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27 p.-7 fig.-7 tab. Campistol, Josep M. et al. ; [EN]Haemolytic uraemic syndrome (HUS) is a clinical entity defined as the triad of nonimmune haemolytic anaemia, thrombocytopenia, and acute renal failure, in which the underlying lesions are mediated by systemic thrombotic microangiopathy (TMA). Different causes can induce the TMA process that characterises HUS. In this document we consider atypical HUS (aHUS) a sub-type of HUS in which the TMA phenomena are the consequence of the endotelial damage in the microvasculature of the kidneys and other organs due to a disregulation of the activity of the complement system. In recent years, a variety of aHUs-related mutations have been identified in genes of the complement system, which can explain approximately 60% of the aHUS cases, and a number of mutations and polymorphisms have been functionally characterised. These findings have stablished that aHUS is a consequence of the insufficient regulation of the activation of the complement on cell surfaces, leading to endotelial damage mediated by C5 and the complement terminal pathway. Eculizumab is a monoclonal antibody that inhibits the activation of C5 and blocks the generation of the pro-inflammatory molecule C5a and the formation of the cell membrane attack complex. In prospective studies in patients with aHUS, the use of Eculizumab has shown a fast and sustained interruption of the TMA process and it has been associated with significative long-term improvements in renal function, the interruption of plasma therapy and important reductions in the need of dialysis. According to the existing literature and the accumulated clinical experience, the Spanish aHUS Group published a consensus document with recommendations for the treatment of aHUs (Nefrologia 2013;33[1]:27–45). In the current online version of this document, we update the aetiological classification of TMAs, the pathophysiology of aHUS, its differential diagnosis and its therapeutic management. ; [ES]El síndrome hemolítico urémico (SHU) es una entidad clínica definida por la tríada anemia hemolítica no inmune, trombocitopenia e insuficiencia renal aguda, en la que las lesiones subyacentes están mediadas por un proceso de microangiopatía trombótica (MAT) sistémico. Distintas causas pueden desencadenar el proceso de MAT que caracteriza el SHU. En este documento consideramos SHU atípico (SHUa) como el subtipo de SHU en el que los fenómenos de MAT son fundamentalmente consecuencia del daño producido en el endotelio de la microvasculatura renal y de otros órganos por desregulación de la actividad del sistema del complemento. En los últimos años se han identificado diversas mutaciones en genes del sistema del complemento asociados a SHUa, que explicarían aproximadamente el 60% de los casos de SHUa, y se han caracterizado funcionalmente numerosas mutaciones y polimorfismos asociados a SHUa que han permitido determinar que la patología se produce como consecuencia de la deficiente regulación de la activación del complemento sobre las superficies celulares y que lleva al daño endotelial mediado por la activación del C5 y de la vía terminal del complemento. Eculizumab es un anticuerpo monoclonal humanizado que inhibe la activación del C5, bloqueando la generación de la molécula proinflamatoria C5a y la formación del complejo de ataque de membrana. En estudios prospectivos en pacientes con SHUa su administración ha demostrado la interrupción rápida y sostenida del proceso de MAT, con una mejora significativa de la función renal a largo plazo y una reducción importante de la necesidad de diálisis y el cese de la terapia plasmática. En función de las evidencias científicas publicadas y la experiencia clínica acumulada, el Grupo Español de SHUa publicamos un documento de consenso con recomendaciones para el tratamiento de la enfermedad (Nefrología 2013;33(1):27–45). En la presente versión online del documento se actualizan los contenidos sobre la clasificación etiológica de las MAT, la fisiopatología del SHUa, su diagnóstico diferencial y su manejo terapéutico. ; Research carried out by Dr. Rodríguez de Córdoba has been funded by the Ministry of Economy and Competitiveness (SAF2011-26583), the Community of Madrid (S2010/BMD2316),the European Union (EURenOmics), the Fundación Renal Ínigo Álvarez de Toledo, and the CIBER of rare diseases. ; Peer reviewed

