Genome-Wide Association Studies of Quantitatively Measured Skin, Hair, and Eye Pigmentation in Four European Populations

<div><p>Pigmentation of the skin, hair, and eyes varies both within and between human populations. Identifying the genes and alleles underlying this variation has been the goal of many candidate gene and several genome-wide association studies (GWAS). Most GWAS for pigmentary traits to date have been based on subjective phenotypes using categorical scales. But skin, hair, and eye pigmentation vary continuously. Here, we seek to characterize quantitative variation in these traits objectively and accurately and to determine their genetic basis. Objective and quantitative measures of skin, hair, and eye color were made using reflectance or digital spectroscopy in Europeans from Ireland, Poland, Italy, and Portugal. A GWAS was conducted for the three quantitative pigmentation phenotypes in 176 women across 313,763 SNP loci, and replication of the most significant associations was attempted in a sample of 294 European men and women from the same countries. We find that the pigmentation phenotypes are highly stratified along axes of European genetic differentiation. The country of sampling explains approximately 35% of the variation in skin pigmentation, 31% of the variation in hair pigmentation, and 40% of the variation in eye pigmentation. All three quantitative phenotypes are correlated with each other. In our two-stage association study, we reproduce the association of rs1667394 at the <em>OCA2/HERC2</em> locus with eye color but we do not identify new genetic determinants of skin and hair pigmentation supporting the lack of major genes affecting skin and hair color variation within Europe and suggesting that not only careful phenotyping but also larger cohorts are required to understand the genetic architecture of these complex quantitative traits. Interestingly, we also see that in each of these four populations, men are more lightly pigmented in the unexposed skin of the inner arm than women, a fact that is underappreciated and may vary across the world.</p> </div

Distribution of skin, hair, and eye pigmentation.

<p>Skin pigmentation histogram (<b>A</b>) and boxplot by country of sampling and sex (<b>B</b>) in 469 individuals showing the normality of the phenotype distribution and the differences between sexes and among countries. Males (M) have consistently lighter pigmentation (lower scored) than females (F) in all four countries. Among countries, the largest pigmentation difference is with Ireland, where, in our sample, individuals have lighter pigmentation or lower M index on average than in Poland, Italy, or Portugal. Hair pigmentation histogram (<b>C</b>) and boxplot by country (<b>D</b>) in 341 individuals showing the distribution of hair pigmentation and the differences among countries. In our sample, individuals from Northern European countries (Ireland, Poland) have on average lighter hair pigmentation than individuals from Southern European countries (Italy, Portugal). The distributions in males are similar to those in females in all countries except Ireland, where, in our sample, males have darker hair color than females (not shown). Eye pigmentation histogram (<b>E</b>) and boxplot by country (<b>F</b>) in 468 individuals showing the bimodal distribution of eye pigmentation and the differences among countries. Comparison with self-reported phenotypes shows that the two modes of the distribution correspond to blue and brown eye color, while individuals reporting green and hazel eye color have intermediate C’ values. As with hair pigmentation, in our sample, individuals from Northern European countries have on average lighter eye pigmentation than individuals from Southern European countries.</p

Population structure of the GWAS samples.

<p>(<b>A</b>) Plots of GWAS individuals on genetic PC1 and PC2 show that individuals largely cluster by country of sampling. PC1 divides the samples according to a North/South geographical axis, while PC2 divides the samples along an East/West geographical axis. Individuals from Ireland, Poland, Italy, and Portugal are colored in yellow, red, blue, and gray, respectively. (<b>B</b>) The plot of individuals on PC1 and PC3 shows that individuals from Portugal tend to have lower values on PC3 than individuals from Italy and Ireland, while individuals from Poland have intermediate values on PC3.</p

GWAS, replication, and combined association results for all signals with p-value<10⁻⁵ in the GWAS.

*<p>Minor/Major allele in the GWAS.</p>†<p>Allele frequencies (AF) and ^‡regression coefficients (beta) are given with respect to the number of copies of the minor allele in the GWAS.</p

GWAS results.

<p>Manhattan plots for the GWAS results for the skin (<b>A</b>), hair (<b>B</b>), and eye (<b>C</b>) pigmentation. The log-transformed p-values from the test of association are plotted as a function of the chromosomal position. Genome-wide significance is defined as the Bonferroni corrected 5% significance threshold (p-value<1.6×10⁻⁷) and is indicated as a red line. For skin pigmentation, one SNP on chromosome 3 in the <i>FLNB</i> gene almost reaches genome-wide significance (p-value = 1.8×10⁻⁷). No SNP achieves genome-wide significance in the hair pigmentation GWAS. For eye pigmentation, two SNPs, one near the pigmentation gene <i>OCA2</i> on chromosome 15 and one in the <i>SCIN</i> gene on chromosome 7 achieve genome-wide significance.</p

Canonical pathway enrichment analysis using Ingenuity.

<p><b>A</b> In the DLPFC. <b>B</b> In the ACC. <b>C</b> In the NAcc.</p

qPCR Confirmation of decreased expression of metallothionein subfamilies 1 and 2.

<p>Significant differences are in bold for one tailed t-test confirming the direction of the change.</p

Heteroplasmy and multiplasmy in mtDNA.

<p>The heteroplasmy was calculated from NGS reads and confirmed by Sanger sequencing (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127280#pone.0127280.g001" target="_blank">Fig 1B</a>). Multiplasmic length polymorphisms were observed in the D-loop region and showed a trend for over-representation of cases at the 514 (CA)_n and the D16189 loci. <b>Multiplasmy was observed about 1.5 times more frequently in cases.</b> Note the tri-allelic multiplasmy results for two subjects (B-76 and S-114) showing 5, 6, and 7 repeat lengths shown in bold. Heteroplasmy/multiplasmy at all these six loci were also confirmed by Sanger sequencing.</p><p>*multiplasmy occurred in all brain regions sequenced.</p><p>#Val313Ile.</p><p>Heteroplasmy and multiplasmy in mtDNA.</p

The number of non-synonymous mutations shown in S4 Table, was extracted when present only in controls and not cases with psychiatric disorders, and is shown in the row for control subjects.

<p>The number of non-synonymous mutations found only in cases (and not controls) is shown on each row for each disorder. The ratio of the number of mutations / subject was calculated for each group, and a z-score for the difference of the observed for each group compared to the entire mean was calculated. There was a significant increase in non-synonymous mutations (p = 0.024, two tailed z-score test) in persons with SZ compared to controls.</p><p>The number of non-synonymous mutations shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127280#pone.0127280.s007" target="_blank">S4 Table</a>, was extracted when present only in controls and not cases with psychiatric disorders, and is shown in the row for control subjects.</p

Demographic and physiological details for the subjects included in the qPCR validation sample (cohort 2).

<p>Mean±standard deviation values (SD) are presented. DLPFC: dorsolateral prefrontal cortex, ACC: anterior cingulate cortex, NAcc: nucleus accumbens, NA: not available.</p