Native American genetic ancestry and pigmentation allele contributions to skin color in a Caribbean population.

Our interest in the genetic basis of skin color variation between populations led us to seek a Native American population with genetically African admixture but low frequency of European light skin alleles. Analysis of 458 genomes from individuals residing in the Kalinago Territory of the Commonwealth of Dominica showed approximately 55% Native American, 32% African, and 12% European genetic ancestry, the highest Native American genetic ancestry among Caribbean populations to date. Skin pigmentation ranged from 20 to 80 melanin units, averaging 46. Three albino individuals were determined to be homozygous for a causative multi-nucleotide polymorphism OCA2NW273KV contained within a haplotype of African origin; its allele frequency was 0.03 and single allele effect size was -8 melanin units. Derived allele frequencies of SLC24A5A111T and SLC45A2L374F were 0.14 and 0.06, with single allele effect sizes of -6 and -4, respectively. Native American genetic ancestry by itself reduced pigmentation by more than 20 melanin units (range 24-29). The responsible hypopigmenting genetic variants remain to be identified, since none of the published polymorphisms predicted in prior literature to affect skin color in Native Americans caused detectable hypopigmentation in the Kalinago.

The variation in skin colour of modern humans is a product of thousands of years of natural selection. All human ancestry can be traced back to African populations, which were dark-skinned to protect them from the intense UV rays of the sun. Over time, humans spread to other parts of the world, and people in the northern latitudes with lower UV developed lighter skin through natural selection. This was likely driven by a need for vitamin D, which requires UV rays for production. Separate genetic mechanisms were involved in the evolution of lighter skin in each of the two main branches of human migration: the European branch (which includes peoples on the Indian subcontinent and Europe) and the East Asian branch (which includes East Asia and the Americas). A variant of the gene SLC24A5 is the primary contributor to lighter skin colour in the European branch, but a corresponding variant driving light skin colour evolution in the East Asian branch remains to be identified. One obstacle to finding such variants is the high prevalence of European ancestry in most people groups, which makes it difficult to separate the influence of European genes from those of other populations. To overcome this issue, Ang et al. studied a population that had a high proportion of Native American and African ancestors, but a relatively small proportion of European ancestors, the Kalinago people. The Kalinago live on the island of Dominica, one of the last Caribbean islands to be colonised by Europeans. Ang et al. were able to collect hundreds of skin pigmentation measurements and DNA samples of the Kalinago, to trace the effect of Native American ancestry on skin colour. Genetic analysis confirmed their oral history records of primarily Native American (55%) ­ one of the highest of any Caribbean population studied to date ­ compared with African (32%) and European (12%) ancestries. Native American ancestry had the highest effect on pigmentation and reduced it by more than 20 melanin units, while the European mutations in the genes SLC24A5 and SLC45A2 and an African gene variant for albinism only contributed 5, 4 and 8 melanin units, respectively. However, none of the so far published gene candidates responsible for skin lightening in Native Americans caused a detectable effect. Therefore, the gene responsible for lighter skin in Native Americans/East Asians has yet to be identified. The work of Ang et al. represents an important step in deciphering the genetic basis of lighter skin colour in Native Americans or East Asians. A better understanding of the genetics of skin pigmentation may help to identify why, for example, East Asians are less susceptible to melanoma than Europeans, despite both having a lighter skin colour. It may also further acceptance of how variations in human skin tones are the result of human migration, random genetic variation, and natural selection for pigmentation in different solar environments.

Prehistoric human migration between Sundaland and South Asia was driven by sea-level rise.

Rapid sea-level rise between the Last Glacial Maximum (LGM) and the mid-Holocene transformed the Southeast Asian coastal landscape, but the impact on human demography remains unclear. Here, we create a paleogeographic map, focusing on sea-level changes during the period spanning the LGM to the present-day and infer the human population history in Southeast and South Asia using 763 high-coverage whole-genome sequencing datasets from 59 ethnic groups. We show that sea-level rise, in particular meltwater pulses 1 A (MWP1A, ~14,500-14,000 years ago) and 1B (MWP1B, ~11,500-11,000 years ago), reduced land area by over 50% since the LGM, resulting in segregation of local human populations. Following periods of rapid sea-level rises, population pressure drove the migration of Malaysian Negritos into South Asia. Integrated paleogeographic and population genomic analysis demonstrates the earliest documented instance of forced human migration driven by sea-level rise.

