- Email: [email protected]
Associations of growth hormone secretagogue receptor (GHSR) genes polymorphisms and protein structure changes with carcass traits in sheep
Gene 505 (2012) 379–383
Contents lists available at SciVerse ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Short Communication
Associations of growth hormone secretagogue receptor (GHSR) genes polymorphisms and protein structure changes with carcass traits in sheep A. Bahrami ⁎, S.R. Miraei-Ashtiani, H. Mehrabani-Yeganeh Department of Animal Science, University college of Agriculture and Natural Resources, University of Tehran, Karaj, Islamic Republic of Iran
a r t i c l e
i n f o
Article history: Accepted 7 June 2012 Available online 23 June 2012 Keywords: Sheep GHSR gene Single-strand conformation polymorphism Single nucleotide polymorphism Growth traits Carcass traits Protein structure
a b s t r a c t Growth hormone secretagogue receptor (GHSR), a G protein-coupled receptor that binds ghrelin, plays an important role in the central regulation of pituitary growth hormone secretion, food intake, and energy homeostasis. Ghrelin receptor (GHSR) modulates many physiological effects and therefore is a candidate gene for sheep production performance. Polymorphism of the GHSR gene was detected by PCR–SSCP and DNA sequencing methods in 463 individuals. Two different structures in protein and nine single nucleotide polymorphisms (SNPs) were identified. The evaluation of the associations between these SSCP patterns with carcass traits suggests a positive effect of genotype TT and B structure on carcass weight, and body length (Pb0.05). In addition, the animal with TC had greater abdominal fat than those with TT and CC (Pb0.05) while CC genotype contributed to low blood cholesterol (P=0.04). The results confirm the hints suggesting that GHSR is a preferential target for further investigation on mutations that influence carcass trait variations. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Ghrelin, the endogenous ligand for the growth hormone secretagogue receptor (GHSR) or ghrelin receptor, is mainly secreted by the stomach in response to fasting, but it is also synthesized locally in the hypothalamus and various peripheral tissues. Ghrelin stimulates growth hormone release via a dual action on hypothalamic growth hormone‐releasing hormone cells and on pituitary somatotrophs (Korbonits et al., 2004). Ghrelin was also shown to have an orexigenic effect and to regulate energy homeostasis, leading to increased body weight and adiposity, in rodents (Tschop et al., 2000). The ghrelin stimulation of appetite involves a complex hypothalamic action through stimulation of the orexigenic neuropeptide Y/agouti-related proteinand orexin-producing neurons in the hypothalamus (Cowley et al., 2003; Korbonits et al., 2004). Although knockout animals for ghrelin and GHSR showed relatively minor changes in growth, weight and food intake under basal conditions (Sun et al., 2003; Wortley et al., 2004), more recent studies have reported that either ghrelin or GHSR deficiency leads to a resistance to diet-induced obesity (Asakawa et al., 2003; De Smet et al., 2006; Shearman et al., 2006; Vizcarra et al., Abbreviations: GHSR, growth hormone secretagogue receptor gene; SNP, single nucleotide polymorphism; PCR–SSCP, polymerase chain reaction single–stranded conformation polymorphism; QTL, quantitative trait loci; SAS, statistical analysis system; Ser, serine acid; Phe, phenylalanine acid; Arg, arginine acid; Leu, leucine acid; MAS, marker-assisted selection. ⁎ Corresponding author at: Department of Animal Science, Tehran University, Karaj, Islamic Republic of Iran. Tel./fax: +98 9187085495. E-mail address: [email protected] (A. Bahrami). 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2012.06.009
2007; Wortley et al., 2005; Zigman et al., 2005). A few studies have reported a linkage between the ghrelin locus or GHSR locus and obesity-related parameters (Luke et al., 2003; Moslehi et al., 2003; Steinle et al., 2002; Wu et al., 2002). A GHSR haplotype was also associated with an increased risk of myocardial infarction (Baessler et al., 2007). Chronically, low circulating ghrelin levels are found in obese patients compared to normal subjects, and also in subjects with insulin resistance and thus high insulin levels: this is probably explained by a direct effect of insulin (Poykko et al., 2003; Purnell et al., 2003). Eating disorders include a broad spectrum of patterns and several have been associated with obesity, for example “snacking” or “grazing” (frequent small meals, producing an almost continuous eating pattern), binge eating (sudden periodic large amounts of food intake), “overeating” (unusually large amount of food consumed at meal times), and night eating (Adair and Popkin, 2005; Cooper and Fairburn, 2003). A reduction in ghrelin levels after food intake is blunted in patients with bulimia nervosa compared to BMI-matched healthy controls (Kojima et al., 2005; Monteleone et al., 2003). Ghrelin is a 28 amino-acid peptide with a fatty acid chain modification on the N-terminal third amino acid by the ghrelin O-acyltransferase enzyme (Yang et al., 2008). Ghrelin and obestatin are encoded by the same gene and propeptide, but different post-translational processes generate two peptides with opposing functions; obestatin was originally proposed to counteract the effects of ghrelin on food intake (Zhang et al., 2005), although ghrelin's original function was depicted in terms of a strong growth hormone releasing effect (Kojima et al., 1999; Seoane et al., 2000). Five novel single nucleotide polymorphisms (SNPs) were detected in the bovine GHSR gene, indicating that the GHSR genotype was significantly
380
A. Bahrami et al. / Gene 505 (2012) 379–383
associated with birth weight and average daily gain up to 6 months of age (Zhang et al., 2009). A general review of the available genomic information uncovers a candidate gene for genetic breeding encoding the GHSR. Polymorphism in the GHSR gene, however, has not been depicted previously in sheep. We therefore screened most exons of the sheep GHSR gene to find sequence variations that might have an effect on protein structure and function and might be related to growth and carcass traits. As well, it is worth mentioning that there has not been any publication regarding the effect of protein structure on production traits.
