Associations between Serum Vitamin D and Genetic Variants inVitamin D Pathways and Age-Related Macular Degeneration in theEuropean Eye StudyMcKay, G. J., Young, I. S., McGinty, A., Bentham, G. C. G., Chakravarthy, U., Rahu, M., ... Fletcher, A. E.(2017). Associations between Serum Vitamin D and Genetic Variants in Vitamin D Pathways and Age-RelatedMacular Degeneration in the European Eye Study. Ophthalmology, 124(1), 90-96.https://doi.org/10.1016/j.ophtha.2016.09.007

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1

Associations between serum vitamin D and genetic variants in vitamin D pathways and age-1

related macular degeneration in the EUREYE study 2 3 Gareth J. McKay

1 PhD 4

Ian S. Young1 MD 5

Ann McGinty1

PhD 6 Graham C.G. Bentham

2 MA 7

Usha Chakravarthy3 MD

8

Mati Rahu4

PhD 9

Johan Seland5 MD 10

Gisele Soubrane6

MD 11 Laura Tomazzoli

7 MD 12

Fotis Topouzis8 MD 13

Jesus Vioque9MD 14

Paulus TVM de Jong10

MD, 15

Astrid E. Fletcher11

PhD 16 17 1

Centre for Public Health, Queen's University Belfast, UK 18 2

School of Environmental Sciences, University of East Anglia, UK 19 3

Centre for Experimental Medicine, Institute of Clinical Science, Queen's University Belfast, 20

UK 21 4 Department of Epidemiology and Biostatistics, National Institute for Health Development, 22

Tallinn, Estonia 23 5Eye Department, University of Bergen, Bergen, Norway 24

6 Department of Ophthalmology, Hotel Dieu de Paris, University Paris Descartes-1, France 25

7 Clinica Oculistica, Università degli Studi di Verona, Italy 26

8 Department of Ophthalmology, Aristotle University of Thessaloniki School of Medicine, 27

Greece 28 9 CIBER de Epidemiologia y Salud Publica, Universidad Miguel Hernandez, Alicante, Spain 29

10The Netherlands Institute of Neuroscience, AMC, Amsterdam, The Netherlands 30

11 Faculty of Epidemiology & Population Health, London School of Hygiene and Tropical 31

Medicine, UK

32 33

Corresponding author 34 Professor Astrid Fletcher 35 Faculty of Epidemiology & Population Health 36 London School of Hygiene and Tropical Medicine 37

United Kingdom 38 Email: astrid.fletcher@lshtm.ac.uk 39 Telephone: +44 (0) 207 927 2253 40

Fax: +44 (0) 207 580 6897 41 42 Fletcher, McGinty, Topouzis, Young report disclosures. 43 This article contains additional online-only material. The following should appear online-only: 44 Table 3,Table 4, Table 5,Table 6. 45 46

Revised manuscript - without track changes

2

Financial support: 47

EUREYE was supported by the European Commission Vth Framework (QLK6-CT-1999-48 02094) with additional funding for cameras provided by the Macular Disease Society UK, for 49 DNA extraction and genotyping (MRC Biomarkers Award G0601354), for dietary data by 50

Thomas Pocklington Trust and for vitamin D analyses by Guide Dogs for the Blind (OR2011-51 05d). M. Rahu is salaried by his home institute (grant IUT5-1 from the Estonian Research 52 Council).Additional funding in Alicante was received from the Fondo de Investigacion Sanitaria, 53 Madrid, Spain (grant nos. FIS 01/1692E, RCESP C 03/09), and Oficina de Ciencia y Tecnologia 54 Generalitat Valenciana, Valencia, Spain (grant no. CTGCA/2002/06). 55

56 The funding organizations had no role in the design or conduct of this research 57 58 Running head: Serum vitamin D, variants in vitamin D genes and age-related macular 59

degeneration 60

3

Abstract 61

Objective: To study associations between early and late age-related macular degeneration 62

(AMD) and the neovascular type of late AMD (nvAMD) with serum 25-hydroxy vitamin D 63

(25(OH)D) and genetic variants in vitamin D pathway genes. 64

Design: Population-based cross-sectional study in a random sample aged 65 and over from seven 65

European countries. 66

Participants: Of 4753 participants, 4496 (2028 men and 2468 women) ,mean age 73, provided a 67

usable blood sample; 2137 had no signs of AMD, 2209 had early AMD, 150 had late AMD of 68

which 104 nvAMD. 69

Methods: Participants were interviewed for smoking and alcohol use, brief medical history, 70

sunlight exposure and a dietary questionnaire, underwent color fundus photography and provided 71

a blood sample. Fundus images were graded using the modified International Classification 72

System for Age Related Maculopathy. 25(OH)D was measured by liquid chromatography-73

tandem mass spectrometry and categorized as deficient (< 30 nmol/L), insufficient (30–50 74

nmol/L), adequate (≥50 nmol/L). Genotyping was performed on a sub-sample of 1284 AMD 75

cases and controls for 93 SNPs from seven genes. Associations were investigated by linear or 76

logistic regression adjusted for potential confounders. 77

Main Outcome Measures: Adjusted odds ratio (AOR) for three outcomes (early AMD, late 78

AMD, nvAMD). 79

Results: No linear association was found with 25(OH)D and early or late AMD or nvAMD. 80

There was no association between insufficient or deficient status with early or late AMD. 81

Deficient status was associated with nvAMD (AOR = 1.27, 95% Confidence Interval 1.1-1.45, p 82

<0.0001). Significant (p<0.05) associations with 25(OH)D were found for SNPs in genes GC, 83

4

VDR ,CYP2R1, CYP27B1. Using a initial threshold of p<0.05, two SNPs (VDR) were associated 84

with early AMD, four SNPs (RXRA) and one (VDR) with nvAMD and one (RXRA), two (VDR) 85

and one (CYP2R1) with late AMD. After Bonferroni correction, no SNPS were associated with 86

early, late or nvAMD. 87

Conclusions: 25(OH)D deficient status was associated with nvAMD but the adjusted OR was 88

small and we cannot exclude residual confounding. The hypothesis of a causal association of 89

vitamin D with AMD is not supported by clear evidence for an association of vitamin D pathway 90

SNPs with early, late or nvAMD. 91

5

Vitamin D is produced in the skin following exposure to ultraviolet B (UVB) radiation as 92

vitamin D3 (cholecaliferol), and to a lesser extent, obtained from the diet (principally oily fish).1 93

The other natural source of vitamin D, Vitamin D2 (ergocalciferol) is found in a few foods and 94

provides a much smaller proportion of dietary vitamin D. Other sources of vitamin D include 95

supplements or, in some countries, fortified foods. In the body the biologically active metabolite 96

of vitamin D3, 1,25-dihydroxyvitamin D (1,25(OH)2D3) is distributed to the tissues and plays an 97

important role in a number of biological functions, including calcium homeostasis, immune 98

response and insulin metabolism.2 Serum 25-hydroxyvitamin D (25(OH)D) measures the total 99

circulating vitamin D (D2 and D3) and is used as a measure of vitamin D status. In countries of 100

the European Union where supplement use is relatively low and foods are not fortified, 25(OH)D 101

deficiency has been found in 13% of the overall population.3 In addition to environmental 102

factors, genetic variants influence 25(OH)D concentrations and single nucleotide polymorphisms 103

(SNPs) in a number of genes related to uptake and metabolism have been identified.4, 5

104

Recommendations for desirable levels of 25(OH)D of at least 50 nmol/L6 go beyond avoiding 105

deficiency to maximizing bone health. Vitamin D has also been postulated to influence disease 106

risk across a wide range of other conditions suggesting the potential benefit from increasing 107

vitamin D by diet or supplementation. However, a clear picture has not emerged. A large body 108

of evidence summarized in recent systematic reviews and meta analyses, based on nearly 300 109

observational studies and 200 randomized controlled trials (RCTs), showed reduced risks of all-110

cause mortality, cardiovascular disease (CVD) incidence and mortality in the observational 111

studies but not in the RCTs except for all-cause mortality.7, 8

112

113

6

Age related macular degeneration (AMD) is characterized by progressive degenerative changes 114

in the retina. Late stage AMD consists of neovascular AMD (nvAMD) and geographic atrophy 115

