| 1. |
- van de Ven, Johannes P. H., et al.
(author)
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A functional variant in the CFI gene confers a high risk of age-related macular degeneration
- 2013
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In: Nature Genetics. - : Springer Science and Business Media LLC. - 1546-1718 .- 1061-4036. ; 45:7, s. 813-813
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Journal article (peer-reviewed)abstract
- Up to half of the heritability of age-related macular degeneration (AMD) is explained by common variants(1-5). Here, we report the identification of a rare, highly penetrant missense mutation in CFI encoding a p.Gly119Arg substitution that confers high risk of AMD (P = 3.79 x 10(-6); odds ratio (OR) = 22.20, 95% confidence interval (CI) = 2.98-164.49). Plasma and sera from cases carrying the p.Gly119Arg substitution mediated the degradation of C3b, both in the fluid phase and on the cell surface, to a lesser extent than those from controls. Recombinant protein studies showed that the Gly119Arg mutant protein is both expressed and secreted at lower levels than wild-type protein. Consistent with these findings, human CFI mRNA encoding Arg119 had reduced activity compared to wild-type mRNA encoding Gly119 in regulating vessel thickness and branching in the zebrafish retina. Taken together, these findings demonstrate that rare, highly penetrant mutations contribute to the genetic burden of AMD.
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| 2. |
- de Jong, Sarah, et al.
(author)
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Effect of rare coding variants in the CFI gene on Factor I expression levels
- 2020
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In: Human Molecular Genetics. - : Oxford University Press (OUP). - 0964-6906 .- 1460-2083. ; 29:14, s. 2313-2324
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Journal article (peer-reviewed)abstract
- Factor I (FI) is one of the main inhibitors of complement activity, and numerous rare coding variants have been reported in patients with age-related macular degeneration, atypical hemolytic uremic syndrome and C3 glomerulopathy. Since many of these variants are of unknown clinical significance, this study aimed to determine the effect of rare coding variants in the complement factor I (CFI) gene on FI expression. We measured FI levels in plasma samples of carriers of rare coding variants and in vitro in the supernatants of epithelial cells expressing recombinant FI. FI levels were measured in 177 plasma samples of 155 individuals, carrying 24 different rare coding variants in CFI. In carriers of the variants p.Gly119Arg, p.Leu131Arg, p.Gly188Ala and c.772G>A (r.685_773del), significantly reduced FI plasma levels were detected. Furthermore, recombinant FI expression levels were determined for 126 rare coding variants. Of these variants 68 (54%) resulted in significantly reduced FI expression in supernatant compared to wildtype (WT). The recombinant protein expression levels correlated significantly with the FI level in plasma of carriers of CFI variants. In this study, we performed the most comprehensive FI expression level analysis of rare coding variants in CFI to date. More than half of CFI variants lead to reduced FI expression, which might impair complement regulation in vivo. Our study will aid the interpretation of rare coding CFI variants identified in clinical practice, which is in particular important in light of patient inclusion in ongoing clinical trials for CFI gene supplementation in AMD.
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| 3. |
- de Jong, Sarah, et al.
(author)
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Functional Analysis of Variants in Complement Factor I Identified in Age-Related Macular Degeneration and Atypical Hemolytic Uremic Syndrome
- 2022
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In: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 12
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Journal article (peer-reviewed)abstract
- Complement factor I (FI) is a central inhibitor of the complement system, and impaired FI function increases complement activation, contributing to diseases such as age-related macular degeneration (AMD) and atypical hemolytic uremic syndrome (aHUS). Genetic variation in complement factor I (CFI) has been identified in both AMD and aHUS, with more than half of these variants leading to reduced FI secretion levels. For many of the variants with normal FI secretion, however, functional implications are not yet known. Here we studied 11 rare missense variants, with FI secretion levels comparable to wildtype, but a predicted damaging effects based on the Combined Annotation Dependent Depletion (CADD) score. Three variants (p.Pro50Ala, p.Arg339Gln, and p.Ser570Thr) were analyzed in plasma and serum samples of carriers affected by AMD. All 11 variants (nine for the first time in this study) were recombinantly expressed and the ability to degrade C3b was studied with the C3b degradation assay. The amount of degradation was determined by measuring the degradation product iC3b with ELISA. Eight of 11 (73%) mutant proteins (p.Pro50Ala, p.Arg339Gln, p.Ile340Thr, p.Gly342Glu, p.Gly349Arg, p.Arg474Gln, p.Gly487Cys, and p.Gly512Ser) showed significantly impaired C3b degradation, and were therefore classified as likely pathogenic. Our data indicate that genetic variants in CFI with a CADD score >20 are likely to affect FI function, and that monitoring iC3b in a degradation assay is a useful tool to establish the pathogenicity of CFI variants in functional studies.
