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Success of genetic association and the prediction of phenotypic traits from DNA are known to depend on the accuracy of phenotype characterization, amongst other parameters. To overcome limitations in the characterization of human iris pigmentation, we introduce a fully automated approach that specifies the areal proportions proposed to represent differing pigmentation types, such as pheomelanin, eumelanin, and non-pigmented areas within the iris. We demonstrate the utility of this approach using high-resolution digital eye imagery and genotype data from 12 selected SNPs from over 3000 European samples of seven populations that are part of the EUREYE study. In comparison to previous quantification approaches, (1) we achieved an overall improvement in eye colour phenotyping, which provides a better separation of manually defined eye colour categories. (2) Single nucleotide polymorphisms (SNPs) known to be involved in human eye colour variation showed stronger associations with our approach. (3) We found new and confirmed previously noted SNP-SNP interactions. (4) We increased SNP-based prediction accuracy of quantitative eye colour. Our findings exemplify that precise quantification using the perceived biological basis of pigmentation leads to enhanced genetic association and prediction of eye colour. We expect our approach to deliver new pigmentation genes when applied to genome-wide association testing.

Purpose: To examine associations between adherence to a Mediterranean diet and prevalence of age-related macular degeneration (AMD) in countries ranging from Southern to Northern Europe., Design: Cross-sectional, population-based epidemiologic study., Participants: Of 5060 randomly sampled people aged 65 years or older from 7 study centers across Europe (Norway, Estonia, United Kingdom, France, Italy, Greece, and Spain), full dietary data were available in 4753. The mean age of participants was 73.2 years (standard deviation, 5.6), and 55% were women., Methods: Participants underwent an eye examination and digital retinal color photography. The images were graded at a single center. Dietary intake during the previous 12 months was assessed by using a semiquantitative food-frequency questionnaire (FFQ). A previously published Mediterranean Diet Score (MDS) was used to classify participants according to their responses on the FFQ. Multivariable logistic regression was used to investigate the association of the MDS score and AMD, taking account of potential confounders and the multicenter study design., Main Outcome Measures: Images were graded according to the International Classification System for age-related maculopathy and stratified using the Rotterdam staging system into 5 exclusive stages (AMD 0-4) and a separate category of large drusen (≥125 μm). Age-related macular degeneration 4 included neovascular AMD (nvAMD) and geographic atrophy (GA)., Results: Increasing MDS was associated with reduced odds of nvAMD in unadjusted and confounder-adjusted analysis. Compared with the lowest MDS adherence (≤4 score), those in the highest category MDS adherence (>6 score) showed lower odds of nvAMD (odds ratio, 0.53; 0.27-1.04; P trend = 0.01). The association with MDS did not differ by Y204H risk allele (P = 0.89). For all early AMD (grade 1-3), there was no relationship with MDS (P trend = 0.9). There was a weak trend (P = 0.1) between MDS and large drusen; those in the highest category of MDS had 20% reduced odds compared with those in the lowest (P = 0.05)., Conclusions: This study adds to the limited evidence of the protective effect of adherence to a Mediterranean dietary pattern in those with late AMD, although it does not support previous reports of a relationship with genetic susceptibility. Interventions to encourage the adoption of the Mediterranean diet should be developed, and methods by which such behavior change can be achieved and maintained investigated., (Copyright © 2016 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Purpose: To study associations between early and late age-related macular degeneration (AMD) and neovascular AMD (nvAMD) with serum 25-hydroxy vitamin D (25(OH)D) and genetic variants in vitamin D pathway genes., Design: Population-based, cross-sectional study in a random sample aged 65 years or older from 7 European countries., Participants: Of 4753 participants, 4496 (2028 men and 2468 women), with a mean age of 73 years, provided a blood sample; 2137 had no