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Maguire MG, Ying G, Jaffe GJ, et al. Single-Nucleotide Polymorphisms Associated With Age-Related Macular Degeneration and Lesion Phenotypes in the Comparison of Age-Related Macular Degeneration Treatments Trials . JAMA Ophthalmol. 2016;134(6):674–681. doi:10.1001/jamaophthalmol.2016.0669
Single-nucleotide polymorphisms (SNPs) associated with the CFH, ARMS2, C3, LIPC, CFB, and C2 genes are associated with age-related macular degeneration (AMD); however, the association of these SNPs with angiographic features of neovascular AMD has been inconsistent in previous studies, and to date, no studies have addressed their association with features on optical coherence tomography.
To evaluate the influence of genotype of SNPs previously associated with AMD on the phenotype of neovascular lesions.
Design, Setting, and Participants
Participants for this cross-sectional study were recruited from the 1185 patients enrolled in the Comparison of Age-Related Macular Degeneration Treatments Trials (CATT), a randomized clinical trial. Eligibility criteria for CATT specified that eyes have choroidal neovascularization and visual acuity between 20/25 and 20/320. A subgroup of 835 patients provided blood samples from July 2010 through September 2011 and were genotyped for the SNPs rs1061170 (CFH), rs10490924 (ARMS2),rs2230199 (C3), rs10468017 (LIPC), rs4151667 (CFB), rs547154 (C2) using TaqMan SNP genotyping assays. Data analysis was initiated in November 2013 and completed in January 2016.
Main Outcomes and Measures
Pretreatment ocular characteristics on fluorescein angiography (lesion type, area of neovascularization and total lesion, retinal angiomatous proliferation) and on time-domain optical coherence tomography (presence of intraretinal, subretinal, and subretinal pigment epithelium fluid; thickness at the foveal center of the retina, subretinal fluid, and subretinal tissue complex), visual acuity, and age.
A total of 835 (73%) of 1150 CATT patients were genotyped. Mean age decreased with the number of risk alleles for CFH (P < .001), ARMS2 (P < .001), and C3 (P = .005). The following results were found as the number of risk alleles increased from 0 to 1 to 2. For CFH, mean total thickness decreased from 476 to 476 to 434 µm (P = .01; adjusted for age, sex, and smoking status). For ARMS2, the mean area of the total lesion increased from 2.0 to 2.8 to 2.4 mm2 (P = .03), the proportion with retinal angiomatous proliferation lesions increased from 8% to 10% to 12% (P = .05), and the proportion with intraretinal fluid increased from 72% to 71% to 82% (P = .008). For C3, the proportion with intraretinal fluid decreased from 78% to 69% to 64% (P = .001), and the mean retinal thickness decreased from 225 to 207 to 197 µm (P = .02).
Conclusions and Relevance
CFH, ARMS2, and C3 were associated with specific features of neovascularization at the time patients were enrolled in CATT. Previously identified associations of ARMS2 and CFH with type of choroidal neovascularization on fluorescein angiography were not confirmed. New associations with OCT features identified in CATT need confirmation to establish whether a true association exists.
clinicaltrials.gov Identifier: NCT00593450.
The features of neovascular lesions vary considerably among patients with newly diagnosed age-related macular degeneration (AMD). Some features, such as angiographic pattern of leakage and size of the lesion, are known to have a strong influence on current visual acuity (VA), prognosis for loss of vision when untreated, change in VA after laser or anti–vascular endothelial growth factor (VEGF) treatment, and development of scar or geographic atrophy after anti-VEGF treatment.1-5
Many single-nucleotide polymorphisms (SNPs) that confer increased risk of developing AMD have been identified; however, SNPs associated with the CFH (OMIM 134370), ARMS2 (OMIM 611313), HTRA1 (OMIM 602194), and C3 (OMIM 120700) genes are among those most consistently associated with neovascular AMD.6,7 Several research groups have investigated the association of these SNPs with features of neovascular lesions apparent on fundus color photography and fluorescein angiography in patients with AMD.8-20 Most of the previous studies8-15,18 have involved 250 or fewer patients, and the results have not been consistent. To our knowledge, there have been no previous studies of the association of these SNPs with features of neovascular AMD on optical coherence tomography (OCT). More recently, SNPs associated with the LIPC (OMIM 151670), CFB (OMIM 138470), and C2 (OMIM 613927) genes have been associated with AMD; however, to date, association studies of these SNPs with the fluorescein angiographic and OCT features of neovascular lesions have not been conducted.21-23
The detailed assessments of color fundus photographs, fluorescein angiograms, and OCT scans by reading centers and genotyping of the large number of patients enrolled in the Comparison of Age-Related Macular Degeneration Treatments Trials (CATT) allow further evaluation of the association of SNPs linked to the development of neovascular AMD with features detectable on photographs and features on OCT. Better understanding of these associations may help in determining how these SNPs affect the pathogenesis of AMD and neovascular lesions.
