The SNPs of Melanocortin 4 Receptor (MC4R) Associated with Body Weight in Beagle Dogs

Melanocortin 4 receptor (MC4R), which is associated with inherited humanobesity, is involoved in food intake and body weight of mammals. To study therelationships between MC4R gene polymorphism and body weight in Beagledogs, we detected and compared the nucleotide sequence of the whole coding region and 3′-and 5′- flanking regions of the dog MC4R gene (1214 bp). In 120 Beagledogs, two SNPs (A420C, C895T) were identified and their relation with body weight wasanalyzed with RFLP-PCR method. The results showed that the SNP at A420C was significantlyassociated with canine body weight trait when it changed amino acid 101 of theMC4R protein from asparagine to threonine,while canine body weightvariations were significant in female dogs when MC4R nonsense mutation atC895T. It suggested that the two SNPs might affect the MC4R gene’sfunction which was relative to body weight in Beagle dogs. Therefore,MC4R was a candidate gene for selecting different size dogs with theMC4R SNPs (A420C, C895T) being potentially valuable as a geneticmarker.     

红鳍东方鲀(Takifugu rubripes)MC4R基因的多态性分析

采用PCR-SSCP(single strand conformation polymorphism)技术和DNA测序方法分析红鳍东方鲀MC4R(Melanocortin-4receptor)基因编码区多态性。在MC4R基因编码区48 nt和264 nt均发生了碱基的转换突变(G→A),两个突变位点分别位于M1和M2引物扩增产物中。引物M1扩增产物SSCP分析得到两种基因型:AA基因型和AB基因型,并且AA基因型和A等位基因频率明显高于AB基因型和B等位基因。引物M2扩增产物也得到两种基因型:CC基因型和CD基因型,CC基因型和C等位基因频率明显高于CD基因型和D等位基因。遗传变异结果分析表明,两个突变位点均属于低度多态性,而且群体遗传杂合度较低,反映了该群体的遗传一致性较高。     

3个猪品种黑素皮质素受体1(MC1R)基因变异研究

利用测序、PCR-RFLP和PCR-SSCP等技术对杜洛克、长白、大白猪MC1R基因进行研究发现了5个多态位点。其中,668位点G→C突变发生在5′UTR,其余4个多态位点nt894insCC(894位点CC插入),1318C→T,1554G→A和1197G→A发生在编码区。nt894insCC导致编码蛋白过早终止。1318C→T,1554G→A和1197G→A突变分别导致a164Val,Ala243Thr和Asp124Asn氨基酸的改变。所有长白、大白猪个体在894位点均存在CC插入,其余多态位点基因型分别为668GG,1197AA,1318CC,1554GG。所有杜洛克个体在894位点均不存在CC插入,其余多态位点基因型分别为668CC,1197GG,1318TT,1554AA。所有突变位点无杂合子出现。由此可以推测,668G→C,1318C→T和1554G→A可能与杜洛克的红毛色存在相关,导致1197G→A突变无意义的894位点CC插入可能与长白、大白猪白毛色存在相关。     

黑素皮质激素受体1（MC1R）基因系统发育树与犬的毛色

犬的驯养迄今约有1万多年,由于不同环境和不同目的人工选择形成了犬品种间或品种内极丰富的毛色多样性,经证实,这些犬的很多毛色类型与MC1R相关 ,MC1R在一些物种中有同源基因 本文阐述了犬MC1R多态性研究进展,并选择其它9个有代表性的哺乳动物物种与犬MC1R同源基因进行了比较,以此建立系统发育树。结果显示,10个物种的MC1R基因的分子进化关系与物种的经典分类学地位基本相符。      

野猪MC4R基因的克隆及变异初步研究

黑素皮质素受体4是在人类肥胖研究中发现的重要调节因子,参与调节动物的体重、采食量和能量稳态,缺失MC4R基因的突变纯合体小鼠出现遗传性肥胖。为了进一步揭示其群体遗传变异,寻找新的遗传标记,本研究对野猪(Sus scrofa ussuricus)MC4R基因进行了克隆(GenBank accession NoDQ388767)和序列分析,并对所发现的错义突变进行了基于限制性内切酶HindⅢ的PCR-RFLP分析。序列分析表明野猪与民猪MC4R基因的编码区序列完全相同,与大白猪相比存在4个SNPs;对14头野猪的酶切多态性分析表明该突变位点是多态位点,并且3种基因型的分布符合Hardy-Weinberg定律。结果表明,野猪具有独特的遗传信息。     