signs of AMD, 2209 had early AMD, and 150 had late AMD, of whom 104 had nvAMD., Methods: Participants were interviewed to determine smoking and alcohol use, sunlight exposure, and diet; underwent fundus photography. Fundus images were graded using the International Classification System for Age-Related Maculopathy. The 25(OH)D was measured by liquid chromatography-tandem mass spectrometry and categorized as deficient (<30 nmol/l), insufficient (30-50 nmol/l), or adequate (≥50 nmol/l). Genotyping was performed on a subsample of 1284 AMD cases and controls for 93 single nucleotide polymorphisms (SNPs) from 7 genes. Associations were investigated by linear or logistic regression adjusted for potential confounders., Main Outcome Measures: Adjusted odds ratio (OR) for 3 outcomes (early AMD, late AMD, nvAMD)., Results: No linear association was found with 25(OH)D and early or late AMD or nvAMD. There was no association between insufficient or deficient status with early or late AMD. Deficient status was associated with nvAMD (adjusted OR, 1.27; 95% confidence interval, 1.1-1.45; P < 0.0001). Significant (P < 0.05) associations with 25(OH)D were found for SNPs in genes GC, VDR, CYP2R1, and CYP27B1. Two SNPs (VDR) were associated with early AMD, 4 SNPs (RXRA) and 1 SNP (VDR) were associated with nvAMD, and 1 SNP (RXRA), 2 SNPs (VDR), and 1 SNP (CYP2R1) were associated with late AMD. After Bonferroni correction, no SNPs were associated with early AMD, late AMD, or nvAMD., Conclusions: Deficiency in 25(OH)D was associated with nvAMD, but the adjusted OR was small, and we cannot exclude residual confounding. The hypothesis of a causal association of vitamin D with AMD is not supported by clear evidence for an association of vitamin D SNPs with early AMD, late AMD, or nvAMD., (Copyright © 2016 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Importance: Myopia is becoming increasingly common globally and is associated with potentially sight-threatening complications. Spending time outdoors is protective, but the mechanism underlying this association is poorly understood., Objective: To examine the association of myopia with ultraviolet B radiation (UVB; directly associated with time outdoors and sunlight exposure), serum vitamin D concentrations, and vitamin D pathway genetic variants, adjusting for years in education., Design, Setting, and Participants: A cross-sectional, population-based random sample of participants 65 years and older was chosen from 6 study centers from the European Eye Study between November 6, 2000, to November 15, 2002. Of 4187 participants, 4166 attended an eye examination including refraction, gave a blood sample, and were interviewed by trained fieldworkers using a structured questionnaire. Myopia was defined as a mean spherical equivalent of -0.75 diopters or less. Exclusion criteria included aphakia, pseudophakia, late age-related macular degeneration, and vision impairment due to cataract, resulting in 371 participants with myopia and 2797 without., Exposures: Exposure to UVB estimated by combining meteorological and questionnaire data at different ages, single-nucleotide polymorphisms in vitamin D metabolic pathway genes, serum vitamin D3 concentrations, and years of education., Main Outcomes and Measures: Odds ratios (ORs) of UVB, serum vitamin D3 concentrations, vitamin D single-nucleotide polymorphisms, and myopia estimated from logistic regression., Result: Of the included 3168 participants, the mean (SD) age was 72.4 (5) years, and 1456 (46.0%) were male. An SD increase in UVB exposure at age 14 to 19 years (OR, 0.81; 95% CI, 0.71-0.92) and 20 to 39 years (OR, 0.7; 95% CI, 0.62-0.93) was associated with a reduced adjusted OR of myopia; those in the highest tertile of years of education had twice the OR of myopia (OR, 2.08; 95% CI, 1.41-3.06). No independent associations between myopia and serum vitamin D3 concentrations nor variants in genes associated with vitamin D metabolism were found. An unexpected finding was that the highest quintile of plasma lutein concentrations was associated with a reduced OR of myopia (OR, 0.57; 95% CI, 0.46-0.72)., Conclusions and Relevance: Increased UVB exposure was associated with reduced myopia, particularly in adolescence and young adulthood. The association was not altered by adjusting for education. We found no convincing evidence for a direct role of vitamin D in myopia risk. The relationship between high plasma lutein concentrations and a lower risk of myopia requires replication.