2.4. Protein structure
2. Materials and methods
Initially, to design the structure of the protein, the sequence of the mRNA of growth hormone secretagogue receptor (GHSR) was retrieved from NCBI GenBank databases and then using the BIOEDIT 7.0 software, the mutations were induced. Later, using the Lasergene 7.1 software, it was then back translated to protein. Further, the sequence of the attained protein was compared with that of available in the UniProtKB site. In order to design the three-dimensional structure of the growth hormone, we used the PyMOL 0.97 software and SWISS-MODEL section available in the ExPASy website. Using the MEGA5 4.3 software, we managed to draw the phylogenetic tree of the relevant proteins.
2.1. Sheep and DNA sources
2.5. Statistical analysis
Genomic DNA samples were obtained from 463 sheep, which were obtained from the Industrial Slaughterhouse in Hamedan Province, Iran. The traits evaluated were back fat thickness (BFT), carcass weight (CW), triglycerides (TG), cholesterol (CL), body length (BL), abdominal fat (AF), fat tail weight (FTW) and fat tail volume (FTV). Approximately 10ml blood per sheep was collected aseptically from the jugular vein and kept in a tube containing anticoagulant EDTA (ethylene diamine tetra acetic acid). All samples were delivered back to the laboratory in an ice box. The genomic DNA was extracted from white blood cells using a salting out procedure (Miller et al., 1988). The DNA samples were dissolved in TE buffer which was made from 10 mM Tris–Cl (pH 7.5) and 1 mM EDTA (pH 8.0) and were stored at −20 °C for future use.
Associations of the animal genotypes with carcass traits were calculated by analyzing variance of quantitative traits, which included BFT, CW, TG, CL, BL, AF, FTW and FTV, using the General Linear Model of SAS (SAS Inst. Inc., Cary, NC.). The model for the analysis was:
2.2. PCR amplification The primers were designed on the basis of DNA sequence of the GHSR gene (accession: NM_001009760) using the oligonucleotide design tool Primer 5.0 software. Twenty five microliters of polymerase chain reaction (PCR) mixture was carried out in 0.5 ml PCR tubes; each tube contained 1.5 units of Taq DNA polymerase, 10 mM of Tris– HCl (pH 9), 50 mM of KCl, 1.5mM of MgCl2 and 200mM of each dNTP. To this mixture, 1 μl of each primer (50 ng/μl), 22μl of water, and 1 μl of DNA (50 ng/μl) were added. The cycling protocol was 5 min at 94 °C, 35 cycles of 94°C for 30s, annealing at 59°C for 35 s, 72 °C for 35s, with a final extension at 72°C for 10min. PCR products were electrophoretically separated on 2% agarose gel (5V/cm), stained with ethidium bromide and excised for sequencing.
− Yijk ¼ μ þ Ai þ Gj þ CGk þ B Wijk− W þ eijk where yijk was the phenotypic value of the trait; μ was the population mean; Gj was the fixed effect of the genotype; CGk was the fixed effect of the contemporary group; Ai was the fixed effect due to the jth age; and eijk was the random residual error. Gene sequences and polymorphisms were assembled and annotated using the VECTOR NTI advance 10.1.1 software (Invitrogen Corporation). Each polymorphism identified in our SNP discovery analysis was compared with the sheep NCBI dbSNP database (http://www. ncbi.nlm.nih.gov/SNP/index.html) using BLAST (Basic Local Alignment Search Tool).
2.3. Single-strand conformation polymorphism analysis Forward primer 5′-CTGGTCGGAGTGGAGCAT-3′ and reverse primer 5′‐ACTGAAGTAGAAGAGGACGAAAGA-3′, amplified PCR products of approximately 404 bp covering complete exon-2 were obtained. PCR products were resolved by SSCP analysis. Several factors were tested for each fragment in order to optimize the methodology: amount of PCR product (8 ml), diluted in 12 ml denaturing solution (A: 95% of formamide, 10 mM NaOH, 0.05% xylene cyanol and 0.05% bromophenol blue; B: same as A, plus 20 mM of EDTA), acrylamide concentration (12%), percentage of crosslinking (1.5% C and 2.5% C), presence (10%) or absence of glycerol, voltage (150 V), running time (16 h) and running temperature (4 °C). Each PCR reaction was diluted in denaturing solution, denatured at 98 °C for 10 min, chilled on ice and resolved on polyacrylamide gel. The electrophoresis was carried in a vertical unit (Bio-rad, 160′140′0.75 mm), in 1× TBE buffer. The gel was stained with 0.1% silver nitrate and visualized with 2% NaOH solution (containing 0.1% formaldehyde) (Zhang et al., 2007). The PCR fragments from different SSCP patterns were sequenced in both directions.
Fig. 1. PCR–SSCP genotypes of complete exon-2 of the growth hormone secretagogue receptor gene in Mehraban sheep.
A. Bahrami et al. / Gene 505 (2012) 379–383 Table 1 Genotypic and gene frequencies of SSCP variants of GHSR‐exon-2 in Mehraban sheep breed. GHSR-exon-2 Genotype
No.
Frequency
Allele
Frequency
TT (sample 362) TC (sample 115) CC (sample 234) Total
157 232 74 463
0.34 0.5 0.16 1
T C
0.59 0.41
3. Results
381
Table 2 Nucleotide substitutions and resulting SNPs in GHSR exon-2 sequences of Mehraban breed of sheep. Nucleotide substitution
Codon no.
