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

Insights into hemolytic uremic syndrome: Segregation of three independent predisposition factors in a large, multiple affected pedigree

Mutations in the complement regulators factor H, membrane cofactor protein (MCP), and factor I are associated with atypical hemolytic uremic syndrome (aHUS, MIM 235400), suggesting that the disease develops as a consequence of the inefficient protection of the renal endothelium from damage by the complement system. Incomplete penetrance of the disease in individuals carrying these mutations is, however, relatively frequent. Here, we report the identification of a large, multiple affected aHUS pedigree in which there is independent segregation of three different aHUS risk factors: a MCP missense mutation (c.-598C > T; Pro165Ser) that decreases MCP expression on the cell surface, a dinucleotide insertion in the coding sequence of factor I (c.-1610insAT) that introduces a premature stop codon in the factor I protein, and the MCPggaac SNP haplotype block that was previously shown to decrease the transcription activity from the MCP promoter. Interestingly, individuals affected by aHUS in the pedigree are only those who have inherited the three aHUS risk factors. These data show an additive effect for mutations in MCP and factor I and provide definitive support to the conclusion that aHUS results from a defective protection of cellular surfaces from complement activation. Furthermore, they help to explain the incomplete penetrance of the disease, illustrating that concurrence of multiple hits in complement regulatory proteins may be necessary to significantly impair host tissue protection and to confer susceptibility to aHUS. © 2005 Elsevier Ltd. All rights reserved.These studies were performed with funds provided by the Ministerio de Educación y Cultura (SAF2002-01085 and SAF03-03485) and from the Fondo de Investigaciones Sanitarias (C03/05; G03/054; G03/011, FIS 03/0621 and FIS 01/A046)Peer Reviewe

The human complement factor H: Functional roles, genetic variations and disease associations

Factor H is an essential regulatory protein that plays a critical role in the homeostasis of the complement system in plasma and in the protection of bystander host cells and tissues from damage by complement activation. Genetic and structural data generated during recent years have been instrumental to delineate the functional domains responsible for these regulatory activities in factor H, which is helping to understand the molecular basis underlying the different pathologies associated to factor H. This review summarises our current knowledge of the role of factor H in health and disease. © 2004 Published by Elsevier Ltd.This work was supported by the Spanish MCyT (SAF2002-1083)Peer Reviewe

Common and rare genetic variants of complement components in human disease

49 p.-2 fig.-2 tab.Genetic variability in the complement system and its association with disease has been known for more than 50 years, but only during the last decade have we begun to understand how this complement genetic variability contributes to the development of diseases. A number of reports have described important genotype-phenotype correlations that associate particular diseases with genetic variants altering specific aspects of the activation and regulation of the complement system. The detailed functional characterization of some of these genetic variants provided key insights into the pathogenic mechanisms underlying these pathologies, which is facilitating the design of specific anti-complement therapies. Importantly, these analyses have sometimes revealed unknown features of the complement proteins. As a whole, these advances have delineated the functional implications of genetic variability in the complement system, which supports the implementation of a precision medicine approach based on the complement genetic makeup of the patients. Here we provide an overview of rare complement variants and common polymorphisms associated with disease and discuss what we have learned from them.SRdeC is supported by the Spanish “Ministerio de Economía y Competitividad/FEDER” [SAF2015-66287-R] and the Autonomous Region of Madrid [S2017/BMD-3673]. SRdeC is member of the "CIB Intramural Program “Molecular Machines for Better Life” (MACBET)". MLT is supported by the Spanish “Ministerio de Economía y Competitividad/FEDER” [PI15-00255]. EGdeJ is supported by the Spanish “Ministerio de Economía y Competitividad/FEDER” [SAF2014-52339-P]. SRdeC and EGdeJ are also supported by the “Fundación Inocente Inocente” (Madrid, Spain).Peer reviewe

Structural and Functional Characterization of Factor H Mutations Associated with Atypical Hemolytic Uremic Syndrome

Genetic studies have demonstrated the involvement of the complement regulator factor H in nondiarrheal, nonverocytotoxin (i.e., atypical) cases of hemolytic uremic syndrome. Different factor H mutations have been identified in 10%–30% of patients with atypical hemolytic uremic syndrome (aHUS), and most of these mutations alter single amino acids in the C-terminal region of factor H. Although these mutations are considered to be responsible for the disease, the precise role that factor H plays in the pathogenesis of aHUS is unknown. We report here the structural and functional characterization of three different factor H proteins purified from the plasma of patients with aHUS who carry the factor H mutations W1183L, V1197A, or R1210C. Structural anomalies in factor H were found only in R1210C carriers; these individuals show, in their plasma, a characteristic high-molecular-weight factor H protein that results from the covalent interaction between factor H and human serum albumin. Most important, all three aHUS-associated factor H proteins have a normal cofactor activity in the proteolysis of fluid-phase C3b by factor I but show very low binding to surface-bound C3b. This functional impairment was also demonstrated in recombinant mutant factor H proteins expressed in COS7 cells. These data support the hypothesis that patients with aHUS carry a specific dysfunction in the protection of cellular surfaces from complement activation, offering new possibilities to improve diagnosis and develop appropriate therapies

