Abstract
Background: There is growing evidence of the importance of nutrition in age-related macular degeneration (AMD), but few studies have explored associations with folate and B vitamins. No effective therapeutic strategy for geographic atrophy (GA) is available, and prevention could be of great value.
Objective: We investigated associations between dietary folate, B vitamins, and progression to GA and whether these associations might be modified by genetic susceptibility.
Design: Among 2525 subjects (4663 eyes) in the Age-Related Eye Disease Study, 405 subjects (528 eyes) progressed to GA over 13 y. Folate and B vitamins were log transformed and calorie adjusted separately for men and women. Ten loci in 7 AMD genes [complement factor H, age-related maculopathy susceptibility 2/high-temperature requirement A serine peptidase 1, complement component 2, complement component 3, complement factor B, collagen type VIII α 1, and RAD51 paralog B] were examined. Survival analysis was used to assess associations between incident GA and dietary intake of folate and B vitamins. Interaction effects between these nutrients and genetic variation on AMD risk were also evaluated. Subjects with at least one eye free of advanced AMD at baseline were included in these analyses.
Results: There was a reduced risk of progression to GA with increasing intake of thiamin, riboflavin, and folate after adjusting for age, sex, and total energy intake (P-trend = 0.01, 0.03, and 0.001, respectively). After adjustment for demographic, behavioral, ocular, and genetic covariates, trends remained statistically significant for folate (P-trend = 0.007) and were borderline for thiamin (P-trend = 0.05). Riboflavin did not retain statistical significance (P-trend = 0.20). Folate was significantly associated with lower risk of incident GA among subjects homozygous for the complement component 3 (C3) R102G rs2230199 nonrisk genotype (CC) (HR = 0.43; 95% CI: 0.27, 0.70; P = 0.0005) but not subjects carrying the risk allele (G) (P = 0.76). Neither folate nor any B vitamin was significantly associated with neovascular AMD.
Conclusions: High folate intake was associated with a reduced risk of progression to GA. This relation could be modified by genetic susceptibility, particularly related to the C3 genotype. This trial was registered at clinicaltrials.gov as NCT00594672.
Keywords: folate, B vitamins, geographic atrophy, macular degeneration, genetics
INTRODUCTION
Age-related macular degeneration (AMD)7 is the leading cause of irreversible vision loss among older adults in industrialized countries (1, 2). Advanced forms of the disease include neovascular AMD and geographic atrophy (GA), which are generally preceded by early and intermediate stages. These nonadvanced stages increase the risk of developing advanced AMD, but the exact mechanisms whereby some subjects go on to develop GA or neovascular AMD are not completely understood. Injections that inhibit vascular endothelial growth factor are available for neovascular AMD (3), although these treatments are not curative.
There are currently no treatments for GA, and risk factors specifically related to this form of advanced AMD are not fully understood. GA is characterized by loss of the neurosensory retina and retinal pigment epithelium (1). Once the foveal center is involved, affected patients are deprived of central vision and may develop legal blindness. It accounts for ∼35–40% of late-stage AMD cases (4, 5) and primarily affects individuals aged >85 y (6). This further emphasizes the impact of GA on the aging population and underscores the need for an effective strategy to prevent and slow the disease.
Genetic variants (7–10) as well as modifiable factors (1, 8, 11–13), including smoking and low intake of omega-3 fatty acids, are known to be associated with higher rates of progression to GA. Attention to modifiable risk factors could be helpful in reducing progression to this form of AMD and associated visual loss. Some potentially modifiable dietary factors have not been extensively explored, and epidemiologic data related to dietary intake of folate and B vitamins are scarce.
Folate and B vitamins play a central role in the methylation and synthesis of DNA and its repair and replication (14). B vitamins could also modify plasma homocysteine, a known risk factor for AMD (11, 15). Subjects with lower concentrations of plasma folate (16) and plasma vitamin B-12 (16–18) have a 2- to 3-fold increased risk of late AMD, whereas those with high dietary intake of folate have ∼50% less risk (16). In one randomized controlled trial, daily supplementation of folic acid and vitamins B-6 and B-12 appeared to reduce risk of AMD by 40% (19). These studies evaluated very small numbers of subjects with advanced AMD, and none accounted for genetic susceptibility.
We hypothesized that higher dietary intake of folate and B vitamins could reduce progression to GA. As some neurodegenerative diseases are associated with dysregulation in gene methylation, we also hypothesized that associations could be modified by genetics. We therefore investigated dietary intake of folate and B vitamins in a prospective study with a large number of incident GA cases, controlling for 10 known AMD variants, and explored gene-diet interactions.
METHODS
Age-Related Eye Disease Study population
The details of the Age-Related Eye Disease Study (AREDS) of the National Eye Institute of the NIH have been reported (20). The AREDS study included a multicenter randomized clinical trial to assess the effect of antioxidant and mineral supplements on risk of AMD and cataracts as well as a longitudinal study of progression to advanced AMD. The protocol was approved by a Data and Safety Monitoring Committee and by each institutional review board for the 11 participating ophthalmic centers before the initiation of the study. Participants were aged 55–80 y at baseline and were required to have at least one eye with a visual acuity no worse than 20/32. In addition, at least one eye of each participant was free from eye disease that could complicate the assessment of AMD, and that eye could not have had previous ocular surgery (except cataract surgery and unilateral photocoagulation for AMD). Potential participants were excluded for illness or disorders that would have made long-term follow-up or compliance with the study protocol unlikely or difficult. Informed consent was obtained from participants before enrollment, and all research followed the tenets of the Declaration of Helsinki. This study enrolled 4757 participants from 1992 to 1998.
Procedures
Data on demographic factors, environmental exposures, medical history, drug use, and habitual diet were obtained through general and ophthalmic examinations in the year before enrollment. Trained fundus graders, masked to clinical and phenotypic information from previous years, ascertained signs of AMD from annual stereoscopic color images by using a standardized and validated protocol at a single reading center. Retinal photographs were taken according to a standardized protocol by AREDS-certified photographers by using AREDS-certified cameras (21). Photographs were scheduled at baseline, at the 2-y visit, and annually thereafter during follow-up.
