Complement C3 Variant and the Risk of Age-Related Macular Degeneration
John R.W. Yates, F.R.C.P., Tiina Sepp, Ph.D., Baljinder K. Matharu, M.Sc., Jane C. Khan, F.R.C.Ophth., Deborah A. Thurlby, R.G.N., M.Sc., Humma Shahid, M.R.C.Ophth., David G. Clayton, M.A., Caroline Hayward, Ph.D., Joanne Morgan, B.Sc., Alan F. Wright, Ph.D., F.R.C.P., Ana Maria Armbrecht, Ph.D., F.R.C.S., Baljean Dhillon, F.R.C.S., F.R.C.Ophth., Ian J. Deary, Ph.D., F.R.C.P.E., Elizabeth Redmond, R.G.N., M.Sc., Alan C. Bird, M.D., F.R.C.S., Anthony T. Moore, F.R.C.S., F.R.C.Ophth., for the Genetic Factors in AMD Study Group
Background Age-related macular degeneration is the most commoncause of blindness in Western populations. Susceptibility isinfluenced by age and by genetic and environmental factors.Complement activation is implicated in the pathogenesis.
Methods We tested for an association between age-related maculardegeneration and 13 single-nucleotide polymorphisms (SNPs) spanningthe complement genes C3 and C5 in case subjects and controlsubjects from the southeastern region of England. All subjectswere examined by an ophthalmologist and had independent gradingof fundus photographs to confirm their disease status. To testfor replication of the most significant findings, we genotypeda set of Scottish cases and controls.
Results The common functional polymorphism rs2230199 (Arg80Gly)in the C3 gene, corresponding to the electrophoretic variantsC3S (slow) and C3F (fast), was strongly associated with age-relatedmacular degeneration in both the English group (603 cases and350 controls, P=5.9x10–5) and the Scottish group (244cases and 351 controls, P=5.0x10–5). The odds ratio forage-related macular degeneration in C3 S/F heterozygotes ascompared with S/S homozygotes was 1.7 (95% confidence interval[CI], 1.3 to 2.1); for F/F homozygotes, the odds ratio was 2.6(95% CI, 1.6 to 4.1). The estimated population attributablerisk for C3F was 22%.
Conclusions Complement C3 is important in the pathogenesis ofage-related macular degeneration. This finding further underscoresthe influence of the complement pathway in the pathogenesisof this disease.
Age-related macular degeneration is the leading cause of visualimpairment in the elderly and the most common cause of blindnessin Western countries.1 It affects the macular region of theretina. The macula has a high density of photoreceptors andprovides detailed central vision. In the early stages of thedisease (referred to as age-related maculopathy), deposits calleddrusen develop between the retinal pigment epithelium and underlyingchoroid.1 Later, the disease is manifested as either extensiveatrophy of the retinal pigment epithelium and overlying photoreceptorcells (geographic atrophy) or aberrant choroidal angiogenesis(choroidal neovascularization).1 Both of these conditions canlead to a loss of central vision. The pathogenesis of age-relatedmacular degeneration is poorly understood. As with other late-onsetchronic diseases, susceptibility is influenced by age, ethnicbackground, and a combination of environmental and genetic factors.1,2Smoking status and family history are well-established determinantsof risk.1,2
Recently, polymorphisms in the genes coding for complement factorH (CFH) and complement factor B (CFB) have been shown to bepredictors of risk for age-related macular degeneration.3,4,5,6,7,8,9,10,11Another susceptibility locus has been mapped to chromosome 10q26;the causative variation probably lies in a hypothetical genecalled LOC387715 or in the promoter of the neighboring geneHTRA1.11,12,13,14 The population attributable risk associatedwith variants in CFH, CFB, and LOC387715/HTRA1 is at least 50%.11
CFH and CFB are key components of the alternative complementpathway. Their involvement in age-related macular degeneration,together with the finding that drusen contain proteins associatedwith inflammation and immune-mediated processes,15 supportsthe hypothesis that inflammation and complement activation influencethe pathogenesis of age-related macular degeneration. To testwhether variants in other genes encoding proteins in the complementpathway influence susceptibility to age-related macular degeneration,we genotyped single-nucleotide polymorphisms (SNPs) spanningthe complement genes C3 and C5, encoding central proteins inthe complement cascade, in subjects with age-related maculardegeneration and in control subjects.