(GA). Hypotheses for an association of vitamin D with AMD are based on the anti-116

inflammatory role of vitamin D, identification of vitamin D receptors in retinal tissues,9, 10

and 117

the major role of inflammation and of complement-related genes and complement activation in 118

the development of AMD.11

CVD and risk factors for CVD such as smoking and diabetes are 119

additionally risk factors for nvAMD.12

There are inconsistent findings from studies investigating 120

the association of 25(OH)D with AMD.9, 13-17

Most studies lacked power to investigate late AMD 121

13-16 or reported an association with early

16 or late

17 AMD only in sub-groups (by gender or age). 122

Early AMD was associated with lower concentrations of 25(OH)D in some13,15

but not all 123

studies.14

Other studies investigating vitamin D and late AMD were based on diagnoses from 124

medical records 18-20

or from a case series.21

Two studies reported inconclusive findings for 125

SNPs in genes influencing 25(OH)D concentrations with early15

or late AMD9. In the absence of 126

clear evidence on this important question, we investigated associations between 25(OH)D and 127

SNPs in seven vitamin D related genes with AMD in the European Eye Study (EUREYE). 128

129

Methods 130

Participants were recruited by random sampling of the population aged 65 years and older in 131

seven centers across Europe: Bergen (Norway), Tallinn (Estonia), Belfast (UK), Paris (France), 132

Verona (Italy), Thessaloniki (Greece) and Alicante, (Spain). Participants were interviewed by 133

trained fieldworkers, underwent fundus photography, and provided blood samples. Information 134

collected at interview included education, smoking and alcohol use, a brief medical history, a 135

semi-quantitative food frequency questionnaire, and a detailed questionnaire to estimate 136

7

ultraviolet radiation exposure (UVR) (see below). Written informed consent was obtained from 137

all study participants. Institutional Review Board (IRB)/Ethics Committee approval was 138

obtained for each center. The study adhered to the tenets of the Declaration of Helsinki. 139

Fundus photography and grading 140

After pupillary dilation with tropicamide 0.5% and phenylephrine 5%, two 35° non-simultaneous 141

stereoscopic digitized color fundus images centered on the fovea were obtained of each eye. The 142

fundus images were sent to a single reading center (Erasmus University Rotterdam) and graded 143

using the International Classification System for Age-Related Maculopathy22

and then 144

categorized into five mutually exclusive grades. Grade 0 was defined as a macula free of drusen 145

or pigmentary irregularities or with hard drusen (< 63 µm) only. Early AMD was subdivided in 146

grade 1, defined as soft distinct drusen (≥ 63 µm) or pigmentary abnormalities, grade 2 as soft 147

indistinct drusen (≥ 125 µm) or reticular drusen only or soft distinct drusen (≥ 63 µm) with 148

pigmentary abnormalities, and grade 3 as soft indistinct drusen (≥ 125 µm) or reticular drusen 149

with pigmentary abnormalities. Grade 4 was defined as presence of either nvAMD (presence of 150

any of the following: serous or hemorrhagic retinal or retinal pigment epithelial detachment, 151

subretinal neovascular membrane, periretinal fibrous scar) or GA (well-demarcated area of 152

retinal pigment atrophy with visible choroidal vessels). 153

Measurement of UV exposure 154

Full details of UV measurement have been published previously23

. We used a detailed 155

questionnaire to ascertain lifetime residence and time spent outdoors (from age 14), for work 156

(including homecare), leisure and retirement up to current age. Information from the 157

questionnaire, residence calendar and geographical co-ordinates for residence were sent to the 158

University of East Anglia to estimate years of exposure for different wavelengths of UVR (UVA, 159

8

UVB and blue light) using published sources that take into account time of day, month, and 160

latitudinal variations. Personal adult lifetime UV exposure were estimated for each of the three 161

wavelengths of light and summed for a mean annual lifetime dose for all day exposures and for 162

exposures in the middle of the day. 163

Blood analyses 164

Non-fasting venous blood samples were separated within 4 hours of collection and serum was 165

stored at -20○C for up to 4 weeks before frozen samples were transferred to a single laboratory 166

(Queens University Belfast) for storage at -80○C. Antioxidant and cholesterol analyses were 167

carried out within 3 months of storage. Plasma lutein, zeaxanthin, beta cryptoxanthin, alpha and 168

beta-carotene, alpha and gamma tocopherol, lycopene and retinol were measured by reverse 169

phase high pressure liquid chromatography. Plasma vitamin C was measured using an enzyme-170

based assay in plasma stabilized with metaphosphoric acid. All assays were standardized against 171

appropriate National Institute of Standards and Technology (NIST) standard reference materials. 172

Serum cholesterol was measured using an enzymatic assay (Randox, Crumlin, UK) on a Cobas 173

FARA centrifugal analyzer (Roche Diagnostics, UK). Serum 25-hydroxy vitamin D2 and D3 174

(25(OH)D2 and 25(OH)D3) were measured by liquid chromatography-tandem mass spectrometry 175

(LC-MS/MS) 8 to 10 years after initial collection. 176

SNP selection and Genotyping 177

For reason of costs, genotyping was undertaken in a sub-sample of the main study. A random 178

sample of controls (AMD grade 0) was frequency matched on age (within a year), sex and center 179

with all cases of (i) late AMD, and (ii) all cases with early AMD grades 2 and 3. Cases with 180

AMD grade 1 were not investigated. Ninety-three common SNPs located across 7 genes 181

involved in the vitamin D metabolic pathway (GC (10), RXRA (14), CYP2R1 (7), DHCR7 (5), 182

9

VDR (29), CYP27B1 (7), CYP24A1 (21) were selected from Phase III, release 2 HapMap 183

(http://www.hapmap.org) CEPH data (Utah residents with ancestry in northern and western 184

Europe; CEU) using Haploview (http://www.broadinstitute.org/haploview) to determine linkage 185

disequilibrium. Tag SNPs were selected using multimarker tagging where r2>0.8 for all 186

downloaded SNPs with a minor allele frequency (MAF) ≥5%, genotype call rate ≥95%, and no 187

significant deviation from Hardy Weinberg equilibrium (HWE). Genotyping was performed by 188

KBiosciences (Hoddesdon, United Kingdom). Quality filters for exclusion of SNPs included call 189

rates below 95% and deviation from HWE (P<0.001). DNA samples were excluded if missing 190

genotypes exceeded 10%. Other quality control measures included duplicates on plates, random 191

sample allocation to plates, independent scoring of problematic genotypes by two individuals 192

and re-sequencing of selected DNAs to validate genotypes. KBiosciences quality control also 193

included validation of all SNP assays on a panel of 44 random Caucasian-derived samples plus 4 194

non-template (negative) controls. 195

Data processing and statistical analysis 196

Statistical analysis was carried out using Stata software version 13 (Stata Corp., College Station, 197

TX) and PLINK (version 1.07) under an additive genotypic model.24

We investigated total 198

25(OH)D i.e. 25(OH)D2 plus 25(OH)D3 as (i) a continuous variable and (ii) categorized by 199

clinical status based on expert guidelines as: deficient (< 30 nmol/L), insufficient (30–50 200

nmol/L), and adequate (≥50 nmol/L).6 We excluded extreme values of ≥150 nmol/L (n=17). 201

25(OH)D was normalized using a square root transformation due to a long right hand tail (110 202

participants had values ≥100 and <150 nmol/L). Dietary vitamin D was estimated using food 203

composition tables25

and adjusted for total energy intake.26

All day UVB exposures were square 204

root transformed and lutein concentrations log transformed. We ran preliminary regression 205

10

analyses to identify factors associated with 25(OH)D as possible confounders of any association 206

with AMD and carried out a similar analysis for vitamin D status. We used univariable and 207

confounder-adjusted logistic regression to investigate associations between 25(OH)D or vitamin 208

D status and early grades of AMD and nvAMD. We also present results for all late AMD 209

(nvAMD and GA combined) as numbers with GA were too small (n=46) to permit meaningful 210

separate analyses. Analyses took account of the study design by including study centre in 211

models and by use of robust errors in Stata.27

We report associations of individual SNPs with 212

early, late and nvAMD at a nominal significance of p<0.05. Since our a priori hypothesis of an 213

association between SNPs in vitamin D-related genes and AMD was based on previously 214

demonstrated associations of vitamin D genes with 25(OH)D,4 5 we applied a Bonferroni 215

correction based on the number of SNPs within each gene in the AMD analyses in preference to 216

a GWAS type correction for all 92 snps. We also investigated whether any association of 217

