthritis demonstrated hyperactive intracellular complement activation and interferon-γ production and CTSL inhibition corrected this deregulated phenotype. Importantly, intracellular C3a was observed in all examined cell populations, suggesting that intracellular complement activation might be of broad physiological significance.

Longitudinal change in proteinuria and kidney outcomes in C3 glomerulopathy.

INTRODUCTION: The association between a change in proteinuria over time and its impact on kidney prognosis has not been analysed in complement component 3 (C3) glomerulopathy. This study aims to investigate the association between the longitudinal change in proteinuria and the risk of kidney failure. METHODS: This was a retrospective, multicentre observational cohort study in 35 nephrology departments belonging to the Spanish Group for the Study of Glomerular Diseases. Patients diagnosed with C3 glomerulopathy between 1995 and 2020 were enrolled. A joint modelling of linear mixed-effects models was applied to assess the underlying trajectory of a repeatedly measured proteinuria, and a Cox model to evaluate the association of this trajectory with the risk of kidney failure. RESULTS: The study group consisted of 85 patients, 70 C3 glomerulonephritis and 15 dense deposit disease, with a median age of 26 years (range 13-41). During a median follow-up of 42 months, 25 patients reached kidney failure. The longitudinal change in proteinuria showed a strong association with the risk of this outcome, with a doubling of proteinuria levels resulting in a 2.5-fold increase of the risk. A second model showed that a ≥50% proteinuria reduction over time was significantly associated with a lower risk of kidney failure (hazard ratio 0.79; 95% confidence interval 0.56-0.97; P < 0.001). This association was also found when the ≥50% proteinuria reduction was observed within the first 6 and 12 months of follow-up. CONCLUSIONS: The longitudinal change in proteinuria is strongly associated with the risk of kidney failure. The change in proteinuria over time can provide clinicians a dynamic prediction of kidney outcomes.

Complement Factor D (adipsin) Levels Are Elevated in Acquired Partial Lipodystrophy (Barraquer-Simons syndrome).

Complement overactivation has been reported in most patients with Barraquer-Simons syndrome (BSS), a rare form of acquired partial lipodystrophy. Complement Factor D (FD) is a serine protease with a crucial role in the activation of the alternative pathway of the complement system, which is mainly synthesized by adipose tissue. However, its role in the pathogenesis of BSS has not been addressed. In this study, plasma FD concentration was measured in 13 patients with BSS, 20 patients with acquired generalized lipodystrophy, 22 patients with C3 glomerulopathy (C3G), and 50 healthy controls. Gene expression and immunohistochemistry studies were assayed using atrophied adipose tissue from a patient with BSS. We found significantly elevated FD levels in BSS cases compared with the remaining cohorts (p < 0.001). There were no significant differences in FD levels between sexes but FD was strongly and directly associated with age in BSS (r = 0.7593, p = 0.0036). A positive correlation between FD and C3 was seen in patients with C3G, characterized by decreased FD levels due to chronic C3 consumption, but no correlation was detected for BSS. Following mRNA quantification in the patient's adipose tissue, we observed decreased CFD and C3 but elevated C5 transcript levels. In contrast, the increased FD staining detected in the atrophied areas reflects the effects of persistent tissue damage on the adipose tissue, thus providing information on the ongoing pathogenic process. Our results suggest that FD could be a reliable diagnostic biomarker involved in the pathophysiology of BSS by promoting unrestrained local complement system activation in the adipose tissue environment.

Noncanonical immunomodulatory activity of complement regulator C4BP(ß-) limits the development of lupus nephritis.

