Eye Color Variation Research Articles

Further development of forensic eye color predictive tests

In forensic analysis predictive tests for external visible characteristics (or EVCs), including inference of iris color, represent a potentially useful tool to guide criminal investigations. Two recent studies, both focused on forensic testing, have analyzed single nucleotide polymorphism (SNP) genotypes underlying common eye color variation (Mengel-From et al., Forensic Sci. Int. Genet. 4:323 and Walsh et al., Forensic Sci. Int. Genet. 5:170). Each study arrived at different recommendations for eye color predictive tests aiming to type the most closely associated SNPs, although both confirmed rs12913832 in HERC2 as the key predictor, widely recognized as the most strongly associated marker with blue and brown iris colors. Differences between these two studies in identification of other eye color predictors may partly arise from varying approaches to assigning phenotypes, notably those not unequivocally blue or dark brown and therefore occupying an intermediate iris color continuum. We have developed two single base extension assays typing 37 SNPs in pigmentation-associated genes to study SNP-genotype based prediction of eye, skin, and hair color variation. These assays were used to test the performance of different sets of eye color predictors in 416 subjects from six populations of north and south Europe. The presence of a complex and continuous range of intermediate phenotypes distinct from blue and brown eye colors was confirmed by establishing eye color populations compared to genetic clusters defined using Structure software. Our study explored the effect of an expanded SNP combination beyond six markers has on the ability to predict eye color in a forensic test without extending the SNP assay excessively - thus maintaining a balance between the test's predictive value and an ability to reliably type challenging DNA with a multiplex of manageable size. Our evaluation used AUC analysis (area under the receiver operating characteristic curves) and naïve Bayesian likelihood-based classification approaches. To provide flexibility in SNP-based eye color predictive tests in forensic applications we modified an online Bayesian classifier, originally developed for genetic ancestry analysis, to provide a straightforward system to assign eye color likelihoods from a SNP profile combining additional informative markers from the predictors analyzed by our study plus those of Walsh and Mengel-From. Two advantages of the online classifier is the ability to submit incomplete SNP profiles, a common occurrence when typing challenging DNA, and the ability to handle physically linked SNPs showing independent effect, by allowing the user to input frequencies from SNP pairs or larger combinations. This system was used to include the submission of frequency data for the SNP pair rs12913832 and rs1129038: indicated by our study to be the two SNPs most closely associated to eye color.

The Genetics of Human Pigmentary Disorders

Interest in the genetics of human and animal pigmentation is longstanding. Variation in human pigmentary form— of skin, hair, and eyes—is one of the most striking polymorphic human traits. Ever since Charles Darwin, biologists have been asking how and why did Nature fashion men so differently across the earth? Moving beyond what we might consider natural physiological variation in skin color to the pathological, human pigmentary disorders such as albinism is often clinically striking. Therefore, some syndromes were described early in modern medical history, and what we would now recognize as modern genetic insights into their nature were present almost as early as the rediscovery of Gregor Mendel’s work just over a century ago. The two decades spanning the start of the twenty-first century have been extraordinarily productive for those interested in human pigment genetics. Of course, the influence and power of what was once called the ‘‘New Genetics’’ has been felt across much of modern biomedicine, but to this skin-watcher, at least, few areas of study have seen such progress with apparently so modest an investment in funding. Recounting the story of this advance is necessary not merely to pay homage to those who laid the foundations that later successes were built on, but also as a lesson for those working in other fields of biomedicine where the problems often appear less tractable. The development of the mouse fancy, and the subsequent institutionalization of this in the twentieth century, provided a fertile resource for those keen to understand mammalian biology (Lamoreux et al., 2010). Interest in this topic, however, was not limited to those, such as skin biologists or dermatologists, whose interests are—professionally speaking—skin deep, but included those far-sighted biologists who recognized that the astonishing diversity of murine coat color mutants offered a way to address the general problem of how genes work. When technological advances in molecular genetics made mammalian gene identification almost routine, the resource of the mouse fancy offered an unrivaled experimental model system both for those interested in human pigmentation and for those whose goals were more general.