Methods
Cases and Controls
We studied three case–control groups, two in England andone in Scotland. English group 1 comprised 446 case subjectswith end-stage age-related macular degeneration (geographicatrophy or choroidal neovascularization) and 267 control subjects,who were spouses of the index patients. All subjects were recruitedfrom ophthalmic clinics in eight hospitals in southeastern Englandfrom 2002 to 2004.7 English group 2 comprised 157 case subjectswith end-stage age-related macular degeneration and 83 controls(67 spouses and 16 friends of index patients) recruited from2003 to 2005, the majority from Moorfields Eye Hospital in Londonand the remainder from southeastern England. All subjects describedthemselves as "white" rather than "other" on a recruitment questionnaire.
The Scottish group comprised 505 case subjects with age-relatedmaculopathy or end-stage age-related macular degeneration and351 control subjects. A total of 337 case subjects from theLothian region were recruited from ophthalmic clinics in Edinburghand 46 case subjects from hospitals in Dundee and Invernessfrom 2004 to 2006. Control subjects, who were recruited fromthe same sources in similar proportions, comprised 32 spousesand 174 subjects who had undergone cataract surgery. Another122 case subjects and 145 controls came from the 1921 Lothianbirth cohort.16
Written informed consent was obtained from all subjects. Theresearch protocol was in keeping with the provisions of theDeclaration of Helsinki, and approval was obtained from a multicenterresearch ethics committee and from research ethics committeesfor each institution. Subjects were examined by an ophthalmologist,and data were collected regarding medical history, lifestyle,and smoking history. Color, stereoscopic fundus photographyof the macular region was performed in all subjects. For Englishsubjects, the images were graded at the Reading Centre, MoorfieldsEye Hospital, with the use of the International Classificationof Age-Related Maculopathy and Macular Degeneration.17 For Scottishsubjects, a study investigator graded images; for validation,images from 100 case subjects and controls were independentlygraded at the Moorfields Reading Centre (kappa statistic, 0.84).Eight prospective English controls with age-related maculardegeneration and 60 prospective Scottish controls with age-relatedmaculopathy were reclassified as case subjects. Data on diseasestatus, sex, age, and smoking history of subjects are providedin Table 1.
Table 1. Disease Status, Sex, Age, and Smoking History of Subjects.
Genotyping
We extracted genomic DNA from peripheral-blood leukocytes. Weselected SNPs spanning the C3 and C5 genes from the InternationalHapMap Project18 data (release 19) for the Centre d'Étudedu Polymorphisme Humain (CEPH) population (Utah residents withancestry from northern and western Europe). Criteria for theselection of SNPs were high heterozygosity with a minor allelefrequency of at least 10%, tagging of the most common haplotypes,and coverage of the main blocks of linkage disequilibrium. TheC3 SNP rs2230199 — which is predicted to result in a substitutionof a glycine residue for arginine at position 80 (Arg80Gly)— generates the "fast" electrophoretic allotype of C3(called C3F); the alternative allotype is "slow" (C3S).19,20We included this SNP in the analysis to provide extra coverageand because of evidence of a functional difference between thetwo alleles. On the basis of our initial analysis, we includedrs1047286 (Pro292Leu), which has a known association with rs2230199.20,21Initial genotyping was carried out in English group 1. Markersof interest were genotyped in group 2 when samples became available.Data from the Scottish group were used for replication.