29 p.-3 fig. ; Hemolytic uremic syndrome (HUS) is a rare, life-threatening disease characterized by thrombocytopenia, microangiopathic hemolytic anemia, and acute renal failure. The atypical form of HUS (aHUS), representing 5 to 10% of cases, lacks the association with infection by Shiga toxin producing Escherichia coli strains that characterizes the commonest clinical presentation of HUS. In the majority of aHUS cases, the disease results from the complement-mediated damage to the microvascular endothelium because of inherited defects in complement genes or autoantibodies against complement regulatory proteins. Incomplete penetrance of aHUS in carriers of mutations is common to all aHUS-associated complement genes and it is now established that the overall genetic predisposition to aHUS of an individual results from the combination of different inherited factors. Moreover, the patient's genotype influences the clinical evolution, the response to plasma therapies, and the recurrence after transplantation. Here, we describe the genetic component of aHUS, the lessons that we have learned from the functional characterization of the aHUS-associated mutations, and the benefits of a comprehensive genetic analysis of the patients. © 2014 by Thieme Medical Publishers, Inc. ; The work of the authors described in this review was supported by the Spanish Ministerio de Economia y Competitividad (SAF2011-26583), the Comunidad de Madrid (S2010/BMD-2316), the Fundación Renal Iñigo Alvarez de Toledo and the Seventh Framework Programme European Union Project EURenOmics (European Consortium for High-Throughtput Research in Rare Kidney Diseases). ; Peer Reviewed

33 p.-4 fig.-2 fig. supl. ; Complement is an essential component of innate immunity. Its activation results in the assembly of unstable protease complexes, denominated C3/C5 convertases, leading to inflammation and lysis. Regulatory proteins inactivate C3/C5 convertases on host surfaces to avoid collateral tissue damage. On pathogen surfaces, properdin stabilizes C3/C5 convertases to efficiently fight infection. How properdin performs this function is, however, unclear. Using electron microscopy we show that the N-and C-terminal ends of adjacent monomers in properdin oligomers conform a curly vertex that holds together the AP convertase, interacting with both the C345C and vWA domains of C3b and Bb, respectively. Properdin also promotes a large displacement of the TED (thioestercontaining domain) and CUB (complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domains of C3b, which likely impairs C3-convertase inactivation by regulatory proteins. The combined effect of molecular cross-linking and structural reorganization increases stability of the C3 convertase and facilitates recruitment of fluid-phase C3 convertase to the cell surfaces. Our model explains how properdin mediates the assembly of stabilized C3/C5-convertase clusters, which helps to localize complement amplification to pathogen surfaces. ; This work was funded by the Autonomous Region of Madrid (S2010/BMD-2316 to S.R.d.C. and O.L.), the Ramón Areces Foundation (O.L.), and the Spanish government (SAF2011-22988 to O.L. and SAF2011-26583 to S.R.d.C.). O.L. is additionally supported by Red Temática de Investigación Cooperativa en Cáncer (RD06/0020/1001), and S.R.d.C. is also supported by the Fundación Renal Iñigo Alvarez de Toledo and the Seventh Framework Programme European Union Project EURenOmics (European Consortium for High-Throughput Research in Rare Kidney Diseases-305608). ; Peer Reviewed

27 p.-3 fig.-1 tab.-3 fig. compl. ; The complement factor H (FH) mutation R1210C, which was described in association with atypical hemolytic uremic syndrome (aHUS), also confers high risk of age-related macular degeneration (AMD) and associates with C3 glomerulopathy (C3G). To reveal the molecular basis of these associations and to provide insight into what determines the disease phenotype in FH-R1210C carriers, we identified FH-R1210C carriers in our aHUS, C3G, and AMD cohorts. Disease status, determined in patients and relatives, revealed an absence of AMD phenotypes in the aHUS cohort and, vice versa, a lack of renal disease in the AMD cohort. These findings were consistent with differences in the R1210C-independent overall risk for aHUS and AMD between mutation carriers developing one pathology or the other. R1210C is an unusual mutation that generates covalent complexes between FH and HSA. Using purified FH proteins and surface plasmon resonance analyses, we demonstrated that formation of these FH-HSA complexes impairs accessibility to all FH functional domains. These data suggest that R1210C is a unique C-terminal FH mutation that behaves as a partial FH deficiency, predisposing individuals to diverse pathologies with distinct underlying pathogenic mechanisms; the final disease outcome is then determined by R1210C-independent genetic risk factors. ; This work was supported by The Spanish "Ministerio de Economía y Competitividad" (SAF2011-26583, PI11/00898 and RETICS RD12/0034) , The Fundación Renal Íñigo Álvarez de Toledo, the Seventh Framework Programme European Union Project EURenOmics (305608) and the Autonomous Region of Madrid S2010/BMD-2316) ; Peer reviewed