AA change
Type of SNP
C/–(686) C/A(695) G/A(705) T/G(708) G/A(711) C/–(716) T/C(724) T/–(730) T/G(886)
207 210 214 215 216 217 220 222 274
– – – – – – F→S – L→R
Synonymous Synonymous Synonymous Synonymous Synonymous Synonymous Non-synonymous Synonymous Non-synonymous
3.1. Single strand confirmation polymorphism analysis We observed 3 band patterns on gel for GHSR exon-2 (Fig. 1). The three genotypes TT, TC and CC in this segment had the genotypic frequencies of 0.34, 0.5 and 0.16, respectively (Table 1). Two different alleles T and C were identified (Fig. 1). From our results, it appears that PCR–SSCP is a potential method for identifying the genetic variants. The advantage of using the SSCP technique is that by neutral polyacrylamide gel electrophoresis, we can separate two single-stranded DNA fragments in which the nucleotide sequences differ at only one position in fragments of genomic DNA (Orita et al., 1989).
3.2. DNA sequence analysis Sequences of PCR amplicons from a representative of the unique PCR–SSCP variants were analyzed and compared with NCBI reference sequence NM_001009760 (Kioka et al., 1989). Only clear chromatographs obtained from ABI sequence files were further analyzed, ensuring that the annotation and variations are not because of PCR or sequencing artifacts. Moreover, the sequences generated from duplicate samples of each SSCP variant were aligned in contigs, using Seqman (Lasergene Inc., USA) for removing any ambiguities. Phylogeny (Fig. 2) shows that all DNA sequences in different species were novel
and distinct from NCBI reference sequence (NM_001009760). different SSCP patterns TT, TC and CC of GHSR gene exon 2 shown in (Fig. 1) were sequenced in both directions, and comparisons among three nucleotide sequences of these genotypes indicated that nine base substitutions (C686−, C695A, G705A, T708G, G711A, C716−, T724C, T730− and T886G NCBI accession no. (NM_001009760)) were detected, in which T724C and T886G substitution led to an amino acid mutation (Ser>Phe and Arg>Leu) (Fig. 3) (Table 2). 3.3. Association of polymorphisms with carcass traits in sheep The genotypes at exon 2 of GHSR of 463 individuals were analyzed for correlation with phenotypic data of carcass traits. Animals with TT genotype had greater carcass weight and body length than those with TC and CC genotypes (Pb0.05) at exon 2. In addition, the animal with TC had greater abdominal fat than those with TT and CC (Pb0.05) while the CC genotype contributed to low blood cholesterol (P= 0.04). The B structure of the growth hormone secretagogue receptor significantly (pb0.05) increased the carcass weight and body length, whereas the A structure resulted in the reduction of cholesterol levels of the blood in this breed (Table 3). 4. Discussion 4.1. The absence of certain genotypes
Fig. 2. Phylogeny of growth hormone secretagogue receptor gene sequences were drawn using the ClustalW and MEGA5 methods of sequence alignment. The novel sequences revealed distinct relationships among them as well as with the other species.
We were led to engage the GHSR gene because (1) the major physiological role of GHSR appears to be in the central regulation of food intake and body weight (Pazos et al., 2008) and (2) it is located within the quantitative trait locus on chromosome 3q26–q29 related to obesity and the metabolic syndrome (Ueda et al., 1999). The combination of location and biological function makes the GHSR gene an excellent candidate gene that may impart to genetic breeding. This study found nine SNPs in exon 2 of the sheep GHSR gene, using
Fig. 3. Comparative alignment of conceptualized protein sequences (366 amino acids) based on the PCR–SSCP haplotype sequence in Mehraban sheep with NCBI reference sequence NP_001009760, drawn based on the MegAlign module of DNAstar software version 7.1.
382
A. Bahrami et al. / Gene 505 (2012) 379–383
Table 3 Association of the GHSR genotypes at exon 2 with growth traits and carcass composition (mean±S.E.). Carcass traits
Back fat thickness (mm) Carcass weight (kg) Blood triglycerides (mg/dl) Blood cholesterol (mg/dl) Body length (cm) Abdominal fat (kg) Fat-tail weight (kg) Fat‐tail volume (lit) a,b,c
Protein structure B
A
A
Genotype TT
Genotype TC
Genotype CC
2.40±0.24 31.50±0.49b 18.85±1.63 53.94±1.54b 51.37±0.54b 1.10±0.80b 3.55±0.20 3.83±0.10
1.95±0.15 29.89±0.29ab 18.09±1.01 59.37±0.93c 49.26±0.37a 1.20±0.04b 3.48±0.13 3.71±0.22
1.89±0.17 28.54±0.54a 18.50±1.20 49.83±1.10a 49.59±0.42a 0.96±0.05a 3.44±0.14 3.63±0.13
Values with different superscripts within the same row differ significantly (Pb0.05).
PCR–SSCP and DNA sequencing methods. The GHSR gene has been demonstrated to be highly preserved throughout evolution, and mutations with an effect on the protein structure have been depicted. It is likely that this receptor has an essential or even vital function in organisms, and the absence of amino acid-changing mutations is therefore surprising. This confirmed the notion that the genotypes at the two loci might be associated with carcass traits. We evaluated the association between genotypes and carcass traits in 463 Mehraban sheep. SNP analysis suggested that C686−, C695A, G705A, T708G, G711A, C716−, T724C, T730− and T886G have varied effects on carcass traits. We observed that it was significantly associated with sheep carcass weight, body length, blood cholesterol and abdominal fat and that there were significant differences between genotypes. Genotype TT resulted in a significant increase in body length and carcass weight (Pb0.05) compared with TC and CC. Moreover, TC and CC individuals tended to have better performance in other traits, such as abdominal fat and blood cholesterol. Genotype CC and TC should not be abandoned in genetic breeding for economic traits. In our study, C686−, C695A, G705A, T708G, G711A, C716−, T724C, T730− and T886G were located in the 3′ UTR of the sheep GHSR gene and showed stronger associations with body length and carcass weight. T724C and T886G substitutions lead to the changes of amino acids (Ser>Phe and Arg>Leu), which have an influence on GHSR protein structure and its biological function, but further confirmation is needed. Therefore, the SNP studied here may not be a causal mutation. It could be in linkage disequilibrium with another SNP in the GHSR gene with greater effects on the traits, which could explicate the effects seen for this SNP. A mutation of this kind could be present in, for example, promoter or intron areas that were not tested in this study. Another possibility is that the SNP might be able to change a binding site for transcription factors and, in this way, may also lead to modified expression levels of the protein product. There is evidence that decreased expression levels of GHSR lead to decreased growth hormone and IGF-I values in transgenic female mice (Shuto et al., 2002). Further investigation of the sheep GHSR gene, including
Fig. 4. The changes of amino acids and resulting changes in GHSR protein structure of Mehraban sheep.