Study subjects
Data were accessed from the NIH Database of Genotypes and Phenotypes. The selection procedures for subjects included in the present study are illustrated in Figure 1. Among the 4757 participants at baseline, we excluded 618 subjects who consented only to “eye research.” For these subjects, phenotype and genetic data could not be linked and therefore could not be included in these analyses. Among the remaining 4139 subjects who consented to “general research,” we excluded 995 subjects for lack of a genetic specimen. Of the 3144 subjects with a genetic specimen, 111 were removed from the data set due to lack of follow-up information. Thirty-nine participants with GA in both eyes at baseline were also removed. Furthermore, an additional 469 subjects were excluded from the data set: 343 because of incomplete genotyping information [genotyping rate of <100% across the 10 single-nucleotide polymorphisms (SNPs) evaluated] and 126 because of an inappropriate total energy intake (TEI) (valid TEI range is 600–3200 kcal for women and 600–4200 kcal for men). Complete data, including AMD grade at baseline and at least one follow-up visit, dietary data, and demographic, behavioral, and genetic covariates, were available for 2525 subjects (4663 eyes) at risk of progression to GA.
FIGURE 1.
Flowchart illustrating the selection of subjects for this study who are at risk of progression to geographic atrophy from the AREDS cohort. AMD, age-related macular degeneration; AREDS, Age-Related Eye Disease Study.
Definition of progression
Eyes were classified by using the clinical age-related maculopathy staging (CARMS) system (22). Conversion from the AREDS to the CARMS grading system was based on all available phenotype data for all follow-up visits, as described in Yu et al. (23). The AREDS system uses the Wisconsin grading classification and, for the purpose of the trial, combined intermediate AMD with noncentral GA into one category (category 3) and central atrophy and neovascular disease along with visual loss due to AMD into another (category 4). When conducting genetic analyses, classification of specific subphenotypes can be informative. We therefore used the CARMS system to reclassify subjects into a separate GA category (central or noncentral) because there is no evidence that these 2 types are different in their etiology and retained neovascular disease as a separate category. Visual loss due to AMD is not part of the CARMS system, and advanced cases were subsequently classified based on their phenotype only, which is useful for the purpose of evaluating progression over time. CARMS grades were defined as follows: no drusen or only a few small drusen (<63 μm) were assigned to grade 1 (no AMD); intermediate drusen (63–124 μm) were assigned to grade 2 (early AMD); large drusen (≥125 μm) were assigned to grade 3 (intermediate AMD) as long there were no signs of advanced AMD; GA (both central and noncentral) was classified as grade 4, if there was GA in the center grid or anywhere within the grid and there was no record of hemorrhage; and neovascular disease, or grade 5, if there were any definitive signs of neovascular AMD such as hemorrhagic retinal detachment, hemorrhage under the retina or retinal pigment epithelium, or subretinal fibrosis, regardless of visual acuity.
Progression was defined as either eye advancing from no, early, or intermediate AMD at baseline to GA at any point during follow-up. Follow-up ended when an eye progressed to GA. Eyes that had no indication of GA were censored when they reached grade 5. Eyes with advanced AMD (either neovascular disease or GA) at baseline were excluded from the analysis.
Dietary data
Data on food consumption were collected at enrollment with a validated, self-administered, semiquantitative 90-item food-frequency questionnaire based on the National Cancer Institute Health Habits and History Questionnaire (version 2.1). Subjects were asked to report how often, on average, they consumed each food or beverage item during the past year. Consumption was classified into 9 levels from “never or less than one per month” to “6 or more per day.” For each item, average serving size was recorded as “small,” “medium,” or “large,” with respect to standard examples. The University of Minnesota Nutrition Coordinating Center Food Composition Database (version 31) was used with the estimated quantity of nutrient intake and DietSys software (version 3.0; Block Dietary Data Systems) to derive individual nutrient values for each questionnaire item, including folate and B vitamins. The instrument was validated through a telephone-administered 24-h dietary recall 3 and 6 mo postenrollment in a sample of 197 randomly selected participants. Dietary folate and B vitamins were evaluated from food sources only and did not include supplemental forms.
Demographic and behavioral covariates
Baseline age, sex, education, smoking, BMI (in kg/m2), AREDS treatment group, multivitamin supplement use, and ocular characteristics were evaluated. Pack-years was defined as the product of smoking duration (years) by average packs of cigarettes smoked per day. The median value among ever smokers was 20 pack-years, and smoking status was classified as follows: never, <20 pack-years, or ≥20 pack-years. AREDS treatment was defined as “any AREDS treatment” for subjects in the “antioxidant-alone,” “zinc-alone,” or the “antioxidant plus zinc” groups and “placebo” for subjects in the placebo group. AREDS treatment groups were randomly assigned in the AREDS clinical trial. Multivitamin use was defined as “never” for subjects who reported never having taken a multivitamin supplement and who were not taking Centrum (Pfizer) during the study and was defined as “ever” for subjects who declared that they had taken a multivitamin supplement in the past or at present or were taking Centrum during the study.
Genotype data
DNA samples were purchased from the AREDS repository. Genotypes for 10 SNPs associated with AMD in 7 different genes were determined. DNA was extracted from blood samples of participants. All variants assessed in this study were genotyped by using array-based and gene-sequencing platforms as previously described (23–25). All SNPs have a high genotype call rate (>98%), none deviate from Hardy-Weinberg equilibrium in the control group, and none failed a differential missing test between case and control groups. PLINK (open-source, http://pngu.mgh.harvard.edu/purcell/plink/) was used to perform all quality control steps (26). The following SNPs were evaluated as covariates: complement factor H (CFH) Y402H (rs1061170), CFH rs1410996, CFH R1210C (rs121913059), age-related maculopathy susceptibility 2/high-temperature requirement A serine peptidase 1 (ARMS2/HTRA1) (rs10490924), complement component 2 (C2) E318D (rs9332739), complement factor B (CFB) R32Q (rs641154), complement component 3 (C3) R102G (rs2230199), C3 K155Q (rs147859257), collagen type VIII α 1 (COL8A1) (rs13095226), and RAD51 paralog B (RAD51B) (rs8017304).
Statistical analysis
Folate and all B vitamins were not normally distributed based on results from the Kolmogorov-Smirnov test for normality (all P < 0.01). Folate and B vitamins (g/d or μg/d) were therefore log transformed and adjusted for TEI (kcal/d) (i.e., calorie-adjusted intake) separately for men and women. Calorie-adjusted dietary folate and B vitamins were ranked into quintiles by sex. Quintiles of folate and B vitamin intake were used in all statistical models with quintile 1 used as the reference group.