Predisposition to atypical hemolytic uremic syndrome involves the concurrence of different susceptibility alleles in the regulators of complement activation gene cluster in 1q32

The efficiency of the complement system as an innate immune defense mechanism depends on a fine control that restricts its action to pathogens and prevents non-specific damage to host tissues. Genetic and functional analyses have shown that this critical control of complement activation may be impaired in atypical hemolytic uremic syndrome (aHUS) patients. Mutations in HF1, MCP or FI have been found in aHUS patients, but incomplete penetrance of the disease in individuals carrying these mutations is relatively frequent and no genetic defect has yet been found in a majority of aHUS patients. We report here the identification of a specific SNP haplotype block, spanning the MCP gene in the regulators of complement activation gene cluster, which is over-represented in aHUS patients and strongly associates with the severity of the disease. Linkage disequilibrium analyses suggest that this SNP haplotype also includes the CR1, DAF and C4BP genes. Initial studies identified two SNPs in the haplotype that influence the transcription activity of the MCP promoter in transient transfection experiments. Notably, the SNP haplotype block was found to be particularly frequent among patients who carry mutations in HF1, MCP or FI. These findings and the identification of aHUS patients carrying mutations in two complement regulatory genes provide an important insight into the etiology of aHUS. Together, they suggest that complement regulatory molecules act as a protein network and that multiple hits, involving plasma- and membrane-associated complement regulatory proteins, are necessary to impair protection to host tissues and to confer significant predisposition to aHUS. © Oxford University Press 2005; all rights reserved.These studies were performed with funds provided by the Ministerio de Educación y Cultura (SAF2002-01085 and SAF2003-03485) and from the Fondo de Investigaciones Sanitarias (C03/05, G03/054, G03/011, FIS 01/3029 and FIS 01/A046). J.E.-G. was supported by a grant from the Comunidad Autónoma de MadridPeer Reviewe

Mutations in proteins of the Alternative Pathway of Complement and the Pathogenesis of Atypical Hemolytic Uremic Syndrome

10 páginas, 5 figuras, 1 tabla -- PAGS nros. 171-180Atypical hemolytic uremic syndrome is associated with mutations in the complement proteins factor H, factor I, factor B, C3, or membrane cofactor protein in about 50% of patients. The evolution and prognosis of the disease in patients carrying mutations in factor H is particularly poor, and renal transplantation most often fails because of recurrence of the disease in the graft. The risk of rapid loss of renal function in patients with functional mutations in factor H requires that effective treatment be initiated as soon as possible, but identification of these patients relies on genetic studies that are time consuming. We describe a case in which an in vitro hemolytic assay proved useful for rapidly assessing factor H dysfunction and for testing whether this dysfunction could be corrected with fresh frozen plasma. In the context of this case, we summarize recent advances in understanding the molecular mechanisms contributing to atypical hemolytic uremic syndrome, including descriptions of DNA- and protein-based analysis. We conclude that functional analysis of factor H should help rationalize the plasma treatment of patients with atypical hemolytic uremic syndromeThis work was funded by the Spanish Ministerio de Sanidad y Consumo (grants FIS 03/0621 and 06/0625 to P.S.C.) and the Spanish Ministerio de Educación y Cultura (grant SAF2005-00913 to S.R.C.)Peer reviewe

Structural and functional characterization of factor H mutations associated with atypical hemolytic uremic syndrome

Genetic studies have demonstrated the involvement of the complement regulator factor H in nondiarrheal, nonverocytotoxin (i.e., atypical) cases of hemolytic uremic syndrome. Different factor H mutations have been identified in 10%-30% of patients with atypical hemolytic uremic syndrome (aHUS), and most of these mutations alter single amino acids in the C-terminal region of factor H. Although these mutations are considered to be responsible for the disease, the precise role that factor H plays in the pathogenesis of aHUS is unknown. We report here the structural and functional characterization of three different factor H proteins purified from the plasma of patients with aHUS who carry the factor H mutations W1183L, V1197A, or R1210C. Structural anomalies in factor H were found only in R1210C carriers; these individuals show, in their plasma, a characteristic high-molecular-weight factor H protein that results from the covalent interaction between factor H and human serum albumin. Most important, all three aHUS-associated factor H proteins have a normal cofactor activity in the proteolysis of fluid-phase C3b by factor I but show very low binding to surface-bound C3b. This functional impairment was also demonstrated in recombinant mutant factor H proteins expressed in COS7 cells. These data support the hypothesis that patients with aHUS carry a specific dysfunction in the protection of cellular surfaces from complement activation, offering new possibilities to improve diagnosis and develop appropriate therapies.This research was supported by the Spanish Comision Interministerial de Ciencia y Tecnologia (SAF2002-01083) and by the Comunidad de Madrid (08.6/0028.1/2000). P.S.-C., D.P.-C., O.H., and E.G. were awarded grants from the Fondo de Investigaciones Sanitarias, Glaxo Wellcome, the i3p program of the Consejo Superior de Investigaciones Científicas, and the Ministerio de Ciencia y Tecnologia, respectivelyPeer Reviewe