upstream and downstream control regions, is required to elucidate the molecular mechanisms causing the quantitative trait effects. In this study, it was shown that the structure of the protein has an impact on carcass traits. The changes of Phenylalanine to Serine and Leucine to Arginine did not bring about any changes in the second structure of the protein (Fig. 4); however, it gave rise to some changes in the third structure of the protein. Here, from the phylogenic study it became clear that ruminant animals were from the same class, which shows high level of similarity in this site. Finally, one can conclude that protein structure can help as markers in selecting animals, though this notion must be further explored. Acknowledgments This work was financially supported by the University of Tehran, Iran. The authors thank all the teams who worked on the experiments and who provided technical assistance in the laboratory during this study. We also thank the anonymous reviewers whose critical comments helped in improving the manuscript. References Adair, L.S., Popkin, B.M., 2005. Are child eating patterns being transformed globally? Obes. Res. 13, 1281–1299. Asakawa, A., et al., 2003. Antagonism of ghrelin receptor reduces food intake and body weight gain in mice. Gut 52, 947–952. Baessler, A., et al., 2007. Epistatic interaction between haplotypes of the ghrelin ligand and receptor genes influence susceptibility to myocardial infarction and coronary artery disease. Hum. Mol. Genet. 16, 887–899. Cooper, Z., Fairburn, C.G., 2003. Refining the definition of binge eating disorder and nonpurging bulimia nervosa. Int. J. Eat. Disord. 34, 89–95. Cowley, M.A., et al., 2003. The distribution and mechanism of action of ghrelin in the CNS demonstrates a novel hypothalamic circuit regulating energy homeostasis. Neuron 37, 649–661. De Smet, B., et al., 2006. Energy homeostasis and gastric emptying in ghrelin knockout mice. J. Pharmacol. Exp. Ther. 316, 431–439. Kioka, N., et al., 1989. Cloning and sequencing of goat growth hormone gene. Agric. Biol. Chem. 53, 1583–1587. Kojima, M., et al., 1999. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402, 656–660. Kojima, S., et al., 2005. Altered ghrelin and peptide YY responses to meals in bulimia nervosa. Clin. Endocrinol. 62, 74–78. Korbonits, M., et al., 2004. Ghrelin — a hormone with multiple functions. Front. Neuroendocrinol. 25, 27–68. Luke, A., et al., 2003. Linkage for BMI at 3q27 region confirmed in an African-American population. Diabetes 52, 1284–1287. Miller, S.A., Dykes, D.D., Polesky, H.F., 1988. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 6, 3–4. Monteleone, P., et al., 2003. Ghrelin and leptin responses to food ingestion in bulimia nervosa: implications for binge eating and compensatory behaviours. Psychol. Med. 33, 1387–1394. Moslehi, R., et al., 2003. A genome-wide linkage scan for body mass index on Framingham Heart Study families. BMC Genet. 4, 95–97. Orita, M., et al., 1989. Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5, 874–879. Pazos, Y., Casanueva, F.F., Camia, J.P., 2008. Basic aspects of ghrelin action. Vitam. Horm. 9, 77–89. Poykko, S.M., et al., 2003. Low plasma ghrelin is associated with insulin resistance, hypertension, and the prevalence of type 2 diabetes. Diabetes 52, 2546–2553. Purnell, J.Q., et al., 2003. Ghrelin levels correlate with insulin levels, insulin resistance, and high-density lipoprotein cholesterol, but not with gender, menopausal status, or cortisol levels in humans. J. Clin. Endocrinol. Metab. 88, 5747–5752. Seoane, L.M., et al., 2000. Ghrelin elicits a marked stimulatory effect on GH secretion in freely-moving rats. Eur. J. Endocrinol. 143, 7–9. Shearman, L.P., et al., 2006. Ghrelin neutralization by a ribonucleic acid-SPM ameliorates obesity in diet-induced obese mice. Endocrinology 147, 1517–1526. Shuto, Y., et al., 2002. Hypothalamic growth hormone secretagogue receptor regulates growth hormone secretion, feeding, and adiposity. J. Clin. Invest. 109, 1429–1436. Steinle, N.I., et al., 2002. Eating behavior in the Old Order Amish: heritability analysis and a genome-wide linkage analysis. Am. J. Clin. Nutr. 75, 1098–1106. Sun, Y., Ahmed, S., Smith, R.G., 2003. Deletion of ghrelin impairs neither growth nor appetite. Mol. Cell. Biol. 23, 7973–7981. Tschop, M., Smiley, D.L., Heiman, M.L., 2000. Ghrelin induces adiposity in rodents. Nature 407, 908–913. Ueda, H., et al., 1999. Genetic analysis of late-onset type 2 diabetes in a mouse model of human complex trait. Diabetes 48, 1168–1174. Vizcarra, J.A., et al., 2007. Active immunization against ghrelin decreases weight gain and alters plasma concentrations of growth hormone in growing pigs. Domest. Anim. Endocrinol. 33, 176–189.