Baseline nondietary characteristics of progressors and nonprogressors were compared by using Cox proportional hazards models to estimate HRs and 95% CIs for progression to GA by using the eye as the unit of analysis. These comparisons were adjusted for age, sex, and AMD grade at baseline (Table 1). Assessments of folate and B vitamins were adjusted for age, sex, and TEI. Each vitamin was analyzed in a separate model (Table 2).
TABLE 1.
Baseline demographic, behavioral, ocular, and genetic characteristics among progressors and nonprogressors to GA: AREDS cohort (n = 2525)1
| Progressors (n = 405), n (%) | Nonprogressors (n = 2120), n (%) | HR (95% CI) | P-trend | |
| Baseline age, y | ||||
| ≤64 | 59 (14.6) | 391 (18.4) | Reference | 0.0002 |
| 65–74 | 238 (58.8) | 1418 (66.9) | 1.23 (0.96, 1.57) | |
| >74 | 108 (26.6) | 311 (14.7) | 1.67 (1.26, 2.21) | |
| Sex | ||||
| Female | 208 (51.4) | 1193 (56.3) | Reference | 0.25 |
| Male | 197 (48.6) | 927 (43.7) | 1.11 (0.93, 1.31) | |
| Education | ||||
| ≤High school | 158 (39.0) | 680 (32.1) | Reference | 0.17 |
| >High school | 247 (61.0) | 1440 (67.9) | 0.86 (0.70, 1.07) | |
| Smoking, pack-years | ||||
| Never | 172 (42.5) | 1011 (47.7) | Reference | 0.14 |
| <20 | 87 (21.5) | 503 (23.7) | 0.98 (0.74, 1.28) | |
| ≥20 | 146 (36.0) | 606 (28.6) | 1.21 (0.95, 1.54) | |
| BMI, kg/m2 | ||||
| <25 | 120 (29.6) | 708 (33.4) | Reference | 0.02 |
| 25–29 | 173 (42.7) | 916 (43.2) | 1.17 (0.91, 1.50) | |
| ≥30 | 112 (27.7) | 496 (23.4) | 1.40 (1.06, 1.84) | |
| AREDS treatment | ||||
| Placebo | 81 (20.0) | 692 (32.6) | Reference | 0.10 |
| Any AREDS treatment | 324 (80.0) | 1428 (67.4) | 1.24 (0.96, 1.61) | |
| Multivitamin supplement use | ||||
| Never | 119 (29.4) | 683 (32.2) | Reference | 0.86 |
| Ever | 286 (70.6) | 1437 (67.8) | 1.02 (0.81, 1.28) | |
| Baseline grade in each eye2 | ||||
| 1,1/1,2/2,2 | 21 (5.2) | 1197 (56.5) | Reference | <0.0001 |
| 1,3/2,3/3,3 | 288 (71.1) | 634 (29.9) | 25.22 (16.69, 38.10) | |
| 1,4/2,4/3,4 | 42 (10.4) | 17 (0.8) | 114.96 (69.45, 190.28) | |
| 1,5/2,5/3,5 | 54 (13.3) | 272 (12.8) | 17.30 (10.66, 28.07) | |
| CFH Y402H rs1061170 | ||||
| TT | 64 (15.8) | 690 (32.6) | Reference | <0.0001 |
| CT | 161 (39.8) | 984 (46.4) | 1.49 (1.09, 2.02) | |
| CC | 180 (44.4) | 446 (21.0) | 2.03 (1.51, 2.74) | |
| CFH rs1410996 | ||||
| TT | 16 (4.0) | 301 (14.2) | Reference | <0.0001 |
| CT | 114 (28.1) | 905 (42.7) | 2.19 (1.27, 3.80) | |
| CC | 275 (67.9) | 914 (43.1) | 3.35 (1.98, 5.67) | |
| CFH R1210C rs121913059 | ||||
| CC | 400 (98.8) | 2113 (99.7) | Reference | 0.10 |
| CT | 5 (1.2) | 7 (0.3) | 2.05 (0.87, 4.84) | |
| ARMS2/HTRA1 rs10490924 | ||||
| GG | 125 (30.9) | 1148 (54.1) | Reference | <0.0001 |
| GT | 202 (49.9) | 786 (37.1) | 1.75 (1.38, 2.21) | |
| TT | 78 (19.2) | 186 (8.8) | 2.01 (1.48, 2.73) | |
| C2 E318D rs9332739 | ||||
| GG | 396 (97.8) | 1952 (92.1) | Reference | 0.006 |
| CG/CC | 9 (2.2) | 168 (7.9) | 0.38 (0.19, 0.76) | |
| CFB R32Q rs641153 | ||||
| CC | 371 (91.6) | 1812 (85.5) | Reference | 0.007 |
| CT/TT | 34 (8.4) | 308 (14.5) | 0.60 (0.41, 0.87) | |
| C3 R102G rs2230199 | ||||
| CC | 199 (49.2) | 1248 (58.9) | Reference | 0.04 |
| CG/GG | 206 (50.8) | 872 (41.1) | 1.25 (1.01, 1.53) | |
| C3 K155Q rs147859257 | ||||
| TT | 387 (95.6) | 2085 (98.4) | Reference | 0.0006 |
| GT | 18 (4.4) | 35 (1.6) | 2.26 (1.42, 3.62) | |
| COL8A1 rs13095226 | ||||
| TT | 309 (76.3) | 1716 (80.9) | Reference | 0.05 |
| CT/CC | 96 (23.7) | 404 (19.1) | 1.28 (1.00, 1.63) | |
| RAD51B rs8017304 | ||||
| AA | 165 (40.7) | 882 (41.6) | Reference | 0.62 |
| AG | 200 (49.4) | 958 (45.2) | 1.07 (0.86, 1.33) | |
| GG | 40 (9.9) | 280 (13.2) | 0.82 (0.57, 1.19) |
Progressors are defined as subjects who progressed in at least one eye, and nonprogressors are defined as subjects who did not progress in either eye. HRs, 95% CIs, and P-trend were calculated by using the Cox proportional hazards model adjusting for age, sex, and AMD grade at baseline. Each variable was analyzed in a separate model. CFH Y402H, CFH rs1410996, ARMS2, and RAD51B were coded with 3 levels (0|1|2) according to the number of risk alleles. Other variants were coded with 2 levels (0|1) as labeled. AMD, age-related macular degeneration; AREDS, Age-Related Eye Disease Study; ARMS2, age-related maculopathy susceptibility 2; CARMS, clinical age-related maculopathy staging system; CFB, complement factor B; CFH, complement factor H; COL8A1, collagen type VIII α 1; C2, complement component 2; C3, complement component 3; GA, geographic atrophy; HTRA1, high-temperature requirement A serine peptidase 1; RAD51B, RAD51 paralog B; 1,1, no AMD; 1,2, no AMD, early AMD; 1,3, no AMD, intermediate AMD; 1,4, no AMD, GA; 1,5, no AMD, neovascular AMD; 2,2, early AMD, early AMD; 2,3, early AMD, intermediate AMD; 2,4, early AMD, GA; 2,5, early AMD, neovascular AMD; 3,3, intermediate AMD, intermediate AMD; 3,4, intermediate AMD, GA; 3,5, intermediate AMD, neovascular AMD.