30 p.-7 fig. ; 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. ; SRdeC is supported by the Spanish Ministerio de Economía y Competitividad/FEDER (grant number SAF2015-66287-R), the Seventh Framework Programme European Union Project EURenOmics (grantnumber 305608), and the Autonomous Region of Madrid (grantnumber S2010/BMD-2316). ; Peer reviewed

33 p.-3 fig. ; During the last decade, the complement field has experienced outstanding advancements in the mechanistic understanding of how complement activators are recognized, what C3 activation means, how protein complexes like the C3 convertases and the membrane attack complex are assembled, and how positive and negative complement regulators perform their function. All of this has been made possible mostly because of the contributions of structural biology to the study of the complement components. The wealth of novel structural data has frequently provided support to previously held knowledge, but often has added alternative and unexpected insights into complement function. Here we will review some of these findings focusing in the alternative and terminal complement pathways. ; SRdeC is supported by the Spanish "Ministerio de Economía y Competitividad-FEDER" (SAF2015-66287R), the Seventh Framework Programme European Union Project EURenOmics (305608) and the Autonomous Region of Madrid (S2010/BMD- 2316). SRdeC is member of the "CIB intramural Program "Molecular Machines for Better Life (MACBET)". EGdeJ is supported by the Spanish "Ministerio de Economía y Competitividad-FEDER" (RYC-2013-13395 and SAF2014-52339P). OL is supported by the Spanish Ministry of Economy, Industry and Competitiveness (SAF2014-52301-R).AT and MS are supported by the Spanish "Ministerio de Economía y Competitividad-FEDER" (IJCI-2015-25222 and IJCI-2015-24388, respectively). ; Peer reviewed

16 p.-1 tab.-1 fig. ; Background: Atypical hemolytic uremic syndrome (aHUS) is a form of thrombotic microangiopathy (TMA) caused by dysregulation of the complement system. Outcomes of kidney transplantation are poor owing to aHUS recurrence and loss of graft. Patients carrying CFH mutations or CFH/CFHR1 hybrid genes present a very high risk of recurrence despite preventive plasmapheresis. Evaluation of recent data suggests that prophylactic eculizumab pretransplant might be the preferred therapy if available. Case-diagnosis/treatment: We report 3-year follow-up data in a 9-year-old boy with aHUS and successful renal transplant treated with prophylactic eculizumab without recurrence. He presented with aHUS at age 3, irreversible renal failure and uncontrolled severe hypertension with concentric left ventricular hypertrophy, recurrent acute pulmonary edema, and congestive heart failure despite five hypotensive agents and bilateral nephrectomy. Complement analysis demonstrated the presence of a CFH/CFHR1 hybrid gene inherited from his mother and a SNP risk CFH haplotype inherited from his father. Kidney transplant was performed with prophylactic eculizumab and subsequent fortnightly administration. Three years post-transplant, graft function remains stable (serum creatinine 0.9 mg/dl), hypertension is controlled, no left ventricular hypertrophy, no opportunistic infections, and negative clinical chemistry parameters for hemolysis. Conclusion: Eculizumab is a safe and effective therapy for preventing TMA recurrence and provides long-term graft function in aHUS with the CFH/CFHR1 hybrid gene. © 2013 IPNA. ; SRdeC is supported by the Spanish Ministerio de Economía y Competitividad [Ministry of Finance and Competition] (SAF2011-26583), the Comunidad de Madrid [Autonomous Region of Madrid] (S2010/BMD-2316), the European Union FP7 (EURENOMICS) and the Fundación Renal Iñigo Alvarez de Toledo. PSC is supported by the Spanish Ministerio de Economía y Competitividad (PS09-00268), and the Comunidad de Madrid (S2010/BMD-2316).MLT is supported by the Spanish Ministerio de Economía y Competitividad (PS09- 00122), and the Comunidad de Madrid (S2010/BMD-2316). ; Peer Reviewed

