MC1R polymorphism associated with plumage color variations in Coturnix chinensis

The melanocortin 1 receptor (MC1R) gene was investigated as a candidate for plumage variations in Chinese painted quail, Coturnix chinensis. Four silent and two missense nucleotide polymorphisms were identified. The correspondent amino acid changes, p.Glu92Lys and p.Pro292Leu, were found in Blue Face and Red Breasted animals respectively. Blue Face is a melanic phenotype similar to the co‐dominant Extended Brown of Japanese quail, and both share the p.Glu92Lys mutation. The association of p.Pro292Leu with the recessive Red Breasted was confirmed in 23 animals from an experimental F2 cross.     

miRNA-338-3p通过靶向抑制MC1R基因表达抑制羊驼黑色素细胞黑色素的生成

黑色素皮质素1受体MC1R）是在黑色素细胞内表达的G蛋白耦合受体（G protein coupled receptor, GPCR）家族成员,参与黑色素细胞中黑色素的生成。微RNAs（miRNAs）是一类非编码RNA,通过与靶基因3′-UTR结合抑制基因表达。已有研究证明,miR-338-3p 在多种人类肿瘤细胞中（过）表达,可通过下调靶基因表达抑制肿瘤细胞的侵袭迁移能力。然而,有关miR-338-3p对羊驼皮肤黑色素细胞的黑色素合成影响却罕见报道。本研究证明,miRNA-338-3p通过靶向抑制MC1R基因表达,抑制羊驼黑色素细胞黑色素的生成。采用生物信息学预测MC1R基因是miRNA-338-3p的靶基因,其基因表达抑制羊驼黑色素细胞黑色素合成。随后构建miR-338-3p真核表达载体。其基因转染结合qPT-PCR和Western印迹结果揭示,与对照细胞比较,过表达miRNA-338-3p的羊驼黑色素细胞的MC1R基因,及其下游与黑色素生成相关的小眼相关性转录因子（MITF)、酪氨酸酶(TYR)、酪氨酸酶相关蛋白1（TYRP1）、酪氨酸酶相关蛋白2（TYRP2）编码基因mRNA及蛋白质表达水平明显下调。酶联免疫吸附分析显示,过表达miRNA-338-3p的羊驼皮肤黑色素细胞的黑色素产量,较对照细胞显著下降（P＜0.01）。综上结果,miR-338-3p可通过抑制靶基因MC1R表达,下调其下游基因MITF、TYR、TYRP1和TYRP2基因的表达,从而抑制羊驼皮肤黑色素细胞黑色素的合成。miRNA-338-3p在羊驼生长发育过程中,是否参与调控体内皮肤黑色素细胞的黑色素生成尚待进一步研究。     

Melanocortin receptor 1 (MC1R) mutations and coat color in pigs.

The melanocortin receptor 1 (MC1R) plays a central role in regulation of eumelanin (black/brown) and phaeomelanin (red/yellow) synthesis within the mammalian melanocyte and is encoded by the classical Extension (E) coat color locus. Sequence analysis of MC1R from seven porcine breeds revealed a total of four allelic variants corresponding to five different E alleles. The European wild boar possessed a unique MC1R allele that we believe is required for the expression of a wild-type coat color. Two different MC1R alleles were associated with the dominant black color in pigs. MC1R*2 was found in European Large Black and Chinese Meishan pigs and exhibited two missense mutations compared with the wild-type sequence. Comparative data strongly suggest that one of these, L99P, may form a constitutively active receptor. MC1R*3 was associated with the black color in the Hampshire breed and involved a single missense mutation D121N. This same MC1R variant was also associated with EP, which results in black spots on a white or red background. Two different missense mutations were identified in recessive red (e/e) animals. One of these, A240T, occurs at a highly conserved position, making it a strong candidate for disruption of receptor function.     