A. Bahrami et al. / Gene 505 (2012) 379–383 Wortley, K.E., et al., 2004. Genetic deletion of ghrelin does not decrease food intake but influences metabolic fuel preference. Proc. Natl. Acad. Sci. U. S. A. 101, 8227–8232. Wortley, K.E., et al., 2005. Absence of ghrelin protects against early-onset obesity. J. Clin. Invest. 115, 3573–3578. Wu, X., et al., 2002. A combined analysis of genomewide linkage scans for body mass index from the National Heart, Lung, and Blood Institute Family Blood Pressure Program. Am. J. Hum. Genet. 70, 1247–1256. Yang, J., et al., 2008. Identification of the acyltransferase that octanoylates ghrelin, an appetite-stimulating peptide hormone. Cell 132, 387–396.
383
Zhang, J.V., et al., 2005. Obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin's effects on food intake. Science 310, 996–999. Zhang, C.L., et al., 2007. Enhance the efficiency of single-strand conformation polymorphism analysis by short polyacrylamide gel and modified silver staining. Anal. Biochem. 365, 286–287. Zhang, B., et al., 2009. Associations of polymorphism within the GHSR gene with growth traits in Nanyang cattle. Mol. Biol. Rep. 36, 2259–2263. Zigman, J.M., et al., 2005. Mice lacking ghrelin receptors resist the development of dietinduced obesity. J. Clin. Invest. 115, 3564–3572.
Contents lists available at SciVerse ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Short Communication
Associations of growth hormone secretagogue receptor (GHSR) genes polymorphisms and protein structure changes with carcass traits in sheep A. Bahrami ⁎, S.R. Miraei-Ashtiani, H. Mehrabani-Yeganeh Department of Animal Science, University college of Agriculture and Natural Resources, University of Tehran, Karaj, Islamic Republic of Iran
a r t i c l e
i n f o
Article history: Accepted 7 June 2012 Available online 23 June 2012 Keywords: Sheep GHSR gene Single-strand conformation polymorphism Single nucleotide polymorphism Growth traits Carcass traits Protein structure
a b s t r a c t Growth hormone secretagogue receptor (GHSR), a G protein-coupled receptor that binds ghrelin, plays an important role in the central regulation of pituitary growth hormone secretion, food intake, and energy homeostasis. Ghrelin receptor (GHSR) modulates many physiological effects and therefore is a candidate gene for sheep production performance. Polymorphism of the GHSR gene was detected by PCR–SSCP and DNA sequencing methods in 463 individuals. Two different structures in protein and nine single nucleotide polymorphisms (SNPs) were identified. The evaluation of the associations between these SSCP patterns with carcass traits suggests a positive effect of genotype TT and B structure on carcass weight, and body length (Pb0.05). In addition, the animal with TC had greater abdominal fat than those with TT and CC (Pb0.05) while CC genotype contributed to low blood cholesterol (P=0.04). The results confirm the hints suggesting that GHSR is a preferential target for further investigation on mutations that influence carcass trait variations. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Ghrelin, the endogenous ligand for the growth hormone secretagogue receptor (GHSR) or ghrelin receptor, is mainly secreted by the stomach in response to fasting, but it is also synthesized locally in the hypothalamus and various peripheral tissues. Ghrelin stimulates growth hormone release via a dual action on hypothalamic growth hormone‐releasing hormone cells and on pituitary somatotrophs (Korbonits et al., 2004). Ghrelin was also shown to have an orexigenic effect and to regulate energy homeostasis, leading to increased body weight and adiposity, in rodents (Tschop et al., 2000). The ghrelin stimulation of appetite involves a complex hypothalamic action through stimulation of the orexigenic neuropeptide Y/agouti-related proteinand orexin-producing neurons in the hypothalamus (Cowley et al., 2003; Korbonits et al., 2004). Although knockout animals for ghrelin and GHSR showed relatively minor changes in growth, weight and food intake under basal conditions (Sun et al., 2003; Wortley et al., 2004), more recent studies have reported that either ghrelin or GHSR deficiency leads to a resistance to diet-induced obesity (Asakawa et al., 2003; De Smet et al., 2006; Shearman et al., 2006; Vizcarra et al., Abbreviations: GHSR, growth hormone secretagogue receptor gene; SNP, single nucleotide polymorphism; PCR–SSCP, polymerase chain reaction single–stranded conformation polymorphism; QTL, quantitative trait loci; SAS, statistical analysis system; Ser, serine acid; Phe, phenylalanine acid; Arg, arginine acid; Leu, leucine acid; MAS, marker-assisted selection. ⁎ Corresponding author at: Department of Animal Science, Tehran University, Karaj, Islamic Republic of Iran. Tel./fax: +98 9187085495. E-mail address: [email protected] (A. Bahrami). 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2012.06.009
2007; Wortley et al., 2005; Zigman et al., 2005). A few studies have reported a linkage between the ghrelin locus or GHSR locus and obesity-related parameters (Luke et al., 2003; Moslehi et al., 2003; Steinle et al., 2002; Wu et al., 2002). A GHSR haplotype was also associated with an increased risk of myocardial infarction (Baessler et al., 2007). Chronically, low circulating ghrelin levels are found in obese patients compared to normal subjects, and also in subjects with insulin resistance and thus high insulin levels: this is probably explained by a direct effect of insulin (Poykko et al., 2003; Purnell et al., 2003). Eating disorders include a broad spectrum of patterns and several have been associated with obesity, for example “snacking” or “grazing” (frequent small meals, producing an almost continuous eating pattern), binge eating (sudden periodic large amounts of food intake), “overeating” (unusually large amount of food consumed at meal times), and night eating (Adair and Popkin, 2005; Cooper and Fairburn, 2003). A reduction in ghrelin levels after food intake is blunted in patients with bulimia nervosa compared to BMI-matched healthy controls (Kojima et al., 2005; Monteleone et al., 2003). Ghrelin is a 28 amino-acid peptide with a fatty acid chain modification on the N-terminal third amino acid by the ghrelin O-acyltransferase enzyme (Yang et al., 2008). Ghrelin and obestatin are encoded by the same gene and propeptide, but different post-translational processes generate two peptides with opposing functions; obestatin was originally proposed to counteract the effects of ghrelin on food intake (Zhang et al., 2005), although ghrelin's original function was depicted in terms of a strong growth hormone releasing effect (Kojima et al., 1999; Seoane et al., 2000). Five novel single nucleotide polymorphisms (SNPs) were detected in the bovine GHSR gene, indicating that the GHSR genotype was significantly
380
A. Bahrami et al. / Gene 505 (2012) 379–383
associated with birth weight and average daily gain up to 6 months of age (Zhang et al., 2009). A general review of the available genomic information uncovers a candidate gene for genetic breeding encoding the GHSR. Polymorphism in the GHSR gene, however, has not been depicted previously in sheep. We therefore screened most exons of the sheep GHSR gene to find sequence variations that might have an effect on protein structure and function and might be related to growth and carcass traits. As well, it is worth mentioning that there has not been any publication regarding the effect of protein structure on production traits.