25(OH)D status and nvAMD or early AMD was modified by the main AMD genetic risk loci, 218

rs1061170 in CFH and rs10490924 in ARMS2,as has been reported previously.15

219

220

Results 221

Serum 25(OH)D 222

Of 4753 participants at the clinical examination, 4496 provided a usable blood sample for the 223

vitamin D assays. Of these 2137 had no signs of AMD, 2209 had early AMD; 1635 (grade 1), 224

460 (grade 2), 114 (grade 3), 46 (GA) and 104 nvAMD. The mean (standard deviation, SD) of 225

25(OH)D concentrations was 49 (23) nmol/L. The highest mean seasonally-adjusted 226

concentration was observed in the Bergen center (63 (20) nmol/L) compared to all other centers 227

with adjusted differences ranging from 20 nmol/L lower concentrations in Paris, approximately 228

11

15 nmol/L lower in Tallinn, Belfast, Verona and Thessaloniki, to 9 nmol/L in Alicante (all 229

comparisons p <0.0001). Vitamin D deficiency was found in 21% (n=944) and 32.8% (1475) 230

were classified as insufficient. Only 13.4% (604) had concentrations of 75 nmol/L or greater. A 231

large number of variables were independently associated with 25(OH)D. In multivariable 232

analyses adjusted for center and season, levels were lower with increasing age, in women, in 233

current smokers, participants with diabetes, and higher in those taking fish oil supplements, and 234

drinking alcohol at least weekly. 25(OH)D was associated with higher UVB exposure, dietary 235

vitamin D intake, serum cholesterol, and plasma concentrations of ascorbate, lutein (or 236

zeaxanthin), and retinol (all associations, p<0.05 or less). There was no association with 237

education. Lutein and zeaxanthin were highly correlated (r=0.85) and therefore associations with 238

25(OH)D were almost identical. The characteristics of people categorized by 25(OH)D status 239

are shown in Table 1 (univariable analyses). Proportionately fewer people classified as 240

insufficient or deficient took fish oil supplements or consumed oily fish at least weekly, were 241

less likely to be in the highest quartile of dietary vitamin D intake, or dietary docosahexaenoic 242

acid intake, and were more likely to be older or obese compared to those with sufficient levels. 243

Associations with AMD 244

Covariate-adjusted models showed no association of 25(OH)D with any grade of early AMD or 245

late or nvAMD (Table 2). 25(OH)D status was not associated with any grade of early or late 246

AMD (Table 2). For nvAMD, deficient status was associated with a covariate adjusted OR of 247

1.27, 95% confidence interval (1.11-1.45) p<0.0001. Although numbers with GA were small 248

there was no indication of increased odds for deficiency status (OR=0.82, 0.36- 1.89). We found 249

no significant interactions with rs1061170 or rs10490924 with 25(OH)D status and nvAMD 250

(p=0.32 and 0.30 respectively) or early AMD (p=0.28 and 0.11). 251

12

Genetic associations 252

Genotype data were available for 93 SNPs from 1284 participants selected for genetic analysis. 253

One SNP in GC was excluded for failing to meet the quality filters of call rates below 95% or 254

deviation from HWE (P<0.001). The average call rate for the remaining SNPs was 99.3%. No 255

participants were excluded due to insufficient genotype data. No duplicate inconsistencies were 256

observed. Results for all 92 SNPs and their age, sex and center adjusted association with 257

25(OH)D (additionally adjusted for season), early,late, nvAMD are provided in Tables 3-6 258

(available at http://www.aaojournal.org). Nominal significant results are summarized in 259

Table7. Our results confirmed that SNPs in GC (3), RXRA (1), CYP2R1 (2), VDR (1) and 260

CYP27B1 (1) were associated with 25(OH)D. Two SNPs both in VDR (rs11574026, rs4516035) 261

were significantly associated with early AMD. Five SNPs were significantly associated with 262

nvAMD: four in RXRA (rs11185644, rs12339187, rs10881582, rs3118536,) and one in VDR 263

(rs11574077). Analysis of late AMD identified four significantly associated SNPs following the 264

inclusion of participants with GA. These were rs3118536 in RXRA, rs10875693 and rs11574077 265

in VDR, and rs11023371 in CYP2R1. After applying Bonferroni corrections of p=0.004 for 14 266

RXRA SNPs and p=0.002 for 29 VDR SNPS, no SNPs were associated with early AMD, nvAMD 267

or late AMD. 268

269

Discussion 270

We found no linear association between 25(OH)D and early or nvAMD. 25(OH)D deficiency 271

status was associated with nvAMD, adjusted OR of 1.27. Insufficient 25(OH)D status was not 272

associated with nvAMD. 273

13

Few studies have investigated associations between 25(OH)D and late AMD with inconsistent 274

results. Several studies have used historical medical records and linked information on vitamin 275

D to a diagnosis of late AMD. No association was found with nvAMD in a Medicare study of 276

7000 people with a diagnosis of Vitamin D deficiency and matched controls followed for 3 277

years.18

Retrospective medical records identification of 146 nvAMD patients tested for vitamin 278

D compared with 100 age, sex and race matched controls with pseudophakia, reported an 279

adjusted OR of 0.37 (CI 0.19- 0.72) between the highest and lowest quintile of 25(OH)D.20

A 280

health records study in Israel of 9267 participants with a routine 25(OH)D measurement 281

identified 1045 individuals with a diagnosis of AMD (grade and severity not available) and 282

found no differences in 25(OH)D compared to non-AMD participants; results were not adjusted 283

for age although AMD cases were older than non-AMD.19

Using records of a mobile geriatric 284

clinic of 26 late AMD cases and 34 with no AMD signs, 25(OH)D deficiency was associated 285

with a 3 fold OR, 95% CI, 1-9, results were adjusted only for age, sex and season of blood 286

collection.28

287

An inverse association was found between early AMD and 25(OH)D in the US National Health 288

and Nutrition Examination survey (NHANES III)13

and in women aged < 75 years in the 289

Carotenoids in Age-Related Eye Disease Study (CAREDS study) but not in those aged 75 and 290

over.16

Neither study found an association with 25(OH) D and late AMD but the number of 291

cases evaluated was small. In the Korean National Health and Nutrition study there was no 292

association with early or late AMD overall; separate analyses by gender found reduced odds of 293

late AMD with increasing 25(OH)D in men but not in women.17

No association of 25(OH)D 294

concentrations categorized as deficient (<25 nmol/L) or insufficient (25–49 nmol/L) compared to 295

14

sufficient (≥50 nmol/L) with either early or late AMD, were observed in a small population 296

based study of elderly residents in France.14

297

In our study, 25(OH)D was measured in stored blood samples collected at the same time as the 298

ascertainment of AMD. We cannot therefore exclude the possibility of reverse causation i.e. that 299

nvAMD, directly or indirectly, influenced the 25(OH)D levels, for example as a result of 300

complement-related or other inflammatory effects.11

We had no information on co-morbidities 301

which may be either consequences of AMD such as depression and anxiety,29

low physical 302

activity30

or other age-related conditions associated with AMD such as cognitive impairment31, 32

303

or complement-related conditions such as diabetic nephropathy,33, 34

some of which have been 304

associated with 25(OH)D.7, 8

The conclusion of one substantial review of vitamin D and a wide 305

range of health outcomes highlighted a lack of highly convincing evidence for a clear role of 306

vitamin D for many health related outcomes in both randomized and observational studies.8 307

Furthermore, Autier and colleagues suggest that low 25(OH)D is a marker of ill health and that 308

inflammatory processes involved in disease manifestation reduce 25(OH)D, explaining why low 309

vitamin D status has been reported for a wide range of disorders7. Restoration of vitamin D 310

deficits due to ageing and lifestyle changes induced by ill health could explain why low-dose 311

supplementation may lead to slight gains in survival in the elderly. 312

Genetic variants in vitamin D pathway members are not subject to concerns of temporality and 313

confounding and therefore provide stronger evidence on the possible association of vitamin D 314

with AMD. SNPs in the CYP24A1 and VDR gene were identified in the discovery phase of a 315

family nvAMD study but in the replication component, only variants in CYP24A1 were found to 316

be associated.9 In the CAREDS study, SNPs in CYP2R1 and VDR but not CYP24A1 were 317

associated with early AMD.15

In our study, we found no significant associations between 318

15

variants in CYP2R1 and early AMD but a nominal association with two VDR SNPs. A VDR 319