Identification of Polymorphisms in the Rabbit Growth Hormone Receptor (GHR) Gene and Association with Finishing Weight in a Commercial Meat Rabbit Line

A shortcut to identify DNA markers associated with economic traits is to use a candidate gene approach that is still useful in livestock species in which molecular tools and resources are not advanced or not well developed. Mutations in the growth hormone receptor (GHR) gene associated with production traits have been already described in several livestock species. For this reason GHR could be an interesting candidate gene in the rabbit. In this study we re-sequenced all exons and non-coding regions of the rabbit GHR gene in a panel of 10 different rabbits and identified 10 single nucleotide polymorphisms (SNPs). One of them (g.63453192C>G or c.106C>G), located in exon 3 was a missense mutation (p.L36V) substituting an amino acid in a highly conserved position across all mammals. This mutation was genotyped in 297 performance tested rabbits of a meat male line and association analysis showed that the investigated SNP was associated with weight at 70 days (P < 0.05). The most frequent genotype (GG) was in animals with higher weight at this age, suggesting that the high directional selection pressure toward this trait since the constitution of the genotyped line might have contributed to shape allele frequencies at this polymorphic site.     

猪黑素皮质素受体1(MC1R)基因与毛色表型的研究

猪的毛色表型虽然与经济性状没有直接相关,但它却对经济效益产生重要影响,在猪育种实践、商品猪生产等方面都有应用。结合PCR—AccⅡ—RFIP、PCR—BspH I—RFLP及PCR-SSCP技术,分析了16个全同胞家系和金华猪、嘉兴黑猪、玉山黑猪、乐平花猪、上高两头乌猪及嵊县花猪等6个地方猪种随机采样个体的黑素皮质素受体1(MCIR)基因型。结果显示,地方猪种在MCIR位点携带高频率的显性黑等位基因E^DI,表明我国地方猪种的黑毛色可能主要由显性黑等位基因E^DI调控。通过对嵊县花猪MCIR位点的分析,首次发现PCR-SSCP证据的新序列,与已知的其他5个等位基因带型不同。家系个体的分析结果进一步验证了E^DI对E^p、e为完全显性,E^p对e为不完全显性。     

比格犬MC4R基因多态性与体重相关性的研究

为了分析比格犬黑素皮质素受体-4基因多态性与犬体重的关系, 根据犬MC4R基因DNA外显子序列, 设计MC4R基因特异PCR引物1对, 犬DNA经PCR扩增, 克隆和测序, 寻找和确定犬MC4R基因的多态性位点, 分析多态性与犬体重的关系。结果在比格犬MC4R基因中发现2处单碱基缺失突变, 1个单碱基颠换变异, 存在Psh AⅠ酶切位点, 并基于PshAⅠ酶切位点建立了PCR-RFLP技术。统计分析显示犬MC4R基因型与体重显著相关, 可以考虑将MC4R基因作为犬体重的候选基因。     

Missense SNP of the MC1R gene is associated with plumage variation in the Gyrfalcon (Falco rusticolus)

A single nucleotide polymorphism (MC1R: c.376A>G) in the MC1R gene was found to be highly correlated with pigment phenotype in the Gyrfalcon. Homozygous genotypes c.376GG and c.376AA were found to dominate the extreme white and dark plumage types respectively, and heterozygotes occurred mainly in intermediate phenotypes. However, some heterozygotes were associated with extreme phenotypes, indicating that melanism/albinism might also involve other loci.     

H-FABP、MC4R、ADD1基因多态性在3个猪群中分布及其与肌内脂肪和背膘的相关研究

具有不同遗传特性的猪种具有不同的肉质性状。尤其是地方品种和引进品种间在肉质性状存在极大的差异。在已有的研究中H-FABP,MC4R,ADD1基因同肌内脂肪或背膘相关。利用梅山猪、苏太猪和杜×长×大猪为试验动物,研究上述3个基因的多态性分布和多态性同肌内脂肪和背膘的相关性。结果表明：3个基因的多态性分布在不同猪种间存在极显著的差异,这种差异可能是肌内脂肪（IMF）或背膘（BF）不同的主要原因之一。连锁分析表明：H-FABP和ADD1基因多态性同IMF有显著的相关,但是同BF没有显著的相关;MC4R基因的多态性同IMF和BF都有显著相关性。说明：H-FABP和ADD1基因多态性有可能应用到提高IMF,同时不影响BF的育种实践中。      

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).     

