Sophie I. Candille

A β-Defensin Mutation Causes Black Coat Color in Domestic Dogs

Genetic analysis of mammalian color variation has provided fundamental insight into human biology and disease. In most vertebrates, two key genes, Agouti and Melanocortin 1 receptor (Mc1r), encode a ligand-receptor system that controls pigment type-switching, but in domestic dogs, a third gene is implicated, the K locus, whose genetic characteristics predict a previously unrecognized component of the melanocortin pathway. We identify the K locus as β-defensin 103 (CBD103) and show that its protein product binds with high affinity to the Mc1r and has a simple and strong effect on pigment type-switching in domestic dogs and transgenic mice. These results expand the functional role of β-defensins, a protein family previously implicated in innate immunity, and identify an additional class of ligands for signaling through melanocortin receptors.

Molecular and Evolutionary History of Melanism in North American Gray Wolves

Morphological diversity within closely related species is an essential aspect of evolution and adaptation. Mutations in the Melanocortin 1 receptor (Mc1r) gene contribute to pigmentary diversity in natural populations of fish, birds, and many mammals. However, melanism in the gray wolf, Canis lupus, is caused by a different melanocortin pathway component, the K locus, that encodes a beta-defensin protein that acts as an alternative ligand for Mc1r. We show that the melanistic K locus mutation in North American wolves derives from past hybridization with domestic dogs, has risen to high frequency in forested habitats, and exhibits a molecular signature of positive selection. The same mutation also causes melanism in the coyote, Canis latrans, and in Italian gray wolves, and hence our results demonstrate how traits selected in domesticated species can influence the morphological diversity of their wild relatives.

Variation and genetic control of protein abundance in humans.

Gene expression differs among individuals and populations and is thought to be a major determinant of phenotypic variation. Although variation and genetic loci responsible for RNA expression levels have been analysed extensively in human populations, our knowledge is limited regarding the differences in human protein abundance and the genetic basis for this difference. Variation in messenger RNA expression is not a perfect surrogate for protein expression because the latter is influenced by an array of post-transcriptional regulatory mechanisms, and, empirically, the correlation between protein and mRNA levels is generally modest. Here we used isobaric tag-based quantitative mass spectrometry to determine relative protein levels of 5,953 genes in lymphoblastoid cell lines from 95 diverse individuals genotyped in the HapMap Project. We found that protein levels are heritable molecular phenotypes that exhibit considerable variation between individuals, populations and sexes. Levels of specific sets of proteins involved in the same biological process covary among individuals, indicating that these processes are tightly regulated at the protein level. We identified cis-pQTLs (protein quantitative trait loci), including variants not detected by previous transcriptome studies. This study demonstrates the feasibility of high-throughput human proteome quantification that, when integrated with DNA variation and transcriptome information, adds a new dimension to the characterization of gene expression regulation.

Dorsoventral patterning of the mouse coat by Tbx15.

Many members of the animal kingdom display coat or skin color differences along their dorsoventral axis. To determine the mechanisms that control regional differences in pigmentation, we have studied how a classical mouse mutation, droopy ear (deH), affects dorsoventral skin characteristics, especially those under control of the Agouti gene. Mice carrying the Agouti allele black-and-tan (at) normally have a sharp boundary between dorsal black hair and yellow ventral hair; the deH mutation raises the pigmentation boundary, producing an apparent dorsal-to-ventral transformation. We identify a 216 kb deletion in deH that removes all but the first exon of the Tbx15 gene, whose embryonic expression in developing mesenchyme correlates with pigmentary and skeletal malformations observed in deH/deH animals. Construction of a targeted allele of Tbx15 confirmed that the deH phenotype was caused by Tbx15 loss of function. Early embryonic expression of Tbx15 in dorsal mesenchyme is complementary to Agouti expression in ventral mesenchyme; in the absence of Tbx15, expression of Agouti in both embryos and postnatal animals is displaced dorsally. Transplantation experiments demonstrate that positional identity of the skin with regard to dorsoventral pigmentation differences is acquired by E12.5, which is shortly after early embryonic expression of Tbx15. Fate-mapping studies show that the dorsoventral pigmentation boundary is not in register with a previously identified dermal cell lineage boundary, but rather with the limb dorsoventral boundary. Embryonic expression of Tbx15 in dorsolateral mesenchyme provides an instructional cue required to establish the future positional identity of dorsal dermis. These findings represent a novel role for T-box gene action in embryonic development, identify a previously unappreciated aspect of dorsoventral patterning that is widely represented in furred mammals, and provide insight into the mechanisms that underlie region-specific differences in body morphology.

Genetic architecture of skin and eye color in an African-European admixed population.