Tortajada, Agustín et alt. ; C3 glomerulopathies (C3G) are a group of severe renal diseases with distinct patterns of glomerular inflammation and C3 deposition caused by complement dysregulation. Here we report the identification of a familial C3G-associated genomic mutation in the gene complement factor H-related 1 (CFHR1), which encodes FHR1. The mutation resulted in the duplication of the N-terminal short consensus repeats (SCRs) that are conserved in FHR2 and FHR5. We determined that native FHR1, FHR2, and FHR5 circulate in plasma as homo- and hetero-oligomeric complexes, the formation of which is likely mediated by the conserved N-terminal domain. In mutant FHR1, duplication of the N-terminal domain resulted in the formation of unusually large multimeric FHR complexes that exhibited increased avidity for the FHR1 ligands C3b, iC3b, and C3dg and enhanced competition with complement factor H (FH) in surface plasmon resonance (SPR) studies and hemolytic assays. These data revealed that FHR1, FHR2, and FHR5 organize a combinatorial repertoire of oligomeric complexes and demonstrated that changes in FHR oligomerization influence the regulation of complement activation. In summary, our identification and characterization of a unique CFHR1 mutation provides insights into the biology of the FHRs and contributes to our understanding of the pathogenic mechanisms underlying C3G. Copyright © 2013, American Society for Clinical Investigation. ; Work in this report was funded by the Spanish Ministerio de Economía y Competitividad (SAF2011-26583), the Fundación Renal Iñigo Alvarez de Toledo and the 7FP European Union project EURenOmics to S. Rodríguez de Córdoba; the "Ramón Areces" Foundation, the Spanish Ministerio de Economía y Competitividad (SAF2011-22988), and the "Red Temática de Investigación Cooperativa en Cáncer(RTICC)" (RD06/0020/1001) to O. Llorca; the Spanish "Ministerio de Economía y Competitividad" (PI0900268) to P. Sánchez-Corral; the MRC UK (G0701298) to C.L. Harris; and the Spanish "Ministerio de Economía y Competitividad" (PS09/00122) to M. López Trascasa. In addition, this work was supported by a grant from the Autonomous Region of Madrid (S2010/BMD-2316) to S. Rodríguez de Córdoba, O. Llorca, M. López Trascasa, and P. Sánchez-Corral. M.C. Pickering is a Wellcome Trust Senior Fellow in Clinical Science (WT098476MA). H. Yébenes was supported by a postdoctoral contract by the RTICC, and C. Abarrategui-Garrido was supported by the "Fundación de Investigación Biomédica del Hospital Universitario La Paz." ; Peer reviewed

C3b, the central component of the alternative pathway (AP) of the complement system, coexists as a mixture of conformations in solution. These conformational changes can affect interactions with other proteins and complement regulators. Here we combine a computational model for electrostatic interactions within C3b with molecular imaging to study the conformation of C3b. The computational analysis shows that the TED domain in C3b is tethered ionically to the macroglobulin (MG) ring. Monovalent counterion concentration affects the magnitude of electrostatic forces anchoring the TED domain to the rest of the C3b molecule in a thermodynamic model. This is confirmed by observing NaCl concentration dependent conformational changes using single molecule electron microscopy (EM). We show that the displacement of the TED domain is compatible with C3b binding to Factor B (FB), suggesting that the regulation of the C3bBb convertase could be affected by conditions that promote movement in the TED domain. Our molecular model also predicts mutations that could alter the positioning of the TED domain, including the common R102G polymorphism, a risk variant for developing age-related macular degeneration. The common C3b isoform, C3bS, and the risk isoform, C3bF, show distinct energetic barriers to displacement in the TED that are related to a network of electrostatic interactions at the interface of the TED and MG-ring domains of C3b. These computational predictions agree with experimental evidence that shows differences in conformation observed in C3b isoforms purified from homozygous donors. Altogether, we reveal an ionic, reversible attachment of the TED domain to the MG ring that may influence complement regulation in some mutations and polymorphisms of C3b. ; Work in this report has been funded by the Spanish Ministry of Economy and Competitiveness (SAF2011-26583 to SRC and SAF2014-52301-R to OL), the Fundación Renal Iñigo Alvarez de Toledo and the Seventh Framework Programme European Union Project EURenOmics (305608) to SRC. In addition, this work has been supported by a grant from the Autonomous Region of Madrid (S2010/BMD-2316) to SRC and OL. ; Peer reviewed

