Identificación y caracterización funcional de variantes genéticas de proteínas del complemento asociadas con patología.

156 p.-16 fig.-3 tab.-anexoDDD (Dense Deposit Disease) y aHUS (atypical Hemolitic Uremic Syndrome) son dos enfermedades renales raras con una importante mortalidad y que frecuentemente causan insuficiencia renal terminal. Al inicio de esta Tesis se conocía la asociación de aHUS y DDD con defectos en la activación o regulación de la vía alternativa del complemento, causados por mutaciones y polimorfismos en los genes del complemento. La caracterización funcional de mutaciones asociadas con aHUS había demostrado que estas alteraciones genéticas resultaban en una regulación defectuosa de la activación del complemento sobre las superficies celulares y que el aHUS, por lo tanto, era seguramente consecuencia del daño causado por el complemento en el endotelio de la microvasculatura glomerular. Por otro lado, estudios animales en DDD sugerían que el mecanismo patogénico en DDD es la activación masiva del C3, consecuencia de la desregulación del complemento en fase fluida. Los objetivos planteados en esta Tesis han sido profundizar en el conocimiento de los factores genéticos de predisposición a aHUS y DDD y de los mecanismos patogénicos responsables de estas patologías.Entre las nuevas variaciones genéticas asociadas a aHUS y DDD descritos en esta Tesis están distintos reordenamientos, polimorfismos y mutaciones en los genes CFHR1‐5. Sin embargo, el descubrimiento más importante en este apartado ha sido la identificación por primera vez de una mutación en el gen C3 asociada con DDD. La caracterización funcional de esta mutación y su comparación con la de otras mutaciones en este mismo gen que se asocian con aHUS ha confirmado que los mecanismos patogénicos de aHUS se corresponden con un defecto de la regulación del complemento en las superficies celulares, mientras que DDD se produce exclusivamente por una desregulación del complemento en fase fluida. La caracterización funcional de la mutación en C3 asociada con DDD ha sido también muy importante para desentrañar algunos aspectos estructurales de la activación de la convertasa del C3 de la vía alternativa, de su regulación por factor H,MCP o DAF, y de cómo se produce el reconocimiento del substrato C3 por esta convertasa. Por último, hemos investigado y confirmado la relevancia de la hipótesis de los “multiple hits” (concurrencia de mutaciones y polimorfismos de riesgo), descrita en el laboratorio hace unos años, para explicar la penetrancia incompleta del aHUS entre los portadores de mutaciones en diversos genes del complemento y justificar la heterogeneidad en la manifestación clínica de la patología.Subvención de la Comunidad de MadridPeer reviewe

C3 glomerulopathy-associated CFHR1 mutation alters FHR oligomerization and complement regulation

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

Human IFN-γ immunity to mycobacteria is governed by both IL-12 and IL-23

Hundreds of patients with autosomal recessive, complete IL-12p40 or IL-12Rß1 deficiency have been diagnosed over the last 20 years. They typically suffer from invasive mycobacteriosis and, occasionally, from mucocutaneous candidiasis. Susceptibility to these infections is thought to be due to impairments of IL- 12–dependent IFN-? immunity and IL-23–dependent IL-17A/IL-17F immunity, respectively. We report here patients with autosomal recessive, complete IL- 12Rß2 or IL-23R deficiency, lacking responses to IL-12 or IL- 23 only, all of whom, unexpectedly, display mycobacteriosis without candidiasis. We show that aß T, ?d T, B, NK, ILC1, and ILC2 cells from healthy donors preferentially produce IFN-? in response to IL-12, whereas NKT cells and MAIT cells preferentially produce IFN-? in response to IL-23. We also show that the development of IFN-?–producing CD4+ T cells, including, in particular, mycobacterium-specific TH1* cells (CD45RA-CCR6+), is dependent on both IL-12 and IL-23. Last, we show that IL12RB1, IL12RB2, and IL23R have similar frequencies of deleterious variants in the general population. The comparative rarity of symptomatic patients with IL-12Rß2 or IL-23R deficiency, relative to IL-12Rß1 deficiency, is, therefore, due to lower clinical penetrance. There are fewer symptomatic IL-23R– and IL-12Rß2–deficient than IL-12Rß1–deficient patients, not because these genetic disorders are rarer, but because the isolated absence of IL-12 or IL-23 is, in part, compensated by the other cytokine for the production of IFN-?, thereby providing some protection against mycobacteria. These experiments of nature show that human IL-12 and IL-23 are both required for optimal IFN-?–dependent immunity to mycobacteria, both individually and much more so cooperatively