Gene–gene interactions contribute to eye colour variation in humans

Prediction of phenotypes from genetic data is considered to be the first practical application of data gained from association studies, with potential importance for medicine and the forensic sciences. Multiple genes and polymorphisms have been found to be associated with variation in human pigmentation. Their analysis enables prediction of blue and brown eye colour with a reasonably high accuracy. More accurate prediction, especially in the case of intermediate eye colours, may require better understanding of gene-gene interactions affecting this polygenic trait. Using multifactor dimensionality reduction and logistic regression methods, a study of gene-gene interactions was conducted based on variation in 11 known pigmentation genes examined in a cohort of 718 individuals of European descent. The study revealed significant interactions of a redundant character between the HERC2 and OCA2 genes affecting determination of hazel eye colour and between HERC2 and SLC24A4 affecting determination of blue eye colour. Our research indicates interactive effects of a synergistic character between HERC2 and OCA2, and also provides evidence for a novel strong synergistic interaction between HERC2 and TYRP1, both affecting determination of green eye colour.

Digital Quantification of Human Eye Color Highlights Genetic Association of Three New Loci

Previous studies have successfully identified genetic variants in several genes associated with human iris (eye) color; however, they all used simplified categorical trait information. Here, we quantified continuous eye color variation into hue and saturation values using high-resolution digital full-eye photographs and conducted a genome-wide association study on 5,951 Dutch Europeans from the Rotterdam Study. Three new regions, 1q42.3, 17q25.3, and 21q22.13, were highlighted meeting the criterion for genome-wide statistically significant association. The latter two loci were replicated in 2,261 individuals from the UK and in 1,282 from Australia. The LYST gene at 1q42.3 and the DSCR9 gene at 21q22.13 serve as promising functional candidates. A model for predicting quantitative eye colors explained over 50% of trait variance in the Rotterdam Study. Over all our data exemplify that fine phenotyping is a useful strategy for finding genes involved in human complex traits.

The eyes have it

In his book, ‘The Astonishing Hypothesis’, Francis Crick describes a simple experiment to demonstrate that while both eyes see, the brain interprets information from only one eye at a time. A small mirror is used to direct the gaze of one eye to an uninteresting surface such as a blank wall, while the other eye focuses on a face ahead of the subject. The brain now sees the face, not the wall. By moving a hand transiently across the field of vision of the misdirected eye, the brain’s attention is redirected by the movement towards the wall. The result is that the subject sees a composite image, frequently just a pair of eyes staring out from a blank wall. The effect is very striking, but it also draws attention to the brain’s priorities: the face is dominant over the wall, and movement is dominant over the face. But the fact that the eyes remain even though the rest of the face is erased by the movement across the field of vision, suggests that eyes hold a special place in the brain’s attention. It is perhaps surprising therefore that the genetics of eye pigmentation has attracted less attention than that of skin or hair. In this issue, Rick Sturm and Mats Larsson redress the balance by reviewing the genetic basis for the variations in iris pigmentation. In particular, they highlight an SNP in an intron of a gene upstream from OCA2 that may account for much of the blue/brown eye colour variation by affecting the regulation of OCA2 expression. Eye colour variations reflect the genetic variation affecting genes implicated in the manufacture of the pigment melanin in melanosomes, specialised lysosome-related organelles. Over the years significant progress has been made in understanding both the genetics and the chemistry behind melanin production, yet many key features of melanin remain to be understood, not least the structure of the polymer. In fact, even using the term melanin is a simplification as birds and mammals make two chemically distinct types of melanin, the black/brown eumelanin and the yellow/reddish brown pheomelanin. The review from Sho Ito and John Simon covers the process of ‘mixed’ melanogenesis, reflecting natural systems in which a combination of pheo- and eumelanin is produced and highlights several outstanding questions relating the presence of combinations of different pigment types within the melanosome. One major role for melanosomes in humans is in photo-protection where pigment transferred from melanocytes to keratinocytes affords protection against UV-induced DNA damage. How this is achieved and regulated is an important issue given the alarming increase in incidence of melanoma. One area that is attracting increasing attention is the role of MC1R in photo protection. This receptor signals primarily via the cAMP pathway and is activated in skin in response to keratinocyte-derived MSH. Increased MC1R signalling in melanocytes leads to elevated pigmentation and transcription of genes implicated in melanin synthesis and consequently contributes to the tanning response. Evidence is also accumulating (see Song et al in the next issue of PCMR for example), that MC1R signalling also facilitates repair of DNA damage. One long-time ambition is to induce a natural tanning response and photoprotection without exposure to UV irradiation, and to this end Zalfa Abdel-Malek et al report the use of short MSH-related peptides that can stimulate the MC1R receptor and reduce UV-induced DNA damage in melanocytes. In fact the use of MSH-related compounds for therapeutic purposes is already bringing clinical benefits to patients with specific photo-sensitivities in the form of Alfamelanotide. However, the dangers of unregulated use by the public of compounds such as ‘Melanotan’ to achieve tanning without UV exposure is highlighted in the letter from Veronique de Marmol et al.