Represents the CARMS grade in each eye.
TABLE 2.
Distribution of dietary intake of B vitamins at baseline among progressors and nonprogressors to GA: AREDS cohort (n = 2525)1
| Quantities consumed/d, median (range) |
||||||
| Men | Women | Progressors (n = 405), n (%) | Nonprogressors (n = 2120), n (%) | OR (95% CI) | P-trend | |
| Thiamin, mg | ||||||
| Quintile 12 | 1.10 (0.57–1.22) | 0.85 (0.51–0.93) | 104 (25.7) | 400 (18.9) | Reference | 0.01 |
| Quintile 2 | 1.31 (1.23–1.38) | 0.99 (0.94–1.05) | 78 (19.2) | 427 (20.1) | 0.71 (0.51, 0.99) | |
| Quintile 3 | 1.43 (1.39–1.51) | 1.10 (1.06–1.15) | 81 (20.0) | 425 (20.1) | 0.72 (0.52, 1.00) | |
| Quintile 4 | 1.60 (1.52–1.68) | 1.21 (1.16–1.28) | 68 (16.8) | 437 (20.6) | 0.57 (0.41, 0.80) | |
| Quintile 5 | 1.90 (1.69–4.91) | 1.43 (1.29–4.26) | 74 (18.3) | 431 (20.3) | 0.65 (0.47, 0.91) | |
| Riboflavin, mg | ||||||
| Quintile 1 | 1.24 (0.63–1.36) | 0.94 (0.47–1.07) | 89 (22.0) | 415 (19.6) | Reference | 0.03 |
| Quintile 2 | 1.49 (1.37–1.60) | 1.17 (1.08–1.26) | 85 (21.0) | 420 (19.8) | 0.91 (0.66, 1.27) | |
| Quintile 3 | 1.72 (1.61–1.80) | 1.35 (1.27–1.44) | 88 (21.7) | 418 (19.7) | 0.93 (0.67, 1.29) | |
| Quintile 4 | 1.95 (1.81–2.11) | 1.55 (1.45–1.70) | 70 (17.3) | 435 (20.5) | 0.69 (0.49, 0.98) | |
| Quintile 5 | 2.41 (2.12–5.37) | 1.93 (1.71–4.63) | 73 (18.0) | 432 (20.4) | 0.74 (0.53, 1.04) | |
| Niacin, mg | ||||||
| Quintile 1 | 14.01 (8.05–15.42) | 10.30 (5.14–11.42) | 97 (24.0) | 407 (19.2) | Reference | 0.31 |
| Quintile 2 | 16.58 (15.43–17.56) | 12.21 (11.43–12.99) | 82 (20.2) | 423 (20.0) | 0.83 (0.60, 1.15) | |
| Quintile 3 | 18.49 (17.57–19.45) | 13.77 (13.00–14.44) | 70 (17.2) | 436 (20.6) | 0.69 (0.49, 0.96) | |
| Quintile 4 | 20.55 (19.46–21.99) | 15.44 (14.45–16.62) | 76 (18.8) | 429 (20.2) | 0.76 (0.55, 1.06) | |
| Quintile 5 | 24.44 (22.00–62.11) | 18.46 (16.63–51.75) | 80 (19.8) | 425 (20.0) | 0.82 (0.59, 1.14) | |
| Vitamin B-6, mg | ||||||
| Quintile 1 | 1.22 (0.66–1.39) | 0.90 (0.39–1.03) | 85 (21.0) | 419 (19.8) | Reference | 0.31 |
| Quintile 2 | 1.51 (1.40–1.60) | 1.13 (1.04–1.22) | 84 (20.7) | 421 (19.9) | 0.96 (0.69, 1.34) | |
| Quintile 3 | 1.72 (1.61–1.83) | 1.33 (1.23–1.41) | 82 (20.2) | 424 (20.0) | 0.93 (0.67, 1.31) | |
| Quintile 4 | 1.96 (1.84–2.11) | 1.53 (1.42–1.65) | 78 (19.3) | 427 (20.1) | 0.89 (0.63, 1.25) | |
| Quintile 5 | 2.46 (2.12–5.74) | 1.89 (1.66–4.98) | 76 (18.8) | 429 (20.2) | 0.85 (0.61, 1.20) | |
| Folate, μg | ||||||
| Quintile 1 | 260.37 (140.11–302.92) | 202.99 (93.54–233.33) | 104 (25.7) | 400 (18.9) | Reference | 0.001 |
| Quintile 2 | 332.89 (302.98–358.31) | 255.44 (233.57–275.63) | 91 (22.5) | 414 (19.5) | 0.85 (0.62, 1.17) | |
| Quintile 3 | 388.58 (358.52–417.63) | 297.29 (275.92–317.48) | 78 (19.2) | 428 (20.2) | 0.70 (0.50, 0.97) | |
| Quintile 4 | 452.97 (417.86–495.66) | 342.81 (317.58–376.00) | 57 (14.1) | 448 (21.1) | 0.49 (0.35, 0.70) | |
| Quintile 5 | 571.66 (495.97–1225.03) | 423.70 (376.11–1091.25) | 75 (18.5) | 430 (20.3) | 0.66 (0.47, 0.91) | |
| Vitamin B-12, μg | ||||||
| Quintile 1 | 2.63 (1.07–3.27) | 1.95 (0.37–2.39) | 87 (21.5) | 417 (19.7) | Reference | 0.18 |
| Quintile 2 | 3.76 (3.28–4.19) | 2.75 (2.40–3.10) | 76 (18.8) | 429 (20.2) | 0.83 (0.59, 1.17) | |
| Quintile 3 | 4.70 (4.20–5.19) | 3.45 (3.11–3.84) | 86 (21.2) | 420 (19.8) | 0.92 (0.66, 1.28) | |
| Quintile 4 | 5.84 (5.20–6.71) | 4.32 (3.85–5.01) | 86 (21.2) | 419 (19.8) | 0.94 (0.67, 1.30) | |
| Quintile 5 | 8.30 (6.72–21.10) | 6.14 (5.02–51.20) | 70 (17.3) | 435 (20.5) | 0.74 (0.53, 1.05) | |
Progressors are defined as subjects who progress in at least one eye, and nonprogressors are defined as subjects who did not progress in either eye. ORs, 95% CIs, and P-trend were calculated by using logistic regression adjusting for age, sex, and total energy intake. Each vitamin was analyzed in a separate model. Median and range are calorie adjusted. AREDS, Age-Related Eye Disease Study; GA, geographic atrophy.