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| 4. |
- Smailhodzic, Dzenita, et al.
(author)
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Zinc supplementation inhibits complement activation in age-related macular degeneration.
- 2014
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In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:11
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Journal article (peer-reviewed)abstract
- Age-related macular degeneration (AMD) is the leading cause of blindness in the Western world. AMD is a multifactorial disorder but complement-mediated inflammation at the level of the retina plays a pivotal role. Oral zinc supplementation can reduce the progression of AMD but the precise mechanism of this protective effect is as yet unclear. We investigated whether zinc supplementation directly affects the degree of complement activation in AMD and whether there is a relation between serum complement catabolism during zinc administration and the complement factor H (CFH) gene or the Age-Related Maculopathy susceptibility 2 (ARMS2) genotype. In this open-label clinical study, 72 randomly selected AMD patients in various stages of AMD received a daily supplement of 50 mg zinc sulphate and 1 mg cupric sulphate for three months. Serum complement catabolism-defined as the C3d/C3 ratio-was measured at baseline, throughout the three months of supplementation and after discontinuation of zinc administration. Additionally, downstream inhibition of complement catabolism was evaluated by measurement of anaphylatoxin C5a. Furthermore, we investigated the effect of zinc on complement activation in vitro. AMD patients with high levels of complement catabolism at baseline exhibited a steeper decline in serum complement activation (p<0.001) during the three month zinc supplementation period compared to patients with low complement levels. There was no significant association of change in complement catabolism and CFH and ARMS2 genotype. In vitro zinc sulphate directly inhibits complement catabolism in hemolytic assays and membrane attack complex (MAC) deposition on RPE cells. This study provides evidence that daily administration of 50 mg zinc sulphate can inhibit complement catabolism in AMD patients with increased complement activation. This could explain part of the mechanism by which zinc slows AMD progression.
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| 5. |
- Geerlings, Maartje J., et al.
(author)
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The functional effect of rare variants in complement genes on C3b degradation in patients with age-related macular degeneration
- 2017
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In: JAMA Ophthalmology. - : American Medical Association (AMA). - 2168-6165. ; 135:1, s. 39-46
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Journal article (peer-reviewed)abstract
- IMPORTANCE In age-related macular degeneration (AMD), rare variants in the complement system have been described, but their functional consequences remain largely unexplored. OBJECTIVES To identify new rare variants in complement genes and determine the functional effect of identified variants on complement levels and complement regulation in serum samples from carriers and noncarriers. DESIGN, SETTING, AND PARTICIPANTS This study evaluated affected (n = 114) and unaffected (n = 60) members of 22 families with AMD and a case-control cohort consisting of 1831 unrelated patients with AMD and 1367 control individuals from the European Genetic Database from March 29, 2006, to April 26, 2013, in Nijmegen, the Netherlands, and Cologne, Germany. Exome sequencing data of families were filtered for rare variants in the complement factor H (CFH), complement factor I (CFI), complement C9 (C9), and complement C3 (C3) genes. The case-control cohort was genotyped with allele-specific assays. Serum samples were obtained from carriers of identified variants (n = 177) and age-matched noncarriers (n = 157). Serum concentrations of factor H (FH), factor I (FI), C9, and C3 were measured, and C3b degradation ability was determined. MAIN OUTCOMES AND MEASURES