TYRP1 and MC1R genotypes and their effects on coat color in dogs

We used PCR amplification of cDNA prepared from skin biopsies to determine the nearly full-length, protein-coding sequence of dog TYRP1, and to define sequence variants potentially responsible for the B locus. One common variant contained a premature stop codon in exon 5 (Q331ter), and the other deleted a proline residue in exon 5 (345delP). A third variant in exon 2 (S41C) occurred less frequently. We genotyped 43 brown (including brown and white) and 34 black (including tricolor, black-and-tan, and black and white) dogs. All 43 of the brown group carried two or more of these sequence variants likely to interfere with TYRP1 function, whereas 0 of 34 in the black group carried two or more of these variants (10 carried one variant). We also genotyped 13 black-nosed and 10 brown-nosed dogs whose coat color was described as red, yellow, gold, apricot, or orange (including various degrees of white). All these dogs were homozygous for a R306X MC1R variant shown to be associated with these coat color phenotypes. The black or brown nose correlated perfectly with the absence or presence of the same three TYRP1 variants described above. TYRP1 was linkage mapped to dog chromosome 11, with a SNP in exon 7.     

Melanocortin 1-receptor (MC1R) mutations are associated with plumage colour in chicken

The co-segregation of plumage colour and sequence polymorphism in the melanocortin 1-receptor gene (MC1R) was investigated using an intercross between the red junglefowl and White Leghorn chickens. The results provided compelling evidence that the Extended black (E) locus controlling plumage colour is equivalent to MC1R. E/MC1R was assigned to chromosome 11 with overwhelming statistical support. Sequence analysis indicated that the E92K substitution, causing a constitutively active receptor in the sombre mouse, is the most likely causative mutation for the Extended black allele carried by the White Leghorn founders in this intercross. The MC1R sequence associated with the recessive buttercup (ebc) allele indicated that this allele evolved from a dominant Extended black allele as it shared the E92K and M71T substitutions with some E alleles. It also carried a third missense mutation H215P which thus may interfere with the constitutive activation of the receptor caused by E92K (and possibly M71T).     

Association of MC3R gene polymorphisms with body weight in the red fox and comparative gene organization in four canids

There are five genes encoding melanocortin receptors. Among canids, the genes have mainly been studied in the dog (MC1R, MC2R and MC4R). The MC4R gene has also been analysed in the red fox. In this report, we present a study of chromosome localization, comparative sequence analysis and polymorphism of the MC3R gene in the dog, red fox, arctic fox and Chinese raccoon dog. The gene was localized by FISH to the following chromosome: 24q24‐25 in the dog, 14p16 in the red fox, 18q13 in the arctic fox and NPP4p15 in the Chinese raccoon dog. A high identity level of the MC3R gene sequences was observed among the species, ranging from 96.0% (red fox – Chinese raccoon dog) to 99.5% (red fox – arctic fox). Altogether, eight polymorphic sites were found in the red fox, six in the Chinese raccoon dog and two in the dog, while the arctic fox appeared to be monomorphic. In addition, association of several polymorphisms with body weight was analysed in red foxes (the number of genotyped animals ranged from 319 to 379). Two polymorphisms in the red fox, i.e. a silent substitution c.957A>C and c.*185C>T in the 3′‐flanking sequence, showed a significant association (P < 0.01) with body weight.     

Two missense mutations in melanocortin 1 receptor (MC1R) are strongly associated with dark ventral coat color in reindeer (Rangifer tarandus)

The protein‐coding region of melanocortin 1 receptor (MC1R) was sequenced to identify potential variation affecting coat color in reindeer (Rangifer tarandus). A T→C sequence variation at nucleotide position 218 (c.218T>C) causing an amino acid (aa) change from methionine to threonine at aa position 73 (p.Met73Thr) was identified. In addition, a T→G sequence variation was found at nucleotide position 839 (c.839T>G), causing phenylalanine to be exchanged by cysteine at aa position 280 (p.Phe280Cys). The two sequence variants (c.218C and c.839G) were found to be closely associated with a darker belly coat compared with animals not having any of these two variants. The aa acid change p.Met73Thr affects the same position as p.Met73Lys previously reported to give constitutive activation of MC1R in black sheep (Ovis aries), whereas p.Phe280Cys is identical to one of two variants previously reported to be associated with dark coat color in Arctic fox (Alopex lagopus), supporting that the two variants found in reindeer are functional. The complete absence of Thr73 and Cys280 among the 51 wild reindeer analyzed provides some evidence that these variants are more common in the domestic herds.     