SNP (rs11574077) was significantly associated with nvAMD in our study but this SNP was not 320

associated with nvAMD in a family study.9 We found the strongest evidence of nominal 321

association for SNPs located within the RXRA gene region (rs12339187, rs3118536, rs11185644, 322

rs10881582), although these did not withstand correction for multiple testing. A limitation of 323

our study was the small number of cases of early and nvAMD for investigation of genetic 324

associations. 325

Genetic determinants of vitamin D bioactivity have been identified previously in the genes 326

encoding VDR and the retinoic acid-X-receptor (RXRA), which forms a complex with 1,25 OH2 327

D bound to VDR and is recognized by vitamin D response elements on target genes.35

We 328

directly genotyped rs1570669 and rs2274130 in CYP24A1, together with rs927651 as a proxy for 329

rs1570670, rs2296239 and rs4809957, all SNPs in high linkage disequilibrium. We found no 330

evidence to support their previously reported association with AMD.13

In addition, previous 331

reports of VDR SNPs associated with AMD offer little support for our study findings and vice 332

versa,19

although lack of support may result from discrepancies in recruitment and composition, 333

study size and characterization of the AMD cases within each study. In contrast to a previous 334

finding we found no evidence that major AMD genetic risk variants (rs1061170 and rs10490924) 335

modified the association between 25(OH)D deficiency and early AMD and nvAMD.19

We did 336

not find a relationship with RXRA SNPs and 25(OH)D; however, a previous association with one 337

RXRA SNP (rs9409929) has been reported only with the metabolite 1,25 OH2 D but not the 338

circulating serum level.36

It is possible that the nominal association we report between RXRA 339

variants and nvAMD may not relate to vitamin D. RXRA receptors are involved in a large 340

number of pathways other than those related to vitamin D including, of relevance to AMD, 341

16

glucose and lipid metabolism37

and the omega 3 fatty acid, docosahexaenoic acid.38

Previously 342

reported genome wide association data evaluated several SNPs across the RXRA gene in 2157 343

mixed early and late AMD cases and 1150 controls (http://www.ncbi.nlm.nih.gov/gap) including 344

rs10881582, p=0.15, but no evidence of association was found.39

345

We conclude therefore that the hypothesis of a causal association of vitamin D with AMD is not 346

supported by clear evidence for an association of vitamin D pathway SNPs with early, late or 347

nvAMD. Moreover concerns about residual confounding and reverse causation make it unlikely 348

that there is an association between vitamin D deficiency and AMD. 349

350

17

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445

Table 1. Characteristics of participants by vitamin D status

1 Mean (SD)

2 % (n)

3 Median, IQR (interquartile range)

4 Energy adjusted

5 p trend based on log lutein

Serum 25(OH)D status

nmol/L)

Sufficient

>50 (nmol/L)

Insufficient

≥30<50 (nmol/L)

Deficient

<30 (nmol/L)

N 2077 1475 944 p trend

Serum 25(OH)D (nmol/L) 1

70.2 (16.0) 39.9 (5.7) 21.2 (5.9)

Age1 72.6 (5.4) 73.3 (5.7) 74.5 (6.0) <0.0001

Women 2

47.9 (1055) 53.7 (823) 61.7 (590) 0.11

Education (lowest tertile of years)2

37.4 (769) 36.5 (561) 39.5 (380) 0.42

Ever smoker 2

49.2 (1020) 44.8 (681) 43.3 (414) 0.13

Alcohol at least weekly2

52.2(903) 44.4 (555) 44.0 (374) 0.18

Annual UVB exposure (MED)3,5

237 (123-393) 229 (112-412) 202 (69-380) 0.17

Obese2 (BMI ≥30) 32.1 (645) 40.4 (567) 40.7 (352) 0.05

Diabetes2

12.9 (218) 17.9 (219) 16.7 (147) 0.02

CVD2 12.5 (268) 13.6 (215) 14.3 (142) 0.29

Fish oil supplement use2

8.2 (381) 3.9 (109) 1.5 (22) 0.22

Blood measures µmol/L

Cholesterol1 5.8 (1.1) 5.6 (1.1) 5.6 (1.0) 0.12

Vitamin C1, 48.4 (24.8) 42.2 (25.4) 40.6 (25.6) 0.02

Lutein3,5

1.26 (0.47-2.79) 0.99 (0.46-2.42) 0.90 (0.46-2.42) 0.05

Alpha- tocopherol1 30.6 (7.0) 30.5 (7.0) 29.9 (6.7) 0.43

Retinol1 2.23 (0.77) 2.19 (0.79) 2.18 (0.74) 0.36

Dietary variables

≥ 1 Oily fish serving per week2

45.8 (892) 36.7 (522) 27.5 (254) 0.02

Vitamin D4 (highest quartile) 30.9 (593) 24.7 (355) 18.2 (153) 0.01

DHA4 (highest quartile) 33.7 (617) 25.9 (350) 17.9 (150) 0.01

Table 1

Table 2. Association of early, late and neovascular AMD with (i) 25(OH)D (nmol/L) distribution (ii) Vitamin D status

a Square root transformed serum 25(OH)D nmol/L

bOdds ratio (OR) and 95% Confidence Interval (95% CI)

cAdjusted for age, sex, center, season, smoking, CVD, diabetes, alcohol consumption, BMI, plasma cholesterol, alpha-tocopherol,

retinol ,lutein, ascorbate.

d Odds ratio (OR) per unit increase of square root transformed serum 25(OH)D

Serum 25(OH)D

(nmol/L)a

Sufficient

>50 nmol/L

Insufficient

≥30 -<50 nmol/L Deficient

<30 nmol/L

OR (95% CI)b,c,d

p Reference OR (95% CI)b,c

p OR (95% CI)b,c

p

Early AMD

Grade 1 0.98 (0.94-1.01) 0.24 1 0.98 (0.88-1.10) 0.78 0.97 (0.79-1.18) 0.73

Grade 2 1.04 (0.93-1.17) 0.36 1 0.94 (0.71-1.23) 0.65 0.88 (0.54-1.45) 0.62

Grade 3 1.03 (0.89-1.20) 0.65 1 0.65 (0.38-1.10) 0.11 1.17 (0.84-1.64) 0.36

Late AMD 1.02 (0.94-1,11) 0.59 1 0.76 (0.48-1.20) 0.19 1.21 (0.88-1.68) 0.19

Neovascular AMD 1.02 (0.95 -1.08) 0.64 1 0.92 (0.57-1.48) 0.73 1.27 (1.11-1.45) <0.0001

Table 2

Table 7. Genetic association of SNPs with P<0.05 for (a) serum 25(OH)D (b) early AMD,

(c) late AMD and (d) neovascular AMD

(a) Vitamin D CHR SNP Gene BP BETA (SE)a P

4 rs2298850 GC 72833131 -0.414 (0.116) <0.001

4 rs1155563 GC 72862352 -0.276 (0.116) 0.018

4 rs11939173 GC 72891022 -0.242 (0.106) 0.023

9 rs11185644 RXRA 136350684 -0.277 (0.133) 0.038

11 rs10832306 CYP2R1 14813670 -0.342 (0.169) 0.043

11 rs7935792 CYP2R1 14866037 -0.556 (0.206) 0.007

12 rs2239186 VDR 46555677 -0.390 (0.125) 0.002

12 rs4760169 CYP27B1 56405114 -0.483 (0.167) 0.004

CHR SNP Gene BP OR (95% CI)b P

(b) Early AMD 12 rs11574026 VDR 46574513 0.74 (0.56-0.98) 0.03

12 rs4516035 VDR 46586093 1.21 (1.02-1.44) 0.03

(c) Late AMD 9 rs3118536 RXRA 136448283 0.60 (0.41-0.90) 0.01

12 rs10875693 VDR 46555917 1.44 (1.08-1.93) 0.01

12 rs11574077 VDR 46539194 0.32 (0.12-0.84) 0.02

11 rs11023371 CYP2R1 14852847 1.67 (1.01-2.74) 0.04

(d) nvAMD 9 rs11185644 RXRA 136350684 0.53 (0.32-0.87) 0.01

9 rs12339187 RXRA 136369148 0.54 (0.33-0.88) 0.01

9 rs10881582 RXRA 136395899 0.64 (0.43-0.97) 0.04

9 rs3118536 RXRA 136448283 0.54 (0.33-0.87) 0.01

12 rs11574077 VDR 46539194 0.18 (0.04-0.76) 0.02

a Regression coefficient (Beta) of square root transformed 25(OH)D and Standard error (SE)

adjusted for age, sex ,center and season.