An effect of a missense mutation in the porcine melanocortin-4 receptor (MC4R) gene on production traits in Polish pig breeds is doubtful

A missense G-A SNP in the porcine melanocortin-4 receptor (MC4R) gene that causes an Asp-Asn substitution at position 298 of the corresponding MC4R protein is considered to be economically important, although published results on its effect are inconsistent. We analysed the association of this MC4R polymorphism with production traits in 679 gilts from two breeds, Polish Large White (PLW) and Polish Landrace (PL), as well as one synthetic line 990. The frequency of the A allele differed significantly among the breeds with frequencies of 0.76, 0.29 and 0.16 in PLW, PL and line 990 respectively. There was no evidence of an effect of this polymorphism on daily food intake, backfat thickness or abdominal fat. The A allele was correlated with higher test daily gains and lower levels of intramuscular fat in PL, and increased levels of intramuscular fat in PLW.     

Single nucleotide polymorphisms in the Melanocortin 1 Receptor gene are linked with lightness of fibre colour in Peruvian Alpaca (Vicugna pacos)

Melanocortin 1 receptor (alpha melanocyte stimulating hormone receptor) (MC1R) is a gene‐controlling melanogenesis in mammals. However, it is not well characterized in alpacas and its association with colour is not known. The aim of this study was to look for polymorphisms in the MC1R gene in Peruvian Huacaya alpacas and to analyse the relationship between MC1R single nucleotide polymorphisms (SNPs) and the variations in the instrumental measurement of colour of alpaca fibre. Sixty alpaca fibre samples from black, brown, cream and white animals (15 for each colour) were used to extract DNA from hair bulbs. Colour was measured with a spectrophotometer to obtain quantitative values (CieL*a*b*). Sixteen samples, four of each colour group, were sequenced. Eighteen SNP mutations, 10 not previously described, were found in these 16 sequences. Three of them were chosen (c.82A>G, c.865C>T, c.901C>T) to analyse genotypes by PCR‐RFLP in the other 44 fibre samples and to determine the association of mutations with instrumental colour. These three polymorphisms showed association with fibre lightness (P < 0.05), although there was no correlation with colour groups.     

Mutations in the melanocortin 1 receptor (MC1R) gene are associated with coat colours in the domestic rabbit (Oryctolagus cuniculus)

We sequenced almost the complete coding region of the MC1R gene in several domestic rabbits (Oryctolagus cuniculus) and identified four alleles: two wild-type alleles differing by two synonymous single nucleotide polymorphisms (c.333A>G;c.555T>C), one allele with a 30-nucleotide in-frame deletion (c.304_333del30) and one allele with a 6-nucleotide in-frame deletion (c.280_285del6). A polymerase chain reaction-based protocol was used to distinguish the wild-type alleles from the other two alleles in 263 rabbits belonging to 37 breeds or strains. All red/fawn/yellow rabbits were homozygous for the c.304_333del30 allele. This allele represents the recessive e allele at the extension locus identified through pioneering genetic studies in this species. All Californian, Checkered, Giant White and New Zealand White rabbits were homozygous for allele c.280_285del6, which was also observed in the heterozygous condition in a few other breeds. Black coat colour is part of the standard colour in Californian and Checkered breeds, in contrast to the two albino breeds, Giant White and New Zealand White. Following the nomenclature established for the rabbit extension locus, the c.280_285del6 allele, which is dominant over c.304_333del30, may be allele E(D) or allele E(S).     

猪资源家系MC4R基因扫描及其与脂肪性状的相关分析

黑素皮质激素受体－4（Melanocortin-4 Receptor,MC4R)是在人类肥胖研究中发现的重要调节因子,它可以与瘦蛋白（Leptin),神经肽Y（Neuropeptide Y,NPY),α－黑素细胞刺激激素（Alpha-melanocyte-stimulating hormone,α-MSH)第一起调节动物体重和采食量。采用PCR－RFLP技术,分析了MC4R基因部分片段在猪资源家系群体中的TaqⅠ酶切片段多态性分析。MC4R基因多态性与生长肥育性状,肉质性状,胴体性状的相关性分析的结果表明,MC4R基因型频率在不同品种群体中的分布不同;MC4R基因与猪胸腰椎间膘厚,臀部膘厚,平均背膘,眼肌宽度,眼肌面积,皮率呈显著性相关。MC4R基因主要以显性作用方式发挥作用,加性作用不显著。     

Melanocortin-4 receptor (MC4R) genotypes have no major effect on fatness in a Large White x Wild Boar intercross

The melanocortin-4 receptor (MC4R), a G-protein coupled receptor, is implicated in mediating the effect of leptin on food intake and energy balance. A previous candidate gene study reported an association between an MC4R missense mutation (Asp298Asn) and fatness, growth and feed intake in pigs. To assess this association further, we analysed the segregation of this missense mutation in relation to variation in fatness traits using a Wild Boar x Large White intercross. The Wild Boar and Large White founders were homozygous for different MC4R alleles. The MC4R was assigned to the expected region on pig chromosome 1. The statistical evaluation did not reveal any indication of a significant effect on fatness related traits in this pedigree.     