We performed genotyping in English subjects with the use ofa single-nucleotide primer extension assay (ABI Prism SNaPshotMultiplex Kit, Applied Biosystems) and a genetic analyzer (ABIPrism 3100, Applied Biosystems) and in Scottish subjects —for rs2230199 and rs1047286 — with the use of competitiveallele-specific polymerase-chain-reaction assays (Taqman SNPGenotyping Assay, Applied Biosystems and KASPar SNP GenotypingSystem, KBiosciences, respectively). Manufacturers' protocolswere followed.
Statistical Analysis
We used the chi-square test for comparisons of categorical variablesand allele and genotype frequencies and to check for Hardy–Weinbergequilibrium. All P values were calculated with two-sided tests,and no correction was made for multiple testing. The Mann–WhitneyU test was used to compare the ages of case subjects and controls.Logistic-regression analysis was used to investigate interactionsbetween genotype and other variables and to estimate odds ratiosand 95% confidence intervals. The covariables of age and smokinghistory were included in the logistic model if univariate analysishad shown a significant difference. Odds ratios for categoricalvariables were estimated in relation to a reference category.Data were analyzed with the use of the SPSS statistical softwarepackage, version 11.0.
The population attributable risk was calculated from the formula100D÷(1+D), in which D was equal to P1(RR1–1) +P2(RR2–1), where P1 and P2 are the frequencies of theat-risk genotypes, and RR1 and RR2 their associated relativerisks, as compared with the low-risk genotype. For the purposesof estimation, odds ratios were equated to relative risks, sincethe disease prevalence is low.
Results
In the initial screening, 12 SNPs spanning C3 and C5 (thoselisted in Table 2, excluding rs1047286) were genotyped in 446case subjects with late-stage age-related macular degenerationand 267 control subjects (English group 1). No evidence of anassociation was found with variants in C5 (Table 2). In C3,the expressed SNP rs2230199 showed strong evidence of an association(P<0.001) and was genotyped in an additional 157 case subjectsand 83 controls (English group 2). The enlarged sample alsoprovided strong evidence of an association (P=5.9x10–5)(Table 2).
To test for replication of this finding, rs2230199 was genotypedby a different laboratory in 244 case subjects with late-stageage-related macular degeneration, 261 case subjects with age-relatedmaculopathy, and 351 controls (Scottish group). Again, therewas a highly significant association between the minor alleleand age-related macular degeneration (P=5.0x10–5) (Table 3).International HapMap Project18 data for the CEPH populationshowed that rs2230199 had an r2 value of 0.75 with rs2230203but a low r2 value with other C3 SNPs in our marker panel andwith other C3 SNPs in the HapMap data set. SNP rs2230203 didnot show a significant association with age-related maculardegeneration in group 1 alone, but there was weak evidence ofan association in groups 1 and 2 combined (Table 2).
Table 3. Genotyping Results for Scottish Replication Group.
Because of the known association between the allotypes of rs2230199and the expressed C3 SNP rs1047286, the English and Scottishsubjects were genotyped for this marker (Table 2 and Table 3).The minor allele frequency was significantly higher in casesubjects than in controls in both groups, but the associationwas not as strong as for rs2230199. Stepwise logistic-regressionanalysis confirmed that rs2230199 is a significantly betterpredictor of risk for age-related macular degeneration. Withthis SNP in the model, adding rs1047286 made no contribution(P=0.90). With rs1047286 in the model, adding rs2230199 produceda significant improvement in fit (P=0.02).
Odds ratios for age-related macular degeneration as a functionof rs2230199 genotype are given in Table 4. Results for theEnglish and Scottish groups were similar. In the combined dataset, with the common CC genotype as the reference, the oddsratio was 1.7 for CG heterozygotes and 2.6 for GG homozygotes.The estimated population attributable risk for this variantwas 22%.
Table 4. Complement C3 rs2230199 Genotype (C3 S/F Allotype) and Odds Ratios for Age-Related Macular Degeneration.