b Odds Ratio (OR) and 95% Confidence Interval (CI) for early, late and nvAMD adjusted for

age, sex, center

Abbreviations: AMD, age related macular degeneration; nvAMD, neovascular age related

macular degeneration; CHR, chromosome; SNP, single nucleotide polymorphism; BP, base pair;

Table 7

Table 3 (online supplement). Genetic association of SNPs with vitamin D concentrations adjusted for age, sex, center and season in participants with no AMD

CHR SNP Gene BP BETA (SE) P

4 rs12512631 GC 72820195 0.118 (0.106) 0.268

4 rs705117 GC 72826979 0.248 (0.146) 0.090

4 rs2298850 GC 72833131 -0.414 (0.116) <0.001

4 rs222020 GC 72855136 0.053 (0.146) 0.716

4 rs1491718 GC 72860143 -0.118 (0.170) 0.487

4 rs1155563 GC 72862352 -0.276 (0.116) 0.018

4 rs16847050 GC 72886195 -0.009 (0.157) 0.956

4 rs1565572 GC 72889055 0.056 (0.132) 0.670

4 rs11939173 GC 72891022 -0.242 (0.106) 0.023

9 rs11185644 RXRA 136350684 -0.277 (0.133) 0.038

9 rs12339187 RXRA 136369148 -0.133 (0.131) 0.310

9 rs914853 RXRA 136378984 0.065 (0.112) 0.560

9 rs11185659 RXRA 136383204 0.049 (0.121) 0.688

9 rs10881582 RXRA 136395899 -0.149 (0.120) 0.214

9 rs7039190 RXRA 136406525 -0.246 (0.254) 0.334

9 rs11103473 RXRA 136427450 -0.193 (0.112) 0.084

9 rs11103482 RXRA 136427681 0.127 (0.170) 0.455

9 rs3118536 RXRA 136448283 -0.254 (0.130) 0.052

9 rs4240705 RXRA 136451221 -0.029 (0.109) 0.789

9 rs6537944 RXRA 136452649 0.337 (0.200) 0.092

9 rs3118571 RXRA 136457999 -0.026 (0.107) 0.805

9 rs4842196 RXRA 136473049 0.224 (0.121) 0.065

9 rs3118526 RXRA 136477178 0.274 (0.149) 0.065

11 rs7117967 CYP2R1 14782654 -0.185 (0.195) 0.343

11 rs12419657 CYP2R1 14783385 0.324 (0.166) 0.051

11 rs1037379 CYP2R1 14809360 -0.072 (0.110) 0.510

11 rs10832306 CYP2R1 14813670 -0.342 (0.169) 0.043

11 rs1496167 CYP2R1 14833083 0.053 (0.112) 0.638

11 rs11023371 CYP2R1 14852847 0.142 (0.215) 0.510

11 rs7935792 CYP2R1 14866037 -0.556 (0.206) 0.007

11 rs1792284 DHCR7 70812614 -0.140 (0.119) 0.240

11 rs4316537 DHCR7 70832777 -0.277 (0.211) 0.191

11 rs12785878 DHCR7 70845097 -0.178 (0.114) 0.118

11 rs949178 DHCR7 70877968 0.046 (0.188) 0.808

11 rs7950649 DHCR7 70898979 0.026 (0.153) 0.864

12 rs11574143 VDR 46521184 0.233 (0.171) 0.172

12 rs731236 VDR 46525024 -0.137 (0.109) 0.209

12 rs7975232 VDR 46525104 0.015 (0.103) 0.886

12 rs1544410 VDR 46526102 -0.134 (0.108) 0.216

12 rs11168268 VDR 46538079 -0.064 (0.104) 0.535

Supplemental Table 3 - Online Only

12 rs11574077 VDR 46539194 -0.398 (0.242) 0.101

12 rs2239182 VDR 46541678 -0.076 (0.101) 0.450

12 rs2107301 VDR 46541837 0.124 (0.110) 0.260

12 rs2283342 VDR 46542126 -0.261 (0.136) 0.055

12 rs2239179 VDR 46544033 -0.163 (0.103) 0.115

12 rs12717991 VDR 46545393 0.032 (0.100) 0.747

12 rs886441 VDR 46549231 0.154 (0.130) 0.237

12 rs2189480 VDR 46550095 -0.152 (0.107) 0.156

12 rs3819545 VDR 46551273 0.089 (0.106) 0.399

12 rs2239186 VDR 46555677 -0.390 (0.125) 0.002

12 rs10875693 VDR 46555917 0.052 (0.113) 0.646

12 rs6580642 VDR 46556863 0.074 (0.131) 0.573

12 rs2254210 VDR 46559981 0.032 (0.104) 0.755

12 rs2238136 VDR 46563980 -0.059 (0.113) 0.605

12 rs2853564 VDR 46564754 0.006 (0.103) 0.955

12 rs4760648 VDR 46566932 -0.043 (0.101) 0.674

12 rs11574027 VDR 46573640 0.013 (0.391) 0.974

12 rs11574026 VDR 46574513 0.009 (0.164) 0.957

12 rs4760655 VDR 46580398 0.021 (0.108) 0.845

12 rs7136534 VDR 46580893 -0.075 (0.126) 0.549

12 rs11574024 VDR 46582488 0.161 (0.169) 0.342

12 rs4516035 VDR 46586093 -0.092 (0.105) 0.380

12 rs11168302 VDR 46598520 -0.097 (0.354) 0.784

12 rs10875702 VDR 46599285 0.060 (0.130) 0.646

12 rs2269720 CYP27B1 56404515 0.028 (0.115) 0.805

12 rs4760169 CYP27B1 56405114 -0.483 (0.167) 0.004

12 rs10877011 CYP27B1 56411259 0.046 (0.110) 0.677

12 rs12368653 CYP27B1 56419523 -0.072 (0.103) 0.487

12 rs1048691 CYP27B1 56439215 -0.103 (0.130) 0.429

12 rs4646537 CYP27B1 56443548 0.080 (0.306) 0.793

12 rs1021469 CYP27B1 56494431 -0.019 (0.111) 0.863

20 rs2762932 CYP24A1 52201798 0.138 (0.142) 0.332

20 rs6068810 CYP24A1 52202758 -0.087 (0.266) 0.744

20 rs11907350 CYP24A1 52203846 -0.062 (0.295) 0.832

20 rs927650 CYP24A1 52206148 -0.004 (0.101) 0.971

20 rs927651 CYP24A1 52206303 -0.036 (0.123) 0.770

20 rs6097809 CYP24A1 52206917 -0.281 (0.217) 0.196

20 rs1570669 CYP24A1 52207834 -0.039 (0.108) 0.718

20 rs2274130 CYP24A1 52208008 -0.046 (0.123) 0.708

20 rs6127119 CYP24A1 52212489 -0.045 (0.130) 0.730

20 rs6068816 CYP24A1 52214498 -0.043 (0.182) 0.813

20 rs3787555 CYP24A1 52216098 -0.008 (0.118) 0.945

20 rs3787557 CYP24A1 52216542 0.016 (0.152) 0.915

20 rs2762941 CYP24A1 52217059 0.004 (0.110) 0.972

20 rs2181874 CYP24A1 52217885 -0.095 (0.121) 0.433

20 rs4809959 CYP24A1 52219266 0.056 (0.101) 0.582

20 rs4809960 CYP24A1 52219480 0.015 (0.124) 0.905

20 rs2296241 CYP24A1 52219626 -0.059 (0.102) 0.563

20 rs2245153 CYP24A1 52219813 -0.003 (0.133) 0.982

20 rs2585428 CYP24A1 52220304 0.084 (0.105) 0.420

20 rs6022999 CYP24A1 52221420 0.038 (0.120) 0.754

20 rs2248359 CYP24A1 52224925 0.045 (0.107) 0.677

Abbreviations: CHR, chromosome; SNP, single nucleotide polymorphism; BP, base pair; SE,

standard error; BETA, beta coefficient; P, P value.