Whole-organism 3D quantitative characterization of zebrafish melanin by silver deposition micro-CT.

We previously described X-ray histotomography, a high-resolution, non-destructive form of X-ray microtomography (micro-CT) imaging customized for three-dimensional (3D), digital histology, allowing quantitative, volumetric tissue and organismal phenotyping (Ding et al., 2019). Here, we have combined micro-CT with a novel application of ionic silver staining to characterize melanin distribution in whole zebrafish larvae. The resulting images enabled whole-body, computational analyses of regional melanin content and morphology. Normalized micro-CT reconstructions of silver-stained fish consistently reproduced pigment patterns seen by light microscopy, and further allowed direct quantitative comparisons of melanin content across wild-type and mutant samples, including subtle phenotypes not previously noticed. Silver staining of melanin for micro-CT provides proof-of-principle for whole-body, 3D computational phenomic analysis of a specific cell type at cellular resolution, with potential applications in other model organisms and melanocytic neoplasms. Advances such as this in whole-organism, high-resolution phenotyping provide superior context for studying the phenotypic effects of genetic, disease, and environmental variables.

Chemical Imaging of Retinal Pigment Epithelium in Frozen Sections of Zebrafish Larvae Using ToF-SIMS.

Variants of the SLC24A5 gene, which encodes a putative potassium-dependent sodium-calcium exchanger (NCKX5) that most likely resides in the melanosome or its precursor, affect pigmentation in both humans and zebrafish (Danio rerio). This finding suggests that genetic variations influencing human skin pigmentation alter melanosome biogenesis via ionic changes. Gaining an understanding of how changes in the ionic environment of organelles impact melanosome morphogenesis and pigmentation will require a spatially resolved way to characterize the chemical environment of melanosomes in pigmented tissue such as retinal pigment epithelium (RPE). The imaging mass spectrometry technique most suited for this type of cell and tissue analysis is time-of-flight secondary ion mass spectrometry (ToF-SIMS) because it is able to detect many biochemical species with high sensitivity and with submicron spatial resolution. Here, we describe chemical imaging of the RPE in frozen-hydrated sections of larval zebrafish using cryo-ToF-SIMS. To facilitate the data interpretation, positive and negative polarity ToF-SIMS image data were transformed into a single hyperspectral data set and analyzed using principal component analysis. The combination of a novel protocol and the use of multivariate data analysis allowed us to discover new marker ions that are attributable to leucodopachrome, a metabolite specific to the biosynthesis of eumelanin. The described methodology may be adapted for the investigation of other classes of molecules in frozen tissues from zebrafish and other organisms.

Computational 3D histological phenotyping of whole zebrafish by X-ray histotomography.

Organismal phenotypes frequently involve multiple organ systems. Histology is a powerful way to detect cellular and tissue phenotypes, but is largely descriptive and subjective. To determine how synchrotron-based X-ray micro-tomography (micro-CT) can yield 3-dimensional whole-organism images suitable for quantitative histological phenotyping, we scanned whole zebrafish, a small vertebrate model with diverse tissues, at ~1 micron voxel resolutions. Micro-CT optimized for cellular characterization (histotomography) allows brain nuclei to be computationally segmented and assigned to brain regions, and cell shapes and volumes to be computed for motor neurons and red blood cells. Striking individual phenotypic variation was apparent from color maps of computed densities of brain nuclei. Unlike histology, the histotomography also allows the study of 3-dimensional structures of millimeter scale that cross multiple tissue planes. We expect the computational and visual insights into 3D cell and tissue architecture provided by histotomography to be useful for reference atlases, hypothesis generation, comprehensive organismal screens, and diagnostics.

Shades of complexity: New perspectives on the evolution and genetic architecture of human skin.

