Genome
3Division of Rheumatology, Orthopaedics and Dermatology, School of Medicine, University of Nottingham, Nottingham, UK.
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4Department of Genetic Identification, Erasmus MC University Medical Center Rotterdam, Rotterdam, Netherlands.
5Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China.
8University of Queensland Diamantina Institute, University of Queensland, Brisbane, Queensland, Australia.
14Epidemiology and Biostatistics Department, Faculty of Medicine, School of Public Health, Imperial College London, London, UK.
16Centre for Ophthalmology and Visual Science, University of Western Australia, Lions Eye Institute, Perth, Australia.
16Centre for Ophthalmology and Visual Science, University of Western Australia, Lions Eye Institute, Perth, Australia.
17Centre for Eye Research Australia, University of Melbourne, Department of Ophthalmology, Royal Victorian Eye and Ear Hospital, Melbourne, Australia.
18School of Medicine, Menzies Research Institute Tasmania, University of Tasmania, Hobart, Australia.
19Department of Epidemiology, Fairbanks School of Public Health, Indiana University, and Indiana University Melvin and Bren Simon Cancer Center, Indianapolis, IN, USA.
20Program in Genetic Epidemiology and Statistical Genetics, Harvard T.H. Chan School of Public Health, Harvard University, Boston, MA, USA.
4Department of Genetic Identification, Erasmus MC University Medical Center Rotterdam, Rotterdam, Netherlands.
5Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China.
5Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China.
22Department of Ophthalmology, Erasmus MC University Medical Center Rotterdam, Rotterdam, Netherlands.
23Department of Epidemiology, Erasmus MC University Medical Center Rotterdam, Rotterdam, Netherlands.
23Department of Epidemiology, Erasmus MC University Medical Center Rotterdam, Rotterdam, Netherlands.
25Department of Internal Medicine, Erasmus MC University Medical Center Rotterdam, Rotterdam, Netherlands.
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23Department of Epidemiology, Erasmus MC University Medical Center Rotterdam, Rotterdam, Netherlands.
26Department of Dermatology, Erasmus MC University Medical Center Rotterdam, Rotterdam, Netherlands.
23Department of Epidemiology, Erasmus MC University Medical Center Rotterdam, Rotterdam, Netherlands.
26Department of Dermatology, Erasmus MC University Medical Center Rotterdam, Rotterdam, Netherlands.
19Department of Epidemiology, Fairbanks School of Public Health, Indiana University, and Indiana University Melvin and Bren Simon Cancer Center, Indianapolis, IN, USA.
16Centre for Ophthalmology and Visual Science, University of Western Australia, Lions Eye Institute, Perth, Australia.
4Department of Genetic Identification, Erasmus MC University Medical Center Rotterdam, Rotterdam, Netherlands.
Results from both the discovery and replication studies were then meta-analyzed including a total of 192,986 Europeans from 10 populations. The Devlin’s genomic factor was relatively unchanged in this analysis (λ = 1.14). With the added statistical power, we found genome-wide significant associations for SNPs in an additional nine separate genomic regions (table ST5). All nine genetic loci were novel, not previously associated with human eye color. Three of these, PDE4D (rs62370541, P = 5.8 × 10−9), JAZF1 (rs849142, 3.0 × 10−9), and SOX6 (rs2351061, P = 4.0 × 10−8), have recently been associated with hair color (31). PDE4D inhibitors have been shown to increase melanin pigment in the skin of mouse models (38), as PDE4D is a target of the melanin-stimulating hormone/cyclic adenosine monophosphate (AMP)/melanocyte-inducing transcription factor (MITF) pathway (38).
