Association of a melanocortin 4 receptor (MC4R) polymorphism with performance traits in Lithuanian White pigs

The melanocortin 4 receptor is expressed in virtually all brain regions of mammals and plays an important role in energy homeostasis. Polymorphisms in this gene may thus be related to growth and obesity. In pigs, a non‐synonymous polymorphic site was described (Asp298Asn) and demonstrated to affect cAMP production and to alter adenylyl cyclase signalling. Association studies revealed significant linkage of this mutation with production trait in pigs. In this study, 207 Lithuanian White pigs were genotyped at the MC4R locus and analysed on relationships between genotype and breeding values for several performance traits. The observed allele and genotype frequencies did not deviate significantly from Hardy–Weinberg equilibrium (wildtype allele 0.59; mutant allele 0.41) and are comparable with those described in other Large White populations. The mutant Asn298 allele of the MC4R gene was significantly associated with increased test daily gain, higher lean meat percentage and lower backfat thickness. There was a trend towards an improved feed conversion ratio (p = 0.065) in animals with the mutant allele whereas no significant effect was found on lifetime daily gain. These results indicate that the MC4R polymorphism should be integrated in selection programmes in the Lithuanian White to improve carcass composition.     

Pharmacological analyses of two naturally occurring porcine melanocortin-4 receptor mutations in domestic pigs

The melanocortin-4 receptor (MC4R) is critical in regulating mammalian food intake and energy expenditure. Numerous mutations in the MC4R gene have been identified from obese humans. So far two naturally occurring porcine MC4R (pMC4R) mutations, D298N and R236H, have been identified from various strains of pigs and D298N is being utilized as a genetic marker to screen performance traits of pigs. In this study, we performed functional analyses of pMC4R D298N and R236H, including their ligand binding and signaling properties in transiently transfected HEK293T cells. Ligand binding assays showed that both D298N and R236H pMC4Rs had similar binding capacities and affinities for the natural agonist -MSH and the natural antagonist Agouti-related protein as wild-type pMC4R. In signaling assays, both mutants had normal EC₅₀ and maximal signaling to -MSH. In summary, pMC4R mutants D298N and R236H do not have any overt functional defects; therefore we suggest caution using these mutations as selection markers in breeding programs.     

Association between melanocortin 4 receptor (MC4R) gene haplotypes and carcass and production traits in Italian Large White pigs evaluated with a selective genotyping approach

Melanocortin 4 receptor (MC4R) plays a key role in controlling energy homeostasis. Several studies have already reported effects on production traits of polymorphisms identified in the porcine MC4R gene. In this study we analysed data on 6 MC4R polymorphisms (c.-780C>G; c.-135C>T; c.175C>T; c.707G>A or p.Arg236His; and c.892G>A or p.Asp298Asn; c.?430A>T) genotyped from (1) two groups of Italian Large White pigs (280+280 animals) with extreme estimated breeding values (EBVs) for back fat thickness (BFT), selected among a performance tested population of about 12,000 pigs, and from (2) 19 Italian Duroc pigs. Two haplotypes, differentiated by the c.892G>A, were identified in the Duroc populations. Four haplotypes were identified in the Italian Large White population, one of which (haplotype 4) was identified for the first time in this study. Single marker and haplotype association analyses for BFT were obtained by comparing allele and haplotype frequency differences from the two extreme tails using χ² and Cochran–Armitage trend tests. Results confirmed the effects of the c.892G>A single nucleotide polymorphism (SNP) on BFT, as also defined by different distributions in the two tails of haplotypes carrying the alternative nucleotides at this polymorphic site (P<0.01). In addition different distributions of haplotype 4 in the two extreme groups suggested that it might affect the same trait (P<0.10). Association analyses for several other traits (average daily gain, ADG; feed gain ratio, FGR; weight of lean cuts; ham weight) were carried out by using EBVs and Random Residuals: significant effects (P<0.05) were only found for the p.Asp298Asn mutation on ADG and FGR. Results did not support any relevant effect of the p.Arg236His mutation on any trait. Data reported in this study contribute to better understand the role of MC4R variants in affecting production traits in pigs, a prerequisite to consider polymorphisms in this gene for marker assisted selection.     

