Complete functional characterization of disease-associated genetic variants in the complement factor H gene
2017; Elsevier BV; Volume: 93; Issue: 2 Linguagem: Inglês
10.1016/j.kint.2017.07.015
ISSN1523-1755
AutoresHéctor Martín Merinero, Sheila Pinto García, Jesús García-Fernández, Emilia Arjona, Agustín Tortajada, Santiago Rodrı́guez de Córdoba,
Tópico(s)Blood groups and transfusion
ResumoGenetic 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. 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. Factor H (FH) is the key regulator of the alternative pathway (AP) of the complement system. FH controls complement activation, both in the fluid phase and on cellular surfaces, preserving complement homeostasis and preventing uncontrolled C3b deposition and host tissue damage. The FH regulatory activities include binding to C3b, blocking C3b-factor B interaction during the assembly of the AP C3-proconvertase complex (C3bB); acceleration of the decay of the AP C3-convertase (C3bBb); and acting as cofactor of factor I (FI) in the proteolytic inactivation of C3b.1Rodriguez de Cordoba S. Esparza-Gordillo J. Goicoechea de Jorge E. et al.The human complement factor H: functional roles, genetic variations and disease associations.Mol Immunol. 2004; 41: 355-367Crossref PubMed Scopus (490) Google Scholar FH is a relatively abundant plasma protein that is secreted as a single-chain glycoprotein of 155 kDa composed of 20 homologous domains of ∼60 amino acids, called short consensus repeats (SCRs). FH concentration in plasma is highly variable, ranging from 76 to 247 μg/ml. Along the 20 SCR of FH, there are different interaction sites for C3b and polyanions. The SCR1-4 region is the only C3b binding site acting as a cofactor for FI to cleave C3b and to accelerate the decay of AP C3-convertase. Similarly, the C3b and polyanion binding site at SCR19-20 determines the ability of FH to bind surface-bound C3b, this region of FH being essential for self-pathogen discrimination.2Sharma A.K. Pangburn M.K. Identification of three physically and functionally distinct binding sites for C3b in human complement factor H by deletion mutagenesis.Proc Natl Acad Sci U S A. 1996; 93: 10996-11001Crossref PubMed Scopus (200) Google Scholar, 3Meri S. Pangburn M.K. Discrimination between activators and nonactivators of the alternative pathway of complement: regulation via a sialic acid/polyanion binding site on factor H.Proc Natl Acad Sci U S A. 1990; 87: 3982-3986Crossref PubMed Scopus (353) Google Scholar In addition to these regions, it was recently postulated that a central FH segment, including SCR14, facilitates FH bending and increases avidity for C3b by enabling the simultaneous binding of FH to different sites in C3b.4Schmidt C.Q. Herbert A.P. Mertens H.D. et al.The central portion of factor H (modules 10-15) is compact and contains a structurally deviant CCP module.J Mol Biol. 2010; 395: 105-122Crossref PubMed Scopus (47) Google Scholar, 5Makou E. Mertens H.D. Maciejewski M. et al.Solution structure of CCP modules 10-12 illuminates functional architecture of the complement regulator, factor H.J Mol Biol. 2012; 424: 295-312Crossref PubMed Scopus (24) Google Scholar Pathogenic variants and polymorphisms in the CFH gene are associated with a number of diseases, including atypical hemolytic uremic syndrome (aHUS) and C3-glomerulopathy (C3G).6de Cordoba S.R. de Jorge E.G. Translational mini-review series on complement factor H: genetics and disease associations of human complement factor H.Clin Exp Immunol. 2008; 151: 1-13Crossref PubMed Scopus (225) Google Scholar aHUS is a thrombotic microangiopathy characterized by thrombocytopenia, hemolytic anemia, and acute renal failure. The primary pathogenic event in aHUS is endothelial cell injury caused by complement dysregulation.7Sanchez-Corral P. Perez-Caballero D. Huarte O. et al.Structural and functional characterization of factor H mutations associated with atypical hemolytic uremic syndrome.Am J Hum Genet. 2002; 71: 1285-1295Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar C3G is a very rare form of glomerulonephritis, characterized by the presence of electron-dense deposits within the glomerular basement membrane.8Fakhouri F. Fremeaux-Bacchi V. Noel L.H. et al.C3 glomerulopathy: a new classification.Nat Rev Nephrol. 