Loci associated with skin pigmentation identified in African populations

Abstract

Despite the wide range of skin pigmentation in humans, little is known about its genetic basis in global populations. Examining ethnically diverse African genomes, we identify variants in or near SLC24A5, MFSD12, DDB1, TMEM138, OCA2, and HERC2 that are significantly associated with skin pigmentation. Genetic evidence indicates that the light pigmentation variant at SLC24A5 was introduced into East Africa by gene flow from non-Africans. At all other loci, variants associated with dark pigmentation in Africans are identical by descent in South Asian and Australo-Melanesian populations. Functional analyses indicate that MFSD12 encodes a lysosomal protein that affects melanogenesis in zebrafish and mice, and that mutations in melanocyte-specific regulatory regions near DDB1/TMEM138 correlate with expression of ultraviolet response genes under selection in Eurasians.

Fig. 1. Correlations between allele frequencies at genes associated with pigmentation and UV exposure in global populations

(A) Global variation in skin pigmentation indicated by Melanin index (Ml). This data was integrated with Ml data for global populations from (1, 102). (B) Mean erythemal dose rate. (C) Manhattan plot of -log10 transformed p-values from GWAS of skin pigmentation with the Illumina Omni5 SNP array. (D) Quantile-Quantile plot of observed vs expected p-values from the GWASIn both (C) and (D) significant SNPs at p < 5 × 10⁻⁸ are highlighted in purple. (E to L) Allele frequencies of genetic variants associated with skin pigmentation in global populations. African populations included are 1. Ethiopia Nilosaharan, 2. Ethiopia Omotic, 3. Ethiopia and Tanzania Cushitic, 4. Ethiopia Semitic, 5. Tanzania Nilosaharan, 6. Tanzania Hadza, 7. Tanzania Sandawe, 8. Botswana Bantu 9. Botswana San/Bantu admixed, and 10. Botswana San. The Melanesian (MEL) samples are from (12) and the Australian Aboriginal and Papua New Guinean samples (merged) are from the SGDP (PNG) (13). All other populations are from the TGP (10). NonAboriginal populations in the Americans are indicated: CEU (European ancestry), ASW (African-American Southwest US) and ACB (African Caribbean in Barbados).

Fig. 2. Melanin distributions

Histograms of melanin index computed from under-arm measurements with a DSM II ColorMeter (68) for all individuals in each population as described in (67). Skin tones were visualized by displaying the mean red, green, and blue values from the ColorMeter for individuals binned by melanin index.

Fig. 3. Genomic context of GWAS loci

Plot of - log10(p-value) versus genomic position for variants near the four regions with most strongly associated SNPs from GWAS, including annotations for genes, MITF ChIP-seq data for melanocytes (45), a CTCF ChIP-seq track for NHEK keratinocytes, and H3K27ac, DNase hypersensitive sites (DHS), and chromHMM tracks for melanocytes and keratinocytes from the Roadmap Epigenomics dataset (29). Genome-wide significant variants are highlighted in red. Circles, squares, and triangles denote noncoding, synonymous, and non-synonymous variants, respectively. (A) SLC24A5 locus, (B) MFSD12 locus, (C) DDB1/TMEM138, locus, and (D) OCA2/HERC2 locus.

Fig. 4. Coalescent Trees and TMRCA dating

Inferred genealogies for regions flanking candidate causal loci. Each leaf corresponds to a single sampled chromosome from one of 278 individuals in the Simons Genome Diversity Project (13). Leaf nodes are colored by the population of origin of the individual and sequences carrying the light allele are indicated with an open dot. Node heights and 95% credible intervals are presented for a subset of internal nodes. Gene genealogies are shown for regions flanking (A) SLC24A5, rs1426654 (15:48426484), (B) MFSD12, rs10424065 (19:3545022), (C) MFSD12, rs6510760 (19:3565253), (D) TMEM138, rs7948623 (11:61137147), (E) DDB1, rs11230664 (11:61076372), (F) OCA2, rs1800404 (15:28235773), (G) HERC2, rs4932620 (15:28514281), and (H) HERC2, rs6497271 (15:28365431).

Fig. 5. Haplotype networks at SLC24A5, MFSD12, DDB1/TMEM138, and OCA2/HERC2

Median joining haplotype networks of regions containing candidate causal variants. Connections between circles indicate genetic relatedness while size is relative to the frequency of haplotypes. Ancestry proportions are displayed as pie charts. Yellow/red subfigures indicate which haplotypes contain the allele associated with dark pigmentation (red) or light pigmentation (yellow). (A) 75kb region flanking the causal variant at SLC24A5. (B and C) 3kb regions flanking rs10424065 in MFSD12 and rs6510760 upstream of MFSD12. (D) 195 kb region flanking DDB1 extending from PGA5 through SDHAF2. (E, F, and G) 50 kb flanking regions 1, 3, and 2 at OCA2 and FIERC2 (ordered based on highest to lowest probability of being causal from CAVIAR analysis).

Fig. 6. MFSD12 suppresses eumelanin production but localizes to lysosomes

Immortalized melan-lnk4a melanocytes expressing non-target (sh NT) shRNA or either of two shRNA plasmid clones (#1 and 2) targeting Mfsd12 were analyzed for Mfsd12 mRNA content by (A) qRT-PCR, (B) melanin content by spectrophotometry, or (C) percent of cell area containing melanin by bright field microscopy; (D) shows quantification. Data in (A) to (C) represent mean ± s.e.m., normalized to sh NT samples, from three separate experiments; in (C), n(sh NT) = 97 cells, n (shMfsd12 #1) = 68 cells, n (shMfsd12 #2) = 71 cells. Bar in (C), 10 μm. (E to G) Melan-ink4a melanocytes transiently expressing MFSD12-HA (E) or not transfected (F) and (G) were fixed, immunolabeled for HA (E) and for LAMP2 to mark lysosomes E) and (G) or for TYRP1 to mark melanosomes (G), and analyzed by immunofluorescence and bright field microscopy. Bright field (melanin) images show pigmented melanosomes (pseudocolored red in the merged images). Insets, 4x magnification of boxed regions. Arrows, MFSD12-containing structures that overlap LAMP2 (E) or TYRP1-containing structures that overlap melanosomes; arrowheads, structures that do not overlap. Bars, 10 μm. (H) Quantification of overlap for structures labeled by MFSD12, TYRP1, LAMP2 and pigment. Data represent mean ± s.e.m. from three independent experiments, n = 17 cells (MFSD12 overlap with LAMP2 and melanin), 33 cells (TYRP1 overlap with melanin), or 23 cells (LAMP2 and melanin).

Fig. 7. In vivo zebrafish and mouse models of MFSD12 deficiency

(A and B) Representative images of methylene blue staining in wild-type TAB5. (A) and compound heterozygous mfsd12a zebrafish (6dpf). Note the absence of stained xanthophores in the mfsd12a mutant (B). (C and D) No difference was observed in the number or distribution of xanthophores detected by mosaic Tg(aox5:PALM-GFP) expression in injected wild-type TAB5 (C) or compound heterozygous mutant mfsd12a (D) zebrafish (5dpf). (E) A wild-type agouti mouse (left) is shown with a gray Mfsd12 targeted littermate (right). (F) Flair from the Mfsd12 targeted mouse has grossly normal eumelanin (lower black region of the hair shaft), however, the upper subapical yellow band in WT (E, left) appears white in the Mfsd12 mutant (E, right) due to a reduction in pheomelanin.