Analysis of Cultured Human Melanocytes Based on Polymorphisms within the SLC45A2/MATP, SLC24A5/NCKX5, and OCA2/P Loci

Single nucleotide polymorphisms (SNPs) within the SLC45A2/MATP, SLC24A5/NCKX5, and OCA2/P genes have been associated with natural variation of pigmentation traits in human populations. Here, we describe the characterization of human primary melanocytic cells genotyped for polymorphisms within the MATP, NCKX5, or OCA2 loci. On the basis of genotype, these cultured cells reflect the phenotypes observed by others in terms of both melanin content and tyrosinase (TYR) activity when comparing skin designated as either "White" or "Black". We found a statistically significant association of MATP-374L (darker skin) with higher TYR protein abundance that was not observed for any NCKX5-111 or OCA2 rs12913832 allele. MATP-374L/L homozygous strains displayed significantly lower MATP transcript levels compared to MATP-374F/F homozygous cells, but this did not reach statistical significance based on NCKX5 or OCA2 genotype. Similarly, we observed significantly increased levels of OCA2 mRNA in rs12913832-T (brown eye) homozygotes compared to rs12913832-C (blue eye) homozygous strains, which was not observed for MATP or NCKX5 gene transcripts. In genotype-phenotype associations performed on a collection of 226 southern European individuals using these same SNPs, we were able to show strong correlations in MATP-L374F, OCA2, and melanocortin-1 receptor with skin, eye, and hair color variation, respectively.

A Single SNP in an Evolutionary Conserved Region within Intron 86 of the HERC2 Gene Determines Human Blue-Brown Eye Color

We have previously demonstrated that haplotypes of three single nucleotide polymorphisms (SNPs) within the first intron of the OCA2 gene are extremely strongly associated with variation in human eye color. In the present work, we describe additional fine association mapping of eye color SNPs in the intergenic region upstream of OCA2 and within the neighboring HERC2 (hect domain and RLD2) gene. We screened an additional 92 SNPs in 300-3000 European individuals and found that a single SNP in intron 86 of HERC2, rs12913832, predicted eye color significantly better (ordinal logistic regression R(2) = 0.68, association LOD = 444) than our previous best OCA2 haplotype. Comparison of sequence alignments of multiple species showed that this SNP lies in the center of a short highly conserved sequence and that the blue-eye-associated allele (frequency 78%) breaks up this conserved sequence, part of which forms a consensus binding site for the helicase-like transcription factor (HLTF). We were also able to demonstrate the OCA2 R419Q, rs1800407, coding SNP acts as a penetrance modifier of this new HERC2 SNP for eye color, and somewhat independently, of melanoma risk. We conclude that the conserved region around rs12913832 represents a regulatory region controlling constitutive expression of OCA2 and that the C allele at rs12913832 leads to decreased expression of OCA2, particularly within iris melanocytes, which we postulate to be the ultimate cause of blue eye color.