41 p.-11 fig. ; 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. ; Work in this report has been funded by the Spanish "Ministerio de Economía y Competitividad" (SAF2011-26583 and SAF2015-66287-R to SRdC and SAF2014- 52301-R to OL) and the Seventh Framework Programme European Union Project EURenOmics (305608) to SRdC. In addition, this work has been supported by a grant from the Autonomous Region of Madrid (S2010/BMD-2316) to SRdeC and OL. ; Peer reviewed

The Gram-positive anaerobic human pathogenic bacterium Atopobium vaginae causes most of the cases of bacterial vaginosis and opportunistic infections in immunocompromised patients. In addition to its well-established role in carbohydrate metabolism, D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Streptococcus pyogenes and S. pneumoniae have been reported to act as an extracellular virulence factor during streptococcal infections. Here, we report the crystal structure of GAPDH from A. vaginae (AvGAPDH) at 2.19 Å resolution. The refined model has a crystallographic Rfree of 22.6%. AvGAPDH is a homotetramer wherein each subunit is bound to a nicotinamide adenine dinucleotide (NAD+) molecule. The AvGAPDH enzyme fulfills essential glycolytic as well as moonlight (non-glycolytic) functions, both of which might be targets of chemotherapeutic intervention. We report that AvGAPDH interacts in vitro with the human C5a anaphylatoxin and inhibits C5a-specific neutrophil chemotaxis, thereby suggesting the participation of AvGAPDH in complement-targeted immunoevasion in a context of infection. The availability of high-quality structures of AvGAPDH and other homologous virulence factors from Gram-positive pathogens is critical for drug discovery programs. In this study, sequence and structural differences between AvGAPDH and related bacterial and eukaryotic GAPDH enzymes are reported in an effort to understand how to subvert the immunoevasive properties of GAPDH and evaluate the potential of AvGAPDH as a druggable target. ; The research leading to these results has received funding from the Spanish Instituto de Salud Carlos III (PI12/01667 to MCV), the Spanish Ministerio de Economía y Competitividad(CTQ2015-66206- C2-2-R and SAF2015-72961-EXP to MCV and SAF2014-54708-R to JRR), CSIC (201620E064), the Regional Government of Madrid (S2010/BD-2316 to JRR, MCV and SRC), and the European Commission (Framework Programme 7 (FP7)) project ComplexINC (Contract No. 279039 to MCV).AVM was supported by the Comunidad de Madrid (S2010/BMD-2316/2326) and the Universidad Complutense de Madrid (CT46/15). ; Peer reviewed

21 p.-6 fig. ; The proteolytic cleavage of C3 to generate C3b is the central and most important step in the activation of Complement, a major component of innate immunity. The comparison of the crystal structures of C3 and C3b illustrates large conformational changes during the transition from C3 to C3b. Exposure of a reactive thio-ester group allows C3b to bind covalently to surfaces such as pathogens or apoptotic cellular debris. The displacement of the thio-ester containing domain (TED) exposes hidden surfaces that mediate the interaction with complement factor B to assemble the C3- convertase of the alternative pathway (AP). In addition, the displacement of the TED domain and its interaction with the macroglobulin 1 (MG1) domain generates a extended surface in C3b where the complement regulators factor H (FH), decay accelerating factor (DAF), membrane cofactor protein (MCP) and complement receptor 1 (CR1) can bind, mediating accelerated decay of the AP C3-convertase and proteolytic inactivation of C3b. In the last few years, evidence has accumulated revealing that the structure of C3b in solution is significantly more flexible than anticipated. We review our current knowledge on C3b structural flexibility to propose a general model where the TED domain can display a collection of conformations around the MG ring, as well as a few specialised positions where the TED is held in one of several fixed locations. Importantly, this conformational heterogeneity in C3b impacts complement regulation, by affecting the interaction with regulators. ; This work was funded by the Spanish Government (SAF2011- 22988 and SAF2014-52301-R to OL;SAF2011-26583 to SRdeC), the EU (Eurenomics to SRdC) and by the Government of the Autonomous Region of Madrid (S2010/BMD-2316 to OL and SRdC). ; Peer reviewed

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. ; Work in this report has been funded by the Spanish "Ministerio de Economía y Competitividad" (SAF2011‐26583 and SAF2015‐66287‐R to SRdC and SAF2014‐52301‐R to OL) and the Seventh Framework Programme European Union Project EURenOmics (305608) to SRdC. In addition, this work has been supported by a grant from the Autonomous Region of Madrid (S2010/BMD‐2316) to SRdeC and OL. ; Sí

