A genome-wide scan study identifies a single nucleotide substitution in ASIP associated with white versus non-white coat-colour variation in sheep (Ovis aries)

In sheep, coat colour (and pattern) is one of the important traits of great biological, economic and social importance. However, the genetics of sheep coat colour has not yet been fully clarified. We conducted a genome-wide association study of sheep coat colours by genotyping 47 303 single-nucleotide polymorphisms (SNPs) in the Finnsheep population in Finland. We identified 35 SNPs associated with all the coat colours studied, which cover genomic regions encompassing three known pigmentation genes (TYRP1, ASIP and MITF) in sheep. Eighteen of these associations were confirmed in further tests between white versus non-white individuals, but none of the 35 associations were significant in the analysis of only non-white colours. Across the tests, the s66432.1 in ASIP showed significant association (P=4.2 × 10⁻¹¹ for all the colours; P=2.3 × 10⁻¹¹ for white versus non-white colours) with the variation in coat colours and strong linkage disequilibrium with other significant variants surrounding the ASIP gene. The signals detected around the ASIP gene were explained by differences in white versus non-white alleles. Further, a genome scan for selection for white coat pigmentation identified a strong and striking selection signal spanning ASIP. Our study identified the main candidate gene for the coat colour variation between white and non-white as ASIP, an autosomal gene that has been directly implicated in the pathway regulating melanogenesis. Together with ASIP, the two other newly identified genes (TYRP1 and MITF) in the Finnsheep, bordering associated SNPs, represent a new resource for enriching sheep coat-colour genetics and breeding.     

Identification of single nucleotide polymorphisms in the agouti signaling protein (ASIP) gene in some goat breeds in tropical and temperate climates

The agouti-signaling protein (ASIP) plays a major role in mammalian pigmentation as an antagonist to melanocortin-1 receptor gene to stimulate pheomelanin synthesis, a major pigment conferring mammalian coat color. We sequenced a 352 bp fragment of ASIP gene spanning part of exon 2 and part of intron 2 in 215 animals representing six goat breeds from Nigeria and the United States: West African Dwarf, predominantly black; Red Sokoto, mostly red; and Sahel, mostly white from Nigeria; black and white Alpine, brown and white Spanish and white Saanen from the US. Twenty haplotypes from nine mutations representing three intronic, one silent and five missense (p.S19R, p.N35K, p.L36V, p.M42L and p.L45W) mutations were identified in Nigerian goats. Approximately 89 % of Nigerian goats carry haplotype 1 (TGCCATCCG) which seems to be the wild type configuration of mutations in this region of the gene. Although we found no association between these polymorphisms in the ASIP gene and coat color in Nigerian goats, in-silico functional analysis predicts putative deleterious functional impact of the p.L45W mutation on the basic amino-terminal domain of ASIP. In the American goats, two intronic mutations, g.293G>A and g.327C>A, were identified in the Alpine breed, although the g.293G>A mutation is common to American and Nigerian goat populations. All Sannen and Sahel goats in this study belong to haplotypes 1 of both populations which seem to be the wild-type composite ASIP haplotype. Overall, there was no clear association of this portion of the ASIP gene interrogated in this study with coat color variation. Therefore, additional genomic analyses of promoter sequence, the entire coding and non-coding regions of the ASIP gene will be required to obtain a definite conclusion.     

The role of MC1R gene in buffalo coat color

Melanocortin-1 receptor (MC1R) plays a major role in pigmentation in many species. To investigate if the MC1R gene is associated with coat color in water buffalo, the coding region of MC1R gene of 216 buffalo samples was sequenced, which included 49 black river buffalo (Murrah and Nili-Ravi), 136 swamp buffalo (Dehong, Diandongnan, Dechang, Guizhou, and Xilin) with white and gray body, and 31 hybrid offspring of river buffalo Nili-Ravi (or Murrah) and swamp buffalo. Among the three variation sites found, SNP684 was synonymous, while SNP310 and SNP384 were nonsynonymous, leading to p.S104G and p.I128M changes, respectively. Only Individuals carrying homozygote EBR/EBR were black. The genotype and phenotype analysis of the hybrid offspring of black river buffalo and gray swamp buffalo further revealed that the river buffalo type allele EBR or the allele carrying the amino acid p.104S was important for the full function of MC1R. The in silico functional analysis showed that the amino acid substitutions p.G104S and p.M128I had significant impact on the function of MC1R. Above results indicate that the allele EBR or the allele carrying the amino acid p.104S was associated with the black coat color in buffalo.     