Nucleotide sequence polymorphisms of beta1‐, beta2‐, and beta3‐adrenergic receptor genes on Jinhua,Meishan, Duroc and Landrace pigs

The full amino acid coding sequences of adrenergic receptor genes beta1, beta2, and beta3 (ADRB1, ADRB2, and ADRB3)were determined for Jinhua, Meishan, Duroc and Landrace pigs. Non‐synonymous substitution of Arg458Pro was found in the porcine ADRB1 gene, resulting in a 469 amino acid sequence. Continuous substitutions of Asn29Asp and Glu30Gln were found in the porcine ADRB2 gene, resulting in a 418 amino acid sequence. Additionally, a Lys30 polymorphism of the ADRB2 gene was found in the Jinhua pigs. There were three non‐synonymous substitutions of Asn24Thr, Arg264Gln and Asn398Asp on the porcine ADRB3 gene. A thymine insertion in the ADRB3 gene, resulting in a protein with two fewer amino acids, was found in the Jinhua and Meishan pigs. To assess the effect of ADRB polymorphisms on porcine subcutaneous fat layer thickness, we calculated the genetic frequency of the variants in fatty and lean groups, each consisting of 24 pigs that were crossbreds of Duroc and Jinhua pigs. The effect of the ADRB3 gene polymorphism was not evaluated, because there was insufficient variation on the ADRB3 gene in the examined groups. Although Fisher's exact test showed no significant difference in the frequency of ADRB1 and ADBR2 variants between the two groups, the Arg458 variant of ADRB1 was higher (P = 0.11) in the lean group, and pigs in that group had a thinner fat layer than did those with the Pro458 variant. These results imply a possibility of ADRB1 polymorphism as a minor factor in porcine fat layer thickness. The Asp29 variant of ADRB2 was higher in the lean group (P = 0.11), and the Glu30 variant was higher in the fatty group (P = 0.15), but the Asp29 variant was found only in the Chinese pigs. Thus, the effect of ADRB2 polymorphisms was not clear in this study.     

吐鲁番黑羊MC1R基因序列分析研究

为研究黑皮质素受体1（MC1R）基因对吐鲁番黑山羊毛色的影响,试验采用PCR扩增和测序技术,对吐鲁番黑羊MC1R基因编码区核苷酸序列及其与毛色的相关性进行了研究。结果表明：MC1R基因编码区核苷酸序列954 bp,编码317个氨基酸,编码区存在2个错义突变（c.218 T〉A,p.73 Met〉Lys;c.361 G〉A,p.121 Asp〉Asn）和3个同义突变（c.429 C〉T,p.143 Tyr〉Tyr;c.600 T〉G,p.200 Leu〉Leu;c.735 C〉T,p.245 Ile〉Ile）。SNPs分析表明,单倍型AATGT数量占62%。初步认为MC1R基因可以作为吐鲁番黑羊黑色被毛调控的候选基因。     

大白猪MC4R、CDC16基因多态性及其与生长性状的关联分析

本研究旨在利用高分辨率熔解曲线(high resolution melt,HRM)分型方法检测大白猪黑素皮质素受体(melanocortin-4 receptor,MC4R)和细胞分裂周期蛋白16(cell division cycle 16,CDC16)基因的多态性,并对其与大白猪的生长性状进行关联性分析。试验共采集246头大白猪的耳组织样提取基因组DNA,对小群体样本进行PCR扩增后,通过测序方法寻找两个基因在该群体中的多态性位点,针对已发现的多态性位点设计HRM专用引物,利用HRM方法对大群体进行多态性检测,将所得结果与大白猪生长性状进行关联性分析。结果表明,在MC4R基因序列中检测到1个突变位点2207A>G,2个等位基因:A和G,3个基因型:AA、AG和GG;在CDC16基因第18外显子上检测到了1个突变位点837T>C,2个等位基因:T和C,2种基因型:TT和CT。χ~2适合性检验结果显示,2个基因的基因型频率和等位基因频率在2个种群中分布差异不显著,符合Hardy-Weinberg平衡定律(P>0.05)。多态信息含量(PIC)分析显示,MC4R基因在2个群体中均处于中度多态(0.25相似文献   

