Caracterización funcional de variantes en genes del sistema del complemento asociadas con patología

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Medicina, leída el 13-07-2023El sistema del complemento es un componente de la inmunidad innata que actúa como una de las primeras barreras en la defensa frente a infecciones. Gran parte de su actividad depende de la vía alternativa, la cual se activa de manera espontánea e inespecífica, y constituye un loop de amplificación de la activación del complemento. Para evitar que por esta vía se genere daño a tejidos propios existe una batería de reguladores de membrana y solubles encargadas de evitar este daño. Variantes en algunos de estos reguladores se han asociado con el desarrollo de patologías, como el síndrome hemolítico urémico atípico y la glomerulopatía de C3. Factor H es el principal regulador de la vía alternativa y para el que se ha identificado el mayor número de variantes en ambas patologías. A pesar de que es una de las proteínas que mejor se conoce del complemento y para la que se han caracterizado funcionalmente un mayor número de variantes, existen muchas otras para las que se desconoce su impacto. Este "vacío" se debe a que aún con todo el conocimiento generado sobre factor H es difícil predecir de manera fiable el impacto de algunas variantes, y a que la caracterización funcional no es un proceso rutinario en los centros de diagnóstico. Además, con estas patologías también se han asociado variantes en las proteínas relacionadas con factor H, originadas por varios eventos de duplicación parcial de factor H. A diferencia del regulador, para estas proteínas se desconoce cuál es su papel en la activación y regulación del complemento y el impacto fisiológico que puedan tener las variantes identificadas. Para abordar estos 2 aspectos en esta tesis se han caracterizado mediante 2 estrategias diferentes el impacto de un total de 108 variantes de factor H de significado incierto asociadas al síndrome hemolítico urémico atípico y/o la glomerulopatía de C3, y se han analizado los ligandos y las propiedades activadoras/reguladoras de la proteína 1 relacionada con factor H y variantes en su extremo C-terminal asociadas al síndrome hemolítico urémico atípico...The complement system is a component of innate immunity that acts as one of the first barriers in the defense against infections. Much of its activity depends on the alternative pathway, which is activated spontaneously and non-specifically, and constitutes an amplification loop of complement activation. To prevent this pathway from causing damage to host tissues, there is a battery of membrane and soluble regulators in charge of avoiding this damage. Variants in some of these regulators have been associated with the development of pathologies such as atypical hemolytic uremic syndrome and C3 glomerulopathy. Factor H is the main regulator of the alternative pathway and for which the largest number of variants has been identified in both pathologies. Although it is one of the best known complement proteins and for which the largest number of variants have been functionally characterized, there are many others for which its impact is unknown. This "gap" is due to the fact that even with all the knowledge generated on factor H it is difficult to reliably predict the impact of some variants, and that functional characterization is not a routine process in diagnostic centers. In addition, variants in factor H related proteins, originated from multiple partial duplication events of factor H, have also been associated with these pathologies. Unlike the regulator, the role of these proteins in the activation and regulation of complement and the physiological impact that the identified variants may have are unknown. To address these 2 aspects in this thesis, the impact of a total of 108 factor H variants of uncertain significance associated with atypical hemolytic uremic syndrome and/or C3 glomerulopathy have been characterized by 2 different strategies, and the ligands and activating/regulatory properties of factor H related protein 1 and variants on its C-terminal end associated with atypical hemolytic uremic syndrome have been analyzed...Fac. de MedicinaTRUEunpu

Hemolysis in eculizumab-treated PNH patients

35 p.-3 fig.-4 tab. Subías, Marta et al.Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia characterized by complement-mediated intravascular hemolysis that is effectively treated with eculizumab. However, treatment responses are reported heterogeneous with some patients presenting residual hemolysis and requiring RBC transfusions. Recent reports have shown that both extravascular hemolysis and incomplete C5 blockade can explain these suboptimal hematological responses. Here we have tested our eculizumab-treated PNH patients (n=12) for signs of hemolysis and assessed complement biomarkers. Patients were also genotyped for complement receptor 1 (CR1, CD35) and C5 polymorphisms and evaluated for free eculizumab in plasma. We report that 10 patients (83%) present parameters suggesting persistent hemolysis, although they did not require additional transfusions. Seven of them (58%) become direct Coombs-test positive as a consequence of treatment, including all patients carrying the low-expression CR1-L allele. CH50 and sC5b-9 assays demonstrate that the persistent low-level hemolysis identified in our treated patients is not a consequence of incomplete C5 blockade, supporting that this hemolysis, as has been suggested previously, results from the extravascular removal of C3 opsonized PNH erythrocytes. We also show that continuous alternative pathway activation in eculizumab-treated individuals carrying the CR1-L allele results in abnormally decreased levels of C3 in plasma that could, potentially, increase their susceptibility to bacterial infections. Finally, we encourage a routine evaluation of free eculizumab levels and terminal pathway activity to personalize eculizumab administration.S.RdeC is supported by the Spanish “Ministerio de Economía y Competitividad” (SAF2011-26583) and the Autonomous Region of Madrid (S2010/BMD-2316).Peer reviewe

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

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 reviewe

Towards a complete functional characterization of the disease-associated genetic variants found in the CFH gene.