Blue eye color in humans may be caused by a perfectly associated founder mutation in a regulatory element located within the HERC2 gene inhibiting OCA2 expression

The human eye color is a quantitative trait displaying multifactorial inheritance. Several studies have shown that the OCA2 locus is the major contributor to the human eye color variation. By linkage analysis of a large Danish family, we finemapped the blue eye color locus to a 166 Kbp region within the HERC2 gene. By association analyses, we identified two SNPs within this region that were perfectly associated with the blue and brown eye colors: rs12913832 and rs1129038. Of these, rs12913832 is located 21.152 bp upstream from the OCA2 promoter in a highly conserved sequence in intron 86 of HERC2. The brown eye color allele of rs12913832 is highly conserved throughout a number of species. As shown by a Luciferase assays in cell cultures, the element significantly reduces the activity of the OCA2 promoter and electrophoretic mobility shift assays demonstrate that the two alleles bind different subsets of nuclear extracts. One single haplotype, represented by six polymorphic SNPs covering half of the 3' end of the HERC2 gene, was found in 155 blue-eyed individuals from Denmark, and in 5 and 2 blue-eyed individuals from Turkey and Jordan, respectively. Hence, our data suggest a common founder mutation in an OCA2 inhibiting regulatory element as the cause of blue eye color in humans. In addition, an LOD score of Z = 4.21 between hair color and D14S72 was obtained in the large family, indicating that RABGGTA is a candidate gene for hair color.

Association of Polymorphic Sites in the OCA2 Gene with Eye Colour Using the Tree Scanning Method

A number of genes are considered to affect normal variation in human pigmentation. Recent studies have indicated that OCA2 is the crucial gene involved in the high variation of iris colour present among populations of European descent. In this study, eleven polymorphisms of the OCA2 gene were examined in search of their association with different pigment traits. The evolutionary tree scanning method indicated that the strongest phenotypic eye colour variation is associated with the branch defined by nonsynonymous change rs1800407, which refers to amino acid causing change Arg419Gln located in exon 13. Single SNP analysis indicated that allele 419Gln is associated with green/hazel iris colour (p < 0.001). According to tree scanning analysis, the proportion of eye colour variation explained by this nucleotide position is merely 4%. Thus, additional variation present in the OCA2 gene and perhaps some other pigment related genes must be taken into account in order to explain the high phenotypic variation in iris colour.

A small and robust Al(III)-chemosensor based on bis-Schiff base N, N′-(1,4-phenylenedimethylidyne)bis-1,4-benzene diamine

A sensitive fluorescent chemosensor based on bis-Schiff base N, N′-(1,4-phenylenedimethylidyne) bis-1,4-benzenediamine (PMBA) was developed for detection of Al 3+ in aqueous solutions. A naked eye color variation from yellow to colorless is observed along with a remarkable fluorescence enhancement. A good linearity between the fluorescence intensity of the coordinate complex Al 3+-PMBA and the concentration of Al 3+ (0–30 μM/L), with a detection limit of 2 × 10 −6 M was established.

A Three–Single-Nucleotide Polymorphism Haplotype in Intron 1 of OCA2 Explains Most Human Eye-Color Variation

We have previously shown that a quantitative-trait locus linked to the OCA2 region of 15q accounts for 74% of variation in human eye color. We conducted additional genotyping to clarify the role of the OCA2 locus in the inheritance of eye color and other pigmentary traits associated with skin-cancer risk in white populations. Fifty-eight synonymous and nonsynonymous exonic single-nucleotide polymorphisms (SNPs) and tagging SNPs were typed in a collection of 3,839 adolescent twins, their siblings, and their parents. The highest association for blue/nonblue eye color was found with three OCA2 SNPs: rs7495174 T/C, rs6497268 G/T, and rs11855019 T/C (P values of 1.02x10(-61), 1.57x10(-96), and 4.45x10(-54), respectively) in intron 1. These three SNPs are in one major haplotype block, with TGT representing 78.4% of alleles. The TGT/TGT diplotype found in 62.2% of samples was the major genotype seen to modify eye color, with a frequency of 0.905 in blue or green compared with only 0.095 in brown eye color. This genotype was also at highest frequency in subjects with light brown hair and was more frequent in fair and medium skin types, consistent with the TGT haplotype acting as a recessive modifier of lighter pigmentary phenotypes. Homozygotes for rs11855019 C/C were predominantly without freckles and had lower mole counts. The minor population impact of the nonsynonymous coding-region polymorphisms Arg305Trp and Arg419Gln associated with nonblue eyes and the tight linkage of the major TGT haplotype within the intron 1 of OCA2 with blue eye color and lighter hair and skin tones suggest that differences within the 5' proximal regulatory control region of the OCA2 gene alter expression or messenger RNA-transcript levels and may be responsible for these associations.