Melanocortin-4 receptor (MC4R) polymorphisms are associated with growth and meat quality traits in sheep

The involvement of melanocortin 4 receptor gene (MC4R) in food intake and body weight regulation is well characterized. MC4R mutations are the most frequent monogenic cause of human obesity. Significant associations have been revealed between MC4R mutations and productive traits in pigs, cattle and poultry. Herein, fluorescence-based conformation sensitive gel electrophoresis was used to identify two single nucleotide polymorphisms (SNPs) in the coding region (93G>A and 292G>A) and two SNPs in the 3′-UTR area (1016G>A and 1240T>C) of MC4R gene in 132 German Merino sheep. We found that the 1016G>A mutation in the 3′-UTR was significantly associated with body weight at 120 and 180 days, average daily gain, back fat thickness and loin-eye area. Allele A located at the 292th position of MC4R gene representing Arg98 was associated with significantly higher loin-eye area in sheep. For the synonymous 93G>A mutation, A allele carrier animals had higher back fat thickness. Our results provide evidence that the MC4R gene may be a candidate gene for growth and meat quality traits with MC4R SNPs being potentially valuable as genetic markers for economic traits in German Merino sheep.     

High diversity in functional properties of melanocortin 1 receptor (MC1R) in divergent primate species is more strongly associated with phylogeny than coat color

We have characterized the biochemical function of the melanocortin 1 receptor (MC1R), a critical regulator of melanin synthesis, from 9 phylogenetically diverse primate species with varying coat colors. There is substantial diversity in melanocyte-stimulating hormone (MSH) binding affinity and basal levels of activity in the cloned MC1Rs. MSH binding was lost independently in lemur and New World monkey lineages, whereas high basal levels of MC1R activity occur in lemurs and some New World monkeys and Old World monkeys. Highest levels of basal activity were found in the MC1R of ruffed lemurs, which have the E94K mutation that leads to constitutive activation in other species. In 3 species (2 lemurs and the howler monkey), we report the novel finding that binding and inhibition of MC1R by agouti signaling protein (ASIP) can occur when MSH binding has been lost, thus enabling continuing regulation of the melanin type via ASIP expression. Together, these findings can explain the previous paradox of a predominantly pheomelanic coat in the red ruffed lemur (Varecia rubra). The presence of a functional, MSH-responsive MC1R in orangutan demonstrates that the mechanism of red hair generation in this ape is different from the prevalent mechanism in European human populations. Overall, we have found unexpected diversity in MC1R function among primates and show that the evolution of the regulatory control of MC1R activity occurs by independent variation of 3 distinct mechanisms: basal MC1R activity, MSH binding and activation, and ASIP binding and inhibition. This diversity of function is broadly associated with primate phylogeny and does not have a simple relation to coat color phenotype within primate clades.     

MC1R expression in HaCaT keratinocytes inhibits UVA-induced ROS production via NADPH oxidase- and cAMP-dependent mechanisms

Ultraviolet A (UVA) radiations are responsible for deleterious effects, mainly due to reactive oxygen species (ROS) production. Alpha-melanocyte stimulating hormone (α-MSH) binds to melanocortin-1 receptor (MC1R) in melanocytes to stimulate pigmentation and modulate cutaneous inflammatory responses. MC1R may be induced in keratinocytes after UV exposure. To investigate the effect of MC1R signaling on UVA-induced ROS (UVA-ROS) production, we generated HaCaT cells that stably express human MC1R (HaCaT-MC1R) or the Arg151Cys (R(151)C) non-functional variant (HaCaT-R(151)C). We then assessed ROS production immediately after UVA exposure and found that: (1) UVA-ROS production was strongly reduced in HaCaT-MC1R but not in HaCaT-R(151)C cells compared to parental HaCaT cells; (2) this inhibitory effect was further amplified by incubation of HaCaT-MC1R cells with α-MSH before UVA exposure; (3) protein kinase A (PKA)-dependent NoxA1 phosphorylation was increased in HaCaT-MC1R compared to HaCaT and HaCaT-R(151)C cells. Inhibition of PKA in HaCaT-MC1R cells resulted in a marked increase of ROS production after UVA irradiation; (4) the ability of HaCaT-MC1R cells to produce UVA-ROS was restored by inhibiting epidermal growth factor receptor (EGFR) or extracellular signal-regulated kinases (ERK) activity before UVA exposure. Our findings suggest that constitutive activity of MC1R in keratinocytes may reduce UVA-induced oxidative stress via EGFR and cAMP-dependent mechanisms.     