2.4. Protein structure
2. Materials and methods
Initially, to design the structure of the protein, the sequence of the mRNA of growth hormone secretagogue receptor (GHSR) was retrieved from NCBI GenBank databases and then using the BIOEDIT 7.0 software, the mutations were induced. Later, using the Lasergene 7.1 software, it was then back translated to protein. Further, the sequence of the attained protein was compared with that of available in the UniProtKB site. In order to design the three-dimensional structure of the growth hormone, we used the PyMOL 0.97 software and SWISS-MODEL section available in the ExPASy website. Using the MEGA5 4.3 software, we managed to draw the phylogenetic tree of the relevant proteins.
2.1. Sheep and DNA sources
2.5. Statistical analysis
Genomic DNA samples were obtained from 463 sheep, which were obtained from the Industrial Slaughterhouse in Hamedan Province, Iran. The traits evaluated were back fat thickness (BFT), carcass weight (CW), triglycerides (TG), cholesterol (CL), body length (BL), abdominal fat (AF), fat tail weight (FTW) and fat tail volume (FTV). Approximately 10ml blood per sheep was collected aseptically from the jugular vein and kept in a tube containing anticoagulant EDTA (ethylene diamine tetra acetic acid). All samples were delivered back to the laboratory in an ice box. The genomic DNA was extracted from white blood cells using a salting out procedure (Miller et al., 1988). The DNA samples were dissolved in TE buffer which was made from 10 mM Tris–Cl (pH 7.5) and 1 mM EDTA (pH 8.0) and were stored at −20 °C for future use.
Associations of the animal genotypes with carcass traits were calculated by analyzing variance of quantitative traits, which included BFT, CW, TG, CL, BL, AF, FTW and FTV, using the General Linear Model of SAS (SAS Inst. Inc., Cary, NC.). The model for the analysis was:
2.2. PCR amplification The primers were designed on the basis of DNA sequence of the GHSR gene (accession: NM_001009760) using the oligonucleotide design tool Primer 5.0 software. Twenty five microliters of polymerase chain reaction (PCR) mixture was carried out in 0.5 ml PCR tubes; each tube contained 1.5 units of Taq DNA polymerase, 10 mM of Tris– HCl (pH 9), 50 mM of KCl, 1.5mM of MgCl2 and 200mM of each dNTP. To this mixture, 1 μl of each primer (50 ng/μl), 22μl of water, and 1 μl of DNA (50 ng/μl) were added. The cycling protocol was 5 min at 94 °C, 35 cycles of 94°C for 30s, annealing at 59°C for 35 s, 72 °C for 35s, with a final extension at 72°C for 10min. PCR products were electrophoretically separated on 2% agarose gel (5V/cm), stained with ethidium bromide and excised for sequencing.
− Yijk ¼ μ þ Ai þ Gj þ CGk þ B Wijk− W þ eijk where yijk was the phenotypic value of the trait; μ was the population mean; Gj was the fixed effect of the genotype; CGk was the fixed effect of the contemporary group; Ai was the fixed effect due to the jth age; and eijk was the random residual error. Gene sequences and polymorphisms were assembled and annotated using the VECTOR NTI advance 10.1.1 software (Invitrogen Corporation). Each polymorphism identified in our SNP discovery analysis was compared with the sheep NCBI dbSNP database (http://www. ncbi.nlm.nih.gov/SNP/index.html) using BLAST (Basic Local Alignment Search Tool).
2.3. Single-strand conformation polymorphism analysis Forward primer 5′-CTGGTCGGAGTGGAGCAT-3′ and reverse primer 5′‐ACTGAAGTAGAAGAGGACGAAAGA-3′, amplified PCR products of approximately 404 bp covering complete exon-2 were obtained. PCR products were resolved by SSCP analysis. Several factors were tested for each fragment in order to optimize the methodology: amount of PCR product (8 ml), diluted in 12 ml denaturing solution (A: 95% of formamide, 10 mM NaOH, 0.05% xylene cyanol and 0.05% bromophenol blue; B: same as A, plus 20 mM of EDTA), acrylamide concentration (12%), percentage of crosslinking (1.5% C and 2.5% C), presence (10%) or absence of glycerol, voltage (150 V), running time (16 h) and running temperature (4 °C). Each PCR reaction was diluted in denaturing solution, denatured at 98 °C for 10 min, chilled on ice and resolved on polyacrylamide gel. The electrophoresis was carried in a vertical unit (Bio-rad, 160′140′0.75 mm), in 1× TBE buffer. The gel was stained with 0.1% silver nitrate and visualized with 2% NaOH solution (containing 0.1% formaldehyde) (Zhang et al., 2007). The PCR fragments from different SSCP patterns were sequenced in both directions.