Next, we compared results obtained from European subjects with association observations from a meta-analysis of 1636 Asian individuals (959 Han Chinese and 677 Indians from Singapore), for which quantitative measurements of the iris color were available (see the Supplementary Methods for information about quantitative phenotyping). Reliable SNP data were available for 44 of the lead autosomal SNPs from the 52 genetic loci identified by conditional analysis in the European discovery cohort. The remaining eight lead SNPs had a MAF smaller than 1% in this cohort and thus were excluded on account of the much smaller sample size. Thirty-one SNPs (70%) had the same direction of effect as the European analysis, and 5 (11%) of these 44 SNPs were significantly associated with eye color in Asians, after adjustment for multiple testing (Bonferroni-adjusted P value: 0.05/44 = 1.13 × 10−3). This included SNPs from two of the newly identified genes GPR157 (rs6693258, P = 3.6 × 10−5) and SIK1 (rs622330, P = 1.7 × 10−4) (table ST6), as well as from the previously known HERC2 gene (rs1129038, P = 2.2 × 10−4), which is the most strongly eye color–associated SNP in Europeans (17). For this marker, we observed considerable MAF differences between Europeans and Asians (T allele: 0.05 in Asians and 0.73 in Europeans). The strongest association in the Singaporean cohorts, however, was found within SLC24A5 (rs1426654, P = 1.9 × 10−7) representing the OCA type 6 (OCA6) locus. Notably, OCA6 was first reported in a Chinese family (39). However, the HERC2 and SLC24A5 SNPs were the only two variants to display heterogeneity between the two Singaporean cohorts (table ST6), with association primarily being driven by the cohort of South Asian ancestry. This is likely a due to South Asian and European populations being more closely related than East Asian and European populations. The MAFs for rs1426654 were considerably different between Asians (0.80 overall, 0.99 in East Asians, and 0.52 in South Asians) and Europeans (0.10) for the G allele, explaining why this SNP is often used as a DNA marker for biogeographic ancestry (40). Previously, SLC24A5 rs1426654 not only explained a considerable proportion of the variation in skin pigmentation between different continental populations (41) but also was associated with skin and eye color variation within a South Asian population (28, 42). Despite such drastic differences in allele frequency for some SNP alleles, the shared eye color effect between populations from two different continents demonstrates the value of our multiethnic study.
DNA variants identified via GWAS are markers of statistical association and not necessarily causative. Because of the differences in LD across continental populations, association may not be universally replicable. Therefore, we next assessed the presence of association not just for the lead SNPs in each region but also by testing other SNPs located within the 52 genomic regions identified in the European discovery analysis. Several SNPs within these regions (table ST7) showed evidence for eye color association in both European and Asian populations, with genome-wide significance in Europeans and suggestive levels of genome-wide association (P −5) in Asians, despite a much smaller sample size. These SNPs also showed no significant heterogeneity between the two Asian cohorts. It is therefore possible that, against the background of much stronger effects of European-only alleles or due to population-specific differences in MAF, some polymorphisms contributing to European eye color variation are also relevant for eye color variability in non-European populations, such as variation of brown eye color in Asians tested here.
In Europeans, the 112 autosomal SNPs identified through conditional analysis (all autosomal SNPs shown in table S1) explained 99.96% (SE = 6.5%, P = 4.8 × 10−279) of the liability scale for blue eyes (against brown eyes) and 38.5% (SE = 5.7%, P = 2.2 × 10−130) for intermediate eyes in the TwinsUK cohort, which was one of the VisiGen cohorts used for replication. Using the same linear scale as the GWAS analysis, these autosomal SNPs explained 53.2% (SE = 4.0%, P = 1.2 × 10−322) of the total phenotypic variation in eye color in TwinsUK.
Last, we performed in silico analyses to explore the putative function of the genetic loci our study highlighted with significant eye color association using the conditional SNPs. Gene set enrichment analysis identified multiple pathways with significant enrichment (table ST8). As expected, this included several pigmentation process pathways, with “Developmental Pigmentation” the most significant (P = 7 × 10−6), followed by “Frizzled Binding” (P = 2.0 × 10−4) and “Melanin Metabolic Process” (P = 2.6 × 10−4). We also examined the potential effects of the identified eye color–associated SNPs on gene expression using data from the GTEx Consortium (43). Despite the lack of iris tissue in the GTEx repository, many SNPs showed significant eQTL (expression quantitative trait loci) effects in multiple cell types and tissues (44), as seen for the associated SNPs across 38 (79%) of the 48 tissues in the GTEx dataset (table ST9). Most of the strongest effects were seen in nerve and sun-exposed skin tissue (P = 8.47 × 10−62 and P = 4.84 × 10−49, respectively) for rs2835660, where the C allele is significantly associated with a decrease in TTC3 expression. TTC3 is in proximity to DSCR9, a gene whose polymorphisms were previously associated with eye color (11). These results implicate TTC3 as a more likely candidate gene influencing eye color at this genetic locus than DSCR9 and that its effect on eye color is likely mediated through variation in gene expression. The lack of iris tissue information in GTEx likely explains the absence of stronger eQTL effects, such as for SNPs with regulatory effects over gene transcription (16).