Question Do single-nucleotide polymorphisms (SNPs) that lead to the development of age-related macular degeneration influence the features of neovascularization?
Findings In this cross-sectional analysis of 835 participants in the Comparison of Age-Related Macular Degeneration Treatments Trials, a greater number of risk alleles for ARMS2 was significantly associated with larger lesions and with retinal angiomatous proliferation lesions.
Meaning Although there were modest associations for some SNPs with neovascular features, no highly predictive genotypes were identified among the 6 SNPs evaluated.
Details of the design and methods for CATT have been published previously.24-27 From February 1, 2008, through December 31, 2009, a total of 1185 patients were recruited for the randomized clinical trial (clinicaltrials.gov Identifier: NCT00593450) through 43 clinical centers in the United States. Inclusion criteria were age of 50 years or older, presence in the study eye of previously untreated active choroidal neovascularization (CNV) secondary to AMD, and VA between 20/25 and 20/320 in the study eye. Active CNV was considered present when leakage on fluorescein angiography and fluid OCT were detected during central review of images. Fluid on OCT could be intraretinal (cystic edema; thickening alone was not considered evidence of fluid), subretinal, or below the retinal pigment epithelium. Neovascularization, fluid, or hemorrhage needed to be under the fovea. For the CNV to be considered secondary to AMD, at least 1 drusen greater than 63 µm needed to be present in the study eye or fellow eye, or the fellow eye needed to have CNV or geographic atrophy. Data analysis for this study was initiated in November 2013. Both the clinical trial and the substudy were approved by an institutional review board associated with each center. Participating patients provided written informed consent for the clinical trial and the substudy.
During the initial visit, patients provided a medical history and were examined by a study-certified ophthalmologist. Patients underwent bilateral color stereoscopic fundus photography and fluorescein angiography that included stereoimages of the macula of the fellow eye at 2 and 10 minutes after dye injection. Study eyes were also imaged at the initial visit with OCT.
Graders at the CATT Fundus Photography Reading Center at the University of Pennsylvania, Philadelphia, and the CATT OCT Reading Center at Duke University, Durham, North Carolina, reviewed images taken at the time of enrollment into the clinical trial. Among the features assessed from the color photographs and fluorescein angiograms were the pattern of fluorescein dye leakage (predominantly classic, minimally classic, or occult only); presence of retinal angiomatous proliferation (RAP); area of the neovascular lesion; area of the total neovascular complex, including the neovascularization and contiguous serous pigment epithelium detachment, scar, hemorrhage, and blocked fluorescence; location of the neovascularization (subfoveal or not subfoveal); presence of hemorrhage associated with the lesion; presence of blocked fluorescence; and CNV in the contralateral eye. Graders at the OCT Reading Center noted the presence of intraretinal, subretinal, and subretinal pigment epithelium fluid, retinal pigment epithelium elevation, epiretinal membrane, and subretinal hyperreflective material. In addition, graders measured the thickness at the foveal center point of the retina, subretinal fluid, and subretinal tissue complex.