Identificación y caracterización funcional de variantes genéticas de proteínas del complemento asociadas con aptología

DDD (Dense Deposit Disease) y aHUS (atypical Hemolitic Uremic Syndrome) son dos enfermedades renales raras con una importante mortalidad y que frecuentemente causan insuficiencia renal terminal. Al inicio de esta Tesis se conocía la asociación de aHUS y DDD con defectos en la activación o regulación de la vía alternativa del complemento, causados por mutaciones y polimorfismos en los genes del complemento. La caracterización funcional de mutaciones asociadas con aHUS había demostrado que estas alteraciones genéticas resultaban en una regulación defectuosa de la activación del complemento sobre las superficies celulares y que el aHUS, por lo tanto, era seguramente consecuencia del daño causado por el complemento en el endotelio de la microvasculatura glomerular. Por otro lado, estudios animales en DDD sugerían que el mecanismo patogénico en DDD es la activación masiva del C3, consecuencia de la desregulación del complemento en fase fluida. Los objetivos planteados en esta Tesis han sido profundizar en el conocimiento de los factores genéticos de predisposición a aHUS y DDD y de los mecanismos patogénicos responsables de estas patologías. Entre las nuevas variaciones genéticas asociadas a aHUS y DDD descritos en esta Tesis están distintos reordenamientos, polimorfismos y mutaciones en los genes CFHR1-5. Sin embargo, el descubrimiento más importante en este apartado ha sido la identificación por primera vez de una mutación en el gen C3 asociada con DDD. La caracterización funcional de esta mutación y su comparación con la de otras mutaciones en este mismo gen que se asocian con aHUS ha confirmado que los mecanismos patogénicos de aHUS se corresponden con un defecto de la regulación del complemento en las superficies celulares, mientras que DDD se produce exclusivamente por una desregulación del complemento en fase fluida. La caracterización funcional de la mutación en C3 asociada con DDD ha sido también muy importante para desentrañar algunos aspectos estructurales de la activación de la convertasa del C3 de la vía alternativa, de su regulación por factor H, MCP o DAF, y de cómo se produce el reconocimiento del substrato C3 por esta convertasa. Por ultimo, hemos investigado y confirmado la relevancia de la hipótesis de los “multiple hits” (concurrencia de mutaciones y polimorfismos de riesgo), descrita en el laboratorio hace unos años, para explicar la penetrancia incompleta del aHUS entre los portadores de mutaciones en diversos genes del complemento y justificar la heterogeneidad en la manifestación clínica de la patología

The molecular and structural bases for the association of complement C3 mutations with atypical hemolytic uremic syndrome