Table 4 (online supplement). Genetic association of SNPs with early AMD adjusted for age, sex and

center

CHR SNP Gene BP OR (95% CI) P

4 rs12512631 GC 72820195 0.92 (0.77-1.09) 0.34

4 rs705117 GC 72826979 1.09 (0.86-1.38) 0.47

4 rs2298850 GC 72833131 0.99 (0.82-1.19) 0.91

4 rs222020 GC 72855136 1.05 (0.83-1.32) 0.69

4 rs1491718 GC 72860143 0.97 (0.74-1.27) 0.81

4 rs1155563 GC 72862352 0.98 (0.81-1.19) 0.85

4 rs16847050 GC 72886195 1.01 (0.79-1.30) 0.92

4 rs1565572 GC 72889055 1.13 (0.92-1.39) 0.24

4 rs11939173 GC 72891022 1.07 (0.90-1.26) 0.45

9 rs11185644 RXRA 136350684 1.09 (0.88-1.35) 0.43

9 rs12339187 RXRA 136369148 0.99 (0.80-1.23) 0.93

9 rs914853 RXRA 136378984 1.12 (0.93-1.33) 0.24

9 rs11185659 RXRA 136383204 0.98 (0.81-1.20) 0.87

9 rs10881582 RXRA 136395899 1.05 (0.86-1.27) 0.65

9 rs7039190 RXRA 136406525 1.24 (0.83-1.85) 0.30

9 rs11103473 RXRA 136427450 1.06 (0.89-1.26) 0.51

9 rs11103482 RXRA 136427681 1.15 (0.88-1.51) 0.30

9 rs3118536 RXRA 136448283 0.92 (0.74-1.14) 0.46

9 rs4240705 RXRA 136451221 0.97 (0.81-1.15) 0.72

9 rs6537944 RXRA 136452649 1.17 (0.85-1.60) 0.34

9 rs3118571 RXRA 136457999 0.97 (0.82-1.16) 0.76

9 rs4842196 RXRA 136473049 0.96 (0.79-1.16) 0.65

9 rs3118526 RXRA 136477178 0.88 (0.69-1.13) 0.31

11 rs7117967 CYP2R1 14782654 1.33 (0.99-1.78) 0.06

11 rs12419657 CYP2R1 14783385 0.88 (0.67-1.15) 0.35

11 rs1037379 CYP2R1 14809360 0.95 (0.80-1.13) 0.59

11 rs10832306 CYP2R1 14813670 1.26 (0.98-1.64) 0.07

11 rs1496167 CYP2R1 14833083 0.98 (0.82-1.18) 0.85

11 rs11023371 CYP2R1 14852847 1.21 (0.86-1.69) 0.28

11 rs7935792 CYP2R1 14866037 1.28 (0.92-1.76) 0.14

11 rs1792284 DHCR7 70812614 1.05 (0.86-1.27) 0.64

11 rs4316537 DHCR7 70832777 1.00 (0.72-1.40) 0.98

11 rs12785878 DHCR7 70845097 1.06 (0.88-1.27) 0.55

11 rs949178 DHCR7 70877968 1.00 (0.74-1.37) 0.99

11 rs7950649 DHCR7 70898979 1.06 (0.83-1.35) 0.66

12 rs11574143 VDR 46521184 1.06 (0.82-1.39) 0.65

12 rs731236 VDR 46525024 0.94 (0.79-1.12) 0.49

12 rs7975232 VDR 46525104 1.01 (0.85-1.19) 0.92

12 rs1544410 VDR 46526102 0.95 (0.80-1.13) 0.54

12 rs11168268 VDR 46538079 1.08 (0.91-1.27) 0.38

12 rs11574077 VDR 46539194 0.87 (0.58-1.30) 0.48

Supplemental Table 4 - Online Only

12 rs2239182 VDR 46541678 1.12 (0.95-1.32) 0.18

12 rs2107301 VDR 46541837 0.89 (0.74-1.07) 0.21

12 rs2283342 VDR 46542126 1.08 (0.86-1.34) 0.51

12 rs2239179 VDR 46544033 1.06 (0.90-1.26) 0.48

12 rs12717991 VDR 46545393 0.95 (0.80-1.11) 0.51

12 rs886441 VDR 46549231 0.94 (0.76-1.16) 0.58

12 rs2189480 VDR 46550095 0.99 (0.83-1.17) 0.90

12 rs3819545 VDR 46551273 0.97 (0.82-1.16) 0.76

12 rs2239186 VDR 46555677 1.05 (0.86-1.29) 0.63

12 rs10875693 VDR 46555917 1.14 (0.96-1.36) 0.14

12 rs6580642 VDR 46556863 0.87 (0.70-1.09) 0.22

12 rs2254210 VDR 46559981 1.13 (0.96-1.34) 0.14

12 rs2238136 VDR 46563980 0.91 (0.75-1.10) 0.32

12 rs2853564 VDR 46564754 1.10 (0.93-1.30) 0.26

12 rs4760648 VDR 46566932 0.97 (0.82-1.14) 0.68

12 rs11574027 VDR 46573640 0.93 (0.49-1.77) 0.83

12 rs11574026 VDR 46574513 0.74 (0.56-0.98) 0.03

12 rs4760655 VDR 46580398 0.93 (0.78-1.11) 0.44

12 rs7136534 VDR 46580893 0.96 (0.79-1.18) 0.72

12 rs11574024 VDR 46582488 0.78 (0.58-1.04) 0.09

12 rs4516035 VDR 46586093 1.21 (1.02-1.44) 0.03

12 rs11168302 VDR 46598520 0.92 (0.51-1.65) 0.78

12 rs10875702 VDR 46599285 0.85 (0.69-1.05) 0.14

12 rs2269720 CYP27B1 56404515 1.08 (0.90-1.30) 0.41

12 rs4760169 CYP27B1 56405114 0.97 (0.74-1.28) 0.83

12 rs10877011 CYP27B1 56411259 0.95 (0.80-1.14) 0.60

12 rs12368653 CYP27B1 56419523 1.02 (0.87-1.21) 0.79

12 rs1048691 CYP27B1 56439215 1.11 (0.90-1.36) 0.35

12 rs4646537 CYP27B1 56443548 1.35 (0.85-2.14) 0.20

12 rs1021469 CYP27B1 56494431 1.05 (0.87-1.25) 0.63

20 rs2762932 CYP24A1 52201798 0.92 (0.73-1.16) 0.47

20 rs6068810 CYP24A1 52202758 1.26 (0.83-1.91) 0.28

20 rs11907350 CYP24A1 52203846 1.15 (0.73-1.83) 0.55

20 rs927650 CYP24A1 52206148 0.98 (0.83-1.15) 0.77

20 rs927651 CYP24A1 52206303 1.05 (0.86-1.28) 0.65

20 rs6097809 CYP24A1 52206917 1.08 (0.77-1.52) 0.65

20 rs1570669 CYP24A1 52207834 1.02 (0.86-1.22) 0.80

20 rs2274130 CYP24A1 52208008 1.04 (0.86-1.27) 0.68

20 rs6127119 CYP24A1 52212489 1.09 (0.89-1.34) 0.39

20 rs6068816 CYP24A1 52214498 0.99 (0.74-1.32) 0.93

20 rs3787555 CYP24A1 52216098 0.94 (0.77-1.13) 0.49

20 rs3787557 CYP24A1 52216542 0.87 (0.68-1.12) 0.29

20 rs2762941 CYP24A1 52217059 0.97 (0.81-1.15) 0.71

20 rs2181874 CYP24A1 52217885 1.02 (0.84-1.24) 0.83

20 rs4809959 CYP24A1 52219266 0.88 (0.75-1.04) 0.13

20 rs4809960 CYP24A1 52219480 0.97 (0.79-1.18) 0.73

20 rs2296241 CYP24A1 52219626 0.91 (0.77-1.07) 0.27

20 rs2245153 CYP24A1 52219813 0.98 (0.79-1.22) 0.84

20 rs2585428 CYP24A1 52220304 1.13 (0.95-1.33) 0.16

20 rs6022999 CYP24A1 52221420 1.08 (0.89-1.31) 0.45

20 rs2248359 CYP24A1 52224925 1.15 (0.97-1.37) 0.11

Abbreviations: CHR, chromosome; SNP, single nucleotide polymorphism; BP, base pair; P, P

value; OR, odds ratio; CI, confidence intervals.