A subgroup of 835 patients from private and institutional practices of retina specialists provided blood samples from July 1, 2010, through September 31, 2011. Blood samples from patients were sent to the CATT Genetics Laboratory of the Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio, for DNA extraction.28 DNA was extracted and purified from leukocytes using the Gentra Systems PUREGENE DNA Purification Kit (Qiagen). For this investigation, 8 SNPs associated with the following genes and previously associated with risk of AMD were tested: CFH Y402H (rs1061170), ARMS2 (also called LOC387715) A69S (rs10490924), HTRA1 (rs11200638), C3 R80G (rs2230199), LIPC (rs10468017), CFB (rs4151667), C2 (rs547154), and C2 (rs9332739). These SNPs were a subset of the SNPs included on a custom-made TaqMan OpenArray loaded with TaqMan SNP genotyping assays (Applied Biosystems) that was used for genotyping.
The association between genotype and each phenotypical feature was assessed with linear regression (features represented as continuous variables) or logistic regression (features represented as binary variables) models. Because of nearly complete linkage disequilibrium between ARMS2 and HTRA1 and between CFB (rs4151667) and C2 (rs9332739) in most populations, including the patients in the CATT genetics substudy, only the results for ARMS2 and CFB (rs4151667) are provided. Genotype was summarized as the number of risk alleles present, where the risk allele is C for CFH and LIPC, T for ARMS2 and CFB, and G for C3 and C2. Patient age and smoking status were included in the models as covariates. Retinal thickness was analyzed as a categorical variable, in addition to as a continuous variable, because values lower than and higher than the range (120-212 µm) are associated with decreased VA due to retinal atrophy or edema.29 Analyses that included only patients homozygous for CFH and ARMS2 were conducted to compare results to a previous study.16 Statistical computations were performed with SAS statistical software, version 9.3 (SAS Institute Inc).
The approach to adjusting P values for multiple comparisons depended on whether the purpose of the analysis of the feature was to confirm the results of previous studies (age, VA, lesion size, lesion type, and RAP lesion for CFH or ARMS2) or to identify new associations. No adjustment for multiple comparisons were made for the confirmatory analyses, whereas a Bonferroni correction for the 6 SNPs under analysis led to considering only P < .008 (or .05 per 6 SNPs) as statistically significant for newly identified associations.
Age at presentation decreased with the number of risk alleles present for CFH, ARMS2, and C3 but not for the other SNPs (Table 1). A higher number of risk alleles was associated with larger total area of the neovascular lesion (P = .03) and with the presence of RAP lesions (P = .05) for ARMS2. No other associations were found among the 6 SNPs and the features listed in Table 1. In addition, no associations were found with subfoveal location of the neovascular lesion (P ≥ .40 for all), blocked fluorescence (P ≥ .49), hemorrhage (P ≥ .11), or CNV in the contralateral eye (P ≥ .89).
When the associations of OCT characteristics, other than thickness measurements, with the genotype of the 6 SNPS were examined, the only associations were with the presence of intraretinal fluid (Table 2). Eyes of patients with a higher number of risk alleles were more likely to have intraretinal fluid for ARMS2 (P = .008), whereas those with a lower number of risk alleles for C3 were more likely to have intraretinal fluid (P = .001). There were no other associations among the 6 SNPs and the features listed in Table 2. In addition, there were no associations with the presence of epiretinal membranes (P ≥ .26) or vitreomacular attachment (P ≥ .29).
The associations of thickness of the retina, subretinal fluid, and subretinal tissue complex and the total thickness (sum of thickness of retina, subretinal fluid, and subretinal tissue complex) with the genotype for the 6 SNPs are given in Table 3. Mean retinal thickness decreased with a higher number of risk alleles (P = .02) for C3. The mean total thickness decreased with a higher number of risk alleles for CFH (P = .01). No other associations were found among the 6 SNPs and the other thickness measurements.
The results of analyses, including only the 228 patients homozygous for risk alleles from CFH and ARMS2, may be compared to the study by Leveziel et al15 for the features that were defined similarly in their study and CATT (Table 4). Patients homozygous for the risk allele for both SNPs were a mean of 6 years younger at study entry than patients homozygous for the wild-type allele for both SNPs (P < .001). No associations among the 4 groups or between the groups homozygous for both SNPs were identified for baseline VA, lesion type, presence of RAP lesion, or bilateral CNV.