猪毛色基因黑色素受体1的基因型分析

为检测黑色素受体1(melanocortin receptor 1,MC1R)基因型在不同毛色猪种中的分布,研究该基因在猪毛色决定中的地位和作用,本试验使用PCR-SSCP和PCR-RFLP方法对军牧1号白猪、杜洛克猪、西藏小型猪和大白猪的MC1R基因型进行了检测。结果显示,长白猪、大白猪存在nt894insCC和G1197A突变;杜洛克猪存在G668C、C1318T和G1554A突变;西藏小型猪存在C1318T和G1554A突变,而在nt894insCC位点则表现出其他基因型。通过对4个猪种的MC1R基因型的检测,为研究MC1R基因对毛色的作用机理奠定了基础。     

Genetic variation within coat color genes of MC1R and ASIP in Chinese brownish red Tibetan pigs

Melanocortin receptor 1 (MC1R) and agouti signaling protein (ASIP) are two major genes affecting coat color phenotypes in mammals, and inactivation mutations in the MC1R gene are responsible for red coat color in European pig breeds. Conversely, the gain‐of‐function ASIP mutations block MC1R signaling and lead to the production of red pheomelanin. Chinese Tibetan pigs have three types of coat color phenotypes, including brownish red, solid black and black with patches of brownish red and white. Herein, we investigated variations of the MC1R and ASIP genes in Tibetan pigs. The results showed that the brownish red Tibet pig had the dominant black MC1R allele (E^D1). No loss‐of‐function mutation in MC1R responsible for red coat color in European breeds was observed in this breed. No causal mutation for the red coat color phenotype was found in the coding sequence of the ASIP gene. A novel missense mutation c.157G > A was firstly identified in exon 2 of ASIP, which was further genotyped in 285 pigs from five Chinese breeds and three Western breeds having different coat color phenotypes. Nearly all pigs were GG homozygotes. In conclusion, no functional variant responsible for brownish red coloration was found in the coding region of MC1R and ASIP in Tibetan pigs.     

Pharmacological characterization of canine melancortin-4 receptor and its natural variant V213F

Dogs have become one of the most important companion animals in modern society. However, it is estimated that 20% to 40% of owned dogs are obese, suggesting that obesity has become one of the most important canine health problem. In addition, obesity in dogs also leads to type II diabetes. Because the melanocortin-4 receptor (MC4R) has been shown to be essential in maintaining energy homeostasis in several different species, including rodents and humans, we initiated studies toward elucidating the roles of MC4R in obesity pathogenesis in dogs. Canine MC4R has been cloned, and a missense variant V213F was identified. We designed primers and successfully cloned canine MC4R and generated the variant V213F by site-directed mutagenesis. The objective of this study was to investigate the pharmacological properties of canine MC4R and its natural variant V213F. We measured ligand binding and signaling properties with the use of both natural and synthetic ligands. Human MC4R was also included in the experiments for comparison. Both wild-type canine MC4R and its natural variant V213F functioned normally in terms of binding and signaling. Of the ligands we used, [Nle⁴, D-Phe⁷]-α-melanocyte-stimulating hormone is the most potent ligand. We conclude that the cloned canine MC4R is a functional receptor, and the natural variant V213F does not have any functional defect and therefore is not likely to cause obesity in dogs.     