Subgroup analysis that was confined to case subjects with onlychoroidal neovascularization showed a highly significant associationin both case–control groups. For case subjects with onlygeographic atrophy, the association was significant in the Englishgroup (P=4.6x10–4) but not in the Scottish group, whichhad fewer subjects with geographic atrophy. The Scottish groupincluded case subjects with age-related maculopathy, and inthis subgroup the association fell just short of significance(Table 3).
Data on other susceptibility loci for age-related macular degenerationwere available for English group 1. Results for CFH Y402H havebeen published previously and are in agreement with other reports.7Odds ratios and population attributable risks for LOC387715(rs10490924) and CFB (rs641153) are given in Table 5. The resultsare similar to those of other studies, except that we founda lower odds ratio for rs10490924 homozygotes. When these variableswere included in the stepwise logistic model, C3 rs2230199 remainedsignificant, with an odds ratio of 1.4 for CG heterozygotesand 3.3 for GG homozygotes (with CC genotype as the reference),confirming that these susceptibility loci are independent riskfactors.
Table 5. Odds Ratios for Age-Related Macular Degeneration and Population Attributable Risk for Variants at the Susceptibility Loci CFH, CFB, and LOC387715 in English Group 1.
Discussion
Our study showed a strong association between the complementC3 S/F (Arg80Gly) polymorphism and age-related macular degeneration,with similar findings for geographic atrophy and choroidal neovascularization.The C3F allele frequency is approximately 20% in white populationsbut lower in other ethnic groups. For age-related maculopathy,the association fell just short of significance, raising thepossibility that this polymorphism has less influence on theearlier stages of the disease.
The complement system comprises more than 30 plasma and cell-surfaceproteins. It mediates the host defense against pathogens andthe elimination of immune complexes and apoptotic cells; italso facilitates adaptive immune responses.22 C3 is the mostabundant complement component, synthesized predominantly inthe liver but to a lesser extent in other cells and tissues.Significant C3 messenger RNA is detectable in the neural retina,choroid, retinal pigment epithelium, and cultured retinal-pigment-epitheliumcells.15
Cleavage of C3 into C3a and C3b is the central step in complementactivation and can be initiated by the classic antibody-mediatedpathway, the lectin pathway, or the alternative complement pathway.22C3b attaches to pathogens or other target surfaces and bindsfactor B, which is then cleaved. The resulting C3bBb complexhas C3 convertase activity, which amplifies the response byfurther cleavage of C3 and leads to the formation of C3b2Bbcomplexes with C5 convertase activity. This brings about cleavageof component C5 and recruitment by C5b of components C6 throughC9 to form a large molecular pore on target membranes (the membraneattack complex), resulting in cell lysis.22
Drusen contain C3 and its activation products, as well as C5,membrane attack complex, and CFH,6,15 supporting the hypothesisthat local inflammation and activation of the complement cascadecontribute to the pathogenesis of age-related macular degeneration.Further support for this hypothesis comes from conclusive evidencethat variants in CFH influence susceptibility to age-relatedmacular degeneration.3,4,5,6,7,8,9 CFH is a key regulator ofthe alternative complement pathway and prevents uncontrolledcomplement activation. Variants in factor B also appear to influencesusceptibility to age-related macular degeneration.10,11 Inmice, activation of complement and formation of the membraneattack complex are essential for the development of laser-inducedchoroidal neovascularization. Indeed, the finding that choroidalneovascularization cannot be induced by laser coagulation inC3–/– mice demonstrates the key role of C3 in thisprocess.23
As a result of cleavage of C3 to form C3b, the molecule undergoesconformational changes that expose several binding sites, includingthe thioester moiety, which is essential for C3b binding totarget surfaces.24 Exposure of this activated acyl-imidazoleintermediate requires a substantial relocation of the thioester-containingdomain to a position adjacent to the first macroglobulin domain.24Arg80 together with Arg72 and Lys82 forms a positively chargedpatch on the surface of this domain, which, in C3b, is broughtinto close proximity with the negatively charged carboxyl groupsof several amino acids on the surface of the thioester-containingdomain (Figure 1). Substitution of an uncharged glycine forthe positively charged Arg80 is predicted to weaken the interactionbetween these oppositely charged surfaces and could potentiallyinfluence thioester activity or other binding interactions ofthe thioester-containing domain, including a probable C3b/C3dbinding site with CFH.25 It follows that there could well befunctional differences between the C3 S/F variants.