Fig. 1. PCR–SSCP genotypes of complete exon-2 of the growth hormone secretagogue receptor gene in Mehraban sheep.
A. Bahrami et al. / Gene 505 (2012) 379–383 Table 1 Genotypic and gene frequencies of SSCP variants of GHSR‐exon-2 in Mehraban sheep breed. GHSR-exon-2 Genotype
No.
Frequency
Allele
Frequency
TT (sample 362) TC (sample 115) CC (sample 234) Total
157 232 74 463
0.34 0.5 0.16 1
T C
0.59 0.41
3. Results
381
Table 2 Nucleotide substitutions and resulting SNPs in GHSR exon-2 sequences of Mehraban breed of sheep. Nucleotide substitution
Codon no.
AA change
Type of SNP
C/–(686) C/A(695) G/A(705) T/G(708) G/A(711) C/–(716) T/C(724) T/–(730) T/G(886)
207 210 214 215 216 217 220 222 274
– – – – – – F→S – L→R
Synonymous Synonymous Synonymous Synonymous Synonymous Synonymous Non-synonymous Synonymous Non-synonymous
3.1. Single strand confirmation polymorphism analysis We observed 3 band patterns on gel for GHSR exon-2 (Fig. 1). The three genotypes TT, TC and CC in this segment had the genotypic frequencies of 0.34, 0.5 and 0.16, respectively (Table 1). Two different alleles T and C were identified (Fig. 1). From our results, it appears that PCR–SSCP is a potential method for identifying the genetic variants. The advantage of using the SSCP technique is that by neutral polyacrylamide gel electrophoresis, we can separate two single-stranded DNA fragments in which the nucleotide sequences differ at only one position in fragments of genomic DNA (Orita et al., 1989).
3.2. DNA sequence analysis Sequences of PCR amplicons from a representative of the unique PCR–SSCP variants were analyzed and compared with NCBI reference sequence NM_001009760 (Kioka et al., 1989). Only clear chromatographs obtained from ABI sequence files were further analyzed, ensuring that the annotation and variations are not because of PCR or sequencing artifacts. Moreover, the sequences generated from duplicate samples of each SSCP variant were aligned in contigs, using Seqman (Lasergene Inc., USA) for removing any ambiguities. Phylogeny (Fig. 2) shows that all DNA sequences in different species were novel
and distinct from NCBI reference sequence (NM_001009760). different SSCP patterns TT, TC and CC of GHSR gene exon 2 shown in (Fig. 1) were sequenced in both directions, and comparisons among three nucleotide sequences of these genotypes indicated that nine base substitutions (C686−, C695A, G705A, T708G, G711A, C716−, T724C, T730− and T886G NCBI accession no. (NM_001009760)) were detected, in which T724C and T886G substitution led to an amino acid mutation (Ser>Phe and Arg>Leu) (Fig. 3) (Table 2). 3.3. Association of polymorphisms with carcass traits in sheep The genotypes at exon 2 of GHSR of 463 individuals were analyzed for correlation with phenotypic data of carcass traits. Animals with TT genotype had greater carcass weight and body length than those with TC and CC genotypes (Pb0.05) at exon 2. In addition, the animal with TC had greater abdominal fat than those with TT and CC (Pb0.05) while the CC genotype contributed to low blood cholesterol (P= 0.04). The B structure of the growth hormone secretagogue receptor significantly (pb0.05) increased the carcass weight and body length, whereas the A structure resulted in the reduction of cholesterol levels of the blood in this breed (Table 3). 4. Discussion 4.1. The absence of certain genotypes
Fig. 2. Phylogeny of growth hormone secretagogue receptor gene sequences were drawn using the ClustalW and MEGA5 methods of sequence alignment. The novel sequences revealed distinct relationships among them as well as with the other species.
We were led to engage the GHSR gene because (1) the major physiological role of GHSR appears to be in the central regulation of food intake and body weight (Pazos et al., 2008) and (2) it is located within the quantitative trait locus on chromosome 3q26–q29 related to obesity and the metabolic syndrome (Ueda et al., 1999). The combination of location and biological function makes the GHSR gene an excellent candidate gene that may impart to genetic breeding. This study found nine SNPs in exon 2 of the sheep GHSR gene, using
Fig. 3. Comparative alignment of conceptualized protein sequences (366 amino acids) based on the PCR–SSCP haplotype sequence in Mehraban sheep with NCBI reference sequence NP_001009760, drawn based on the MegAlign module of DNAstar software version 7.1.
382
A. Bahrami et al. / Gene 505 (2012) 379–383
Table 3 Association of the GHSR genotypes at exon 2 with growth traits and carcass composition (mean±S.E.). Carcass traits
Back fat thickness (mm) Carcass weight (kg) Blood triglycerides (mg/dl) Blood cholesterol (mg/dl) Body length (cm) Abdominal fat (kg) Fat-tail weight (kg) Fat‐tail volume (lit) a,b,c
Protein structure B
A
A
Genotype TT
Genotype TC
Genotype CC
2.40±0.24 31.50±0.49b 18.85±1.63 53.94±1.54b 51.37±0.54b 1.10±0.80b 3.55±0.20 3.83±0.10
1.95±0.15 29.89±0.29ab 18.09±1.01 59.37±0.93c 49.26±0.37a 1.20±0.04b 3.48±0.13 3.71±0.22
1.89±0.17 28.54±0.54a 18.50±1.20 49.83±1.10a 49.59±0.42a 0.96±0.05a 3.44±0.14 3.63±0.13
Values with different superscripts within the same row differ significantly (Pb0.05).