Lupus nephritis is a chronic autoimmune-inflammatory condition that can lead to end-stage kidney disease. Presently available immunosuppressive treatments for lupus nephritis are suboptimal and can induce significant side effects. Recently, we characterized a novel immunomodulatory activity of the minor isoform of the classical pathway complement inhibitor, C4BP(ß-). We show here that C4BP(ß-) treatment prevented the development of proteinuria and albuminuria, decreased significantly the formation of anti-dsDNA antibodies and, locally, mitigated renal glomerular IgG and C3 deposition and generation of apoptotic cells. There was a consequent histological improvement and increased survival in lupus-prone mice. The therapeutic efficacy of C4BP(ß-) was analogous to that of the broad-acting immunosuppressant cyclophosphamide. Remarkably, a comparative transcriptional profiling analysis revealed that the kidney gene expression signature resulting from C4BP(ß-) treatment turned out to be 10 times smaller than that induced by cyclophosphamide treatment. C4BP(ß-) immunomodulation induced significant downregulation of transcripts relevant to lupus nephritis indicating immunopathogenic cell infiltration, including activated T cells (Lat), B cells (Cd19, Ms4a1, Tnfrsf13c), inflammatory phagocytes (Irf7) and neutrophils (Prtn3, S100a8, S100a9). Furthermore, cytokine profiling and immunohistochemistry confirmed that C4BP(ß-), through systemic and local CXCL13 downregulation, was able to prevent ectopic lymphoid structures neogenesis in aged mice with lupus nephritis. Thus, due to its anti-inflammatory and immunomodulatory activities and high specificity, C4BP(ß-) could be considered for further clinical development in patients with systemic lupus erythematosus.

Absence of CD59 in Guinea Pigs: Analysis of the Cavia porcellus Genome Suggests the Evolution of a CD59 Pseudogene.

CD59 is a membrane-bound regulatory protein that inhibits the assembly of the terminal membrane attack complex (C5b-9) of complement. From its original discovery in humans almost 30 years ago, CD59 has been characterized in a variety of species, from primates to early vertebrates, such as teleost fish. CD59 is ubiquitous in mammals; however, we have described circumstantial evidence suggesting that guinea pigs (Cavia porcellus) lack CD59, at least on erythrocytes. In this study, we have used a combination of phylogenetic analyses with syntenic alignment of mammalian CD59 genes to identify the only span of genomic DNA in C. porcellus that is homologous to a portion of mammalian CD59 and show that this segment of DNA is not transcribed. We describe a pseudogene sharing homology to exons 2 through 5 of human CD59 present in the C. porcellus genome. This pseudogene was flanked by C. porcellus homologs of two genes, FBXO3 and ORF91, a relationship and orientation that were consistent with other known mammalian CD59 genes. Analysis using RNA sequencing confirmed that this segment of chromosomal DNA was not transcribed. We conclude that guinea pigs lack an intact gene encoding CD59; to our knowledge, this is the first report of a mammalian species that does not express a functional CD59. The pseudogene we describe is likely the product of a genomic deletion event during its evolutionary divergence from other members of the rodent order.

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.

Secondary atypical hemolytic uremic syndromes in the era of complement blockade.

Le Clech et al. found rare variants in complement genes in only 5% of patients among a large cohort of secondary hemolytic uremic syndrome cases. The absence of genetic abnormalities does not exclude, however, the implication of complement in the pathogenesis of secondary hemolytic uremic syndrome. Prospective studies should be performed to evaluate the efficacy of complement blockers in patients with secondary atypical hemolytic uremic syndrome who do not respond to the treatment of the triggering condition.

Severe and malignant hypertension are common in primary atypical hemolytic uremic syndrome.

Malignant hypertension is listed among the causes of secondary thrombotic microangiopathy, but pathogenic mutations in complement genes have been reported in patients with hypertension-induced thrombotic microangiopathy. Here we investigated the frequency and severity of hypertension in 55 patients with primary atypical hemolytic uremic syndrome (aHUS). A genetic analysis was performed in all patients, and funduscopic examination was performed in all the patients with Grades 2 and 3 hypertension. A cohort of 110 patients with malignant hypertension caused by diseases other than aHUS served as control. Thirty-six patients with aHUS presented Grade 2 or Grade 3 hypertension and funduscopic examination showed malignant hypertension in 19. Genetic abnormalities in complement were found in 19 patients (37% among patients with malignant hypertension). Plasmapheresis was performed in 46 patients and 26 received eculizumab. Renal and hematological responses were significantly lower after plasmapheresis (24%) than after eculizumab (81%). Renal survival was significantly higher in patients treated with eculizumab (85% at one, three and five years) compared to patients who did not receive this treatment (54%, 46% and 41%), respectively. Response to eculizumab was independent of hypertension severity and the presence of complement genetic abnormalities. Among patients with malignant hypertension caused by other diseases the prevalence of thrombotic microangiopathy was very low (5%). Thus, severe and malignant hypertension are common among patients with aHUS and eculizumab treatment leads to a higher renal survival when compared to plasmapheresis. However, thrombotic microangiopathy is uncommon among patients presenting with malignant hypertension caused by diseases other than aHUS.

