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Original Contribution |

Complement Factor H Polymorphism, Complement Activators, and Risk of Age-Related Macular Degeneration FREE

Dominiek D. G. Despriet, MD; Caroline C. W. Klaver, MD, PhD; Jacqueline C. M. Witteman, PhD; Arthur A. B. Bergen, PhD; Isabella Kardys, MD; Moniek P. M. de Maat, PhD; Sharmila S. Boekhoorn, MD; Johannes R. Vingerling, MD, PhD; Albert Hofman, MD, PhD; Ben A. Oostra, PhD; André G. Uitterlinden, PhD; Theo Stijnen, PhD; Cornelia M. van Duijn, PhD; Paulus T. V. M. de Jong, MD, PhD
[+] Author Affiliations

Author Affiliations: Departments of Epidemiology and Biostatistics (Drs Despriet, Klaver, Witteman, Kardys, Boekhoorn, Vingerling, Hofman, Uitterlinden, Stijnen, Van Duijn, and De Jong), Ophthalmology (Drs Despriet, Klaver, and Vingerling), Hematology (Dr De Maat), Clinical Genetics (Dr Oostra), and Internal Medicine (Dr Uitterlinden), Erasmus Medical Center, Rotterdam, the Netherlands; and the Department of Molecular and Clinical Ophthalmogenetics, the Netherlands Institute for Neuroscience, Amsterdam, the Netherlands (Drs Despriet, Klaver, Bergen, and De Jong); and the Departments of Clinical Genetics (Dr Bergen) and Ophthalmology (Dr De Jong), Academic Medical Centre, Amsterdam, the Netherlands.

More Author Information
JAMA. 2006;296(3):301-309. doi:10.1001/jama.296.3.301.
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Published online

Context The evidence that inflammation is an important pathway in age-related macular degeneration (AMD) is growing. Recent case-control studies demonstrated an association between the complement factor H (CFH) gene, a regulator of complement, and AMD.

Objectives To assess the associations between the CFH gene and AMD in the general population and to investigate the modifying effect of smoking, serum inflammatory markers, and genetic variation of C-reactive protein (CRP).

Design, Setting, and Participants Population-based, prospective cohort study of individuals aged 55 years or older (enrollment between March 20, 1990, and July 31, 1993, and 3 follow-up examinations that were performed between September 1, 1993, and December 31, 2004) in Rotterdam, the Netherlands. The CFH Y402H polymorphism was determined in a total of 5681 individuals. Information on smoking, erythrocyte sedimentation rate, CRP serum levels, and haplotypes of the CRP gene were assessed at baseline.

Main Outcome Measures All severity stages of prevalent and incident AMD, graded according to the international classification and grading system for AMD.

Results The frequency of CFH Y402H was 36.2% (4116/11 362 alleles). At baseline, there were 2062 persons (36.3%) with any type of AMD (prevalent cases), including 78 (1.4%) with late AMD (stage 4). During follow-up (mean, 8 years; median, 10 years), 1649 (35.5%) of 4642 participants progressed to a higher stage of AMD (incident cases), including 93 (5.6%) who developed late AMD. The odds ratio (OR) of AMD increased in an allele-dose manner with 2.00 (95% confidence interval [CI], 1.56-2.55) for stage 2 AMD, 4.58 (95% CI, 2.82-7.44) for stage 3 AMD, and 11.02 (95% CI, 6.82-11.81) for stage 4 (late, vision threatening) AMD for homozygous persons. Cumulative risks calculated by Kaplan-Meier analysis of late AMD by age 95 years were 48.3% for homozygotes, 42.6% for heterozygotes, and 21.9% for noncarriers. The population-attributable risk for CFH Y402H was 54.0%. Elevated erythrocyte sedimentation rates further increased the OR to 20.2 (95% CI, 9.5-43.0), elevated serum CRP levels to 27.7 (95% CI, 10.7-72.0), and smoking to 34.0 (95% CI, 13.0-88.6) for homozygotes compared with noncarriers without these determinants. The CRP haplotypes conferring high levels of CRP significantly increased the effect of CFH Y402H (P<.01).

Conclusions The CFH Y402H polymorphism may account for a substantial proportion of AMD in individuals similar to those in the Rotterdam Study and may confer particular risk in the presence of environmental and genetic stimulators of the complement cascade.

Figures in this Article

Age-related macular degeneration (AMD) is the most important cause of irreversible visual loss in the elderly of the Western world.1 This late-onset disorder causes focal deposition of extracellular material (drusen) underneath the retinal pigment epithelium, ultimately leading to geographic atrophy or subretinal neovascularization. Recent studies provide increasing evidence that inflammation is an important disease mechanism. Drusen were shown to contain complement components and regulators, immunoglobulins, and anaphylatoxins2; C-reactive protein (CRP) was associated with AMD3; and a mouse model lacking the gene for monocyte chemoattractant protein appeared to develop hallmarks of AMD.4

It has long been recognized that hereditary factors play a role in AMD. First-degree relatives were shown to have an increased risk,5,6 and segregation analysis suggested the presence of a major gene.7 Genome-wide linkage analyses identified a disease locus on 1q25-q31,814 and case-control studies recently identified complement factor H (CFH) as the responsible gene.1521 This gene has many frequent polymorphic variants that relate to AMD.18 The CFH Y402H variant, located within a binding site for CRP, was consistently shown to have the strongest association in the coding region.1521

Complement factor H is an important inhibitor of the complement pathway. Activation of this pathway initiates a proteolytic cascade that releases proinflammatory anaphylatoxins and causes formation of a membrane-attack complex ultimately leading to cell lysis. Complement factor H preferentially binds and inactivates complement component C3b, and prevents the production of C3 convertase and progression of the cascade.22 The association between CFH and AMD emphasizes an inflammatory pathogenesis of AMD and suggests that triggering the complement cascade in genetically predisposed individuals promotes development of AMD.