Variation in human skin and eye color is substantial and especially apparent in admixed populations, yet the underlying genetic architecture is poorly understood because most genome-wide studies are based on individuals of European ancestry. We study pigmentary variation in 699 individuals from Cape Verde, where extensive West African/European admixture has given rise to a broad range in trait values and genomic ancestry proportions. We develop and apply a new approach for measuring eye color, and identify two major loci (HERC2[OCA2] P = 2.3×10−62, SLC24A5 P = 9.6×10−9) that account for both blue versus brown eye color and varying intensities of brown eye color. We identify four major loci (SLC24A5 P = 5.4×10−27, TYR P = 1.1×10−9, APBA2[OCA2] P = 1.5×10−8, SLC45A2 P = 6×10−9) for skin color that together account for 35% of the total variance, but the genetic component with the largest effect (∼44%) is average genomic ancestry. Our results suggest that adjacent cis-acting regulatory loci for OCA2 explain the relationship between skin and eye color, and point to an underlying genetic architecture in which several genes of moderate effect act together with many genes of small effect to explain ∼70% of the estimated heritability.

Genetics of shoulder girdle formation: roles of Tbx15 and aristaless-like genes

The diverse cellular contributions to the skeletal elements of the vertebrate shoulder and pelvic girdles during embryonic development complicate the study of their patterning. Research in avian embryos has recently clarified part of the embryological basis of shoulder formation. Although dermomyotomal cells provide the progenitors of the scapular blade, local signals appear to have an essential guiding role in this process. These signals differ from those that are known to pattern the more distal appendicular skeleton. We have studied the impact of Tbx15, Gli3, Alx4 and related genes on formation of the skeletal elements of the mouse shoulder and pelvic girdles. We observed severe reduction of the scapula in double and triple mutants of these genes. Analyses of a range of complex genotypes revealed aspects of their genetic relationship, as well as functions that had been previously masked due to functional redundancy. Tbx15 and Gli3 appear to have synergistic functions in formation of the scapular blade. Scapular truncation in triple mutants of Tbx15, Alx4 and Cart1 indicates essential functions for Alx4 and Cart1 in the anterior part of the scapula, as opposed to Gli3 function being linked to the posterior part. Especially in Alx4/Cart1 mutants, the expression of markers such as Pax1, Pax3 and Scleraxis is altered prior to stages when anatomical aberrations are visible in the shoulder region. This suggests a disorganization of the proximal limb bud and adjacent flank mesoderm, and is likely to reflect the disruption of a mechanism providing positional cues to guide progenitor cells to their destination in the pectoral girdle.

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

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 OCA2/HERC2 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.

Linkage and Segregation Analysis of Black and Brindle Coat Color in Domestic Dogs

Mutations of pigment type switching have provided basic insight into melanocortin physiology and evolutionary adaptation. In all vertebrates that have been studied to date, two key genes, Agouti and Melanocortin 1 receptor (Mc1r), encode a ligand-receptor system that controls the switch between synthesis of red–yellow pheomelanin vs. black–brown eumelanin. However, in domestic dogs, historical studies based on pedigree and segregation analysis have suggested that the pigment type-switching system is more complicated and fundamentally different from other mammals. Using a genomewide linkage scan on a Labrador × greyhound cross segregating for black, yellow, and brindle coat colors, we demonstrate that pigment type switching is controlled by an additional gene, the K locus. Our results reveal three alleles with a dominance order of black (KB) > brindle (kbr) > yellow (ky), whose genetic map position on dog chromosome 16 is distinct from the predicted location of other pigmentation genes. Interaction studies reveal that Mc1r is epistatic to variation at Agouti or K and that the epistatic relationship between Agouti and K depends on the alleles being tested. These findings suggest a molecular model for a new component of the melanocortin signaling pathway and reveal how coat-color patterns and pigmentary diversity have been shaped by recent selection.

Integrative analysis of RNA, translation, and protein levels reveals distinct regulatory variation across humans

Elucidating the consequences of genetic differences between humans is essential for understanding phenotypic diversity and personalized medicine. Although variation in RNA levels, transcription factor binding, and chromatin have been explored, little is known about global variation in translation and its genetic determinants. We used ribosome profiling, RNA sequencing, and mass spectrometry to perform an integrated analysis in lymphoblastoid cell lines from a diverse group of individuals. We find significant differences in RNA, translation, and protein levels suggesting diverse mechanisms of personalized gene expression control. Combined analysis of RNA expression and ribosome occupancy improves the identification of individual protein level differences. Finally, we identify genetic differences that specifically modulate ribosome occupancy--many of these differences lie close to start codons and upstream ORFs. Our results reveal a new level of gene expression variation among humans and indicate that genetic variants can cause changes in protein levels through effects on translation.

New ligands for melanocortin receptors

Named originally for their effects on peripheral end organs, the melanocortin system controls a diverse set of physiological processes through a series of five G-protein-coupled receptors and several sets of small peptide ligands. The central melanocortin system plays an essential role in homeostatic regulation of body weight, in which two alternative ligands, α-melanocyte-stimulating hormone and agouti-related protein, stimulate and inhibit receptor signaling in several key brain regions that ultimately affect food intake and energy expenditure. Much of what we know about the relationship between central melanocortin signaling and body weight regulation stems from genetic studies. Comparative genomic studies indicate that melanocortin receptors used for controlling pigmentation and body weight regulation existed more than 500 million years ago in primitive vertebrates, but that fine-grained control of melanocortin receptors through neuropeptides and endogenous antagonists developed more recently. Recent studies based on dog coat-color genetics revealed a new class of melanocortin ligands, the β-defensins, which reveal the potential for cross talk between the melanocortin and the immune systems.