Intakes were log transformed and calorie-adjusted separately for men and women.
For analysis of incident outcomes, we assessed progression to GA over 13 y by using survival analysis methodology (Table 3). The associations of dietary folate and B vitamin intake with progression to GA were analyzed by using Cox proportional hazards models with the individual eye as the unit of analysis [PROC PHREG with the covariance aggregate option in SAS 9.3 (SAS Institute)] (27). In the multivariate models, we adjusted for age (≤64, 65–74, and >74 y), sex, education (≤high school, >high school), smoking (never smoker, <20 pack-years, and ≥20 pack-years), BMI (in kg/m2; <25, 25–29, and ≥30), AREDS treatment (placebo, any AREDS treatment), multivitamin supplement use (never, ever), AMD grade at baseline for both the study and fellow eye (CARMS grade), TEI (kcal/d), and the 10 AMD SNPs reported above. The P-trend was calculated for multivariate models by using the median intake for each quintile.
TABLE 3.
Multivariate associations between dietary B vitamins and progression to GA: AREDS cohort (n = 4663 eyes)1
| Quintiles of B vitamins, HR (95% CI) |
||||||
| 1 | 2 | 3 | 4 | 5 | P-trend | |
| Thiamin | Reference | 0.81 (0.60, 1.09) | 0.85 (0.62, 1.16) | 0.70 (0.51, 0.97) | 0.74 (0.55, 0.99) | 0.05 |
| Riboflavin | Reference | 0.82 (0.61, 1.12) | 1.03 (0.76, 1.39) | 0.75 (0.54, 1.03) | 0.83 (0.61, 1.12) | 0.20 |
| Niacin | Reference | 0.77 (0.57, 1.04) | 0.74 (0.53, 1.03) | 0.70 (0.51, 0.95) | 0.78 (0.57, 1.06) | 0.18 |
| Vitamin B-6 | Reference | 0.98 (0.72, 1.35) | 0.92 (0.66, 1.28) | 0.94 (0.68, 1.30) | 0.87 (0.63, 1.19) | 0.36 |
| Folate | Reference | 0.94 (0.71, 1.25) | 0.75 (0.55, 1.02) | 0.66 (0.46, 0.93) | 0.70 (0.52, 0.95) | 0.007 |
| Vitamin B-12 | Reference | 0.84 (0.61, 1.16) | 0.88 (0.65, 1.20) | 0.93 (0.69, 1.25) | 0.77 (0.56, 1.06) | 0.19 |
P-trend was calculated by using median values within each quintile. Cox proportional hazards models were adjusted for age, sex, education, smoking, BMI, AREDS treatment, multivitamin supplement use, age-related macular degeneration grade at baseline for both eyes, total energy intake, and 10 genetic variants [CFH: rs1061170 (Y402H), CFH: rs1410996, CFH: rs121913059 (R1210C), ARMS2/HTRA1: rs10490924, C2: rs9332739 (E318D), CFB: rs641153 (R32Q), C3: rs2230199 (R102G), C3: rs147859257 (K155Q), COL8A1: rs13095226, and RAD51B: rs8017304]. Each vitamin was analyzed in a separate model. For eyes that progressed to GA, n = 528; eyes at risk, n = 4663. AREDS, Age-Related Eye Disease Study; ARMS2, age-related maculopathy susceptibility 2; CFB, complement factor B; CFH, complement factor H; COL8A1, collagen type VIII α 1; C2, complement component 2; C3, complement component 3; GA, geographic atrophy; HTRA1, high-temperature requirement A serine peptidase 1; RAD51B, RAD51 paralog B.
Secondary analyses
Interactions between all AMD genes and folate and B vitamin intake were analyzed (Table 4). Using a multiplicative model, interaction terms for the number of risk alleles and folate and B vitamin quintile were assessed separately for each genetic variant. Because of the small number of subjects in each genotype group, CFH R1210C (CC compared with CT), C2 E318D (GG compared with CG/CC), CFB (CC compared with CT/TT), C3 R102G (CC compared with CG/GG), C3 K155Q (TT compared with GT), and COL8A1 (TT compared with CT/CC) variants were analyzed as binary variables, as presented in Table 1. Other genes (CFH Y402H, CFH rs1410996, ARMS2/HTRA1, and RAD51B) were analyzed as continuous variables according to the number of risk alleles (0–2).
TABLE 4.