Objective: To study associations between severity stages of early and late age-related macular degeneration (AMD) and genetic variations in age-related maculopathy susceptibility 2 (ARMS2) and complement factor H (CFH) and to investigate potential interactions between smoking and ARMS2., Design: Population-based, cross-sectional European Eye Study in 7 countries in Europe., Participants: Four thousand seven hundred fifty participants, 65 years of age and older, recruited through random sampling., Methods: Participants were classified on the basis of the more severely affected eye into 5 mutually exclusive AMD severity stages ranging from no AMD, 3 categories of early AMD, and late AMD. History of cigarette smoking was available and allowed classification into never, former, and current smokers, with the latter 2 groups combined into a single category of ever smokers for analysis. Genotyping was performed for single nucleotide polymorphisms rs10490924 and rs4146894 in ARMS2 and rs1061170 in CFH. Associations were analyzed by logistic regression., Main Outcome Measures: Odds ratios (ORs) for stage of AMD associated with genetic variations in ARMS2 and CFH and interactions between ARMS2 and smoking status., Results: Early AMD was present in 36.4% and late AMD was present in 3.3% of participants. Data on both genotype and AMD were available for 4276 people. The ORs for associations between AMD stage and ARMS2 increased monotonically with more severe stages of early AMD and were altered little by adjustment for potential confounders. Compared with persons with no AMD, carriers of the TT genotype for rs10490924 in ARMS2 had a 10-fold increase in risk of late AMD (P<3 × 10(-20)). The ORs for associations with CFH were similar for stage 3 early AMD and late AMD. Interactions between rs10490924 in ARMS2 and smoking status were significant in both unadjusted and adjusted models (P = 0.001). The highest risk was observed in those doubly homozygous for rs10490924 and rs1061170 in CFH (OR, 62.3; 95% confidence interval, 16-242), with P values for trend ranging from 0.03 (early AMD, stage 1) to 1 × 10(-26) (late AMD)., Conclusions: A strong association was demonstrated between all stages of AMD and genetic variation in ARMS2, and a significant gene-environment interaction with cigarette smoking was confirmed., (Copyright © 2013 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

The ability to predict Externally Visible Characteristics (EVCs) from DNA, also referred to as Forensic DNA Phenotyping (FDP), is an exciting new chapter in forensic genetics holding great promise for tracing unknown individuals who are unidentifiable via standard forensic short tandem repeat (STR) profiling. For the purpose of DNA-based eye colour prediction, we previously developed the IrisPlex system consisting of a multiplex genotyping assay and a prediction model based on genotype and phenotype data from 3804 Dutch Europeans. Recently, we performed a forensic developmental validation study of the highly sensitive IrisPlex assay, which currently represents the only validated tool available for DNA-based prediction of eye colour in forensic applications. In the present study, we validate the IrisPlex prediction model by extending our initially described model towards genotype and phenotype data from multiple European populations. We performed IrisPlex analysis on 3840 individuals from seven sites across Europe as part of the European Eye (EUREYE) study for which DNA and high-resolution eye images were available. The accuracy rate of correctly predicting an individual's eye colour as being blue or brown, above the empirically established probability threshold of 0.7, was on average 94% across all seven European populations, ranging from 91% to 98%, despite the large variation in eye colour frequencies between the populations. The overall prediction accuracies expressed by the area under the receiver characteristic operating curves (AUC) were 0.96 for blue and 0.96 for brown eyes, which is considerably higher than those established before. The IrisPlex prediction model parameters generated from this multi-population European dataset, and thus its prediction capabilities, were highly comparable to those previously established. Therefore, the increased information regarding eye colour phenotype and genotype distributions across Europe, and the system's ability to provide eye colour predictions across Europe accurately, both highlight additional evidence for the utility of the IrisPlex system in forensic casework., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)