2010; 6: 494-499Crossref PubMed Scopus (268) Google Scholar C3G is associated with complement abnormalities that lead to a persistent reduction of C3 serum levels and to intense deposition of degradation products of C3 in the glomerular basement membrane.9Dragon-Durey M.A. Fremeaux-Bacchi V. Loirat C. et al.Heterozygous and homozygous factor h deficiencies associated with hemolytic uremic syndrome or membranoproliferative glomerulonephritis: report and genetic analysis of 16 cases.J Am Soc Nephrol. 2004; 15: 787-795Crossref PubMed Scopus (309) Google Scholar, 10Licht C. Fremeaux-Bacchi V. Hereditary and acquired complement dysregulation in membranoproliferative glomerulonephritis.Thromb Haemost. 2009; 101: 271-278Crossref PubMed Scopus (52) Google Scholar Over the past 15 years, genetic analyses in aHUS and C3G patients have shown that variants in CFH are the most common genetic alteration associated with these disorders. However, we have a limited understanding of the functional consequences of almost one-third of the genetic variants identified in CFH, which is a potential cause of misinterpretations with important consequences for the patients and their relatives. We report here the development of a comprehensive analytical procedure for the functional characterization of FH and demonstrate its effectiveness in identifying the expression and functional consequences of a large number of novel aHUS and C3G-associated CFH variants. The data allowed us to categorize them as pathogenic or benign and reinforced our current understanding of the pathogenic mechanisms underlying aHUS and C3G. A total of 707 aHUS and 234 C3G patients included in the Spanish aHUS/C3G Registry (https://www.aHUSC3G.es) were screened for CFH variants, which resulted in the identification of 101 CFH variants, 87 in aHUS and 35 in C3G; 21 variants were associated with both pathologies. As a whole, 123 aHUS patients (17.4%) and 27 C3G patients (11.5%) carried CFH gene variants. Notably, 28 of these CFH variants (27.5%) were novel variants for which the functional consequences were unknown (Table 1). In addition, these patients were screened for all aHUS and C3G candidate genes. A summary of the clinical and genetic characteristics of the patients carrying these novel CFH variants is presented in Table 2 to illustrate that most patients presented with severe disease phenotypes. All other CFH variants (N = 73) found in our patients were previously described, and their functional consequences are known.11Rodriguez de Cordoba S. Hidalgo M.S. Pinto S. et al.Genetics of atypical hemolytic uremic syndrome (aHUS).Semin Thromb Hemost. 2014; 40: 422-430Crossref PubMed Scopus (101) Google Scholar We named those variants located in SCR1-4 (R53C, R53P, R53S, S58A, C66Y, L98F, A110S, Y118Ifs*4, C192W, and Y235C) N-terminal, those in SCR19-20 (D1119N, P1166L, R1182K, C1218R, and c.3493+1G>A) were named C-terminal, and variants located in SCR5-18 (C309W, C320R, K331E, C597*, K670T, M823T, R830W, C853R, C853Y, R885Sfs*13, T956M, W1037*, and c.2957-1G>A) were named central region. A number of asymptomatic relatives carrying the CFH gene variants were also identified (Table 1). A search for these CFH variants in the Spanish and European control populations illustrated, that with only 3 exceptions (S58A, R830W, and T956M), all other CFH variants have not been previously encountered (Supplementary Table S1).Table 1Novel uncharacterized CFH variants associated with aHUS and C3G in Spanish patientsVariantProteinExon (SCR)Carrier IDStatusTyr402His genotypesPlasma levels (μg/ml)Tyr402His402N-terminal regionc.157C>TR53C2 (1)1aHUSHaAllele carrying the disease-associated variant./HaAllele carrying the disease-associated variant.cIndicates the absence of an allele because the individual is homozygous for the other allele.172c.157C>AR53S2 (1)2aHUSYaAllele carrying the disease-associated variant./Y242cIndicates the absence of an allele because the individual is homozygous for the other