Investigation of the role of the agouti signaling protein gene (ASIP) in coat color evolution in primates

We investigated variation in the gene encoding the agouti signaling protein (ASIP) in relation to coat color evolution in primates. We found little evidence that mutations in the coding region of ASIP have been involved in color changes among closely related primate species. Among many closely related species with differing coat color, the coding region of ASIP was identical. In two cases (Sulawesi macaque and black lion tamarin) where species with almost completely black coat color had derived point mutations in exon 4 of the ASIP coding sequence, the same mutations did not alter coloration in other mammals and so probably do not affect ASIP function. Evolutionary reconstructions of two key phenotypes that are typically related to ASIP function—transverse phaeomelanin bands on hairs and pale ventral coloration—showed that these usually evolved concurrently, suggesting that loci acting downstream of ASIP may be involved. Analysis of dN/dS ratios revealed a likely change in functional constraint on ASIP following loss of agouti-banded hairs + pale ventral coloration, particularly in catarrhine primates (humans, apes, and Old World monkeys). Together with previous results on a lack of association of coat color with MC1R variation, these results suggest that other loci probably have an important role in primate coat color evolution.     

Skin Transcriptome Profiles Associated with Skin Color in Chickens

Nutritional and medicinal benefits have been attributed to the consumption of tissues from the black-boned chickens in oriental countries. Lueyang black-boned chicken is one of the native chicken breeds. However, some birds may instead have white or lighter skin, which directly causes economic losses every year. Previous studies of pigmentation have focused on a number of genes that may play important roles in coat color regulation. Illumina2000 sequencing technology was used to catalog the global gene expression profiles in the skin of the Lueyang chicken with white versus black skin. A total of 18,608 unigenes were assembled from the reads obtained from the skin of the white and black chickens. A total of 649 known genes were differentially expressed in the black versus white chickens, with 314 genes that were up regulated and 335 genes that were down-regulated, and a total of 162 novel genes were differentially expressed in the black versus white chickens, consisting of 73 genes that were up-regulated (including 4 highly expressed genes that were expressed exclusively in the skin of the black chickens) and 89 genes that were down-regulated. There were also a total of 8 known coat-color genes expressed in previous studies (ASIP, TYR, KIT, TYRP1, OCA2, KITLG, MITF and MC1R). In this study, 4 of which showed greater expression in the black chickens, and several were up-regulated, such as KIT, ASIP, TYR and OCA2. To our surprise, KITLG, MITF and MC1R showed no significant difference in expression between the black- and white-skinned chickens, and the expression of TYRP1 was not detected in either skin color. The expression of ASIP, TYR, KIT, TYRP1, OCA2, KITLG, MITF and MC1R was validated by real-time quantitative polymerase chain reaction (qPCR), and the results of the qPCR were consistent with the RNA-seq. This study provides several candidate genes that may be associated with the development of black versus white skin. More importantly, the fact that the MC1R gene showed no significant difference in expression between the black and white chickens is of particular interest for future studies that aim to elucidate its functional role in the regulation of skin color.     

Triplex forming oligonucleotide targeted to 3'UTR downregulates the expression of the bcl-2 proto-oncogene in HeLa cells

The bcl-2 proto-oncogene is overexpressed in a variety of human cancers and plays an important role in programmed cell death. Recent reports implied that the 3′-untranslated region (3′UTR) functions effectively in the regulation of gene expression. Here, we attempt to assay the ability of triplex forming oligonucleotides (TFOs) to inhibit expression of a target gene in vivo and to examine the potential of the 3′UTR of the bcl-2 proto-oncogene in the regulation of bcl-2 gene expression. To do this, we have developed a novel cellular system that involves transfection of a Doxycyclin inducible expression plasmid containing the bcl-2 ORF and the 3′UTR together with a TFO targeted to the 3′UTR of the bcl-2 proto-oncogene. Phosphorothioate-modified TFO targeted to the 3′UTR of the bcl-2 gene significantly downregulated the expression of the bcl-2 gene in HeLa cells as demonstrated by western blotting. Our results indicate that blocking the functions of the 3′UTR using the TFO can downregulate the expression of the targeted gene, and suggest that triplex strategy is a promising approach for oligonucleotide-based gene therapy. In addition, triplex-based sequence targeting may provide a useful tool for studying the regulation of gene expression.     