1 p. SI: XXVI ICW Kanazawa 2016Peer reviewe

Functional characterization of 105 Factor H variants associ-ated with atypical HUS: lessons for variant classification

1 p.Background: Atypical hemolytic uremic syndrome (aHUS) isa life-threatening thrombotic microangiopathy that can progress,when untreated, to end-stage renal disease. Most frequently, aHUSis caused by complement dysregulation due to pathogenic vari-ants in genes that encode complement components and regulators.Amongst these genes, the Factor H (FH) gene, CFH, presents withthe highest frequency (15-20%) of variants and is associated withthe poorest prognosis. Correct classification of CFH variants aspathogenic or benign is essential to clinical care but remains chal-lenging owing to the dearth of functional studies. As a result,significant numbers of variants are reported as variants of uncertainsignificance.Methods: To address this knowledge gap, we expressed andfunctionally characterized 105 aHUS-associated FH variants witha battery of hemolytic and plate-based assays to determine theirimpact on its regulatory and binding properties. We used 26 pre-viously characterized FH variants to validate the robustness of thefunctional assays used.Results: All FH variants were categorized as pathogenic orbenign, and for each, we fully documented the nature of thepathogenicity. Among the 79 uncharacterized variants, only 29(36.7%) alter FH in vitro expression or function and are thereforeproposed to be pathogenic. We show that rarity in control databasesis not informative for variant classification, and we identify impor-tant limitations in applying prediction algorithms to FH variants.Based on structural and functional data, we suggest ways to circum-vent these difficulties and thereby improve variant classification.Conclusions: Our work reveals limitations of routinely usedvariant classification methods. Rarity in control databases can bemisleading and prediction algorithms fails classifying up to 17%of the variants. This highlights the need for functional assays tointerpret FH variants accurately if clinical care of patients withaHUS is to be individualized and optimized.Peer reviewe

Short title: Molecular understanding of FHR-1 function

33 p.-7 fig.Factor H-related proteins (FHRs) are a group of partly characterized complement proteins that are thought to promote complement activation by competing binding of factor H (FH) to surface-bound C3b. Among them, FHR-1 is remarkable because is associated with atypical hemolytic uremic syndrome (aHUS) and other important diseases. Using a combination of biochemical, immunological, nuclear magnetic resonance and computational approaches, we have characterized a series of FHR-1 mutants (including two associated with aHUS) and have unraveled the molecular bases of the so-called de-regulation 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. Whilst C3b-binding competition with FH is limited to aHUS-associated mutants, all surfacebound FHR-1 promote complement activation, which is delimited by the FHR-1/FH activity ratio. Our data indicate that the FHR-1 de-regulation 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 a pathological complement dysregulation at complement activating surfaces, which may explain the association of FHR-1 quantitative variations with diseases.SRdeC, FJC and SMS are supported by the Spanish “Ministerio de Economía y Competitividad-FEDER” grants (SAF2015-66287-R, RTI2018-094751-B-C22, and CTQ2017-88353-R, respectively) and the Autonomous Region of Madrid (S2017/BMD-3673). EGdeJ is supported by the Spanish “Ministerio de Ciencia, Innovación y Universidades-FEDER (RYC-2013-13395 and RTI2018-095955-B-100).Peer reviewe

Functional characterization of 105 Factor H variants associated with HUS:lessons for variant classification

36 p.-3 fig.-3 tab.Atypical hemolytic uremic syndrome (aHUS) is alife-threatening thrombotic microangiopathy that can progress, when untreated, to end-stage renal disease. Most frequently, aHUS is caused by complement dysregulationdue topathogenic variants in genes that encode complement components and regulators. Amongst these genes, the Factor H (FH) gene, CFH, presents with the highest frequency (15-20%) of variantsand is associated with the poorest prognosis. Correct classification of CFH variants as pathogenic or benign is essential to clinical care but remainschallenging owing to the dearth of functional studies. As a result, significant numbers of variants are reported as variants of uncertain significance. Toaddress this knowledge gap, we expressed and functionally characterized105 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-sixpreviously characterizedFH variantswere 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 in vitroexpression or function and are therefore proposed to be pathogenic. We show that rarityin control databases isnot informative for variant classification,andwe identifyimportant limitations in applying prediction algorithms toFH variants. Based on structural and functional data, we suggest ways to circumvent these difficultiesand thereby improve variant classification.Our work highlights the need for functional assays to interpret FH variants accuratelyif clinical care of patients with aHUS is to be individualized and optimized.SRdC was supported by grants from the Spanish Ministerio de Economía y Competitividad-FEDER (PID2019-104912RB-I00) and Autonomous Region of Madrid (S2017/BMD-3673). RJHS was supported, in part, by a grant from the National Institute of Diabetes and Digestive and Kidney Diseases (R01 110023). Conception, design, data collection and analysis, as well as writing of the manuscript, was performed with support from Alexion Pharmaceuticals, Inc. Editorial support, funded by Alexion Pharmaceuticals Inc., Boston, MA, was provided by Oxford PharmaGenesis, Oxford, UK.Peer reviewe

Factor H competitor generated by gene conversion events associates with atypical hemolytic uremic syndrome

10 p.-6 fig.-1 tab.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.S.R.d.C. is supported by the Spanish “Ministerio de Economía y Competitividad-Federación Española de Enfermedades Raras (FEDER)” (SAF2015-66287R), the Seventh Framework Programme European Union Project EURenOmics (305608), and the Autonomous Region of Madrid (S2010/BMD-2316). S.R.d.C. is member of the “Centro de Investigaciones Biológicas Intramural Program “Molecular Machines for Better Life” (MACBET).” E.G.d.J. is supported by the Spanish “Ministerio de Economía y Competitividad” (RYC-2013-13395 and SAF2014-52339P). M.N. is supported by the Seventh Framework Programme European Union Project EURenOmics (305608). This study was also partially supported by Redes Tematicas De Investigacion Cooperativa En Salud, Red de Investigación Renal (RD06/0016/1002 and RD12/0028), the Instituto de Salud Carlos III, and the Fondo Europeo de Desarrollo Regional (FEDER) “Una manera de hacer Europa.”Peer reviewe