Effect of folate on progression to GA according to genotypes1
| Dietary folate, quintile 5 vs. quintile 1 |
|||
| HR (95% CI) | P value | P-interaction2 | |
| CFH Y402H rs1061170 | |||
| TT | 0.75 (0.31, 1.79) | 0.52 | 0.13 |
| CT | 0.87 (0.52, 1.47) | 0.61 | |
| CC | 0.59 (0.38, 0.92) | 0.02 | |
| CFH rs1410996 | |||
| TT | 0.30 (0.05, 1.74) | 0.18 | 0.13 |
| CT | 1.20 (0.66, 2.18) | 0.55 | |
| CC | 0.58 (0.40, 0.83) | 0.003 | |
| CFH R1210C rs121913059 | |||
| CC | 0.69 (0.51, 0.94) | 0.02 | 0.64 |
| CT | — | — | |
| ARMS2/HTRA1 rs10490924 | |||
| GG | 0.84 (0.48, 1.48) | 0.55 | 0.71 |
| GT | 0.61 (0.40, 0.95) | 0.03 | |
| TT | 0.66 (0.32, 1.37) | 0.26 | |
| C2 E318D rs9332739 | |||
| GG | 0.72 (0.53, 0.98) | 0.03 | 0.14 |
| CG/CC | — | — | |
| CFB R32Q rs641153 | |||
| CC | 0.66 (0.48, 0.90) | 0.009 | 0.12 |
| CT/TT | 2.40 (0.54, 10.61) | 0.25 | |
| C3 R102G rs2230199 | |||
| CC | 0.43 (0.27, 0.70) | 0.0005 | 0.0025 |
| CG/GG | 0.94 (0.62, 1.42) | 0.76 | |
| C3 K155Q rs147859257 | |||
| TT | 0.69 (0.51, 0.95) | 0.02 | 0.74 |
| GT | 1.01 (0.12, 8.57) | 0.99 | |
| COL8A1 rs13095226 | |||
| TT | 0.73 (0.52, 1.05) | 0.09 | 0.53 |
| CT/CC | 0.53 (0.28, 1.00) | 0.05 | |
| RAD51B rs8017304 | |||
| AA | 0.97 (0.58, 1.62) | 0.90 | 0.06 |
| AG | 0.56 (0.37, 0.85) | 0.007 | |
| GG | 0.53 (0.18, 1.58) | 0.26 | |
| Composite genetic risk score | |||
| Low (<median) | 0.88 (0.41, 1.86) | 0.73 | 0.89 |
| High (≥median) | 0.73 (0.52, 1.02) | 0.06 | |
Cox proportional hazards models were adjusted for age, sex, education, smoking, BMI, AREDS treatment, multivitamin supplement use, age-related macular degeneration grade at baseline for both eyes, total energy intake, and genetic variants [CFH: rs1061170 (Y402H), CFH: rs1410996, CFH: rs121913059 (R1210C), ARMS2/HTRA1: rs10490924, C2: rs9332739 (E318D), CFB: rs641153 (R32Q), C3: rs2230199 (R102G), C3: rs147859257 (K155Q), COL8A1: rs13095226, RAD51B: rs8017304]. AREDS, Age-Related Eye Disease Study; ARMS2, age-related maculopathy susceptibility 2; CFB, complement factor B; CFH, complement factor H; COL8A1, collagen type VIII α 1; C2, complement component 2; C3, complement component 3; GA, geographic atrophy; HTRA1, high-temperature requirement A serine peptidase 1; RAD51B, RAD51 paralog B.
Each interaction was assessed in a separate model. Each interaction term is based on a cross product of folate quintile by number of risk alleles as continuous variables for CFH Y402H, CFH rs1410996, ARMS2, and RAD51B and as binary variables for CFH R1210C, C2 E318D, CFB, C3 R102G, C3 K155Q, and COL8A1. We applied a Bonferroni correction for interaction analyses. Interaction terms were considered significant for P values <0.005.
Each interaction term was introduced independently into a demographic, behavioral, ocular, and genetic-adjusted Cox proportional hazards model (as described above). To assess the combined effect of the 10 genetic variants, we calculated a composite genetic risk score by using the sum of the regression coefficients multiplied by the corresponding number of risk alleles and summed over the 10 genetic variants used in our comprehensive prediction model (25). Interactions between this genetic risk score (classified as below the median, greater than or equal to the median) and folate and B vitamin intake were analyzed by using the same model as for single SNPs described above.
We compared subjects who were included with those who were not included in the analytic sample by using χ2 or Fisher’s exact tests (Supplemental Table 1). Associations between food sources and dietary folate and B vitamins were assessed by using linear regression. Food intakes were log transformed, and a stepwise method was applied with a significance level of P ≤ 0.05 for entry and P ≤ 0.10 for exiting the model. Age, sex, and TEI were forced into the model. Each vitamin was examined in a separate model (Supplemental Table 2).
We evaluated whether associations between folate, B vitamins, and progression to GA differ between users and nonusers of multivitamin supplements (Supplemental Tables 3 and 4), as well as participants who had taken an AREDS treatment and participants who had not (Supplemental Table 5). These associations were evaluated separately for multivitamin supplement use (never, ever) and each AREDS treatment group (any AREDS treatment, placebo) by using a Cox proportional hazards model adjusted for demographic, behavioral, ocular, and genetic factors. P-trend was calculated by using the median value for each quintile of folate or B vitamin.
Associations between dietary folate and B vitamins and progression to neovascular AMD were estimated by using the same methods as previously described for progression to GA (Supplemental Table 6).
P values <0.05 were considered statistically significant for all analyses, excluding those related to nutrient-gene interactions. We applied a Bonferroni correction for analyses of interactions (28). Interaction terms were considered significant for P values <0.005. All statistical analyses were performed using SAS software, version 9.3 (SAS Institute).
RESULTS
Baseline demographic, behavioral, ocular, and genetic characteristics for progressors and nonprogressors, adjusted for age, sex, and AMD grade at baseline, are shown in Table 1. The mean follow-up time was 8.7 y (range: 0.5–13 y). Among 2525 subjects, 405 (16.0%) progressed to GA. Progressors to GA tended to be older (P-trend = 0.0002) and to have a higher BMI (P-trend = 0.02). Sex (P = 0.25), education (P = 0.17), smoking (P-trend = 0.14), AREDS treatment (P = 0.10), and multivitamin use (P = 0.86) did not significantly differ between progressors and nonprogressors. Subjects with intermediate or advanced AMD in the worst eye were at higher risk of progression to GA (P-trend < 0.0001). The risk alleles for CFH Y402H and CFH rs1410996, ARMS2/HTRA1, and both C3 variants were significantly associated with an increased risk of progression to GA, and the protective alleles of C2 and CFB were significantly associated with a decreased risk of progression. CFH R1210C, COL8A1, and RAD51B were not significantly associated with risk of progression to GA (P = 0.10, P = 0.05, and P = 0.62, respectively), although HRs trended in the direction previously reported (25).