Figure 1. Structure of Complement C3b, Showing the Location of Arg80.
Ribbon representation of the structure of complement C3b, as proposed by Janssen et al.,24 shows the interface region between the macroglobulin 1 domain (MG1) (light blue) and the thioester-containing domain (orange). The residues participating in the formation of the thioester bond are shown in yellow. Arg80 (red) is located in MG1, adjacent to two other positively charged amino acids, Arg72 and Lys82 (purple). These residues are approximately 4 Å from the negatively charged amino acids Asp1007, Glu1008, Glu1010, and Glu1013 (dark blue) in the thioester-containing domain. The first three of these residues contribute to a probable C3b/C3d binding site with complement factor H.25 Arg80 may also have interactions with negatively charged residues (not shown) in the complement C1r/C1s–Uegf–Bmp1–containing domain, adjacent to the thioester-containing domain.
Direct experimental evidence of functional differences in vitrobetween the C3 S/F allotypes is not conclusive. Arvilommi26reported that erythrocytes coated with C3F showed greater rosettingwith peripheral-blood mononuclear cells than those coated withC3S. Welch et al.27 studied uptake on sheep erythrocytes, hemolyticactivity, conversion to inactive C3b, and capacity to solubilizepreformed immune complexes. The only significant differencewas that C3F had lower activity than C3S in a hemolytic assayusing sensitized sheep erythrocytes as a result of a small differencein cell-surface binding. Bartók and Walport28 found nodifferences between binding of C3S and C3F and the major complementreceptor types 1, 2, and 3.
On the other hand, there is compelling indirect evidence ofa functional difference between C3S and C3F. A recent studyhas shown that the C3 S/F genotype is an important determinantof the long-term outcome of renal transplantation.29 In recipientswho were C3S homozygotes, graft survival was substantially prolongedand renal function significantly better with C3 F/F and C3 F/Sdonor kidneys than with C3 S/S kidneys.
Several associations of disease with C3F have been reported,including IgA nephropathy,30 systemic vasculitis,31 partiallipodystrophy, and membranoproliferative glomerulonephritistype II (MPGNII).32,33 The association with MPGNII is particularlyrelevant. This is a rare disease characterized by complement-containingdense deposits in the glomerular basement membrane of the kidney.34The condition is caused by uncontrolled activation of the alternativecomplement pathway. In the majority of patients, the conditionis associated with serum C3 nephritic factor,34 an autoantibodydirected against the C3bBb complex, but rare cases associatedwith mutations in CFH have been reported.35 A similar form ofglomerulonephritis develops in CFH-deficient pigs36 and CFHknockout mice.37 The interface of the capillary tuft, the glomerularbasement membrane, and the glomerular epithelial cells in thekidney is similar in structure to the interface involving choriocapillaris,Bruch's membrane, and retinal pigment epithelium in the eye,and macular drusen similar to those in age-related macular degenerationdevelop in patients with MPGNII, but at a much younger age.38These lesions are structurally and compositionally identicalto those in patients with age-related macular degeneration andshow immunoreactivity to complement C5 and C5b-9 complexes.39Drusen have also been reported in patients with partial lipodystrophy.40The association of MPGNII and partial lipodystrophy with C3Ffits well with our current findings.