allele.2-MHealthyYaAllele carrying the disease-associated variant./H84982-P1HealthyYaAllele carrying the disease-associated variant./Y138cIndicates the absence of an allele because the individual is homozygous for the other allele.c.158G>CR53P2 (1)3aHUSYaAllele carrying the disease-associated variant./Y168cIndicates the absence of an allele because the individual is homozygous for the other allele.3-MHealthyYaAllele carrying the disease-associated variant./Y206cIndicates the absence of an allele because the individual is homozygous for the other allele.3-P1HealthyYaAllele carrying the disease-associated variant./H14481c.172T>GS58A2 (1)4aHUSHaAllele carrying the disease-associated variant./H (null)bCompound heterozygote with a null CFH allele.cIndicates the absence of an allele because the individual is homozygous for the other allele.514-P4HealthyY/HaAllele carrying the disease-associated variant.112574-P8HealthyY/HaAllele carrying the disease-associated variant.128585aHUSY/HaAllele carrying the disease-associated variant.9662c.197G>AC66Y2 (1)6aHUSYaAllele carrying the disease-associated variant./H0646-PHealthyYaAllele carrying the disease-associated variant./Y47cIndicates the absence of an allele because the individual is homozygous for the other allele.c.292C>TL98F3 (2)7aHUSYaAllele carrying the disease-associated variant./Y75cIndicates the absence of an allele because the individual is homozygous for the other allele.c.328G>TA110S3 (2)8C3GHaAllele carrying the disease-associated variant./HaAllele carrying the disease-associated variant.cIndicates the absence of an allele because the individual is homozygous for the other allele.388-P1HealthyY/HaAllele carrying the disease-associated variant.114188-P2HealthyY/HaAllele carrying the disease-associated variant.74238-P3HealthyY/HaAllele carrying the disease-associated variant.68128-P4HealthyY/HaAllele carrying the disease-associated variant.94178-P5HealthyHaAllele carrying the disease-associated variant./HaAllele carrying the disease-associated variant.cIndicates the absence of an allele because the individual is homozygous for the other allele.338-P6HealthyHaAllele carrying the disease-associated variant./HaAllele carrying the disease-associated variant.cIndicates the absence of an allele because the individual is homozygous for the other allele.288-P7HealthyY/HaAllele carrying the disease-associated variant.75188-P8HealthyY/HaAllele carrying the disease-associated variant.8816c.350delGY118Ifs*43 (2)9C3GYaAllele carrying the disease-associated variant./H0789-P1HealthyYaAllele carrying the disease-associated variant./Y62cIndicates the absence of an allele because the individual is homozygous for the other allele.10aHUSYaAllele carrying the disease-associated variant./Y58cIndicates the absence of an allele because the individual is homozygous for the other allele.c.576T>GC192W5 (3)11C3GYaAllele carrying the disease-associated variant./H065c.704A>GY235C6 (4)12aHUSY/HaAllele carrying the disease-associated variant.1280Central regionc.927C>GC309W7 (5)13aHUSY/YaAllele carrying the disease-associated variant.75cIndicates the absence of an allele because the individual is homozygous for the other allele.13-P2HealthyYaAllele carrying the disease-associated variant./H09913-P4HealthyY/YaAllele carrying the disease-associated variant.89cIndicates the absence of an allele because the individual is homozygous for the other allele.c.958T>CC320R7 (5)14aHUSYaAllele carrying the disease-associated variant./Y58cIndicates the absence of an allele because the individual is homozygous for the other allele.14-P4HealthyYaAllele carrying the disease-associated variant./H089c.991A>GK331E8 (6)15C3GNY/HaAllele carrying the disease-associated variant.1089015-PHealthyH/HaAllele carrying the disease-associated variant.cIndicates the absence of an allele because the individual is homozygous for the other allele.145c.1791C>AC597*13 (10)16aHUSY/HaAllele carrying the disease-associated variant.510c.2009A>CK670T14 (11)17aHUSY/HaAllele carrying the disease-associated variant.90102c.2468T>CM823T17 (14)18aHUSYaAllele carrying the disease-associated variant./Y161cIndicates the absence of an