The purpose of this study was 3-fold. First, we examined the associations between the CFH Y402H polymorphism and early (less severe) as well as late (vision threatening) AMD in a general population. Second, we investigated whether smoking and other proinflammatory markers may modify the relationship between CFH and AMD. And third, we assessed whether genetic variants of CRP interact with this CFH polymorphism. We investigated these issues in the population-based Rotterdam Study. The large study sample, the variety of risk factors determined at baseline, and the unbiased diagnosis of AMD during a long follow-up particularly addressed the multifactorial origin of AMD and facilitated the study of gene-environment interaction.

Study Population

The Rotterdam Study is a prospective, population-based cohort study of chronic diseases in the elderly. The eligible population comprised all 10 275 inhabitants aged 55 years or older living in Ommoord, a suburb of Rotterdam, the Netherlands. Inhabitants were ascertained from the municipal register, were invited by mail, and contacted by telephone for a home interview and examinations at the research center. Of the eligible population, 7983 (78%) individuals participated (58% female and 98% white).23 The ophthalmologic part of the study became operational after the pilot phase of the study had started and consisted of 9774 eligible individuals, of whom 7598 (78%) participated. The investigation was approved by the medical ethics committee of Erasmus University (Rotterdam, the Netherlands), and all participants provided signed, informed consent for participation in the study, publication of obtained data, retrieval of medical records, and the use of blood and DNA for scientific purposes.

Baseline examinations took place from March 20, 1990, through July 31, 1993. One follow-up examination was performed between September 1, 1993, and December 31, 1994, and had a mean (SD) time between baseline and follow-up of 1.98 (0.64) years; another examination was performed between April 15, 1997, and December 31, 2000, and had a mean (SD) time of 6.50 (0.35) years; and the third examination was performed between April 23, 2002, and December 31, 2004, and had a mean (SD) time of 11.08 (0.53) years. At baseline, 6418 participants underwent an eye examination and had gradable fundus photographs; 5681 of these had a successful assessment of the CFH gene polymorphism (88.6% of persons with AMD and 88.4% of those without AMD) and were therefore available for prevalence analyses. Seventy-eight persons with prevalent late AMD (stage 4) were excluded from further incidence analyses. At first follow-up examination, 270 persons had died and 691 were not included in the analyses due to refusal, lost to follow-up, or ungradable fundus photographs, resulting in 4642 individuals with complete data of whom 12 had late AMD. At the second follow-up examination, 663 persons had died and 561 were not included due to refusal, lost to follow-up, or ungradable photographs, leaving 3406 with complete data of whom 32 had late AMD. At the third follow-up examination, 738 persons had died, 249 were not included due to refusal, lost to follow-up, or ungradable photographs, and 2387 individuals had complete data of whom 49 had late AMD. The total number of person-years on which incidence analyses were based was 30 621. Data analysis took place from May 10, 2005, through May 30, 2006.

Genotyping

All participants were genotyped for the CFH Y402H polymorphism (1277 T→C, rs1061170) in 2-ng genomic DNA extracted from leukocytes with the Taqman assay (Applied Biosystems, Foster City, Calif).

To assess variation in the CRP gene, we genotyped single-nucleotide polymorphisms (rs1130864 C→T, rs1205 C→T, rs3093068 C→G), enabling stratification into the 4 haplotypes that are present in persons of European descent (SeattleSNPs, http://pga.gs.washington.edu).24 Overlap between these 3 tagging single-nucleotide polymorphisms was only present in 9 of 10 800 alleles (<0.001%). Haplotypes were estimated with the PHASE program25 and the probability of the estimated haplotypes was 0.999 or higher in all individuals. Haplotypes were coded as 1 to 4 in order of decreasing frequency in the population (coding from rs1130864 C→T, rs1205 C→T, and rs3093068 C→G; haplotype 1 = C-T-C; haplotype 2 = T-C-C; haplotype 3 = C-C-C; and haplotype 4 = C-C-G). In the analysis of interaction between the CRP gene and CFH, not all strata had sufficient cases for haplotype 4. These analyses were restricted to the 3 most common haplotypes, which describe 94.2% of the study population.

Inflammatory Mediators

The inflammatory mediators erythrocyte sedimentation rate (ESR), CRP serum level, and leucocyte count were evaluated at baseline. Blood was drawn by venous puncture and the ESR was read after 60 minutes. Leukocyte count was directly assessed using a Coulter Counter T540 (Coulter Electronics, Luton, England). Serum samples were initially stored at −20°C, and were thawed and assayed for CRP by Rate Near Infrared Particle Immunoassay method (IMMAGE high sensitive CRP, Beckman Coulter, Fullerton, Calif). Information on cigarette smoking (never, past, and current) was obtained by interview at baseline.23

Diagnosis of AMD

Fundus photographs covering a 35° field centered on the macula were taken at each visit (Topcon TRV-50VT fundus camera, Topcon Optical Co, Tokyo, Japan) after pharmacologic mydriasis. All signs of AMD were graded according to the international classification and grading system for AMD under the supervision of senior ophthalmologists (J.R.V. and P.T.V.M.D.J.), and graders were tested regularly for interrater and intrarater agreement.26,27 We categorized these signs into 5 exclusive stages that represent an increasing severity of AMD. In short, stage 0 contains no or only small hard drusen; stage 1, either small soft distinct drusen or pigmentary irregularities; stage 2, either soft distinct drusen with pigmentary irregularities or soft indistinct drusen without pigmentary irregularities; stage 3, soft indistinct drusen with pigmentary irregularities; and stage 4, geographic atrophy (dry AMD), neovascular AMD (wet AMD), or mixed AMD (both dry and wet AMD). Persons were classified in stage 0 to 4 based on the eye with the more severe stage. Unaffected participants were defined as those who remained in stage 0 throughout the follow-up period. Incident cases were defined as the absence of a stage in both eyes at baseline and the first appearance of AMD in at least 1 eye at follow-up.