In summary, our study shows a strong association between theC3F variant and age-related macular degeneration, and thereis evidence of functional differences between the C3 S/F allotypes.It follows that C3F is likely to have a causal role in the disorder.The estimated population attributable risk in the white populationis 22%. These findings add to our growing understanding of thegenetics of age-related macular degeneration and provide conclusiveevidence that the complement pathway has a key role in the pathogenesisof this common and debilitating condition.
Supported by grants from the Medical Research Council, UnitedKingdom (to Drs. Yates, Clayton, Bird, and Moore), the ChiefScientist Office, Scotland (to Dr. Dhillon), the Macular VisionResearch Foundation (to Dr. Wright), and the Wellcome Trustand the Juvenile Diabetes Research Foundation (to Dr. Clayton).The Lothian Birth Cohort collection was supported by the Biotechnologyand Biological Sciences Research Council. Dr. Deary is the recipientof a Royal Society–Wolfson Research Merit Award.
Dr. Dhillon reports receiving consulting fees from Novartisand Pfizer. No other potential conflict of interest relevantto this article was reported.
We thank members of the Scottish Macula Society Study Group(M. Gavin, F. Imrie, N. Lois, R. Murray, A. Purdie, A. Pyott,S. Roxburgh, C. Styles, M. Virdi, and W. Wykes) for their helpin the recruitment of patients for our study; clinic staff andmedical photographers at the participating clinics for theirhelp; Tunde Peto and colleagues at the Reading Centre, MoorfieldsEye Hospital, London, for grading the fundus photographs; thestaff at Tepnel Life Sciences for performing the DNA extractions;Roger Williams for his helpful discussion about C3b proteinstructure; the International HapMap Consortium for the use ofdata; and all the patients and their families who participatedin the study.
* Other members of the Genetic Factors in Age-Related MacularDegeneration (AMD) Study Group are listed in the Appendix.
Source Information
From the Cambridge Institute for Medical Research, University of Cambridge, Cambridge (J.R.W.Y., T.S., B.K.M., J.C.K., D.A.T., H.S., D.G.C.); the Medical Research Council Human Genetics Unit, Edinburgh (C.H., J.M., A.F.W.); the Princess Alexandra Eye Pavilion, Edinburgh (A.M.A., B.D.); the University of Edinburgh, Edinburgh (I.J.D.); the Institute of Ophthalmology, University College London (E.R., A.C.B., A.T.M.); and Moorfields Eye Hospital, London (A.C.B., A.T.M.) — all in the United Kingdom. Drs. Yates and Sepp contributed equally to this article. This article (10.1056/NEJMoa072618) was published at www.nejm.org on July 18, 2007.
Address reprint requests to Dr. Yates at the Department of Medical Genetics, University of Cambridge, Cambridge Institute for Medical Research, Wellcome Trust/MRC Bldg., Box 139, Addenbrooke's Hospital, Cambridge CB2 0XY, United Kingdom, or at jrwy1{at}cam.ac.uk.
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Appendix
The following investigators are members of the Genetic Factorsin Age-Related Macular Degeneration Study Group: S.S. Bhattacharya,P. Bishop, P. Black, Z. Butt, V. Chong, N.E. Day, C. Edelsten,A. Fitt, D.W. Flanagan, A. Glenn, S. Harding, C. Jakeman, C.Jones, R.J. Lamb, A. Lotery, V. Moffatt, C.M. Moorman, T. Peto,R.J. Pushpanathan, and T. Rimmer.
Yang, P., Tyrrell, J., Han, I., Jaffe, G. J.
(2009). Expression and Modulation of RPE Cell Membrane Complement Regulatory Proteins. IOVS
50: 3473-3481
[Abstract][Full Text]
Park, K. H., Fridley, B. L., Ryu, E., Tosakulwong, N., Edwards, A. O.
(2009). Complement Component 3 (C3) Haplotypes and Risk of Advanced Age-Related Macular Degeneration. IOVS
50: 3386-3393
[Abstract][Full Text]
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