Association of rare variants in the CFH, CFI, C9, and C3 genes with AMD, serum levels of corresponding proteins, and C3b degradation ability of CFH and CFI variant carriers. RESULTS The 1831 unrelated patients with AMD had a mean (SD) age of 75.0 (9.4) years, and 60.5%were female. The 1367 unrelated control participants had a mean (SD) age of 70.4 (7.0), and 58.7%were female. All individuals were of European descent. Rare variants in CFH, CFI, C9, and C3 contributed to an increased risk of developing AMD (odds ratio, 2.04; 95%CI, 1.47-2.82; P < .001). CFI carriers had decreased median FI serum levels (18.2 μg/mL in Gly119Arg carriers and 16.2 μg/mL in Leu131Arg carriers vs 27.2 and 30.4 μg/mL in noncarrier cases and controls, respectively; both P < .001). Elevated C9 levels were observed in Pro167Ser carriers (10.7 μg/mL vs 6.6 and 6.1 μg/mL in noncarrier cases and controls, respectively; P < .001). The median FH serum levels were 299.4 μg/mL for CFH Arg175Gln and 266.3 μg/mL for CFH Ser193Leu carriers vs 302.4 and 283.0 μg/mL for noncarrier cases and controls, respectively. The median C3 serum levels were 943.2 μg/mL for C3 Arg161Trp and 946.7 μg/mL for C3 Lys155Gln carriers vs 874.0 and 946.7 μg/mL for noncarrier cases and controls, respectively. The FH and FI levels correlated with C3b degradation in noncarriers (R2 = 0.35 and R2 = 0.31, respectively; both P < .001). CONCLUSIONS AND RELEVANCE Reduced serum levels were associated with C3b degradation in carriers of CFI but not CFH variants, suggesting that CFH variants affect functional activity of FH rather than serum levels. Carriers of CFH (Arg175Gln and Ser193Leu) and CFI (Gly119Arg and Leu131Arg) variants have an impaired ability to regulate complement activation and may benefit more from complement-inhibiting therapy than patients with AMD in general.
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| 6. |
- Kremlitzka, Mariann, et al.
(author)
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Functional analyses of rare genetic variants in complement component C9 identified in patients with age-related macular degeneration
- 2018
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In: Human Molecular Genetics. - : Oxford University Press (OUP). - 0964-6906 .- 1460-2083. ; 27:15, s. 2678-2688
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Journal article (peer-reviewed)abstract
- Age-related macular degeneration (AMD) is a progressive disease of the central retina and the leading cause of irreversible vision loss in the western world. The involvement of abnormal complement activation in AMD has been suggested by association of variants in genes encoding complement proteins with disease development. A low-frequency variant (p.P167S) in the complement component C9 (C9) gene was recently shown to be highly associated with AMD; however, its functional outcome remains largely unexplored. In this study, we reveal five novel rare genetic variants (p.M45L, p.F62S, p.G126R, p.T170I and p.A529T) in C9 in AMD patients, and evaluate their functional effects in vitro together with the previously identified (p.R118Wand p.P167S) C9 variants. Our results demonstrate that the concentration of C9 is significantly elevated in patients' sera carrying the p.M45L, p.F62S, p.P167S and p.A529T variants compared with non-carrier controls. However, no difference can be observed in soluble terminal complement complex levels between the carrier and non-carrier groups. Comparing the polymerization of the C9 variants we reveal that the p.P167S mutant spontaneously aggregates, while the other mutant proteins (except for C9 p.A529T) fail to polymerize in the presence of zinc. Altered polymerization of the p.F62S and p.P167S proteins associated with decreased lysis of sheep erythrocytes and adult retinal pigment epithelial-19 cells by carriers' sera. Our data suggest that the analyzed C9 variants affect only the secretion and polymerization of C9, without influencing its classical lytic activity. Future studies need to be performed to understand the implications of the altered polymerization of C9 in AMD pathology.
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