Table 5 (online supplement). Genetic association of SNPs with late AMD adjusted for age, sex and

center

CHR SNP Gene BP OR (95% CI) P

4 rs12512631 GC 72820195 1.03 (0.79-1.36) 0.81

4 rs705117 GC 72826979 0.88 (0.58-1.32) 0.53

4 rs2298850 GC 72833131 1.06 (0.78-1.44) 0.72

4 rs222020 GC 72855136 0.81 (0.54-1.21) 0.31

4 rs1491718 GC 72860143 0.92 (0.59-1.44) 0.72

4 rs1155563 GC 72862352 1.05 (0.77-1.43) 0.77

4 rs16847050 GC 72886195 0.98 (0.65-1.49) 0.93

4 rs1565572 GC 72889055 0.98 (0.69-1.40) 0.91

4 rs11939173 GC 72891022 1.02 (0.77-1.34) 0.89

9 rs11185644 RXRA 136350684 0.70 (0.47-1.03) 0.07

9 rs12339187 RXRA 136369148 0.69 (0.47-1.01) 0.06

9 rs914853 RXRA 136378984 0.87 (0.64-1.18) 0.38

9 rs11185659 RXRA 136383204 0.76 (0.54-1.08) 0.12

9 rs10881582 RXRA 136395899 0.76 (0.55-1.07) 0.11

9 rs7039190 RXRA 136406525 1.20 (0.63-2.30) 0.58

9 rs11103473 RXRA 136427450 0.74 (0.55-1.00) 0.06

9 rs11103482 RXRA 136427681 1.05 (0.68-1.63) 0.81

9 rs3118536 RXRA 136448283 0.60 (0.41-0.90) 0.01

9 rs4240705 RXRA 136451221 0.77 (0.58-1.04) 0.09

9 rs6537944 RXRA 136452649 0.99 (0.58-1.68) 0.96

9 rs3118571 RXRA 136457999 0.81 (0.61-1.08) 0.14

9 rs4842196 RXRA 136473049 0.94 (0.68-1.30) 0.70

9 rs3118526 RXRA 136477178 0.84 (0.55-1.28) 0.42

11 rs7117967 CYP2R1 14782654 1.07 (0.65-1.76) 0.80

11 rs12419657 CYP2R1 14783385 0.68 (0.41-1.13) 0.14

11 rs1037379 CYP2R1 14809360 1.09 (0.81-1.45) 0.57

11 rs10832306 CYP2R1 14813670 1.05 (0.67-1.63) 0.83

11 rs1496167 CYP2R1 14833083 1.10 (0.82-1.47) 0.51

11 rs11023371 CYP2R1 14852847 1.67 (1.01-2.74) 0.04

11 rs7935792 CYP2R1 14866037 0.99 (0.57-1.74) 0.98

11 rs1792284 DHCR7 70812614 0.86 (0.62-1.18) 0.35

11 rs4316537 DHCR7 70832777 1.35 (0.83-2.19) 0.23

11 rs12785878 DHCR7 70845097 1.01 (0.75-1.36) 0.95

11 rs949178 DHCR7 70877968 1.43 (0.92-2.22) 0.11

11 rs7950649 DHCR7 70898979 0.95 (0.63-1.43) 0.81

12 rs11574143 VDR 46521184 1.24 (0.81-1.90) 0.32

12 rs731236 VDR 46525024 0.91 (0.68-1.21) 0.50

12 rs7975232 VDR 46525104 0.99 (0.75-1.31) 0.96

12 rs1544410 VDR 46526102 0.92 (0.69-1.23) 0.57

12 rs11168268 VDR 46538079 1.01 (0.76-1.33) 0.97

12 rs11574077 VDR 46539194 0.32 (0.12-0.84) 0.02

Supplemental Table 5 - Online Only

12 rs2239182 VDR 46541678 1.14 (0.88-1.49) 0.32

12 rs2107301 VDR 46541837 0.86 (0.63-1.17) 0.34

12 rs2283342 VDR 46542126 0.93 (0.65-1.34) 0.71

12 rs2239179 VDR 46544033 1.12 (0.85-1.47) 0.41

12 rs12717991 VDR 46545393 0.97 (0.74-1.26) 0.81

12 rs886441 VDR 46549231 0.83 (0.58-1.20) 0.32

12 rs2189480 VDR 46550095 0.91 (0.68-1.21) 0.51

12 rs3819545 VDR 46551273 0.94 (0.71-1.25) 0.66

12 rs2239186 VDR 46555677 0.92 (0.66-1.28) 0.62

12 rs10875693 VDR 46555917 1.44 (1.08-1.93) 0.01

12 rs6580642 VDR 46556863 0.84 (0.58-1.24) 0.39

12 rs2254210 VDR 46559981 0.96 (0.73-1.27) 0.80

12 rs2238136 VDR 46563980 1.13 (0.83-1.54) 0.43

12 rs2853564 VDR 46564754 0.91 (0.69-1.22) 0.54

12 rs4760648 VDR 46566932 0.97 (0.74-1.29) 0.86

12 rs11574027 VDR 46573640 1.83 (0.72-4.63) 0.20

12 rs11574026 VDR 46574513 0.70 (0.44-1.11) 0.13

12 rs4760655 VDR 46580398 1.08 (0.81-1.44) 0.59

12 rs7136534 VDR 46580893 0.86 (0.61-1.22) 0.41

12 rs11574024 VDR 46582488 0.84 (0.52-1.36) 0.49

12 rs4516035 VDR 46586093 1.04 (0.78-1.39) 0.77

12 rs11168302 VDR 46598520 1.75 (0.75-4.11) 0.20

12 rs10875702 VDR 46599285 0.82 (0.58-1.17) 0.27

12 rs2269720 CYP27B1 56404515 1.07 (0.79-1.44) 0.68

12 rs4760169 CYP27B1 56405114 1.08 (0.69-1.69) 0.73

12 rs10877011 CYP27B1 56411259 1.03 (0.77-1.38) 0.82

12 rs12368653 CYP27B1 56419523 0.99 (0.75-1.30) 0.94

12 rs1048691 CYP27B1 56439215 0.97 (0.69-1.37) 0.86

12 rs4646537 CYP27B1 56443548 0.61 (0.22-1.66) 0.33

12 rs1021469 CYP27B1 56494431 1.09 (0.81-1.46) 0.57

20 rs2762932 CYP24A1 52201798 1.12 (0.78-1.60) 0.55

20 rs6068810 CYP24A1 52202758 0.91(0.45-1.85) 0.80

20 rs11907350 CYP24A1 52203846 1.25 (0.56-2.80) 0.59

20 rs927650 CYP24A1 52206148 0.98 (0.74-1.29) 0.86

20 rs927651 CYP24A1 52206303 0.98 (0.70-1.36) 0.89

20 rs6097809 CYP24A1 52206917 1.06 (0.59-1.93) 0.84

20 rs1570669 CYP24A1 52207834 0.97 (0.73-1.30) 0.85

20 rs2274130 CYP24A1 52208008 1.01 (0.72-1.40) 0.97

20 rs6127119 CYP24A1 52212489 1.02 (0.72-1.43) 0.93

20 rs6068816 CYP24A1 52214498 0.97 (0.59-1.57) 0.89

20 rs3787555 CYP24A1 52216098 0.91 (0.66-1.24) 0.54

20 rs3787557 CYP24A1 52216542 0.77 (0.51-1.18) 0.23

20 rs2762941 CYP24A1 52217059 1.00 (0.74-1.34) 0.99

20 rs2181874 CYP24A1 52217885 1.01 (0.73-1.39) 0.96

20 rs4809959 CYP24A1 52219266 0.77 (0.59-1.02) 0.07

20 rs4809960 CYP24A1 52219480 0.85 (0.61-1.18) 0.33

20 rs2296241 CYP24A1 52219626 0.80 (0.61-1.06) 0.12

20 rs2245153 CYP24A1 52219813 0.94 (0.67-1.34) 0.74

20 rs2585428 CYP24A1 52220304 1.32 (0.99-1.74) 0.06

20 rs6022999 CYP24A1 52221420 1.13 (0.82-1.54) 0.46

20 rs2248359 CYP24A1 52224925 1.17 (0.88-1.56) 0.27

Abbreviations: CHR, chromosome; SNP, single nucleotide polymorphism; BP, base pair; P, P

value; OR, odds ratio; CI, confidence intervals.