Like many highly variable human traits, more than a dozen genes are known to contribute to the full range of skin color. However, the historical bias in favor of genetic studies in European and European-derived populations has blinded us to the magnitude of pigmentation's complexity. As deliberate efforts are being made to better characterize diverse global populations and new sequencing technologies, better measurement tools, functional assessments, predictive modeling, and ancient DNA analyses become more widely accessible, we are beginning to appreciate how limited our understanding of the genetic bases of human skin color have been. Novel variants in genes not previously linked to pigmentation have been identified and evidence is mounting that there are hundreds more variants yet to be found. Even for genes that have been exhaustively characterized in European populations like MC1R, OCA2, and SLC24A5, research in previously understudied groups is leading to a new appreciation of the degree to which genetic diversity, epistatic interactions, pleiotropy, admixture, global and local adaptation, and cultural practices operate in population-specific ways to shape the genetic architecture of skin color. Furthermore, we are coming to terms with how factors like tanning response and barrier function may also have influenced selection on skin throughout human history. By examining how our knowledge of pigmentation genetics has shifted in the last decade, we can better appreciate how far we have come in understanding human diversity and the still long road ahead for understanding many complex human traits.

Functional assessment of human coding mutations affecting skin pigmentation using zebrafish.

A major challenge in personalized medicine is the lack of a standard way to define the functional significance of the numerous nonsynonymous, single nucleotide coding variants that are present in each human individual. To begin to address this problem, we have used pigmentation as a model polygenic trait, three common human polymorphisms thought to influence pigmentation, and the zebrafish as a model system. The approach is based on the rescue of embryonic zebrafish mutant phenotypes by "humanized" zebrafish orthologous mRNA. Two hypomorphic polymorphisms, L374F in SLC45A2, and A111T in SLC24A5, have been linked to lighter skin color in Europeans. The phenotypic effect of a second coding polymorphism in SLC45A2, E272K, is unclear. None of these polymorphisms had been tested in the context of a model organism. We have confirmed that zebrafish albino fish are mutant in slc45a2; wild-type slc45a2 mRNA rescued the albino mutant phenotype. Introduction of the L374F polymorphism into albino or the A111T polymorphism into slc24a5 (golden) abolished mRNA rescue of the respective mutant phenotypes, consistent with their known contributions to European skin color. In contrast, the E272K polymorphism had no effect on phenotypic rescue. The experimental conclusion that E272K is unlikely to affect pigmentation is consistent with a lack of correlation between this polymorphism and quantitatively measured skin color in 59 East Asian humans. A survey of mutations causing human oculocutaneous albinism yielded 257 missense mutations, 82% of which are theoretically testable in zebrafish. The developed approach may be extended to other model systems and may potentially contribute to our understanding the functional relationships between DNA sequence variation, human biology, and disease.

Skin color variation in Orang Asli tribes of Peninsular Malaysia.

Pigmentation is a readily scorable and quantitative human phenotype, making it an excellent model for studying multifactorial traits and diseases. Convergent human evolution from the ancestral state, darker skin, towards lighter skin colors involved divergent genetic mechanisms in people of European vs. East Asian ancestry. It is striking that the European mechanisms result in a 10-20-fold increase in skin cancer susceptibility while the East Asian mechanisms do not. Towards the mapping of genes that contribute to East Asian pigmentation there is need for one or more populations that are admixed for ancestral and East Asian ancestry, but with minimal European contribution. This requirement is fulfilled by the Senoi, one of three indigenous tribes of Peninsular Malaysia collectively known as the Orang Asli. The Senoi are thought to be an admixture of the Negrito, an ancestral dark-skinned population representing the second of three Orang Asli tribes, and regional Mongoloid populations of Indo-China such as the Proto-Malay, the third Orang Asli tribe. We have calculated skin reflectance-based melanin indices in 492 Orang Asli, which ranged from 28 (lightest) to 75 (darkest); both extremes were represented in the Senoi. Population averages were 56 for Negrito, 42 for Proto-Malay, and 46 for Senoi. The derived allele frequencies for SLC24A5 and SLC45A2 in the Senoi were 0.04 and 0.02, respectively, consistent with greater South Asian than European admixture. Females and individuals with the A111T mutation had significantly lighter skin (p = 0.001 and 0.0039, respectively). Individuals with these derived alleles were found across the spectrum of skin color, indicating an overriding effect of strong skin lightening alleles of East Asian origin. These results suggest that the Senoi are suitable for mapping East Asian skin color genes.