Statistical Analysis

Baseline characteristics of participants were compared among CFH genotypes with analysis of covariance for continuous variables and with logistic regression analysis for discrete variables and were adjusted for age and sex. Natural, log-transformed CRP levels, ESRs, and leukocyte counts were used to normalize a skewed distribution. P<.05 was considered statistically significant along with 95% confidence intervals (CIs) excluding the null hypothesis.

Prevalences of AMD stages were calculated as percentages of the study population at baseline and incidence rates were calculated by dividing the number of incident cases by the number of person-years of follow-up. Duration of follow-up was calculated from the date of fundus photography at baseline until diagnosis of AMD, lost to follow-up, last visit before death, or end of follow-up, whichever came first.

Genotype distributions for CFH were tested for Hardy-Weinberg equilibrium using the χ2 test. Prevalence odds ratios (ORs) were estimated with logistic regression analysis and incidence relative risks were estimated with Cox proportional hazards analysis, both analyses were adjusted for age and sex. The proportional hazards assumption was tested by drawing log minus log plots of the survival function, which was verified by visual inspection. The ORs based on both prevalent and incident cases were estimated with logistic regression analyses with age, sex, and follow-up time in the model. Cumulative risks of late AMD were calculated using Kaplan-Meier product-limit analysis in the presence of competing risks. Participants who died, and those who were free of AMD and were lost to follow-up, were censored at the time of their last visit. P values from log-rank tests of equality were used to test significant differences between CFH genotypes. The formula to calculate population attributable risk is: population attributable risk = (relative risk −1/relative risk) × proportion of exposed. Relative risk of late AMD in this formula is estimated by the OR. The proportion exposed is the proportion of participants with late AMD carrying the Y402H allele.

To maximize contrast between affected and healthy individuals, the disease outcome for the interaction analyses was defined as late AMD (stage 4) vs no AMD (stage 0). Risk assessments were initially performed for prevalent and incident cases separately. However, because risk estimates were similar, interaction analyses were subsequently performed using pooled data of prevalent and incident cases to increase statistical power. For the pooled data, ORs were used as representative for relative risks because pooling allows a case-control analysis. The ESRs were evaluated using categories of normal and elevated rates (cutoff of 12 mm per hour for men and 20 mm per hour for women). Because CRP levels and leukocyte counts were within normal limits (CRP<8 mg/L; leukocyte count, 3.5-10.0 × 109/L) for all participants, we evaluated these mediators in tertiles. Smoking was stratified as never, past, and current. Biological interaction with CFH was assessed by calculating the synergy index.28 The synergy index measures deviation from additivity of 2 risk factors and is based on the ratio of the combined effect to the sum of the separate effects. A synergy index exceeding 1.00 suggests the presence of at least 1 pathway in the pathogenesis of the disease for which both risk factors are required.

Interaction with the CRP gene was studied with homozygous haplotype 1 carriers (1-1) as the reference category because this genotype was associated with the lowest CRP levels. We pooled all haplotype 2 carriers (2*) in 1 group and all haplotype 3 carriers in another group (3*). Individuals with haplotypes 2 and 3 (2-3) were present in both groups 2* and 3*.

At baseline, we identified 2062 (36.3%) of 5681 persons with any type of AMD and 78 (1.4%) with late AMD (stage 4). During follow-up (mean, 7.85 years; median, 10.31 years), we identified 1858 (40.0%) of 4642 persons who had remained in stage 0 and 1649 (35.5%) who progressed to a higher stage of AMD. Of these 1649, 93 persons (5.6%) had late AMD. The baseline characteristics and frequency of AMD stratified by CFH genotypes are presented in Table 1.

Table Graphic Jump LocationTable 1. Baseline Characteristics Stratified by CFH Y402H Genotype*

Genotype frequencies of CFH were analogous for participants and nonparticipants and were in Hardy-Weinberg equilibrium. The risks of AMD for the CFH Y402H genotypes appear in Table 2. The prevalence of the CFH Y402H allele was 36.2% (4116/11 362). Risks were consistently higher for homozygous than for heterozygous persons, representing an allele-dose effect. Overall, carriers of the CFH Y402H allele had higher risks of all types of AMD than noncarriers and risks increased with each stage of AMD.

Table Graphic Jump LocationTable 2. Risks of Age-Related Macular Degeneration for Complement Factor H Genotypes*

The ORs of bilateral late AMD (17.93; 95% CI, 9.00-35.70) were higher than of unilateral late AMD (6.58; 95% CI, 3.47-12.48) in homozygotes and the same trend was seen in heterozygotes. Within AMD subtypes adjusted for bilaterality, homozygotes had higher ORs of geographic atrophy (17.2; 95% CI, 6.7-44.2) and mixed AMD (19.2; 95% CI, 6.1-60.2) than of neovascular AMD (12.4; 95% CI, 2.4-65.4), although differences between subtypes did not reach statistical significance (geographic atrophy vs neovascular AMD, P = .63; mixed vs neovascular AMD, P = .66; geographic atrophy vs mixed AMD, P = .56). The population attributable risk of late AMD for CFH Y402H was 54.0% (ie, 23.2% for those carrying 1 allele and 30.8% for those carrying 2 alleles).

The cumulative incidence of late AMD for the CFH genotypes appears in Figure 1. Risk differences between carriers and noncarriers became statistically significant after age 75 years (log-rank P<.01). At age 95 years, risks had increased to 48.3% (95% CI, 28.8%-67.7%) for homozygous and 42.6% (95% CI, 24.3%-60.8%) for heterozygous carriers compared with 21.9% (95% CI, 5.4%-38.4%) for noncarriers.