Baseline demographic, behavioral, ocular, and nutritional characteristics among subjects included (n = 2525) and not included (n = 619) in the analytic sample are shown in Supplemental Table 1. The 2 groups did not differ by age, sex, education, AREDS treatment, multivitamin supplement use, AMD grade at baseline, TEI, folate, and B vitamins. Subjects who were not included were somewhat more likely to be smokers and to have a higher BMI (P = 0.02 and P = 0.03, respectively). These variables were accounted for by adjustment in multivariate models.
The baseline distribution of dietary B vitamins between progressors and nonprogressors is displayed in Table 2. After adjustment for age, sex, and TEI, progressors had a lower intake of thiamin, riboflavin, and folate (P-trend = 0.01, 0.03, and 0.001, respectively). No statistically significant variation was found between progressors and nonprogressors for niacin, vitamin B-6, and vitamin B-12.
Supplemental Table 2 shows major food sources of dietary folate and B vitamins in our study population. Major plant sources are cereals (refined, whole-grain, and fortified cereals), citrus fruits, and bananas. The major animal source is dairy products, which provide vitamin B-12. Folate is mainly provided by cereals, dark-green vegetables, citrus fruits, bananas, and dairy products.
Multivariate associations between dietary folate, B vitamins, and progression to GA in a model adjusted for age, sex, education, smoking, BMI, AREDS treatment, multivitamin use, baseline AMD grade in both eyes, TEI, and 10 genetic variants are shown in Table 3. The median time interval between baseline food-frequency questionnaire administration and endpoint attainment was 10 y (range: 0.5–13 y). This model revealed a statistically significant trend for a lower risk of progression to GA with increasing intake of dietary folate (P-trend = 0.007). Quintiles 4 and 5 were significantly associated with a decreased risk of progression, with an HR of 0.66 (95% CI: 0.46, 0.93) for quintile 4 and 0.70 (95% CI: 0.52, 0.95) for quintile 5. The trend for thiamin was borderline (P-trend = 0.053), and quintile 4 (HR = 0.70; 95% CI: 0.51, 0.97) and quintile 5 (HR = 0.74; 95% CI: 0.55, 0.99) of thiamin were significantly associated with a decreased risk of progression compared with quintile 1. Quintile 4 of niacin intake was significantly associated with a decreased risk of progression compared with quintile 1, although the overall trend was not statistically significant. Associations between riboflavin and progression did not retain statistical significance after adjustment for the covariates reported above (P-trend = 0.20). Vitamins B-6 and B-12 were not significantly associated with the risk of progression to GA.
The effect of folate on progression to GA according to risk and nonrisk genotypes after controlling for demographic, behavioral, and ocular factors as well as genetic variants is displayed in Table 4. We found a statistically significant interaction between C3 R102G and folate (P = 0.0025). There was a significant protective effect of folate among subjects homozygous for the C3 R102G nonrisk allele (C) (HR = 0.43; 95% CI = 0.27, 0.70; P = 0.0005, quintile 5 compared with quintile 1). No association was observed among subjects who were heterozygous or homozygous for the risk allele (G). Additional interactions between folate and other AMD genes, as well as our composite genetic score, were not statistically significant. The HRs for each quintile of folate intake according to C3 R102G genotype, with quintile 1 used as the referent, are illustrated in Figure 2.
FIGURE 2.
Effect of dietary folate on progression to geographic atrophy according to C3 R102G genotype. HRs, 95% CIs, and P-trend were calculated by using Cox proportional hazards models adjusted for age, sex, education, smoking, BMI, AREDS treatment, multivitamin supplement use, AMD grade at baseline for both eyes, TEI, and 9 genetic variants [CFH: rs1061170 (Y402H), CFH: rs1410996, CFH: rs121913059 (R1210C), ARMS2/HTRA1: rs10490924, C2: rs9332739 (E318D), CFB: rs641153 (R32Q), C3: rs147859257 (K155Q), COL8A1: rs13095226, RAD51B: rs8017304]. AMD, age-related macular degeneration; AREDS, Age-Related Eye Disease Study; ARMS2, age-related maculopathy susceptibility 2; CFB, complement factor B; CFH, complement factor H; COL8A1, collagen type VIII α 1; C2, complement component 2; C3, complement component 3; HTRA1, high-temperature requirement A serine peptidase 1; Q, quintile; RAD51B, RAD51 paralog B; TEI, total energy intake.
The proportion of subjects reporting multivitamin use in each quintile of folate and B vitamin intake is shown in Supplemental Table 3. There was a slight trend toward a higher percentage of multivitamin users in higher quintiles of B vitamin intake. Associations between folate, B vitamins, and progression to GA according to multivitamin supplement use are reported in Supplemental Table 4. There was a trend toward a reduced risk of progression with higher concentrations of folate intake for both multivitamin users and nonusers. This trend was statistically significant only in the supplement users (P = 0.02), possibly due to the smaller sample size for the nonusers. Interactions between folate and B vitamins and the use of multivitamins were not statistically significant.
Associations between folate, B vitamins, and progression to GA according to AREDS treatment group are reported in Supplemental Table 5. We found a protective effect of dietary folate intake in the placebo group (P-trend = 0.04), although no statistically significant association was observed in the AREDS treatment group (P-trend = 0.06). In this subgroup, however, quintile 4 of thiamin was significantly associated with progression. In addition, quintiles 3 and 4 of niacin intake were significantly associated with a decreased risk of progression compared with quintile 1, but the overall trend was not statistically significant (P-trend = 0.34). Other B vitamins were not significantly associated with progression to GA according to AREDS treatment group.
Associations between folate and B vitamins and progression to neovascular AMD are reported in Supplemental Table 6. Neither folate nor any B vitamin was significantly associated with progression to neovascular AMD (n = 610 eyes).