allele because the individual is homozygous for the other allele.18-MHealthyYaAllele carrying the disease-associated variant./Y166cIndicates the absence of an allele because the individual is homozygous for the other allele.c.991A>GR830W17 (14)19C3GYaAllele carrying the disease-associated variant./Y149cIndicates the absence of an allele because the individual is homozygous for the other allele.19-PHealthyYaAllele carrying the disease-associated variant./Y161cIndicates the absence of an allele because the individual is homozygous for the other allele.c.2557T>CC853R17 (14)20aHUSY/HaAllele carrying the disease-associated variant.113020-P2HealthyY/HaAllele carrying the disease-associated variant.100020-P3HealthyY/HaAllele carrying the disease-associated variant.820c.2558G>AC853Y17 (14)21aHUSYaAllele carrying the disease-associated variant./Y52cIndicates the absence of an allele because the individual is homozygous for the other allele.21-P4HealthyYaAllele carrying the disease-associated variant./H0101c.2655delR885Sfs*1318 (15)22aHUSY/HaAllele carrying the disease-associated variant.93022-P1HealthyH/HaAllele carrying the disease-associated variant.cIndicates the absence of an allele because the individual is homozygous for the other allele.6223C3GHaAllele carrying the disease-associated variant./HaAllele carrying the disease-associated variant.cIndicates the absence of an allele because the individual is homozygous for the other allele.0c.2940C>TT956M19 (16)24aHUSH/HaAllele carrying the disease-associated variant.cIndicates the absence of an allele because the individual is homozygous for the other allele.24124-MHealthyY/HaAllele carrying the disease-associated variant.5979c.2957-1G>A-Intron 1925aHUSYaAllele carrying the disease-associated variant./Y75cIndicates the absence of an allele because the individual is homozygous for the other allele.25-P1HealthyYaAllele carrying the disease-associated variant./Y79cIndicates the absence of an allele because the individual is homozygous for the other allele.25-P7HealthyYaAllele carrying the disease-associated variant./Y72cIndicates the absence of an allele because the individual is homozygous for the other allele.c.3110G>AW1037*20 (17)26aHUSY/HaAllele carrying the disease-associated variant.800C-terminal regionc.3355G>AD1119N22 (19)27aHUSYaAllele carrying the disease-associated variant./Y178cIndicates the absence of an allele because the individual is homozygous for the other allele.27-PHealthyYaAllele carrying the disease-associated variant./Y91cIndicates the absence of an allele because the individual is homozygous for the other allele.c.3493+1G>A—Intron 2228aHUSYaAllele carrying the disease-associated variant./Y43cIndicates the absence of an allele because the individual is homozygous for the other allele.c.3497C>TP1166L23 (20)29aHUSYaAllele carrying the disease-associated variant./Y139cIndicates the absence of an allele because the individual is homozygous for the other allele.30aHUSYaAllele carrying the disease-associated variant./H608230-MHealthyYaAllele carrying the disease-associated variant./H90103c.3545G>AR1182K23 (20)31aHUSYaAllele carrying the disease-associated variant./Y183cIndicates the absence of an allele because the individual is homozygous for the other allele.31-MHealthyYaAllele carrying the disease-associated variant./Y172cIndicates the absence of an allele because the individual is homozygous for the other allele.31-P1HealthyYaAllele carrying the disease-associated variant./Y164cIndicates the absence of an allele because the individual is homozygous for the other allele.31-P2HealthyYaAllele carrying the disease-associated variant./H969231-P3HealthyYaAllele carrying the disease-associated variant./H6769c.3652T>CC1218R23 (20)32aHUS/C3GY/HaAllele carrying the disease-associated variant.600a Allele carrying the disease-associated variant.b Compound heterozygote with a null CFH allele.c Indicates the absence of an allele because the individual is homozygous for the other allele. Open table in a new tab Table 2Clinical and genetic data of patients carrying CFH variantsCarrier IDVariantAge, yrSexOnset age (yr)OutcomeTransplantRecurrencesTreatmentAdditional