Genetic alterations of KIT during clonal expansion and subsequent acquisition of resistance to toceranib in a canine mast cell tumor cell line

One of the potential mechanisms underlying acquired resistance to toceranib in canine mast cell tumor (MCT) is the emergence of a secondary mutation in the KIT gene. Here, genetic alterations of KIT during clonal expansion and subsequent acquisition of resistance to toceranib were investigated in the toceranib‐susceptible canine MCT cell line VI‐MC, which carries a KIT‐activating mutation resulting in a predicted p.(Asn508Ile) amino acid change in the receptor tyrosine kinase protein KIT. Two sublines were cloned from VI‐MC and toceranib‐resistant sublines then were established by continuous exposure to toceranib. The mutation status of KIT in parental VI‐MC and its sublines was investigated using next‐generation sequencing (NGS). Additionally, effects of secondary mutations on toceranib sensitivity in p.(Asn508Ile)‐mutant KIT were examined. KIT secondary mutations, including those encoding p.(Asn679Lys)‐, p.(Asp819Val)‐, and p.(Asp819Gly)‐mutant KIT, that confer toceranib insensitivity to p.(Asn508Ile)‐mutant KIT emerged only in toceranib‐resistant VI‐MCs. These mutations were not detected by NGS in the parental VI‐MC line or in the toceranib‐naive cloned VI‐MCs, although the parental line and sublines exhibited genetic heterogeneity in KIT that may have been caused by genetic evolution during clonal expansion. VI‐MC clones with these secondary mutations in KIT appear to have arisen from subclones during treatment with toceranib rather than being pre‐existing. However, further study using a higher resolution technique will be needed to confirm the developmental mechanism of KIT secondary mutation in canine MCT cells with acquired resistance to toceranib.     

Structural analysis and haplotype diversity in swine LEP and MC4R genes.

Knowledge about structural variation of candidate genes could be important to improve breeding selection scheme and preserve genetic variability in livestock species. Leptin (LEP) and melanocortin-4 receptor (MC4R) genes are involved in the energetic pathway and are obvious candidate genes for fatness. By sequencing LEP and MC4R genes in 72 pigs belonging to lean (Large White and Duroc), fat (Meishan and Casertana) breeds and also Wild Boar, 98 polymorphic sites, of which 91 were novel, were found in the Leptin sequence while only the previously described mutation was found in the MC4R gene. A total of 18 LEP haplotypes were observed and their distribution was unequal among the breeds. The phylogenetic analysis showed two haplotype branches distinguishing between lean and fat breeds.     

四环素诱导的猪黑素皮质激素受体-4基因真核表达载体的构建

猪黑素皮质激素受体-4(melanocortin-4 receptor,MC4R)基因是影响猪生长肥育的主效基因之一,为了进一步研究MC4R基因的生物学功能,制备高成活率转基因猪,通过采用条件性诱导表达载体系统,PCR方法扩增了猪MC4R基因完整编码区,四环素诱导表达系统(Tet-on)的启动子调控区与转录调节元件。采用双酶切和连接等方法将上述元件连接到pcDNA3.1(+)上,构建了重组质粒pcDNA3.1(+)-rtTA-P_Tight-MC4R。经双酶切和测序鉴定,结果显示片段正向连接且片段全长测序正确。试验成功构建了四环素诱导的pcDNA3.1(+)-rtTA-P_Tight-MC4R真核表达载体, 并能在时间特异性方面调控MC4R基因的表达。     

新民猪RYR1和MC4R基因的多态性检测分析

为了调查兰尼定1型受体(RYR1)基因和黑素皮质素受体4(MC4R)基因[其目前公认的数量性状基因座(QTN)]在新组建的民猪群体内的分布情况,试验采用PCR-RFLP的方法,对黑龙江省农业科学院畜牧研究所收集整理的23头民猪的RYR1基因和MC4R基因进行检测,并与其他已报道的猪种进行比较。结果表明:RYR1基因的N等位基因频率为86.96%,n等位基因频率为13.04%;MC4R的A等位基因频率为76.19%,G等位基因频率为23.81%,与其他猪种相比,n等位基因频率偏高。     

Mutations in the melanocortin 1 receptor, β-defensin103 and agouti signaling protein genes, and their association with coat color phenotypes in Akita-inu dogs