敲减MC4R表达对牛胎儿成纤维细胞CMS系统关键因子的影响

为获得敲减黑素皮质素4受体（melanocortin 4 receptor,MC4R）基因的牛胎儿成纤维细胞,并探讨其在能量平衡神经调节系统中的作用,将构建成功并已鉴定为有效序列的短发夹状RNA (short hairpin RNA, shRNA)真核表达载体pGSH1 GFP MC4R,利用阳离子脂质体转染牛胎儿成纤维细胞并使用G418筛选稳定转染细胞株.利用实时荧光定量和Western印迹检测MC4R及中枢黑素皮质素系统(central melanocortin system, CMS)关键因子的表达水平变化.结果表明,在稳定转染的牛胎儿成纤维细胞系中, MC4R表达显著抑制,瘦蛋白(leptin)和阿黑色素原(POMC)表达下调,黑素皮质素拮抗物agouti相关蛋白(AGRP)和MC3R表达上调,而神经肽Y (NPY)表达无明显改变.综上所述,本研究成功获得了敲减MC4R基因表达的牛胎儿成纤维细胞.相关基因表达水平检测结果提示, MC4R的表达水平对CMS系统中的各关键基因的表达有不同的抑制或促进影响.     

Genetic Diversity of the Melanocortin 4 Receptor (MC4R) Gene and its Association with Slaughter Traits in the Landes Goose

Melanocortin 4 receptor (MC4R) plays a crucial part in regulating feeding behavior in humans and rodents. We detected two single nucleotide polymorphisms (SNPs; c.108G → A and c.627C → T) in the goose MC4R gene and genotyped 94 Landes geese for association analysis with several carcass traits. Significant associations (P < 0.05) were obtained for c.108G → A with carcass weight, breast muscle percentage, and leg muscle percentage, and for c.627C → T with body weight, carcass weight, semi-eviscerated weight, and eviscerated weight. We re-constructed haplotypes based on the two SNPs and analyzed diplotypes in association with carcass traits, obtaining significant associations with several of the traits. These results suggest that polymorphisms in the MC4R gene could have effects on carcass traits in Landes geese. More study is required to confirm these results.     

The Asp298Asn polymorphism of melanocortin-4 receptor (MC4R) in pigs: evidence for its potential effects on MC4R constitutive activity and cell surface expression

In humans and mice, melanocortin receptor 4 (MC4R) and melanocortin receptor accessory protein 2 (MRAP2) can form a complex and control energy balance, thus regulating body weight and obesity. In pigs, a missense variant (p.Asp298Asn) of MC4R has been suggested to be associated with growth and fatness; however, the effect of Asp298Asn substitution on MC4R function is controversial, limiting its application in animal breeding. Here we examined the effect of this polymorphism on MC4R constitutive activity, cell surface expression and signaling, and its interaction with MRAP2 in pigs. We found that: (i) both pig MC4R^Asp and MC4R^Asn can be activated by its ligands (α-MSH and ACTH) and stimulate cAMP/PKA signaling pathway, as detected by pGL3–CRE–luciferase reporter assay, indicating that, like pMC4R^Asp, pMC4R^Asn is coupled to the cAMP/PKA signaling pathway; (ii) compared with pMC4R^Asp, pMC4R^Asn loses the basal constitutive activity and shows a decreased surface expression, as detected by dual-luciferase reporter assay and Nano-HiBiT system; (iii) as in other vertebrates, both pMC4R^Asp and pMC4R^Asn can interact with pMRAP2, thus decreasing receptor surface expression and enhancing ligand sensitivity, although, in contrast to pMC4R^Asp, the basal constitutive activity of pMC4R^Asn cannot be affected by pMRAP2; and (iv) RNA-seq data analysis revealed a co-expression of MC4R and MRAP2 in pig hypothalamus. Taken together, our data provide convincing evidence that Asp298Asn substitution decreases the constitutive activity and cell surface expression of MC4R or MC4R–MRAP2 complex, which may affect energy balance and be a valuable selection marker for breeding programs in pigs.