44 p.-7 fig.-1 tab.Martínez-Barricarte, Rubén et alt.Atypical hemolytic uremic syndrome (aHUS) associates with complement dysregulation caused by mutations and polymorphisms in complement activators and regulators. However, the reasons why some mutations in complement proteins predispose to aHUS are poorly understood. Here, we have investigated the functional consequences of three aHUS-associated mutations in C3, R592W, R161W and I1157T. First, we provide evidence that penetrance and disease severity for these mutations is modulated by inheritance of documented “risk” haplotypes as has been observed with mutations in other complement genes. Next, we show that all three mutations markedly reduce the efficiency of factor I-mediated C3b cleavage when catalysed by membrane cofactor protein (MCP), but not when catalysed by factor H. Biacore analysis showed that each mutant C3b bound sMCP (recombinant soluble MCP; CD46) at reduced affinity, providing a molecular basis for its reduced cofactor activity. Lastly, we show by electron microscopy structural analysis a displacement of the TED domain from the MG ring in C3b in two of the C3 mutants that explains these defects in regulation. As a whole our data suggest that aHUS-associated mutations in C3 selectively affect regulation of complement on surfaces and provide a structural framework to predict the functional consequences of the C3 genetic variants found in patients.Work in this report has been funded by the Spanish “Ministerio de Economía y Competitividad” (SAF2011-26583 to SRdeC, PI12-00597 to PS-C and SAF2011-22988 to OL), the Fundación Renal Iñigo Alvarez de Toledo, the Seventh Framework Programme European Union Project EURenOmics (305608) to SRdeC, by a grant from the Autonomous Region of Madrid (S2010/BMD-2316) to SRdeC and OL and the Medical Research Council, United Kingdom (G0701298) to CLH and BPM.Peer reviewe

Método para la predicción del riesgo de desarrollar la enfermedad de degeneración macular asociada a la edad en la población española

Método para predecir el riesgo de desarrollar la enfermedad de degeneración macular asociada a la edad en población caucásica española que se caracteriza porque se utilizan para el cálculo marcadores genéticos prevalentes en población afectada por dicha enfermedad en relación a población sana.Peer reviewedSegugen S.L., Universidad de Navarra, Consejo Superior de Investigaciones Científicas (España)B1 Patente sin examen previ

Relevance of complement factor H-related 1 (CFHR1) genotypes in age- related macular degeneration (AMD)

31 p.-4 fig.-4 tab.Purpose. Age-related macular degeneration (AMD) has a strong genetic component with a major locus at 1q31, including the complement factor H (CFH) gene. Detailed analyses of this locus have demonstrated the existence of one SNP haplotype block, carrying the CFH 402His allele, which confers increased risk for AMD, and two protective SNP haplotypes, one of them carrying a deletion of the CFHR1 and CFHR3 genes (ΔCFHR3-CFHR1). The purpose of these studies was to evaluate the contribution of newly described CFHR1 alleles to the association of the 1q31 locus with AMD. Methods. Two hundred fifty-nine patients and 191 age-matched controls of Spanish origin were included in a transversal case–control study using multivariate logistic regression analysis and ROC (receiver operating characteristic) statistics to generate and test models predictive of the development of AMD. Results. This study showed for the first time that a particular CFHR1 allotype, CFHR1*A, is strongly associated with AMD (odds ratio, 2.08; 95% confidence interval, 1.59–2.73; P < 0.0001) and illustrate a peculiar genotype–phenotype correlation between the CFHR1 alleles and different diseases that may have important implications for understanding the pathophysiology of AMD. It also shows that CFHR1*A is in strong linkage disequilibrium with the CFH 402His allele, which provides additional candidate variants within the major risk haplotype at 1q31, promoting its association with AMD. Further, using the Spanish population as a model, the results showed that analysis of the CFHR1 genotypes provide sufficient information to delineate the individual risk of developing AMD. Conclusions. The results support a relevant role of CFHR1 in the pathogenesis of AMDSupported by Grant SAF2008-00226 from the Spanish Ministerio de Ciencia e Innovación, the Ciber de Enfermedades Raras, and the Fundación Renal Iñigo Alvarez de Toledo (SRdeC); Grants RTICS RD07/0062, FIS PI 08/1705, and FIS 11/00898 from the Instituto de Salud Carlos III (AG-L); and Grant 35/2008 from the Comunidad Autónoma de Madrid, Dirección General de Innovación Tecnológica of the Consejeria Economía e Innovavión Tecnológica (JP-P)Peer reviewe

Spontaneous hemolytic uremic syndrome triggered by complement factor H lacking surface recognition domains