Figure 1. Cumulative Incidence of Late Age-Related Macular Degeneration for Homozygous and Heterozygous Carriers vs Noncarriers of the CFH Y402H Polymorphism
Graphic Jump Location

CFH indicates complement factor H. Cumulative risks of late age-related macular degeneration were calculated using Kaplan-Meier product-limit analysis in the presence of competing risks. Differences in risks between the CFH genotypes were statistically significant (log-rank P<.001).

Interaction between ESR, CRP level, leukocyte count, smoking, and CFH Y402H is demonstrated in Figure 2. The joint effect of each determinant with CFH Y402H was significantly greater than the sum of the independent effects except for leukocyte count. An elevated ESR augmented the association to an OR of 20.2 (95% CI, 9.5-43.0). Higher serum CRP levels increased the association for the second tertile to an OR of 27.7 (95% CI, 10.7-72.0) while the third tertile did not further increase the OR. Neither ESR nor CRP levels were significantly associated with AMD in noncarriers.

Figure 2. Risk of Late Age-Related Macular Degeneration for CFH Y402H Genotypes, Stratified by C-Reactive Protein Serum Level, Erythrocyte Sedimentation Rate, Leukocyte Count, and Smoking
Graphic Jump Location

AMD indicates age-related macular degeneration; CFH, complement factor H; CI, confidence interval; SI, synergy index.
*Normal and elevated rates defined as cutoff of 12 mm per hour for men and 20 mm per hour for women.

Smoking had a large influence on the risk of AMD related to CFH. Current smokers homozygous for CFH Y402H had an OR of 34.0 (95% CI, 13.0-88.6) for late AMD compared with individuals who never smoked without the risk allele. In the absence of CFH Y402H, current smoking increased the OR for AMD to 3.36 (95% CI, 1.14-9.86).

Serum levels of CRP varied among the CRP haplotypes in this study population. Haplotype 1 (the most common) had a frequency of 32.7% and the lowest CRP levels; haplotype 2 had a frequency of 31.6% and the highest CRP levels; and haplotype 3 had a frequency of 29.9% and intermediate levels of CRP.29 The CRP haplotypes per se were not related to AMD. We tested the hypothesis whether these haplotypes influenced the effect of CFH Y402H on AMD (Figure 3). Compared with noncarriers of CFH Y402H with CRP haplotype 1, noncarriers with CRP haplotype 2 had an OR of AMD of 0.17 (95% CI, 0.06-0.46; P<.001) and noncarriers with CRP haplotype 3 had an OR of 0.25 (95% CI, 0.09-0.64; P = .004). In contrast, in homozygous CFH Y402H carriers, the OR of AMD was 3.32 (95% CI, 1.38-8.01; P = .007) for haplotype 2 and 3.86 (95% CI, 1.56-9.53; P = .003) for haplotype 3. The highest difference in ORs between homozygous carriers and noncarriers was observed for CRP haplotype 2, which is the haplotype with the highest CRP levels. Our results show that those participants homozygous for CFH Y402H with an additional genetic predisposition to high serum CRP levels were at higher risk of developing AMD.

Figure 3. Risk of Late Age-Related Macular Degeneration for C-Reactive Protein (CRP ) Haplotypes Stratified by Complement Factor H Genotype
Graphic Jump Location

AMD indicates age-related macular degeneration; CI, confidence interval. Risk of late AMD estimated by logistic regression analysis and adjusted for age and sex. Haplotype 2 carriers (2*) were grouped, as were haplotype 3 carriers (3*). Individuals with haplotypes 2 and 3 (2-3) were present in both the 2* and 3* group. P<.01 for comparison with homozygous haplotype 1 carriers (1-1).

In this prospective study, which was based on an older, white population in the middle socioeconomic class in the Netherlands, we find that the CFH gene is a major risk factor for AMD. The gene was implicated in all stages of AMD from early hallmarks such as drusen to vision-disabling late AMD and the risks increased with each successive stage to a high of 11 for late AMD. We calculated that individuals homozygous for the CFH Y402H polymorphism had a 48% risk of developing late AMD by age 95 years while this risk did not exceed 22% for noncarriers. These data suggest that CFH Y402H may be a causal factor in more than 50% of all AMD cases in the general population.

Previous reports on the association between CFH and AMD were from clinic or family-based, case-control studies with cross-sectional designs. This hampers extrapolation of the role of CFH in AMD development for the population at large. Strengths of our current study are the population-based prospective design, the large study sample, and the use of standardized procedures for AMD diagnosis by experienced graders.27 We restricted the analyses of potential modifiers to individuals with late AMD (stage 4) and those who remained free of any type of AMD (stage 0) throughout the study. To maximize statistical power and enable precise risk estimates, we pooled prevalent and incident cases with late AMD. Prevalent cases showed similar risk estimates as incident cases so the exposures to inflammation and smoking likely preceded AMD and pooling did not jeopardize causal inference. Although not negligible, our a priori hypothesis and assessment of only 1 single-nucleotide polymorphism in the CFH gene make it unlikely that our findings are falsely positive.

Complement factor H was associated with both late AMD subtypes in this study. Homozygous CFH Y402H carriers had a higher risk of bilateral than of unilateral late AMD, and risks of geographic atrophy and mixed AMD were slightly but not significantly higher than neovascular AMD. This is in agreement with other studies that reported higher frequencies of CFH Y402H carriers in persons with geographic atrophy,19,30 and 1 study that suggested a lower risk of geographic atrophy for a CFH haplotype containing the nonrisk allele.18 Nevertheless, the high risk for both subtypes signifies a common inflammatory pathogenesis.

