Fig 1.
Manhattan plot showing the results of the GWAS for eyebrow thickness in Han Chinese.
Manhattan plot illustrating the results of the genome-wide scan for eyebrow thickness in 2961 Han Chinese after adjusting for the top four PCs, gender and age. The red line indicates the threshold for genome-wide statistical significance (P<5×10−8). Red dots represent SNPs that are close (<5 kb) to the genome-wide significant signals.
Table 1.
GWAS results for lead SNPs significantly associated with eyebrow thickness.
Fig 2.
Manhattan plot showing the results of the GWAS meta-analysis for eyebrow thickness in Han Chinese, Uyghurs, and Latin Americans.
A) Manhattan plot illustrating results of the transethnic meta-analysis of 2961 Han Chinese, 721 Uyghurs and 2301 Latin Americans. The red line indicates the threshold for genome-wide statistical significance (P<5×10−8). Red dots represent SNPs that are close (<5 kb) to the genome-wide significant signals. Regional association and linkage disequilibrium plots are shown for the significantly associated regions around B) rs1866188, C) rs112458845, D) rs1345417 and E) rs12651896 (gray bar). Different colors denote different populations. Increasing color intensities represent an increasing degree of linkage disequilibrium (r2) with the top SNP in each panel. The recombination rate (right-hand y axis) is plotted in blue and is based on the ASN population from the 1000 Genome Project. Exons for each gene are represented by vertical bars below the x axis, based on all isoforms available from the hg19 UCSC Genome Browser.
Fig 3.
Epigenetic annotation suggests the presence of putative regulatory regions at loci associated with eyebrow thickness.
Epigenetic annotation at the significantly associated regions around A) rs1345417 (3q26.33), B) rs12651896 (5q13.2) and C) rs1866188 (2q12.3), based on ENCODE and REMC project data. Regions of significant association (P<5×10−8) are denoted by a yellow box, with the top signal indicated by a black dashed line. The region exhibits distinct active enhancer signatures defined by epigenetic marks, such as H3K4me1 (red) and H3K27ac (blue) histone modifications and DNase hypersensitivity (green) in foreskin melanocyte primary cells, based on two independent biological replicates. Phastcons (pink) indicates the evolutionary conservation. ChIA-PET indicates long-range chromatin interaction. The different tracks were overlaid with physical positions using the WashU Epigenome Browser.
Fig 4.
Cas9-mediated gene editing provided evidence that rs1345417 and rs12651896 affect the transcriptional activity of the nearby SOX2 and FOXD1.
(A-D) qRT-PCR analysis of relative expression of nearby genes near (A) rs1345417, (B) rs12651896, (C) rs10051469 and (D) rs1866188. Scr denotes lentiCRISPR-Scramble sgRNA infected cells. 417mix, 896mix, 469mix and188mix denote CRISPR-Cas9 edited mixed clones for rs1345417, rs12651896, rs10051469 and rs1866188, respectively. (E-H) qRT-PCR analysis of relative SOX2 and FOXD1 expression in the CRISPR-Cas9 edited A375 cell clones. Especially, E) 417sg1m1-2: clone 1 and 2 edited by sgRNA1 for rs1345417; F) 417sg2m1-3: clone 1–3 edited by sgRNA2 for rs1345417. G) 896sg1m1-2: clone 1 and 2 edited by sgRNA1 for rs12651896. H) 896sg2m1-2: clone 1–2 edited by sgRNA2 for rs12651896. Detailed DNA sequencing results for each clone are shown in Supplementary Fig 4. Vector (lentiCRISPR vector infected cells), Scr (lentiCRISPR-Scramble sgRNA infected cells), sg1nm and sg2nm (cells infected with lentiCRISPR-sgRNA1 or sgRNA2 but not detectably modified near the rs1345417 and rs12651896 sites). (I) qRT-PCR analysis of relative SOX2 expression in heterozygously edited A375 cell clones, as above. 417-G/G (Original A375 cell line), Scr (lentiCRISPR-Scramble sgRNA infected cells), 417-G/C (the G/C-heterozygous A375 clone) Error bars represent standard errors. *p<0.05, **p<0.01, ***p<0.001, t-test comparing to vector control. N = 3 for each experiment.