To identify factors that control coat color in Akita-inu dogs, we sequenced all the exons of the melanocortin 1 receptor (MC1R), β-defensin103 (CBD103) and agouti signaling protein (ASIP) genes of dogs with four distinct coat colors, namely, brindle, sesame, red and white. Then we examined correlations among specific alleles and coat color. In the case of the MC1R gene, all white dogs were homozygous for a nonsense mutation, R306ter, while brindle, sesame, and red dogs had at least one R306 allele. In the case of the CBD103 gene, all brindle dogs were heterozygous for the G23del mutation (deletion of codon 23, encoding glycine), while all sesame and red dogs were homozygous for G23. In the case of the ASIP gene, all dogs, regardless of coat color, had at least one S82 H83 allele. A missense mutation in the ASIP gene, P87L, was identified for the first time in some Akita-inu dogs but was not associated with any specific coloration. Our results indicate that the 2 key mutations, R306ter in the MC1R gene and G23del in the CBD103 gene, are associated with the phenotypic discriminations among brindle, red/sesame, and white coats, while no mutation that might potentially be associated with the discrimination of a sesame coat from a red coat is present in the coding sequences of these three genes.     

猪肉质性状功能基因在野猪群体内的变异研究

本试验旨在研究猪肉质性状功能基因突变位点在野猪群体里面的遗传变异规律。以MC4R基因(D298N G>A)、RYR1基因(c.1843 C>T)、PRKAG3基因(1849 G>A)和IGF2基因(3072 G>A)突变位点为基础,通过PCR-RFLP、PCR-SSCP技术,对72头圈养野猪群体进行突变位点的多态性检测和群体遗传变异分析。结果表明,RYR1基因在c.1843 C>T突变位点的CC基因型为野猪群体内的优势基因型,等位基因C是野猪群体内的优势基因,没有TT基因型,该突变位点处于不平衡状态(P<0.01和P<0.05)。在MC4R基因D298N G>A突变位点上,GG基因型为野猪群体内的优势基因型,等位基因G是野猪群体内的优势基因。而PRKAG3基因(1849 G>A)的突变位点检测结果是GG基因型为野猪群体内的优势基因型,等位基因G是野猪群体内的优势基因,没有发现AA基因型。IGF2基因3072 G>A在野猪群体内没有发现多态性,经测序验证全部为GG基因型。这3个基因突变都处于Hardy-Weinberg平衡状态(P>0.05)。本研究得出该野猪种群体遗传多样性和变异较小,符合其品种特性。     

不同羽色鸽种MC1R基因的扩增与序列分析

黑素皮质素受体1(Melanocortin-1 Receptor,MC1R)是一种7跨膜结构G蛋白耦联受体,在鸟类羽色形成中起着重要作用。为探讨MC1R对太湖鸽特殊羽色形成的影响,本实验利用PCR和直接测序法对太湖鸽、乌鸽和白卡奴鸽MC1R基因编码区进行扩增,并对不同鸽种MC1R基因序列进行差异性分析。结果表明:鸽MC1R基因编码区全长942 bp,共编码313个氨基酸,存在7个跨膜结构;白卡奴鸽与乌鸽的MC1R基因编码区核酸序列基本一致,黑羽和棕羽太湖鸽的MC1R基因序列没有差异,而太湖鸽与其他2个鸽种分别于279bp(A>G)和520bp(G>A)处存在碱基差异,其中G520A造成了氨基酸(Ser174Gly)的改变,推测该位点突变与太湖鸽特殊羽色形成有关。     

Effects of weaning and syndyphalin-33 on expression of melanocortinergic appetite-regulating genes in swine