In CATT, the analysis of genotype for CFH, ARMS2, and C3 confirmed some previously identified associations with angiographic features of neovascular AMD, did not support some other associations, and yielded new associations with features of neovascular AMD on OCT. No associations with LIPC, CFB, or C2 were identified. Results from previous studies10,11,15,18,20 identified patients with a higher number of risk alleles for ARMS2 or the closely associated SNP HTRA1 as having a larger area of the total area of the neovascular lesion. In CATT, the mean total area of CNV was larger when risk alleles were present (P = .03), confirming the previous results. However, the magnitude of the effect on the lesion area was modest, with the mean area of 2.42 mm2 for patients with 2 ARMS2 risk alleles and 1.99 mm2 for patients with no risk alleles (Table 1).
The association of lesion type on fluorescein angiography with genotype for CFH, ARMS2, and/or HTRA1 has been examined in at least 9 previous studies with between 84 and 264 patients with neovascular AMD in the analyses.8-10,12-16,18CFH has been reported as not being associated with classic or occult CNV in 5 studies,10,13-15,18 associated with classic CNV in 2 studies,8,9 and associated with occult CNV in 1 study,12 although ARMS2 has been reported as being associated with occult CNV in 2 studies12,18 and classic CNV in 2 studies.8,16 In CATT with 835 patients, no association was detected with either SNP for classic or occult CNV.
Although the association of RAP lesions with CFH and ARMS2 has been examined in a number of studies, a low number (<40) of patients with RAP lesions among the patients with neovascular AMD has limited the statistical power in several of the analyses.12,14,16,17, In a larger study, Caramoy et al19 reported that among 108 patients with RAP and 258 patients with CNV without RAP, the proportion of patients with RAP decreased when the CFH risk alleles were present (43% for 0 risk alleles, 28% for 1 risk allele, and 27% for 2 risk alleles; P = .03). In addition, Wegschieder et al9 and Seitsonsen et al14 reported that among patients with CNV, the percentage with RAP lesions was less when risk alleles for CFH were present. The percentage of patients with RAP lesions in CATT also decreased with more CFH risk alleles present (13% for 0 risk alleles, 10% for 1 risk allele, and 6% for 2 risk alleles; P = .07) (Table 1), consistent with these 3 previous studies.9,14,19
Fewer genotype-phenotype studies have been conducted for ARMS2 and RAP lesions; 2 studies12,16 with fewer than 30 patients with RAP had no association with ARMS2. In a study reported by Hayashi et al17 that involved 36 patients with RAP and 408 patients with CNV but neither RAP nor polypoidal choroidal vasculopathy, RAP was present in 31 (14.5%) of 214 patients having the TT genotype, 3 (1.9%) of 158 having 1 risk allele, and 2 (2.9%) of 69 having no risk alleles (P < .0001). Consistent with this result, the proportion with RAP was similar (10%-12%) in patients with 1 or 2 risk alleles and lower (7.5%) in patients with no risk alleles (P = .05) (Table 1).
Although previous researchers have addressed the association of CFH, ARMS2, and C3 with features of neovascular AMD on fluorescein angiography, they have not addressed the association with features on OCT. Although intraretinal fluid was present in most eyes at baseline, the proportion of eyes with intraretinal fluid increased with the number of risk alleles for ARMS2 (P = .008) and decreased with the number of risk alleles for C3 (P = .001) (Table 2). The associations for intraretinal fluid were reflected in the retinal thickness measurements; however, the associations were not as strong for ARMS2 (P = .09) or C3 (P = .02) (Table 3). The mean total thickness of the retina, subretinal fluid, and subretinal tissue complex decreased with the number of risk alleles for CFH (P = .01). None of the other associations with the presence of subretinal fluid, subretinal pigment epithelium fluid, retinal pigment epithelium elevation, subretinal hyperreflective material, epiretinal membrane, vitreomacular attachment, or thickness of the subretinal fluid or subretinal tissue complex were statistically significant after application of the Bonferroni correction to account for analysis of the 6 SNPs.