FHR-1 Binds to C-Reactive Protein and Enhances Rather than Inhibits Complement Activation.

Factor H-related protein (FHR) 1 is one of the five human FHRs that share sequence and structural homology with the alternative pathway complement inhibitor FH. Genetic studies on disease associations and functional analyses indicate that FHR-1 enhances complement activation by competitive inhibition of FH binding to some surfaces and immune proteins. We have recently shown that FHR-1 binds to pentraxin 3. In this study, our aim was to investigate whether FHR-1 binds to another pentraxin, C-reactive protein (CRP), analyze the functional relevance of this interaction, and study the role of FHR-1 in complement activation and regulation. FHR-1 did not bind to native, pentameric CRP, but it bound strongly to monomeric CRP via its C-terminal domains. FHR-1 at high concentration competed with FH for CRP binding, indicating possible complement deregulation also on this ligand. FHR-1 did not inhibit regulation of solid-phase C3 convertase by FH and did not inhibit terminal complement complex formation induced by zymosan. On the contrary, by binding C3b, FHR-1 allowed C3 convertase formation and thereby enhanced complement activation. FHR-1/CRP interactions increased complement activation via the classical and alternative pathways on surfaces such as the extracellular matrix and necrotic cells. Altogether, these results identify CRP as a ligand for FHR-1 and suggest that FHR-1 enhances, rather than inhibits, complement activation, which may explain the protective effect of FHR-1 deficiency in age-related macular degeneration.

Factor H Competitor Generated by Gene Conversion Events Associates with Atypical Hemolytic Uremic Syndrome.

Atypical hemolytic uremic syndrome (aHUS), a rare form of thrombotic microangiopathy caused by complement pathogenic variants, mainly affects the kidney microvasculature. A retrospective genetic analysis in our aHUS cohort (n=513) using multiple ligation probe amplification uncovered nine unrelated patients carrying a genetic abnormality in the complement factor H related 1 gene (CFHR1) that originates by recurrent gene conversion events between the CFH and CFHR1 genes. The novel CFHR1 mutants encode an FHR-1 protein with two amino acid substitutions, L290S and A296V, converting the FHR-1 C terminus into that of factor H (FH). Next-generation massive-parallel DNA sequencing (NGS) analysis did not detect these genetic abnormalities. In addition to the CFHR1 mutant, six patients carried the previously uncharacterized CFH-411T variant. In functional analyses, the mutant FHR-1 protein strongly competed the binding of FH to cell surfaces, impairing complement regulation, whereas the CFH-411T polymorphism lacked functional consequences. Carriers of the CFHR1 mutation presented with severe aHUS during adulthood; 57% of affected women in this cohort presented during the postpartum period. Analyses in patients and unaffected carriers showed that FH plasma levels determined by the nonmutated chromosome modulate disease penetrance. Crucially, in the activated endothelial (HMEC-1) cell assay, reduced FH plasma levels produced by the nonmutated chromosome correlated inversely with impairment of complement regulation, measured as C5b-9 deposition. Our data advance understanding of the genetic complexities underlying aHUS, illustrate the importance of performing functional analysis, and support the use of complementary assays to disclose genetic abnormalities not revealed by current NGS analysis.

Complete functional characterization of disease-associated genetic variants in the complement factor H gene.