Complement factor H is an important regulator of the complement system. Three enzyme cascades exist: the classical complement pathway, initiated by antigen-antibody complexes and surface-bound CRP; the lectin, turned on by mannose groups of microbial carbohydrates; and the alternative complement pathway, activated by surface-bound C3b. The pathways converge at the point in which C3 is cleaved into C3a and C3b by C3 convertase, which initiates C5 convertase, resulting in the formation of the membrane-attack complex with the terminal components (C5b-C9). Complement factor H specifically inhibits the alternative complement cascade but also regulates the common pathway. It binds C3b and acts as a cofactor in the proteolysis of C3b by factor I, resulting in an inactive C3b molecule. This prevents the production of C3 convertase in the alternative cascade as well as the production of C5 convertase in the common pathway. As a result, CFH interferes with progression of the entire cascade.22,31

Genetic predisposition to a malfunctioning CFH can only be of importance when the complement system is switched on. Our data provide strong evidence that onset of this cascade leads to AMD in persons with the CFH Y402H polymorphism. This is demonstrated by the significant interaction between chronic as well as acute inflammation and CFH Y402H. Elevated baseline ESRs considerably increased the risk of AMD in carriers and a similar trend was observed for serum CRP levels. Neither ESRs nor CRP levels increased the risk significantly in noncarriers. Earlier studies reported a relationship between serum CRP level and progression of AMD.3 However, our results imply that this relationship is mostly determined by the CFH polymorphism. Increased leukocyte counts did not contribute to an additional effect, possibly due to the absence of clinically elevated levels in our study.

Smoking was considered the highest risk factor for AMD prior to the introduction of CFH in AMD. Our data show that the combined effect of both exposures exceeds the sum of the independent effects. Compared with no exposure, smoking increased the risk of AMD 3.3 times, the presence of 2 CFH Y402H alleles increased the risk 12.5 times, while the combination of both determinants increased this risk 34-fold. Smoking increases cytokines and inflammatory cells and has been shown to activate the complement pathway by weakening the susceptibility of C3 to CFH and factor I.32,33 When CFH function is genetically impaired, progression may be further accelerated. However, the elevated risk of smoking in noncarriers suggests that smoking may have an alternative mechanism in AMD pathogenesis.

We further explored the relationship with CRP for 2 reasons. First, the CFH Y402H variant represents an amino acid change in the SCR7 domain, which contains a binding site for CRP, heparin, and M-protein, prompting a functional interaction with these proteins.22 Second, CRP not only triggers the classical complement cascade by binding to C1q, it also limits the amount of complement activation by its ability to interact with CFH, thereby reducing the complement-associated damaging effect.34 Because serum CRP levels are known to fluctuate, a single measurement of CRP may not accurately reflect a continuous baseline level nor adequately represent the possible response after an inflammatory stimulus. This motivated us to examine the CRP gene. Our data suggest that CRP haplotypes, which increase serum levels, modify the effect of CFH. These haplotypes decrease the risk of AMD in noncarriers but increase the risk in persons homozygous for CFH Y402H. We propose the following as a plausible biological mechanism. Genetic variants of CRP have been shown to determine serum levels especially in response to inflammatory stimuli.35 The CFH Y402H polymorphism may impair CRP binding, decrease CFH inhibition, and lead to destruction of host cells in particular in those individuals who are genetically predisposed to high CRP levels. By contrast, normal binding of CFH with CRP may increase inhibition and decelerate complement activation in those who are hyperresponsive to inflammation.31

In conclusion, CFH, an inhibitory gene of the complement pathway, is a major risk factor for AMD in this population. It is involved in early as well as late disease pathogenesis and markedly increases risk of late AMD in the very old. The effect of CFH is significantly influenced by environmental and genetic factors that determine the inflammatory response and activate the complement pathway. Future research on therapeutic modalities that help regulate the terminal complement pathway, thereby sparing host tissue, may provide an approach for preventing sight-threatening AMD in genetically predisposed individuals.

Corresponding Author: Paulus T. V. M. de Jong, MD, PhD, Netherlands Institute for Neuroscience, Meibergdreef 47, 1105 BA Amsterdam, the Netherlands (p.dejong@nin.knaw.nl).

Authors Contributions: Dr Despriet had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: Klaver, Hofman, Oostra, Uitterlinden, Van Duijn, De Jong.

Acquisition of data: Klaver, Kardys, Boekhoorn, Vingerling, Uitterlinden.

Analysis and interpretation of data: Despriet, Klaver, Bergen, Witteman, De Maat, Vingerling, Stijnen, Van Duijn, De Jong.

Drafting of the manuscript: Despriet, Klaver.

Critical revision of the namuscript for important intellectual content: Klaver, Witteman, Bergen, Kardys, De Maat, Boekhoorn, Vingerling, Hofman, Oostra, Uitterlinden, Stijnen, Van Duijn, De Jong.

Statistical analysis: Despriet, Klaver, Witteman, Stijnen.

Obtained funding: Bergen, Hofman, De Jong.

Administrative, technical, or material support: De Maat, Hofman, Uitterlinden.

Study supervision: Klaver, Vingerling, Hofman, Van Duijn, De Jong.

Financial Disclosures: None reported.

Funding/Support: This study was supported by the Netherlands Organization for Scientific Research (the Hague), Optimix (Amsterdam), Swart van Essen (Rotterdam), Neyenburgh (Bunnik), Physico Therapeutic Institute (Rotterdam), Blindenpenning (Amsterdam), Sint Laurens Institute (Rotterdam), Bevordering van Volkskracht (Rotterdam), Blindenhulp (the Hague), Rotterdamse Blindenbelangen Association (Rotterdam), OOG Foundation (the Hague), Ooglijders (Rotterdam), Prins Bernhard Cultuurfonds (Amsterdam), Van Leeuwen Van Lignac (Rotterdam), Verhagen (Rotterdam), the Netherlands Society for the Prevention of Blindness (Doorn), and Elise Mathilde (Maarn). An unrestricted grant was obtained from Topcon Europe BV (Capelle aan de IJssel).