DISCUSSION
We report the results of the largest GWAS for human eye color to date. In addition to confirming the association of SNPs in 11 previously known eye color genes (11, 13, 14, 17, 28), the identification of 50 novel eye color–associated genetic loci helps explain previously missing heritability of eye color variability in European populations. Moreover, because of the multiethnic design of our study, we demonstrate that several of the genetic loci discovered in Europeans also have an effect on eye color in Asians.
Eight of the genes in or near the loci newly associated with eye color in our study were previously reported for genetic associations with other pigmentation traits, such as hair and skin color, for instance, TPCN2, MITF, and DCT (27, 30, 32, 45). The commonality of associated DNA variants across the three pigmentation traits helps explain why the different pigmentation traits frequently (but not completely) intercorrelate in European populations. While many significant genetic associations are shared between iris color and other pigmentation traits, there are also notable differences. Although DNA variants within the MC1R gene are strongly associated with light skin and red hair color (27), no detectable association with eye color was found in our large GWAS, in line with previous albeit smaller-sized GWASs of more limited statistical power (11, 12, 14). Similarly, other DNA variants strongly associated with skin and hair color within genes, such as SILV, ASIP, and POMC (30), showed no statistically significant effect on eye color in this study, nor in previous studies. Moreover, we also identified 34 genetic loci that were significantly associated with eye color, but for which there is no report of significant association with hair and/or skin color. This is remarkable as the statistical power of the recent GWASs on hair color (31, 46) and sun sensitivity (32) were similar to that of our current eye color GWAS. Significant associations for SNPs in/near genes involved in iris structure, such as TRAF3IP1 and SEMA3A, suggest that they exert their effects with changes in Tyndall scattering, rather than through alterations of melanin metabolism. Overall, this demonstrates that although many genes overlap between eye, hair, and skin color, the different human pigmentation traits are not completely determined by the same genes as we showed.
The major strengths of our study compared with previous eye color GWASs arise from the larger sample size, which translated into increased statistical power and also the ability to lower the threshold of MAF for which sufficient power to detect association is available. Rare SNPs are often a source of considerable phenotypic variation (47). For instance, seven (6%) of the independently associated SNPs identified by conditional analysis in the discovery cohort had a MAF between 0.1 and 1%. Despite their low frequency, however, five (71%) of these rare SNPs were in the same region as other, more common conditional SNPs that did replicate. The remaining two loci (DAB2 and an intronic region on chromosome 4) that were not formally replicated should therefore be considered only as strong candidates with respect to their association with eye color, pending independent validation in future studies.
Another strength of this work is the inclusion of European and non-European populations. Non-European populations are underrepresented in the GWAS literature in general, including in pigmentation GWASs, but their study is important for the understanding of the genetic basis of human phenotypes (48). Although eye color variation is typically attributed to individuals of (at least partial) European descent, or those originating from areas nearby Europe, more subtle variation in brown eyes is also observed in Asian populations without European admixture (9). Our results from the Asian cohorts showed remarkable consistence in the genetic architecture of eye color among individuals of different continental ancestries with Asian replication for the two major European genes OCA2 and HERC2. Moreover, our findings also suggest that while a single regulatory variant in HERC2 is responsible for most blue/brown variation in Europeans (16), many additional DNA variants across both OCA2 and HERC2 seem to have independent effects. This hypothesis is further supported by our conditional analysis in the European discovery cohort, identifying independent associations spanning ~14 mbp across both genes rather than a concentrated cluster centered at HERC2 rs1129038. This is remarkable given the large eye color variation from the lightest blue to the darkest brown in Europeans, compared with the more limited variation within brown eye color in Asians.
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In conclusion, our work has identified numerous novel genetic loci associated with human eye color in Europeans, of which a subset also shows effects in Asians, despite their largely reduced phenotypic eye color variation compared with Europeans. The genetic loci we identified explain the majority (53.2%) of eye color phenotypic variation (classified using a three-category scale) in Europeans and a large proportion of the previously noted missing heritability of eye color. Our findings clearly demonstrate that eye color is a genetically highly complex human trait, similar to hair (31) and skin color (32), as highlighted recently in large European GWASs. The large number of novel eye color–associated genetic loci identified here provide a valuable resource for future functional studies, aiming to understand the molecular mechanisms that explain their eye color association, and for future genetic prediction studies, aiming to improve DNA-based eye color prediction in anthropological and forensic applications.
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