Preliminary study on eye colour in Japanese macaques (Macaca fuscata) in their natural habitat

Eye colour in Japanese macaques shows apparent differences between individuals, continuously ranging from orange (bright), through shades of yellow and hazel-blue to dark blue (dark). We arbitrarily classified them into either 'yellow' eyes or 'blue' eyes based on the yellow area occupying in the iris' peripupillary ring. Most Japanese macaques have yellow eyes after infant phase, whilst 19, 17, 12, and 15% of monkeys (>6 months, sexes combined) have blue-eye in studied two groups of Shodoshima and two groups of Takasakiyama, respectively. Frequency of eye colour did not differ between males and females, but significantly differed in each age class. Blue eyes significantly more frequently occurred in newborns, infants and aged monkeys than in juveniles and prime adults. Data from mother-infant pairs indicated eye colour could be inherited from their parents. A case of asymmetric eye colour in Japanese macaques was found from a sample of 1962 individuals. Eye colour variation of Japanese macaques was discussed in relation to those of humans and rhesus macaques. A possible evolutionary model of eye colour in Japanese macaques was discussed.

A Genome Scan for Eye Color in 502 Twin Families: Most Variation is due to a QTL on Chromosome 15q

We have rated eye color on a 3-point scale (1 = blue/grey, 2 = hazel/green, 3 = brown) in 502 twin families and carried out a 5–10 cM genome scan (400–757 markers). We analyzed eye color as a threshold trait and performed multipoint sib pair linkage analysis using variance components analysis in Mx. A lod of 19.2 was found at the marker D15S1002, less than 1 cM from OCA2, which has been previously implicated in eye color variation. We estimate that 74% of variance in eye color liability is due to this QTL and a further 18% due to polygenic effects. However, a large shoulder on this peak suggests that other loci affecting eye color may be telomeric of OCA2 and inflating the QTL estimate. No other peaks reached genome-wide significance, although lods &gt; 2 were seen on 5p and 14q and lods &gt;1 were additionally seen on chromosomes 2, 3, 6, 7, 8, 9, 17 and 18. Most of these secondary peaks were reduced or eliminated when we repeated the scan as a two locus analysis with the 15q linkage included, although this does not necessarily exclude them as false positives. We also estimated the interaction between the 15q QTL and the other marker locus but there was only minor evidence for additive [.dotmath] additive epistasis. Elaborating the analysis to the full two-locus model including non-additive main effects and interactions did not strengthen the evidence for epistasis. We conclude that most variation in eye color in Europeans is due to polymorphism in OCA2 but that there may be modifiers at several other loci.

A genome scan for eye color in 502 twin families: most variation is due to a QTL on chromosome 15q.

We have rated eye color on a 3-point scale (1 = blue/grey, 2 = hazel/green, 3 = brown) in 502 twin families and carried out a 5-10 cM genome scan (400-757 markers). We analyzed eye color as a threshold trait and performed multipoint sib pair linkage analysis using variance components analysis in Mx. A lod of 19.2 was found at the marker D15S1002, less than 1 cM from OCA2, which has been previously implicated in eye color variation. We estimate that 74% of variance in eye color liability is due to this QTL and a further 18% due to polygenic effects. However, a large shoulder on this peak suggests that other loci affecting eye color may be telomeric of OCA2 and inflating the QTL estimate. No other peaks reached genome-wide significance, although lods > 2 were seen on 5p and 14q and lods >1 were additionally seen on chromosomes 2, 3, 6, 7, 8, 9, 17 and 18. Most of these secondary peaks were reduced or eliminated when we repeated the scan as a two locus analysis with the 15q linkage included, although this does not necessarily exclude them as false positives. We also estimated the interaction between the 15q QTL and the other marker locus but there was only minor evidence for additive x additive epistasis. Elaborating the analysis to the full two-locus model including non-additive main effects and interactions did not strengthen the evidence for epistasis. We conclude that most variation in eye color in Europeans is due to polymorphism in OCA2 but that there may be modifiers at several other loci.