Role of the Sponsors: The study's sponsors had no role in the design and conduct of the study; in the collection, analysis, and interpretation of the data; or in the preparation, review, and approval of the manuscript.

Previous Presentations: Part of this study was presented at the 29th Annual Macula Society Meeting in Carlsbad, Calif, on February 23, 2006; and at the Association for Research in Vision and Ophthalmology Annual Meeting in Fort Lauderdale, Fla, on May 2, 2006.

Acknowledgments: We thank Ada Hooghart and Corina Brussee for grading of fundus photographs, Pascal Arp for genotyping, and statistician Paul Mulder, PhD, for his excellent advice on statistical analyses. All of these individuals were paid by different groups (and partly by grants) within the departments of epidemiology, biostatistics, and internal medicine at Erasmus Medical Center, Rotterdam, the Netherlands, and also volunteered to help with this study.

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Heiba IM, Elston RC, Klein BE, Klein R. Sibling correlations and segregation analysis of age-related maculopathy: the Beaver Dam Eye Study.  Genet Epidemiol. 1994;11:51-67
PubMed   |  Link to Article
Klein ML, Schultz DW, Edwards A.  et al.  Age-related macular degeneration: clinical features in a large family and linkage to chromosome 1q.  Arch Ophthalmol. 1998;116:1082-1088
PubMed   |  Link to Article
Majewski J, Schultz DW, Weleber RG.  et al.  Age-related macular degeneration—a genome scan in extended families.  Am J Hum Genet. 2003;73:540-550
PubMed   |  Link to Article
Seddon JM, Santangelo SL, Book K, Chong S, Cote J. A genomewide scan for age-related macular degeneration provides evidence for linkage to several chromosomal regions.  Am J Hum Genet. 2003;73:780-790
PubMed   |  Link to Article
Abecasis GR, Yashar BM, Zhao Y.  et al.  Age-related macular degeneration: a high-resolution genome scan for susceptibility loci in a population enriched for late-stage disease.  Am J Hum Genet. 2004;74:482-494
PubMed   |  Link to Article
Iyengar SK, Song D, Klein BE.  et al.  Dissection of genomewide-scan data in extended families reveals a major locus and oligogenic susceptibility for age-related macular degeneration.  Am J Hum Genet. 2004;74:20-39
PubMed   |  Link to Article
Weeks DE, Conley YP, Tsai HJ.  et al.  Age-related maculopathy: a genomewide scan with continued evidence of susceptibility loci within the 1q31, 10q26, and 17q25 regions.  Am J Hum Genet. 2004;75:174-189
PubMed   |  Link to Article
Weber JL, Broman KW. Genotyping for human whole-genome scans: past, present, and future.  Adv Genet. 2001;42:77-96
PubMed
Klein RJ, Zeiss C, Chew EY.  et al.  Complement factor H polymorphism in age-related macular degeneration.  Science. 2005;308:385-389
PubMed   |  Link to Article
Edwards AO, Ritter R III, Abel KJ, Manning A, Panhuysen C, Farrer LA. Complement factor H polymorphism and age-related macular degeneration.  Science. 2005;308:421-424
PubMed   |  Link to Article
Haines JL, Hauser MA, Schmidt S.  et al.  Complement factor H variant increases the risk of age-related macular degeneration.  Science. 2005;308:419-421
PubMed   |  Link to Article
Hageman GS, Anderson DH, Johnson LV.  et al.  A common haplotype in the complement regulatory gene factor H (HF1/CFH) predisposes individuals to age-related macular degeneration.  Proc Natl Acad Sci U S A. 2005;102:7227-7232
PubMed   |  Link to Article
Zareparsi S, Branham KE, Li M.  et al.  Strong association of the Y402H variant in complement factor H at 1q32 with susceptibility to age-related macular degeneration.  Am J Hum Genet. 2005;77:149-153
PubMed   |  Link to Article
Souied EH, Leveziel N, Richard F.  et al.  Y402H complement factor H polymorphism associated with exudative age-related macular degeneration in the French population.  Mol Vis. 2005;11:1135-1140
PubMed
Magnusson KP, Duan S, Sigurdsson H.  et al.  CFH Y402H confers similar risk of soft drusen and both forms of advanced AMD.  PLoS Med. 2006;3:e5
PubMed   |  Link to Article
Rodriguez de Cordoba S, Esparza-Gordillo J, Goicoechea de Jorge E, Lopez-Trascasa M, Sanchez-Corral P. The human complement factor H: functional roles, genetic variations and disease associations.  Mol Immunol. 2004;41:355-367
PubMed   |  Link to Article
Hofman A, Grobbee DE, de Jong PT, van den Ouweland FA. Determinants of disease and disability in the elderly: the Rotterdam Elderly Study.  Eur J Epidemiol. 1991;7:403-422
PubMed   |  Link to Article
Carlson CS, Aldred SF, Lee PK.  et al.  Polymorphisms within the C-reactive protein (CRP) promoter region are associated with plasma CRP levels.  Am J Hum Genet. 2005;77:64-77
PubMed   |  Link to Article
Stephens M, Smith NJ, Donnelly P. A new statistical method for haplotype reconstruction from population data.  Am J Hum Genet. 2001;68:978-989
PubMed   |  Link to Article
Bird AC, Bressler NM, Bressler SB.  et al. International ARM Epidemiological Study Group.  An international classification and grading system for age-related maculopathy and age-related macular degeneration.  Surv Ophthalmol. 1995;39:367-374
PubMed   |  Link to Article
van Leeuwen R, Klaver CC, Vingerling JR, Hofman A, de Jong PT. Risk and natural course of age-related maculopathy: follow-up at 6 1/2 years in the Rotterdam study.  Arch Ophthalmol. 2003;121:519-526
PubMed   |  Link to Article
Rothman KJ, Greenland S. Modern Epidemiology. 2nd ed. Philadelphia, Pa: Lippincott-Raven; 1998
Kardys I, de Maat MP, Uitterlinden AG, Hofman A, Witteman JC. C-reactive protein gene haplotypes and risk of coronary heart disease: the Rotterdam Study.  Eur Heart J. 2006;27:1331-1337
PubMed   |  Link to Article
Rivera A, Fisher SA, Fritsche LG.  et al.  Hypothetical LOC387715 is a second major susceptibility gene for age-related macular degeneration, contributing independently of complement factor H to disease risk.  Hum Mol Genet. 2005;14:3227-3236
PubMed   |  Link to Article
Giannakis E, Male DA, Ormsby RJ.  et al.  Multiple ligand binding sites on domain seven of human complement factor H.  Int Immunopharmacol. 2001;1:433-443
PubMed   |  Link to Article
van der Vaart H, Postma DS, Timens W.  et al.  Acute effects of cigarette smoking on inflammation in healthy intermittent smokers.  Respir Res. 2005;6:22
PubMed   |  Link to Article
Kew RR, Ghebrehiwet B, Janoff A. Cigarette smoke can activate the alternative pathway of complement in vitro by modifying the third component of complement.  J Clin Invest. 1985;75:1000-1007
PubMed   |  Link to Article
Black S, Kushner I, Samols D. C-reactive protein.  J Biol Chem. 2004;279:48487-48490
PubMed   |  Link to Article
Brull DJ, Serrano N, Zito F.  et al.  Human CRP gene polymorphism influences CRP levels: implications for the prediction and pathogenesis of coronary heart disease.  Arterioscler Thromb Vasc Biol. 2003;23:2063-2069
PubMed   |  Link to Article