Table 6 (online supplement). Genetic association of SNPs with neovascular AMD adjusted for age,

sex and center

CHR SNP Gene BP OR (95% CI) P

4 rs12512631 GC 72820195 1.05 (0.76-1.43) 0.78

4 rs705117 GC 72826979 0.72 (0.43-1.21) 0.22

4 rs2298850 GC 72833131 1.21 (0.85-1.72) 0.30

4 rs222020 GC 72855136 0.88 (0.56-1.40) 0.60

4 rs1491718 GC 72860143 0.91 (0.54-1.53) 0.72

4 rs1155563 GC 72862352 1.13 (0.79-1.61) 0.50

4 rs16847050 GC 72886195 0.95 (0.58-1.54) 0.82

4 rs1565572 GC 72889055 1.03 (0.69-1.55) 0.88

4 rs11939173 GC 72891022 1.10 (0.79-1.51) 0.58

9 rs11185644 RXRA 136350684 0.53 (0.32-0.87) 0.01

9 rs12339187 RXRA 136369148 0.54 (0.33-0.88) 0.01

9 rs914853 RXRA 136378984 0.87 (0.60-1.24) 0.43

9 rs11185659 RXRA 136383204 0.71 (0.47-1.07) 0.10

9 rs10881582 RXRA 136395899 0.64 (0.43-0.97) 0.04

9 rs7039190 RXRA 136406525 1.21 (0.57-2.58) 0.62

9 rs11103473 RXRA 136427450 0.72 (0.51-1.03) 0.07

9 rs11103482 RXRA 136427681 1.30 (0.80-2.10) 0.29

9 rs3118536 RXRA 136448283 0.54 (0.33-0.87) 0.01

9 rs4240705 RXRA 136451221 0.72 (0.51-1.02) 0.07

9 rs6537944 RXRA 136452649 0.98 (0.53-1.83) 0.96

9 rs3118571 RXRA 136457999 0.78 (0.56-1.10) 0.16

9 rs4842196 RXRA 136473049 0.98 (0.67-1.42) 0.90

9 rs3118526 RXRA 136477178 0.78 (0.47-1.29) 0.33

11 rs7117967 CYP2R1 14782654 0.94 (0.51-1.74) 0.85

11 rs12419657 CYP2R1 14783385 0.66 (0.37-1.20) 0.17

11 rs1037379 CYP2R1 14809360 1.00 (0.71-1.40) 0.99

11 rs10832306 CYP2R1 14813670 0.97 (0.57-1.66) 0.92

11 rs1496167 CYP2R1 14833083 1.10 (0.78-1.54) 0.60

11 rs11023371 CYP2R1 14852847 1.56 (0.88-2.77) 0.13

11 rs7935792 CYP2R1 14866037 1.00 (0.52-1.92) 0.99

11 rs1792284 DHCR7 70812614 0.82 (0.57-1.19) 0.30

11 rs4316537 DHCR7 70832777 1.48 (0.86-2.57) 0.16

11 rs12785878 DHCR7 70845097 0.98 (0.69-1.38) 0.89

11 rs949178 DHCR7 70877968 1.50 (0.91-2.47) 0.12

11 rs7950649 DHCR7 70898979 1.10 (0.69-1.75) 0.68

12 rs11574143 VDR 46521184 1.31 (0.80-2.16) 0.29

12 rs731236 VDR 46525024 0.76 (0.53-1.07) 0.11

12 rs7975232 VDR 46525104 1.08 (0.78-1.48) 0.65

12 rs1544410 VDR 46526102 0.76 (0.54-1.07) 0.12

12 rs11168268 VDR 46538079 1.11 (0.80-1.53) 0.53

12 rs11574077 VDR 46539194 0.18 (0.04-0.76) 0.02

Supplemental Table 6 - Online Only

12 rs2239182 VDR 46541678 1.01 (0.74-1.37) 0.97

12 rs2107301 VDR 46541837 0.98 (0.69-1.40) 0.93

12 rs2283342 VDR 46542126 1.05 (0.70-1.57) 0.83

12 rs2239179 VDR 46544033 1.06 (0.78-1.46) 0.70

12 rs12717991 VDR 46545393 1.07 (0.79-1.45) 0.67

12 rs886441 VDR 46549231 0.77 (0.50-1.20) 0.25

12 rs2189480 VDR 46550095 0.93 (0.66-1.30) 0.66

12 rs3819545 VDR 46551273 1.06 (0.77-1.47) 0.72

12 rs2239186 VDR 46555677 1.05 (0.72-1.53) 0.79

12 rs10875693 VDR 46555917 1.36 (0.97-1.90) 0.08

12 rs6580642 VDR 46556863 0.71 (0.44-1.15) 0.16

12 rs2254210 VDR 46559981 0.98 (0.71-1.36) 0.93

12 rs2238136 VDR 46563980 1.20 (0.84-1.71) 0.33

12 rs2853564 VDR 46564754 0.90 (0.64-1.25) 0.51

12 rs4760648 VDR 46566932 1.01 (0.73-1.39) 0.95

12 rs11574027 VDR 46573640 2.10 (0.73-6.06) 0.17

12 rs11574026 VDR 46574513 0.83 (0.50-1.39) 0.48

12 rs4760655 VDR 46580398 1.13 (0.81-1.58) 0.47

12 rs7136534 VDR 46580893 1.03 (0.70-1.52) 0.88

12 rs11574024 VDR 46582488 0.91 (0.53-1.57) 0.74

12 rs4516035 VDR 46586093 0.91 (0.65-1.27) 0.57

12 rs11168302 VDR 46598520 2.12 (0.82-5.51) 0.12

12 rs10875702 VDR 46599285 0.95 (0.64-1.41) 0.78

12 rs2269720 CYP27B1 56404515 1.10 (0.78-1.56) 0.57

12 rs4760169 CYP27B1 56405114 1.20 (0.72-1.99) 0.49

12 rs10877011 CYP27B1 56411259 1.04 (0.74-1.44) 0.84

12 rs12368653 CYP27B1 56419523 1.04 (0.76-1.44) 0.80

12 rs1048691 CYP27B1 56439215 0.95 (0.63-1.43) 0.81

12 rs4646537 CYP27B1 56443548 0.79 (0.26-2.38) 0.68

12 rs1021469 CYP27B1 56494431 1.13 (0.81-1.58) 0.46

20 rs2762932 CYP24A1 52201798 1.11 (0.73-1.68) 0.63

20 rs6068810 CYP24A1 52202758 0.94 (0.42-2.11) 0.89

20 rs11907350 CYP24A1 52203846 1.68 (0.71-3.97) 0.24

20 rs927650 CYP24A1 52206148 0.94 (0.68-1.30) 0.73

20 rs927651 CYP24A1 52206303 0.98 (0.67-1.44) 0.93

20 rs6097809 CYP24A1 52206917 0.83 (0.39-1.75) 0.62

20 rs1570669 CYP24A1 52207834 0.98 (0.70-1.38) 0.90

20 rs2274130 CYP24A1 52208008 1.02 (0.70-1.50) 0.90

20 rs6127119 CYP24A1 52212489 1.10 (0.74-1.63) 0.64

20 rs6068816 CYP24A1 52214498 1.10 (0.64-1.90) 0.73

20 rs3787555 CYP24A1 52216098 0.92 (0.64-1.33) 0.66

20 rs3787557 CYP24A1 52216542 0.70 (0.42-1.15) 0.16

20 rs2762941 CYP24A1 52217059 1.06 (0.75-1.49) 0.75

20 rs2181874 CYP24A1 52217885 1.04 (0.71-1.51) 0.86

20 rs4809959 CYP24A1 52219266 0.78 (0.56-1.07) 0.12

20 rs4809960 CYP24A1 52219480 0.83 (0.57-1.22) 0.35

20 rs2296241 CYP24A1 52219626 0.84 (0.61-1.16) 0.28

20 rs2245153 CYP24A1 52219813 0.91 (0.61-1.37) 0.65

20 rs2585428 CYP24A1 52220304 1.28 (0.92-1.76) 0.14

20 rs6022999 CYP24A1 52221420 1.24 (0.87-1.78) 0.24

20 rs2248359 CYP24A1 52224925 1.27 (0.91-1.77) 0.16

Abbreviations: CHR, chromosome; SNP, single nucleotide polymorphism; BP, base pair; P, P

value; OR, odds ratio; CI, confidence intervals.