genetic risk factorsaHUS 1R53C24F2CKD0NoEculizumabNo 2R53S40F36CR0YesPlasma treatmentNo 3R53P38F35N/A0N/AN/ANo 4S58A58F47CKDN/ANoN/ACFH: c.88dup (p.T30Nfs*10) 5S58A6M3CR0N/AN/ANo 6C66Y36F12CKD3YesEculizumabNo 7L98F36M4CKD2YesEculizumabMCP: c.800_820del (p.T267_N273del) 10Y118Ifs*461F42CR0YesPlasma treatmentNo 12Y235C52M34CKD2YesN/ANo 13C309W51F48N/AN/AN/AEculizumabNo 14C320R3F1CR0NoEculizumabNo 16C597*16F13CR0N/AEculizumabNo 17K670T8F4CR0NoN/ANo 18M823T10F1CR0YesEculizumabCFI: c.1149-2 A>G;c.1643A>G (p.Q548G) 20C853R67M53CKD1N/APlasma treatmentNo 21C853Y49M41CKD0N/AN/ANo 22R885Sfs*1362M59N/A0N/AEculizumabC3: c.193A>C (p.K65Q) 24T956M30M1N/AN/AN/APlasma treatmentNo 25c.2957-1G>A50M46CR0YesN/AMCP; c.286+1G>C 26W1037*49F47CKD0N/AEculizumabTHBD: c.1502C>T (p.P501L) 27D1119N9F1N/A0N/AN/ANo 28c.3493+1G>A36F30N/AN/AN/AN/ANo 29P1166L56M45CKD1N/AN/ACFI: c.1322A>G (p.K441R) 30P1166L38F29CKD1N/AEculizumabNo 31R1182K50F36CKD0N/AN/ANo 32C1218R39F29CR0YesPlasma treatmentNoC3G 8A110S64M57N/A1YesEculizumab, prednisoneNo 9Y118Ifs*460M25CKD0N/AN/ANo 11C192W45M1N/A0YesPrednisoneNo 15K331E10MNAN/AN/AN/APrednisoneNo 19R830W14FNAN/AN/AN/AN/ANo 23R885Sfs*1353F17CKD2YesPrednisoneC3: c.193A>C (p.K65Q)aHUS, atypical hemolytic uremic syndrome; CKD, chronic kidney disease; CR, complete remission; F, female; M, male; N/A, data no available. Open table in a new tab aHUS, atypical hemolytic uremic syndrome; CKD, chronic kidney disease; CR, complete remission; F, female; M, male; N/A, data no available. To predict the functional consequences of the 28 novel CFH variants detected in this study, we used several prediction algorithms available in ANNOVAR software. We considered likely pathogenic those variants in which a deleterious impact of the genetic variation was anticipated by a majority of these prediction algorithms. According to these analyses, the CFH variants R53C, R53S, C66Y, C192W, Y235C, C309W, C320R, C853R, C853Y, D1119N, and C1218R were considered likely pathogenic, whereas R53P, S58A, L98F, A110S, K331E, K670T, M823T, R830W, T956M, P1166L, and R1182K were predicted to be benign or variants of uncertain significance (Supplementary Table S2). For the C597*, W1037*, Y118Ifs*4, and R885Sfs*13 variants, some of the prediction algorithms failed to give a result. However, the nature of these CFH variants, introducing a stop codon, justifies their assignment to the category of likely pathogenic variants. Similarly, c.2957-1G>A and c.3493+1G>A were predicted to be pathogenic because they alter the splicing sequences. As a whole, the analyses to predict pathogenicity indicate that 11 of the 28 CFH variations (39%) are predicted to be either benign or variants of uncertain significance (Supplementary Table S2). To formally categorize the disease-associated CFH variants and to establish their causal relationship with the pathology, we determined their expression levels in plasma and purified those that were expressed to perform a complete functional characterization. To do this, we benefited from having serum or plasma samples from all patients and relatives and the Y402H genotypes available for all individuals (Table 1). Twenty-one of the 28 CFH variants are carried by Y402H heterozygote individuals (patients and/or relatives). In all of these cases, a direct measurement of the FH produced by the mutated CFH allele was obtained. These analyses allowed us to conclude that the C66Y, Y118Ifs*4, C192W, Y235C, C309W, C320R, C597*, C853R, C853Y, R885Sfs*13, W1037*, and C1218R CFH variants are not expressed or result in very low levels of FH in plasma. We also concluded that the L98F, c.2957-1G>A, and c.3493+1G>A variants are not expressed because total plasma levels in heterozygote carriers were ANoPathogenicPathogenicW1037*NoPathogenicPathogenicC-terminal regionD1119NYesAffectedAffectedAffectedPathogenicPathogenicc.3493+1G>ANoPathogenicPathogenicP1166LYesAffectedAffectedAffectedPathogenicVUSR1182KYesAffectedAffectedAffectedPathogenicBenignC1218RNoPathogenicPathogenicVUS, variants of uncertain significance. Open table in a new tab VUS, variants of uncertain sig
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