Syndyphalin-33 (SD-33) increases feed intake in sheep and recently weaned pigs. To assess the effects of SD-33 on hypothalamic gene expression, hypothalami were collected from unweaned pigs (n=19; 21±3 d of age) on day 0. Remaining pigs received an intramuscular injection of 0.5 μmole/kg SD-33 (SD) or saline (VEH) and weaned into individual pens. On days 1, 4, and 7 after weaning, hypothalami were collected from subsets of pigs (n=8 or 9) within each treatment group. Expression of μ-opioid receptor (MOR) was less in SD pigs than in VEH pigs on day 1 and day 4, suggesting down-regulation of the receptor by SD-33. Expression of hypothalamic melanocortin 4 receptor (MC4R) at 1 d after weaning was increased in VEH pigs (but not SD pigs) relative to levels before weaning. Expression of AGRP was not significantly altered by weaning or treatment at 1 d after weaning. At 4 d after weaning, expression of AGRP was greater in SD pigs than in VEH pigs, but at day 7 expression was less in SD pigs than in VEH pigs. A strong positive correlation was noted between expression levels of MOR and MC4R across treatment and time. Treatment with SD-33 appeared to partially abrogate the effects of weaning on expression of two key appetite-regulating genes within 24 h. Effects of SD-33 appear to be mediated at least in part by the μ-opioid receptor and include actions on the melanocortinergic pathway.     

陆川猪MC1R和KIT基因型鉴定

为了检测陆川猪种群的纯度,本研究以4个华南地方猪种（莆田黑猪、粤东黑猪、大花白猪、巴马香猪）和3个外来猪种（杜洛克猪、大白猪、长白猪）作为对照,利用PCR测序法对陆川猪群体56个样本的MC1R和KIT基因进行基因型鉴定。测序结果表明,大白猪和长白猪KIT基因内含子17的第1个碱基发生G>A剪接突变,而陆川猪和4个华南地方猪种,以及杜洛克猪中KIT基因型一样,均为野生型的GG基因型;MC1R基因编码区分析表明,以海南野猪MC1R基因型作为参考序列,陆川猪和4个华南地方猪种在MC1R基因编码区存在95Val > Met和102Leu > Pro两个错义突变;MC1R基因非编码区分析表明,与大白猪、长白猪和杜洛克猪相比,陆川猪和4个华南地方猪种MC1R基因的5'UTR分别存在5~6个SNPs,3'UTR存在1个A碱基的缺失。另外,试验对一窝毛色异常的陆川猪仔猪及其亲本的KIT和MC1R基因进行测序分析,结果表明,仔猪及其亲本的KIT基因型均为GG野生型,仔猪和母本的MC1R基因均存在E^D1和E^p两种不同的基因型,父本的MC1R基因型为E^D1,因E^p对应于皮特兰猪的斑块表型,所以初步判定母本渗入了外来猪种的血统。本研究通过检测陆川猪等8个猪种的毛色相关基因KIT和MC1R基因的基因型,证实了中外猪种间在毛色遗传上的分子差异,为今后深入研究猪毛色遗传的分子调控机理提供参考。     

乌骨绵羊MC1R基因多态性研究

酪氨酸酶(TYR)、酪氨酸酶相关蛋白1(TYRP1)和酪氨酸酶相关蛋白2(TYRP2)是动物黑色素生物合成过程的重要酶,黑素皮质素1受体(MC1R)一旦与配体α-促黑素细胞激素(α-MSH)结合,对TYR、TYRP1和TYRP2酶活性的表达水平均有重要影响,因此认为MC1R是黑素合成模式的控制点。本研究以MC1R基因为候选基因,对乌骨绵羊MC1R基因多态性进行了研究,希望揭示MC1R基因与乌骨绵羊乌质性状之间的关系。结果表明,乌骨绵羊MC1R基因存在2个多态位点,分别为A12G和G144C。翻译表明这2处突变都为同义突变,对应的氨基酸分别是丝氨酸(Ser)和亮氨酸(Leu)。PCR-RFLP分析发现绵羊MC1R存在2个等位基因,即MC1R*1和MC1R*2,对应11型、22型和12型3种基因型。乌骨绵羊和兰坪本地绵羊MC1R等位基因的频率差异不显著,而与罗姆尼羊差异显著,推测MC1R基因可能不是控制乌骨绵羊乌质性状的主效基因。