We are grateful to all patients and the collaborating clinicians for their participation in this study. We thank Mr. Ian Shore for his technical assistance with the preparation of tissue for electron microscopy, Mrs. Margarita Lewis for technical assistance with the processing of histological specimens, and the staff of the Biological Services Unit at Imperial College for the care of the animals involved in this study. We also thank the members of the DNA sequencing laboratory at the Centro de Investigaciones Biologicas, as well as Dr. Elena Aller and Ms. Sheila Pinto for invaluable technical assistance with patient genotypingFactor H (FH) is an abundant serum glycoprotein that regulates the alternative pathway of complement-preventing uncontrolled plasma C3 activation and nonspecific damage to host tissues. Age-related macular degeneration (AMD), atypical hemolytic uremic syndrome (aHUS), and membranoproliferative glomerulonephritis type II (MPGN2) are associated with polymorphisms or mutations in the FH gene (Cfh), suggesting the existence of a genotype–phenotype relationship. Although AMD and MPGN2 share pathological similarities with the accumulation of complement-containing debris within the eye and kidney, respectively, aHUS is characterized by renal endothelial injury. This pathological distinction was reflected in our Cfh association analysis, which demonstrated that although AMD and MPGN2 share a Cfh at-risk haplotype, the haplotype for aHUS was unique. FH-deficient mice have uncontrolled plasma C3 activation and spontaneously develop MPGN2 but not aHUS. We show that these mice, transgenically expressing a mouse FH protein functionally equivalent to aHUS-associated human FH mutants, regulate C3 activation in plasma and spontaneously develop aHUS but not MPGN2. These animals represent the first model of aHUS and provide in vivo evidence that effective plasma C3 regulation and the defective control of complement activation on renal endothelium are the critical events in the molecular pathogenesis of FH-associated aHUS.These studies were funded by the Wellcome Trust and the Spanish Ministerio de Educación y Cultura (grant SAF2005-00913). M.C. Pickering is a Wellcome Trust Research Fellow (fellowship GR071390).Peer reviewe

Biochemical and X-ray diffraction analysis of the interaction between iC3b and the CR3 alfa I domain

1 p.Background: Communication between the humoral and cellu-lar branches and the innate and the adaptive branches of vertebrateimmunity is essential for mounting an effective immune response,for surveying the presence of pathogens, immune complexes andapoptotic cell debris and for maintaining homeostasis (Merle et al.,2015). Complement activation on cell surfaces results in theiropsonization and subsequent presentation to immune cells bearingcomplement receptors. The complex formed by the complement’siC3b opsonin and the integrin complement receptor type 3 (CR3)is thought to mediate this communication and stimulate adaptiveimmune cells (Xu et al., 2017). Despite the centrality of the iC3b-CR3 complex to immune signaling, a high-resolution structure ofthe complex between the complete opsonin and the I domain orthe entire headpiece of CR3 is still lacking. Furthermore, the poten-tial involvement of regions of iC3b other than the TED domain inthe interaction remains unclear.Methods: The iC3b-CR3 I domain complex was purifiedby size-exclusion chromatography and subjected to crystalliza-tion screenings. Diffraction-quality crystals were obtained, whichyielded a complete diffraction data set to 3.4˚A resolution on thePX2A beamline at the SOLEIL synchrotron (Paris, France). Small-angle X-ray scattering (SAXS) profiles of the complex were collectedon the BioSAXS beamline at the ESRF (Grenoble, France). KD valueswere measured by surface plasmon resonance (SPR). MD simula-tions were carried out with GROMACSResults: Here we present the high-resolution (3.4˚A) crystallo-graphic structure of human iC3b and the von Willebrand A inserted domain of the CR3 ( I) receptor and a model built from crystal-lographic, biochemical, biophysical and SAXS data. In addition tothe known interaction between the thioester-domain (TED) of iC3band the I domain, the crystal structure reveals the involvement ofnovel interaction surfaces on the C3c macroglobulin ring in secur-ing and orientating the complex.Conclusions: Based on available data and novel insights wehave built an integrative structural biology model that reconciles previously known information about the iC3b-CR3 interaction,and which allows us to predict the dynamics of the iC3b-CR3interaction across biological cell surfaces.Peer reviewe