Patterns and Possible Roles of LINE-1 Methylation Changes in Smoke-Exposed Epithelia

Tobacco smoking and reduced methylation of long interspersed element-1 (LINE-1) are crucial in oral carcinogenesis. 5′UTR of human LINE-1 sequence contains several CpG dinucleotides which are methylated in various proportions (0–100%). Methylation levels of many LINE-1s in cancer were reduced, hypomethylated. The hypomethylation of each LINE-1 locus can promote instability of genome and repress expression of a gene located on that same chromosome. This study investigated if cigarette smoking influences LINE-1 methylation of oral mucosal cells. The methylation of human LINE-1 in clinically normal oral mucosa of current smokers was compared to non-smokers. By using the combined bisulphite restriction analysis, each LINE-1 sequence was categorised into 4 patterns depending on the methylation status and location of the two 18-bp successive CpG from 5′ to 3′ including ^mC^mC, ^uC^uC, ^mC^uC and ^uC^mC. Of these, ^mC and ^uC represent methylated and unmethylated CpG, respectively. The DNA bisulphite sequence demonstrated that most CpGs of ^mC^mC and ^uC^uC were methylated and unmethylated, respectively. Nevertheless, some CpGs of each ^mC^uC or ^uC^mC allele were methylated. Imaging of the digestion products was used to generate %methylation value. No significant difference in the overall LINE-1 methylation level but the differences in percentages of some methylation patterns were discovered. The %^mC^mC and %^uC^uC increased, while the %^mC^uC decreased in current smokers (p = 0.002, 0.015, and <0.0001, respectively). Additionally, the lower %^mC^uC still persisted in persons who had stopped smoking for over 1 year (p = 0.001). The %^mC^uC also decreased in the higher pack-year smokers (p = 0.028). Smoking possibly altered ^mC^uC to ^mC^mC and ^uC^uC forms, and changes ^uC^mC to ^uC^uC forms. In conclusion, smoking changes methylation levels of partial methylated LINE-1s and increased the number of hypo- and hypermethylated loci. These hypomethylated LINE-1s may possess carcinogenesis potential. Moreover, LINE-1 methylation patterns may be useful for monitoring oral carcinogenesis in smokers.     

The 8818G allele of the agouti signaling protein (ASIP) gene is ancestral and is associated with darker skin color in African Americans

Skin color, a predictor of social interactions and risk factor for several types of cancer, is due to two contrasting forms of melanin, the darker eumelanin and lighter phaeomelanin. The lighter pigment phaeomelanin is the product of the antagonistic function of the agouti signaling protein (ASIP) on the -melanocyte stimulating hormone receptor (MC1R). Studies have shown that a single-nucleotide polymorphism (SNP) in the 3UTR of the ASIP gene is associated with dark hair and eyes; however, little is known about its role in inter-individual variation in skin color. Here we examine the relationship between the ASIP g.8818A>G SNP and skin color (M index) as assessed by reflectometry in 234 African Americans. Analyses of variance (ANOVA) were performed to evaluate the effects of ASIP genotypes, age, individual ancestry, and sex on skin color variation. Significant effects on M index variation were observed for ASIP genotypes (F(2,236)=4.37, P=0.01), ancestry (F(1,243)=37.2, P<0.001), and sex (F(1,244)=4.08, P=0.05). Subsequent analyses revealed a strong effect on M index from ASIP genotypes in African American females (P<0.001). Our study suggests that the ASIP G>A polymorphism exhibits a dominant effect leading to lighter skin color and that variation in the ASIP gene may have been one of several factors contributing to reductions in pigmentation in some populations. Further study is needed to reveal how interactions between ASIP and several other genes, such as MC1R and P, predict human pigmentation.     

Rare BRCA1 haplotypes including 3′UTR SNPs associated with breast cancer risk

Genetic markers identifying women at an increased risk of developing breast cancer exist, yet the majority of inherited risk remains elusive. While numerous BRCA1 coding sequence mutations are associated with breast cancer risk, BRCA1 mutations account for less then 5% of breast cancer risk. Since 3′ untranslated region (3′UTR) polymorphisms disrupting microRNA (miRNA) binding can be functional and can act as genetic markers of cancer risk, we tested the hypothesis that such polymorphisms in the 3′UTR of BRCA1 and haplotypes containing these functional polymorphisms may be associated with breast cancer risk. We sequenced the BRCA1 3′UTR from breast cancer patients to identify miRNA disrupting polymorphisms. We further evaluated haplotypes of this region including the identified 3′UTR variants in a large population of controls and breast cancer patients (n = 221) with known breast cancer subtypes and ethnicities. We identified three 3′UTR variants in BRCA1 that are polymorphic in breast cancer populations, and haplotype analysis including these variants revealed that breast cancer patients harbor five rare haplotypes not generally found among controls (9.50% for breast cancer chromosomes, 0.11% for control chromosomes, p = 0.0001). Three of these rare haplotypes contain the rs8176318 BRCA1 3′UTR functional variant. These haplotypes are not biomarkers for BRCA1 coding mutations, as they are found rarely in BRCA1 mutant breast cancer patients (1/129 patients = 0.78%). These rare BRCA1 haplotypes and 3′UTR SNPs may represent new genetic markers of breast cancer risk.Key words: BRCA1, haplotype, microRNA, SNP, 3′UTR, breast cancer, triple negative breast cancer