牛黑素皮质素受体1(MC1R)基因与毛色表形的研究

牛MC1R基因不仅与毛色有关, 而且与牛乳中乳蛋白的含量有关。利用PCR-RFLP和DNA测序技术分析了中国荷斯坦黑白花牛, 中国荷斯坦红白花牛, 鲁西黄牛和渤海黑牛共4个品种的MC1R基因。共检测出3种等位基因(E^D, E⁺, e)。中国荷斯坦黑白花牛主要是E^D和E⁺等位基因(E^D=0.12、E⁴=0.80); 渤海黑牛也主要是E^D和E⁺等位基因(E^D=0.52、E⁺=0.47); 中国荷斯坦红白花牛和鲁西黄牛大多为e等位基因(e=0.95)。中国荷斯坦红白花牛和鲁西黄牛还存在E⁺/e基因型。由此推测E^D和E⁺等位基因导致黑色素合成。另外发现牛MC1R基因编码区725处存在一重要的SNP(单核苷酸多态性)。     

Endotoxemia is associated with altered innate and adaptive immune responses in untreated HIV-1 infected individuals

Background

Microbial translocation may contribute to the immunopathogenesis in HIV infection. We investigated if microbial translocation and inflammation were associated with innate and adaptive immune responses in adults with HIV.

Methodology/Principal Findings

This was an observational cohort study. Sera from HIV-infected and HIV-uninfected individuals were analyzed for microbial translocation (soluble CD14, lipopolysaccharides [LPS], endotoxin core antibody, and anti-α-galactosyl antibodies) and inflammatory markers (high sensitivity C-reactive protein, IL-6, IL-1 receptor antagonist, soluble tumor necrosis factor receptor II, and IL-10) with enzyme-linked immunosorbent assays. Peripheral blood mononuclear cells (PBMC) from HIV-infected persons and healthy controls (primed with single-stranded HIV-1-derived RNA) were stimulated with LPS, and cytokine production was measured. Finally, HIV-infected patients were immunized with Prevnar 7vPnC±CpG 7909 followed by Pneumo Novum PPV-23. Effects of microbial translocation and inflammation on immunization were analyzed in a predictive regression model. We included 96 HIV-infected individuals, 76 on highly active antiretroviral therapy (HAART), 20 HAART-naive, and 50 healthy controls. Microbial translocation and inflammatory markers were higher among HIV-infected persons than controls. Cytokine levels following LPS stimulation were increased in PBMCs from HAART-naive compared to HAART-treated HIV-infected persons. Further, RNA-priming of PBMCs from controls acted synergistically with LPS to augment cytokine responses. Finally, high serum LPS levels predicted poor vaccine responses among HAART-naive, but not among HAART-treated HIV-infected individuals.

Conclusions/Significance

LPS acts synergistically with HIV RNA to stimulate innate immune responses in vitro and increasing serum LPS levels seem to predict poor antibody responses after vaccination among HAART-naive HIV-infected persons. Thus, our results suggest that microbial translocation may be associated with innate and adaptive immune dysfunction in untreated HIV infection.     