There are limitations and advantages to the analyses of the CATT data. Only images at 1 time, the time of enrollment into CATT, are available for characterization of the dynamic process of neovascularization. Although prompt referral was the general practice during the recruitment phase of CATT, undoubtedly there was variation in time since the development of the lesion that would be expected to have an effect on lesion size, fluid, thickness of the retina, or angiographic pattern. However, there is no reason to believe that patients with a particular genotype were referred earlier or later than other patients to induce artificial associations with these features. The SNPs examined were limited to a subset of the SNPs that are associated with the prevalence of AMD. Given the wide variability in the AMD phenotype and in the types of neovascular lesions, it is reasonable to hypothesize that the SNPs that lead to initiation of AMD might influence the nature of neovascular lesions. Selecting specific SNPs for association studies because of such hypotheses does not require adopting the very high thresholds for strength of associations needed to protect genome-wide association studies from identifying false associations, although genome-wide association studies have the ability to identify SNPs that affect the nature of neovascular lesions via any number of pathways. Finally, the associations identified are modest; neovascular lesions did not segregate neatly by any of the genotypes examined.
The analyses of the CATT data for genotype-phenotype associations for features of neovascular AMD confirmed the association of larger lesions in patients with risk alleles for ARMS2 and fewer RAP lesions in patients with risk alleles for CFH. Previously reported associations of CFH and ARMS2 with classic and occult types of neovascularization on fluorescein angiography were not confirmed. Newly identified associations of CFH, ARMS2, and C3 with retinal fluid, retinal thickness, or total thickness require confirmation in other studies. Although baseline lesion size, RAP features, retinal fluid and thickness, and total thickness have prognostic importance, anti-VEGF treatments currently are used for nearly all patients with neovascular AMD, regardless of features on angiography and OCT. In addition, previous analyses of the CATT data and analyses of the data from the Alternative Treatments to Inhibit VEGF in Patients With Age-Related Choroidal Neovascularisation trial28,30 did not identify an association of CFH, ARMS2, or C3 with any of several measures of response to anti-VEGF treatment. Although genotype-phenotype association may aid in understanding the effects of SNPs in the pathogenesis of AMD, results from genetic testing currently do not affect patient care for neovascular AMD.
Submitted for Publication: October 26, 2015; final revision received February 23, 2016; accepted February 23, 2016.
Corresponding Author: Maureen G. Maguire, PhD, Department of Ophthalmology, University of Pennsylvania, 3535 Market St, Ste 700, Philadelphia, PA 19104-3309 (firstname.lastname@example.org).
Published Online: April 21, 2016. doi:10.1001/jamaophthalmol.2016.0669.
Author Contributions: Drs Maguire and Ying had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.
Study concept and design: Maguire, Martin.
Acquisition, analysis, or interpretation of data: Maguire, Jaffe, Toth, Daniel, Grunwald, Martin, Hagstrom.
Drafting of the manuscript: Maguire, Grunwald.
Critical revision of the manuscript for important intellectual content: Ying, Jaffe, Toth, Daniel, Martin, Hagstrom.
Statistical analysis: Maguire, Ying.
Obtained funding: Maguire, Ying, Jaffe, Martin.
Administrative, technical, or material support: Maguire, Daniel, Hagstrom
Study supervision: Maguire, Ying, Jaffe, Grunwald, Martin.
Conflict of Interest Disclosures: All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Dr Toth reported receiving research grants from Bioptigen. Dr Jaffe reported receiving personal fees from Heidelberg Engineering. No other disclosures were reported.
Funding/Support: The Comparison of Age-related Macular Degeneration Treatment Trials is supported by grants U10 EY017823, U10 EY017825, U10 EY017826, and U10 EY017828 from the National Eye Institute of the National Institutes of Health, US Department of Health and Human Services.
Role of the Funder/Sponsor: The funding source had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and the decision to submit the manuscript for publication.
Previous Presentation: This study was presented in part at the Association for Research in Vision and Ophthalmology Meeting; May 4, 2014; Ft Lauderdale, Florida.
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