Figures

Figure 1. Cumulative Incidence of Late Age-Related Macular Degeneration for Homozygous and Heterozygous Carriers vs Noncarriers of the CFH Y402H Polymorphism
Graphic Jump Location

CFH indicates complement factor H. Cumulative risks of late age-related macular degeneration were calculated using Kaplan-Meier product-limit analysis in the presence of competing risks. Differences in risks between the CFH genotypes were statistically significant (log-rank P<.001).

Figure 2. Risk of Late Age-Related Macular Degeneration for CFH Y402H Genotypes, Stratified by C-Reactive Protein Serum Level, Erythrocyte Sedimentation Rate, Leukocyte Count, and Smoking
Graphic Jump Location

AMD indicates age-related macular degeneration; CFH, complement factor H; CI, confidence interval; SI, synergy index.
*Normal and elevated rates defined as cutoff of 12 mm per hour for men and 20 mm per hour for women.

Figure 3. Risk of Late Age-Related Macular Degeneration for C-Reactive Protein (CRP ) Haplotypes Stratified by Complement Factor H Genotype
Graphic Jump Location

AMD indicates age-related macular degeneration; CI, confidence interval. Risk of late AMD estimated by logistic regression analysis and adjusted for age and sex. Haplotype 2 carriers (2*) were grouped, as were haplotype 3 carriers (3*). Individuals with haplotypes 2 and 3 (2-3) were present in both the 2* and 3* group. P<.01 for comparison with homozygous haplotype 1 carriers (1-1).

Tables

Table Graphic Jump LocationTable 1. Baseline Characteristics Stratified by CFH Y402H Genotype*
Table Graphic Jump LocationTable 2. Risks of Age-Related Macular Degeneration for Complement Factor H Genotypes*