PTH-1R responses to PTHrP and regulation by vitamin D in keratinocytes and adjacent fibroblasts

Vitamin D and PTHrP are essential for the differentiation of keratinocytes and epidermal development. The action of PTHrP on skin is mediated via its PTH-1R receptors present in both epidermal and dermal cells. This suggests that PTHrP may have a paracrine/autocrine role, and its receptors may act in association or in negative cooperativity. We compared the intracellular signaling pathways in response to PTHrP (1-34) and to various PTHrP peptides, the N-terminal (1-34), Mid region (67-89), and C-terminal (107-139) fragments, and the possible modulation of PTHrP and its receptor mRNA expressions by vitamin D. Adjacent dermal fibroblasts as freshly isolated keratinocytes expressed both PTHrP and PTH-1R mRNAs, and responded to the various PTHrP fragments. bPTH and PTHrP(1-34) increased both cellular cAMP and [Ca(2+)]i in keratinocytes and fibroblasts. In contrast, PTHrP (107-139) increased [Ca(2+)]i but not cAMP in the two cell types. PTHrP (67-89) had no effect in keratinocytes, and only increased [Ca(2+)]i in fibroblasts. Vitamin D deficiency in weaned rats increased the expression of PTHrP mRNA in keratinocytes, and decreased it in fibroblasts and kidneys. Vitamin D deficiency increased PTH-1R mRNA expression in keratinocytes and kidneys, but not in fibroblasts. Although keratinocytes and skin fibroblasts are target cells for PTHrP and express PTH-1R, the two adjacent cell types differ as regards their intracellular signaling in response to PTHrP peptides. Moreover vitamin D regulates PTHrP and PTH-1R in a cell-specific manner.     

Identification of a major locus interacting with MC1R and modifying black coat color in an F2 Nellore-Angus population

Background

In cattle, base color is assumed to depend on the enzymatic activity specified by the MC1R locus, i.e. the extension locus, with alleles coding for black (E ^D ), red (e), and wild-type (E ⁺ ). In most mammals, these alleles are presumed to follow the dominance model of E ^D  > E ⁺  > e, although exceptions are found. In Bos indicus x Bos taurus F₂ cattle, some E ^D E ⁺ heterozygotes are discordant with the dominance series for MC1R and display various degrees of red pigmentation on an otherwise predicted black background. The objective of this study was to identify loci that modify black coat color in these individuals.

Results

Reddening was classified with a subjective scoring system. Interval analyses identified chromosome-wide suggestive (P < 0.05) and significant (P < 0.01) QTL on bovine chromosomes (BTA) 4 and 5, although these were not confirmed using single-marker association or Bayesian methods. Evidence of a major locus (F = 114.61) that affects reddening was detected between 60 and 73 Mb on BTA 6 (Btau4.0 build), and at 72 Mb by single-marker association and Bayesian methods. The posterior mean of the genetic variance for this region accounted for 43.75% of the genetic variation in reddening. This region coincided with a cluster of tyrosine kinase receptor genes (PDGFRA, KIT and KDR). Fitting SNP haplotypes for a 1 Mb interval that contained all three genes and centered on KIT accounted for the majority of the variation attributed to this major locus, which suggests that one of these genes or associated regulatory elements, is responsible for the majority of variation in degree of reddening.

Conclusions

Recombinants in a 5 Mb region surrounding the cluster of tyrosine kinase receptor genes implicated PDGFRA as the strongest positional candidate gene. A higher density marker panel and functional analyses will be required to validate the role of PDGFRA or other regulatory variants and their interaction with MC1R for the modification of black coat color in Bos indicus influenced cattle.     

Leukotriene pathway polymorphisms are associated with altered cysteinyl leukotriene production in children with acute asthma