DISCUSSION
High dietary intake of folate was associated with a decreased risk of progression to GA. We also found a significant interaction between folate intake and the C3 R102G variant. Risk of progression was significantly reduced among subjects homozygous for the C3 R102G nonrisk allele (C) but not those carrying the risk allele (G). In our study, thiamin, riboflavin, niacin, and vitamins B-6 and B-12 were not significantly associated with progression. To our knowledge, this study is the first to report associations between progression to GA and the dietary intake of folate and B vitamins. It is also the first to evaluate genetic factors and their interactions with those nutrients in a large prospective cohort with a high number of incident cases.
The Blue Mountains Eye Study (BMES) reported an inverse association between high dietary intake of folate and 10-y incidence of combined advanced forms of AMD (n = 57 cases) (16). Subjects within the third tertile of folate intake (≥475.3 μg/d) had a decreased risk of incident late AMD (HR = 0.45; 95% CI: 0.21, 0.98; P-trend = 0.05). In our study, the risk of subjects in the highest quintiles of folate intake is very similar to those observed in the BMES. Similar to our findings on GA, dietary intake of vitamin B-12 was not significantly associated with the incidence of combined forms of advanced AMD. Another study of 137 AMD cases, including early and intermediate disease, suggested that daily intake of a supplement formula combining folic acid and vitamins B-6 and B-12 could help reduce the risk of AMD (19). To our knowledge, the BMES is the only other observational study that reports associations between dietary folate, vitamin B-12, and AMD. However, this well-conducted study had a small number of participants who developed incident advanced AMD, and analyses did not separate the advanced subtypes or include genetic factors.
Our results suggest a threshold effect of dietary folate on progression to GA (Figure 2). Subjects in the fourth and the fifth quintiles appear to have a similar reduced risk of progression. In the Dietary Guidelines for Americans, 2010 (29), the USDA and the US Department of Health and Human Services suggested an Adequate Intake and a Recommended Dietary Allowance of 400 μg/d for men and women aged ≥51 y. In our study, subjects in the highest quintiles for dietary folate reported a daily intake in accordance with these recommendations. Our results suggest that adhering to the USDA’s recommendations (29) for adequate folate intake may help to reduce progression to GA.
Associations between folate and vitamin B-12 in plasma (17) and in serum (16, 18, 30) have also been reported. Subjects with a serum deficiency for vitamin B-12 were at higher risk of both forms of advanced AMD using cross-sectional (18) and prospective BMES data (16). The NHANES III, however, had a small sample of participants with AMD and did not show an association for serum vitamin B-12 (30). A clinical case-control study also reported a significantly lower concentration of plasma vitamin B-12 among 30 patients with neovascular AMD (17). Each of these studies reported significant associations between circulating folate and advanced AMD (16–18, 30).
The biological mechanisms underlying the beneficial effect of folate on AMD risk are not well understood. They may include a direct antioxidant effect as well as enhancement of endothelial nitric oxide concentrations in the choroidal vasculature, both of which are associated with an increase in vascular reactivity (19). Another plausible explanation could be the putative role of folate and B vitamins in DNA methylation processes. These nutrients are involved in the one-carbon metabolism, and a deficiency can significantly decrease DNA methylation (31–34). One-carbon metabolism dysregulations are linked to numerous age-related and neurodegenerative diseases (33), and folate deficiency might also have deleterious effects on cells by permitting the accumulation of homocysteine (35, 36). Hyperhomocysteinemia is a risk factor for several age-related diseases, and observational studies have shown that subjects with advanced AMD have higher plasma homocysteine concentrations (15–17).
To our knowledge, this study is the first to suggest an interaction between folate intake and C3 R102G. Literature related to this topic is scarce, and biological mechanisms underlying these results have not been explored. To reduce the possibility of a chance finding, we applied a Bonferroni correction for multiple testing in all interaction analyses. Additional experimental and observational studies are needed to better understand the mechanisms implied by these results.
This report expanded on previous research by assessing progression to GA, accounting for progression in both eyes, controlling for 10 AMD variants, and assessing interactions between dietary folate, B vitamins, and genetics. We also examined the association of folate and B vitamins according to multivitamin supplement use. Strengths of our study include standardized data collection for a large and well-defined cohort, extensive follow-up time, and a large number of subjects who progressed to GA. Dietary data were collected before the occurrence of GA and therefore minimized the impact of changes in eating habits due to knowledge of the disease or the induced disability. Our findings are applicable to an older American Caucasian population and could also be relevant for other developed countries.
Residual confounding is a common limitation in epidemiologic studies, and the potential benefit of folate might be explained by other factors. For instance, subjects with high dietary intake of folate are more likely to have a healthier lifestyle. In nutritional epidemiology, intercorrelations between nutrients cannot be completely eliminated. Folate is mainly provided by green vegetables, fruits, nuts, beans, and peas, and it shares common food sources with nutrients such as lutein and zeaxanthin, which have a protective effect on progression to advanced stages of AMD (37–41). We therefore adjusted for numerous diet- and AMD-related risk factors. Data collected from food-frequency questionnaires may result in over- or underestimating the evaluation of folate and B vitamins consumed and do not distinguish between regular and fortified or enriched foods. To strengthen our analyses and minimize reporting bias, we excluded subjects with unusually low or high TEI.
In conclusion, this study suggests that high dietary folate intake is associated with a lower risk of progression to GA. This association might be moderated by genetic susceptibility related to C3 R102G. Additional research is needed to confirm our findings and to better understand the role of folate and B vitamins in progression from early and intermediate disease to advanced stages of AMD. Eating a healthy diet rich in folate could potentially contribute to the reduction and prevention of visual loss due to atrophic macular degeneration.
Acknowledgments
The authors’ responsibilities were as follows—BMJM, BR, and JMS: designed and conducted the research; BMJM and BR: analyzed the data or performed the statistical analysis; and all authors: wrote the manuscript and have primary responsibility for the final content. The authors declared no conflicts of interest.
Footnotes
Abbreviations used: AMD, age-related macular degeneration; AREDS, Age-Related Eye Disease Study; ARMS2, age-related maculopathy susceptibility 2; BMES, Blue Mountains Eye Study; CARMS, clinical age-related maculopathy staging system; CFB, complement factor B; CFH, complement factor H; C2, complement component 2; C3, complement component 3; COL8A1, collagen type VIII α 1; GA, geographic atrophy; HTRA1, high-temperature requirement A serine peptidase 1; RAD51B, RAD51 paralog B; SNP, single-nucleotide polymorphism; TEI, total energy intake.
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