References

Friedman DS, O'Colmain BJ, Munoz B.  et al.  Prevalence of age-related macular degeneration in the United States.  Arch Ophthalmol. 2004;122:564-572
PubMed   |  Link to Article
Hageman GS, Luthert PJ, Victor Chong NH, Johnson LV, Anderson DH, Mullins RF. An integrated hypothesis that considers drusen as biomarkers of immune-mediated processes at the RPE-Bruch's membrane interface in aging and age-related macular degeneration.  Prog Retin Eye Res. 2001;20:705-732
PubMed   |  Link to Article
Seddon JM, George S, Rosner B, Rifai N. Progression of age-related macular degeneration: prospective assessment of C-reactive protein, interleukin 6, and other cardiovascular biomarkers.  Arch Ophthalmol. 2005;123:774-782
PubMed   |  Link to Article
Ambati J, Anand A, Fernandez S.  et al.  An animal model of age-related macular degeneration in senescent Ccl-2- or Ccr-2-deficient mice.  Nat Med. 2003;9:1390-1397
PubMed   |  Link to Article
Seddon JM, Ajani UA, Mitchell BD. Familial aggregation of age-related maculopathy.  Am J Ophthalmol. 1997;123:199-206
PubMed
Klaver CC, Wolfs RC, Assink JJ, van Duijn CM, Hofman A, de Jong PT. Genetic risk of age-related maculopathy. Population-based familial aggregation study.  Arch Ophthalmol. 1998;116:1646-1651
PubMed   |  Link to Article
Heiba IM, Elston RC, Klein BE, Klein R. Sibling correlations and segregation analysis of age-related maculopathy: the Beaver Dam Eye Study.  Genet Epidemiol. 1994;11:51-67
PubMed   |  Link to Article
Klein ML, Schultz DW, Edwards A.  et al.  Age-related macular degeneration: clinical features in a large family and linkage to chromosome 1q.  Arch Ophthalmol. 1998;116:1082-1088
PubMed   |  Link to Article
Majewski J, Schultz DW, Weleber RG.  et al.  Age-related macular degeneration—a genome scan in extended families.  Am J Hum Genet. 2003;73:540-550
PubMed   |  Link to Article
Seddon JM, Santangelo SL, Book K, Chong S, Cote J. A genomewide scan for age-related macular degeneration provides evidence for linkage to several chromosomal regions.  Am J Hum Genet. 2003;73:780-790
PubMed   |  Link to Article
Abecasis GR, Yashar BM, Zhao Y.  et al.  Age-related macular degeneration: a high-resolution genome scan for susceptibility loci in a population enriched for late-stage disease.  Am J Hum Genet. 2004;74:482-494
PubMed   |  Link to Article
Iyengar SK, Song D, Klein BE.  et al.  Dissection of genomewide-scan data in extended families reveals a major locus and oligogenic susceptibility for age-related macular degeneration.  Am J Hum Genet. 2004;74:20-39
PubMed   |  Link to Article
Weeks DE, Conley YP, Tsai HJ.  et al.  Age-related maculopathy: a genomewide scan with continued evidence of susceptibility loci within the 1q31, 10q26, and 17q25 regions.  Am J Hum Genet. 2004;75:174-189
PubMed   |  Link to Article
Weber JL, Broman KW. Genotyping for human whole-genome scans: past, present, and future.  Adv Genet. 2001;42:77-96
PubMed
Klein RJ, Zeiss C, Chew EY.  et al.  Complement factor H polymorphism in age-related macular degeneration.  Science. 2005;308:385-389
PubMed   |  Link to Article
Edwards AO, Ritter R III, Abel KJ, Manning A, Panhuysen C, Farrer LA. Complement factor H polymorphism and age-related macular degeneration.  Science. 2005;308:421-424
PubMed   |  Link to Article
Haines JL, Hauser MA, Schmidt S.  et al.  Complement factor H variant increases the risk of age-related macular degeneration.  Science. 2005;308:419-421
PubMed   |  Link to Article
Hageman GS, Anderson DH, Johnson LV.  et al.  A common haplotype in the complement regulatory gene factor H (HF1/CFH) predisposes individuals to age-related macular degeneration.  Proc Natl Acad Sci U S A. 2005;102:7227-7232
PubMed   |  Link to Article
Zareparsi S, Branham KE, Li M.  et al.  Strong association of the Y402H variant in complement factor H at 1q32 with susceptibility to age-related macular degeneration.  Am J Hum Genet. 2005;77:149-153
PubMed   |  Link to Article
Souied EH, Leveziel N, Richard F.  et al.  Y402H complement factor H polymorphism associated with exudative age-related macular degeneration in the French population.  Mol Vis. 2005;11:1135-1140
PubMed
Magnusson KP, Duan S, Sigurdsson H.  et al.  CFH Y402H confers similar risk of soft drusen and both forms of advanced AMD.  PLoS Med. 2006;3:e5
PubMed   |  Link to Article
Rodriguez de Cordoba S, Esparza-Gordillo J, Goicoechea de Jorge E, Lopez-Trascasa M, Sanchez-Corral P. The human complement factor H: functional roles, genetic variations and disease associations.  Mol Immunol. 2004;41:355-367
PubMed   |  Link to Article
Hofman A, Grobbee DE, de Jong PT, van den Ouweland FA. Determinants of disease and disability in the elderly: the Rotterdam Elderly Study.  Eur J Epidemiol. 1991;7:403-422
PubMed   |  Link to Article
Carlson CS, Aldred SF, Lee PK.  et al.  Polymorphisms within the C-reactive protein (CRP) promoter region are associated with plasma CRP levels.  Am J Hum Genet. 2005;77:64-77
PubMed   |  Link to Article
Stephens M, Smith NJ, Donnelly P. A new statistical method for haplotype reconstruction from population data.  Am J Hum Genet. 2001;68:978-989
PubMed   |  Link to Article
Bird AC, Bressler NM, Bressler SB.  et al. International ARM Epidemiological Study Group.  An international classification and grading system for age-related maculopathy and age-related macular degeneration.  Surv Ophthalmol. 1995;39:367-374
PubMed   |  Link to Article
van Leeuwen R, Klaver CC, Vingerling JR, Hofman A, de Jong PT. Risk and natural course of age-related maculopathy: follow-up at 6 1/2 years in the Rotterdam study.  Arch Ophthalmol. 2003;121:519-526
PubMed   |  Link to Article
Rothman KJ, Greenland S. Modern Epidemiology. 2nd ed. Philadelphia, Pa: Lippincott-Raven; 1998
Kardys I, de Maat MP, Uitterlinden AG, Hofman A, Witteman JC. C-reactive protein gene haplotypes and risk of coronary heart disease: the Rotterdam Study.  Eur Heart J. 2006;27:1331-1337
PubMed   |  Link to Article
Rivera A, Fisher SA, Fritsche LG.  et al.  Hypothetical LOC387715 is a second major susceptibility gene for age-related macular degeneration, contributing independently of complement factor H to disease risk.  Hum Mol Genet. 2005;14:3227-3236
PubMed   |  Link to Article
Giannakis E, Male DA, Ormsby RJ.  et al.  Multiple ligand binding sites on domain seven of human complement factor H.  Int Immunopharmacol. 2001;1:433-443
PubMed   |  Link to Article
van der Vaart H, Postma DS, Timens W.  et al.  Acute effects of cigarette smoking on inflammation in healthy intermittent smokers.  Respir Res. 2005;6:22
PubMed   |  Link to Article
Kew RR, Ghebrehiwet B, Janoff A. Cigarette smoke can activate the alternative pathway of complement in vitro by modifying the third component of complement.  J Clin Invest. 1985;75:1000-1007
PubMed   |  Link to Article
Black S, Kushner I, Samols D. C-reactive protein.  J Biol Chem. 2004;279:48487-48490
PubMed   |  Link to Article
Brull DJ, Serrano N, Zito F.  et al.  Human CRP gene polymorphism influences CRP levels: implications for the prediction and pathogenesis of coronary heart disease.  Arterioscler Thromb Vasc Biol. 2003;23:2063-2069
PubMed   |  Link to Article
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