Cysteinyl leukotrienes (cysLTs) are pro-inflammatory mediators with increasing evidence for a role in childhood acute asthma. This study examined the influence of polymorphisms in cysLT pathway genes on urinary leukotriene E₄ (uLTE₄) levels and clinical status in acute asthmatic children. Children aged 2–16 years were recruited during an asthma attack (n=205). Where possible, asthma severity scores were assigned, ALOX5AP G-336A, ALOX5 G-1708A, LTC4S A-444C and G-1072A, GPX4 C718T, and CYSTLTR1 T927C genotypes were determined and uLTE₄ was measured in acute and convalescent samples. uLTE₄ levels were higher acutely compared with convalescence (acute GM: 115.7 pg/mg creatinine; 95% CI 88.6–151.1, convalescence GM: 66.4 pg/mg creatinine; 95% CI 51.5–85.6; n=50 paired samples, p=0.003) and paired sample analysis showed genotype-specific effects with significantly increased uLTE₄ for LTC₄S-444AA (acute GM: 127.9 pg/mg creatinine; 95% CI 91.8–178.3, convalescence GM: 68.2 pg/mg creatinine; 95% CI 50.5–92.0; n=32, p=0.002), LTC₄S-1072 GG (acute GM: 126.7 pg/mg creatinine; 95% CI 95.4–168.3, convalescence GM: 78.9 pg/mg creatinine; 95% CI 59.7–104.1; n=39, p=0.019) and CYSLTR1 927 TT/T_ (acute GM: 96.8 pg/mg creatinine; 95% CI 73.8–126.9, convalescence GM: 62.4 pg/mg creatinine; 95% CI 46.8–83.3; n=28, p=0.036) but not AC/CC, GA/AA, or TC/CC/C_, respectively. When we compared the allele frequencies of the CYSLTR1 SNP between asthmatics and non-asthmatics, the 927C allele was found to be a risk allele for asthma (OR=2.13, 95% CI: 1.06–4.26, p=0.033). Genotypes were not associated with acute or convalescent uLTE₄ levels alone and neither the SNPs nor uLTE₄ correlated with acute asthma severity. Leukotriene pathway gene polymorphisms may influence the magnitude of cysLT production during an attack, yet their influence alone may not be substantial enough to alter the severity of exacerbations.     

Detecting low frequent loss-of-function alleles in genome wide association studies with red hair color as example

Multiple loss-of-function (LOF) alleles at the same gene may influence a phenotype not only in the homozygote state when alleles are considered individually, but also in the compound heterozygote (CH) state. Such LOF alleles typically have low frequencies and moderate to large effects. Detecting such variants is of interest to the genetics community, and relevant statistical methods for detecting and quantifying their effects are sorely needed. We present a collapsed double heterozygosity (CDH) test to detect the presence of multiple LOF alleles at a gene. When causal SNPs are available, which may be the case in next generation genome sequencing studies, this CDH test has overwhelmingly higher power than single SNP analysis. When causal SNPs are not directly available such as in current GWA settings, we show the CDH test has higher power than standard single SNP analysis if tagging SNPs are in linkage disequilibrium with the underlying causal SNPs to at least a moderate degree (r²>0.1). The test is implemented for genome-wide analysis in the publically available software package GenABEL which is based on a sliding window approach. We provide the proof of principle by conducting a genome-wide CDH analysis of red hair color, a trait known to be influenced by multiple loss-of-function alleles, in a total of 7,732 Dutch individuals with hair color ascertained. The association signals at the MC1R gene locus from CDH were uniformly more significant than traditional GWA analyses (the most significant P for CDH = 3.11×10⁻¹⁴² vs. P for rs258322 = 1.33×10⁻⁶⁶). The CDH test will contribute towards finding rare LOF variants in GWAS and sequencing studies.     

MC1R studies in dogs with melanistic mask or brindle patterns

Black mask is a characteristic pattern in which red, yellow, tan, fawn, or brindle dogs exhibit a melanistic muzzle which may extend up onto the ears. Melanistic mask is inherited in several breeds as an autosomal dominant trait, and appears to be a fixed trait in a few breeds of dogs. A MC1R nonsense mutation, R306ter, has been shown to cause a completely red or yellow coat color in certain breeds such as Irish setters, yellow Labrador retrievers, and golden retrievers. The amino acid sequence for the melanocortin receptor 1 gene (MC1R) was examined in 17 dogs with melanistic masks from seven breeds, 19 dogs without melanistic masks, and 7 dogs in which their coat color made the mask difficult to distinguish. We also examined nine brindle dogs of four breeds, including three dogs who also had a black mask. No consistent amino acid change was observed in the brindle dogs. All dogs with a melanistic mask had at least one copy of a valine substitution for methionine at amino acid 264 (M264V) and none were homozygous for the premature stop codon (R306ter). These results suggest that black mask, but not brindle, is caused by a specific MC1R allele.