Genetic analyses in atypical hemolytic uremic syndrome (aHUS) and C3-glomerulopathy (C3G) patients have provided an excellent understanding of the genetic component of the disease and informed genotype-phenotype correlations supporting an individualized approach to patient management and treatment. In this context, a correct categorization of the disease-associated gene variants is critical to avoid detrimental consequences for patients and their relatives. Here we describe a comprehensive procedure to measure levels and functional activity of complement regulator factor H (FH) encoded by CFH, the commonest genetic factor associated with aHUS and C3G, and present the results of the analysis of 28 uncharacterized, disease-associated, FH variants. Sixteen variants were not expressed in plasma and eight had significantly reduced functional activities that impact on complement regulation. In total, 24 of 28 CFH variants were unambiguously categorized as pathogenic and the nature of the pathogenicity fully documented for each. The data also reinforce the genotype-phenotype correlations that associate specific FH functional alterations with either aHUS or C3G and illustrate important drawbacks of the prediction algorithms dealing with variants located in FH functional regions. We also report that the novel aHUS-associated M823T variant is functionally impaired. This was unexpected and uncovered the important contribution of regions outside the N-terminal and C-terminal functional domains to FH regulatory activities on surfaces. Thus, our work significantly advances knowledge towards a complete functional understanding of the CFH genetic variability and highlights the importance of functional analysis of the disease-associated CFH variants.

A retrospective study of pregnancy-associated atypical hemolytic uremic syndrome.

Pregnancy-associated atypical hemolytic uremic syndrome (aHUS) refers to the thrombotic microangiopathy resulting from uncontrolled complement activation during pregnancy or the postpartum period. Pregnancy-associated aHUS is a devastating disease for which there is a limited clinical understanding and treatment experience. Here we report a retrospective study to analyze the clinical and prognostic data of 22 cases of pregnancy-associated aHUS from the Spanish aHUS Registry under different treatments. Sixteen patients presented during the first pregnancy and as many as nine patients required hemodialysis at diagnosis. Identification of inherited complement abnormalities explained nine of the 22 cases, with CFH mutations and CFH to CFHR1 gene conversion events being the most prevalent genetic alterations associated with this disorder (66%). In thirteen of the cases, pregnancy complications were sufficient to trigger a thrombotic microangiopathy in the absence of genetic or acquired complement alterations. The postpartum period was the time with highest risk to develop the disease and the group shows an association of cesarean section with pregnancy-associated aHUS. Seventeen patients underwent plasma treatments with a positive renal response in only three cases. In contrast, ten patients received eculizumab with an excellent renal response in all, independent of carrying or not inherited complement abnormalities. Although the cohort is relatively small, the data suggest that pregnancy-associated aHUS is not different from other types of aHUS and suggest the efficacy of eculizumab treatment over plasma therapies. This study may be useful to improve prognosis in this group of aHUS patients.

Functional and structural characterization of four mouse monoclonal antibodies to complement C3 with potential therapeutic and diagnostic applications.

C3 is the central component of the complement system. Upon activation, C3 sequentially generates various proteolytic fragments, C3a, C3b, iC3b, C3dg, each of them exposing novel surfaces, which are sites of interaction with other proteins. C3 and its fragments are therapeutic targets and markers of complement activation. We report the structural and functional characterization of four monoclonal antibodies (mAbs) generated by immunizing C3-deficient mice with a mixture of human C3b, iC3b and C3dg fragments, and discuss their potential applications. This collection includes three mAbs interacting with native C3 and inhibiting AP complement activation; two of them by blocking the cleavage of C3 by the AP C3-converase and one by impeding formation of the AP C3-convertase. The interaction sites of these mAbs in the target molecules were determined by resolving the structures of Fab fragments bound to C3b and/or iC3b using electron microscopy. A fourth mAb specifically recognizes the iC3b, C3dg, and C3d fragments. It binds to an evolutionary-conserved neoepitope generated after C3b cleavage by FI, detecting iC3b/C3dg deposition over opsonized surfaces by flow cytometry and immunohistochemistry in human and other species. Because well-characterized anti-complement mAbs are uncommon, the mAbs reported here may offer interesting therapeutic and diagnostic opportunities.