5 p.-2 fig. ; [Background] Glomerulonephritis is one of the most severe complications of lupus, a systemic disease with multi-organ involvement, with tissue damage produced mainly by complement activation. As a result of this activation, patients with active lupus present hypocomplementemia during disease flares, but C3 and C4 levels are recovered between episodes. ; [Case presentation] We present a patient who suffered two lupus nephritis episodes in 5 years, achieving complete remission with treatment after both of them, but with C3 levels persistently below normal range. Genetic study revealed that the patient carried a mutation in heterozygosis in the C3 gene. Serial sera samples were analyzed, and autoantibodies to complement alternative pathway proteins (Factor I, Factor B, C3 and Properdin) were found. Functional assays showed that these autoantibodies cause alternative pathway activation. ; [Conclusion] This case is the first reported of a heterozygous C3 mutation associated with lupus nephritis and autoantibodies against complement alternative pathway proteins (Factor I, Factor B, C3 and Properdin).These autoantibodies cause activation of this pathway and this fact could explain that the tissue damage is restricted to the kidney. ; This work was funded by Spanish government, Ministerio de Economía y Competitividad (grant SAF2012-38636), Autonomous region of Madrid (S2010/BMD-2316 to SRdeC y MLT) and Sociedad Española de Nefrología Fundación Senefro to MLT. ; Peer reviewed

32 p.-4 tab-3 fig.-2 tab. suppl. ; Background and objectives Atypical hemolytic uremic syndrome is characterized by vascular endothelial damage caused by complement dysregulation. Consistently, complement inhibition therapies are highly effective in most patients with atypical hemolytic uremic syndrome. Recently, it was shown that a significant percentage of patients with early-onset atypical hemolytic uremic syndrome carry mutations in diacylglycerol kinase-«, an intracellular protein with no obvious role in complement. These data support an alternative, complement-independent mechanism leading to thrombotic microangiopathy that has implications for treatment of early-onset atypical hemolytic uremic syndrome. To get additional insights into this new form of atypical hemolytic uremic syndrome, the diacylglycerol kinase-« gene in a cohort with atypical hemolytic uremic syndrome was analyzed. Design, setting, participants, & measurements Eighty-three patients with early-onset atypical hemolytic uremic syndrome (,2 years) enrolled in the Spanish atypical hemolytic uremic syndrome registry between 1999 and 2013 were screened for mutations in diacylglycerol kinase-«. These patients were also fully characterized for mutations in the genes encoding factor H, membrane cofactor protein, factor I, C3, factor B, and thrombomodulin CFHRs copy number variations and rearrangements, and antifactor H antibodies. Results Four patients carried mutations in diacylglycerol kinase-«, one p.H536Qfs*16 homozygote and three compound heterozygotes (p.W322*/p.P498R, two patients; p.Q248H/p.G484Gfs*10, one patient). Three patients also carried heterozygous mutations in thrombomodulin or C3. Extensive plasma infusions controlled atypical hemolytic uremic syndrome recurrences and prevented renal failure in the two patients with diacylglycerol kinase-« and thrombomodulin mutations. A positive response to plasma infusions and complement inhibition treatment was also observed in the patient with concurrent diacylglycerol kinase-« and C3 mutations. Conclusions Data suggest that complement dysregulation influences the onset and disease severity in carriers of diacylglycerol kinase-« mutations and that treatments on the basis of plasma infusions and complement inhibition are potentially useful in patients with combined diacylglycerol kinase-« and complement mutations. A comprehensive understanding of the genetic component predisposing to atypical hemolytic uremic syndrome is, therefore, critical to guide an effective treatment. ; Work in this report was funded by Spanish "Ministerio de Economía y Competitividad" Grants SAF2011-26583, PS09/00122 (to M.L.T.), SAF2012-38636 (to M.L.T.), and PI1200597 (to P.S.-C.) and Fundación Renal Inigo Alvarez de Toledo and Seventh Framework ~Programme European Union Project EURenOmics Grant 305608 (to S.R.d.C.). In addition, this work was supported by Autonomous Region of Madrid Grant S2010/BMD-2316 (to M.L.T., P.S.-C., and S.R.d.C.). ; Peer reviewed