PCR–SSCP and DNA sequencing methods. The GHSR gene has been demonstrated to be highly preserved throughout evolution, and mutations with an effect on the protein structure have been depicted. It is likely that this receptor has an essential or even vital function in organisms, and the absence of amino acid-changing mutations is therefore surprising. This confirmed the notion that the genotypes at the two loci might be associated with carcass traits. We evaluated the association between genotypes and carcass traits in 463 Mehraban sheep. SNP analysis suggested that C686−, C695A, G705A, T708G, G711A, C716−, T724C, T730− and T886G have varied effects on carcass traits. We observed that it was significantly associated with sheep carcass weight, body length, blood cholesterol and abdominal fat and that there were significant differences between genotypes. Genotype TT resulted in a significant increase in body length and carcass weight (Pb0.05) compared with TC and CC. Moreover, TC and CC individuals tended to have better performance in other traits, such as abdominal fat and blood cholesterol. Genotype CC and TC should not be abandoned in genetic breeding for economic traits. In our study, C686−, C695A, G705A, T708G, G711A, C716−, T724C, T730− and T886G were located in the 3′ UTR of the sheep GHSR gene and showed stronger associations with body length and carcass weight. T724C and T886G substitutions lead to the changes of amino acids (Ser>Phe and Arg>Leu), which have an influence on GHSR protein structure and its biological function, but further confirmation is needed. Therefore, the SNP studied here may not be a causal mutation. It could be in linkage disequilibrium with another SNP in the GHSR gene with greater effects on the traits, which could explicate the effects seen for this SNP. A mutation of this kind could be present in, for example, promoter or intron areas that were not tested in this study. Another possibility is that the SNP might be able to change a binding site for transcription factors and, in this way, may also lead to modified expression levels of the protein product. There is evidence that decreased expression levels of GHSR lead to decreased growth hormone and IGF-I values in transgenic female mice (Shuto et al., 2002). Further investigation of the sheep GHSR gene, including
Fig. 4. The changes of amino acids and resulting changes in GHSR protein structure of Mehraban sheep.
upstream and downstream control regions, is required to elucidate the molecular mechanisms causing the quantitative trait effects. In this study, it was shown that the structure of the protein has an impact on carcass traits. The changes of Phenylalanine to Serine and Leucine to Arginine did not bring about any changes in the second structure of the protein (Fig. 4); however, it gave rise to some changes in the third structure of the protein. Here, from the phylogenic study it became clear that ruminant animals were from the same class, which shows high level of similarity in this site. Finally, one can conclude that protein structure can help as markers in selecting animals, though this notion must be further explored. Acknowledgments This work was financially supported by the University of Tehran, Iran. The authors thank all the teams who worked on the experiments and who provided technical assistance in the laboratory during this study. We also thank the anonymous reviewers whose critical comments helped in improving the manuscript. References Adair, L.S., Popkin, B.M., 2005. Are child eating patterns being transformed globally? Obes. Res. 13, 1281–1299. Asakawa, A., et al., 2003. Antagonism of ghrelin receptor reduces food intake and body weight gain in mice. Gut 52, 947–952. Baessler, A., et al., 2007. Epistatic interaction between haplotypes of the ghrelin ligand and receptor genes influence susceptibility to myocardial infarction and coronary artery disease. Hum. Mol. Genet. 16, 887–899. Cooper, Z., Fairburn, C.G., 2003. Refining the definition of binge eating disorder and nonpurging bulimia nervosa. Int. J. Eat. Disord. 34, 89–95. Cowley, M.A., et al., 2003. The distribution and mechanism of action of ghrelin in the CNS demonstrates a novel hypothalamic circuit regulating energy homeostasis. Neuron 37, 649–661. De Smet, B., et al., 2006. Energy homeostasis and gastric emptying in ghrelin knockout mice. J. Pharmacol. Exp. Ther. 316, 431–439. Kioka, N., et al., 1989. Cloning and sequencing of goat growth hormone gene. Agric. Biol. Chem. 53, 1583–1587. Kojima, M., et al., 1999. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402, 656–660. Kojima, S., et al., 2005. Altered ghrelin and peptide YY responses to meals in bulimia nervosa. Clin. Endocrinol. 62, 74–78. Korbonits, M., et al., 2004. Ghrelin — a hormone with multiple functions. Front. Neuroendocrinol. 25, 27–68. Luke, A., et al., 2003. Linkage for BMI at 3q27 region confirmed in an African-American population. Diabetes 52, 1284–1287. Miller, S.A., Dykes, D.D., Polesky, H.F., 1988. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 6, 3–4. Monteleone, P., et al., 2003. Ghrelin and leptin responses to food ingestion in bulimia nervosa: implications for binge eating and compensatory behaviours. Psychol. Med. 33, 1387–1394. Moslehi, R., et al., 2003. A genome-wide linkage scan for body mass index on Framingham Heart Study families. BMC Genet. 4, 95–97. Orita, M., et al., 1989. Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5, 874–879. Pazos, Y., Casanueva, F.F., Camia, J.P., 2008. Basic aspects of ghrelin action. Vitam. Horm. 9, 77–89. Poykko, S.M., et al., 2003. Low plasma ghrelin is associated with insulin resistance, hypertension, and the prevalence of type 2 diabetes. Diabetes 52, 2546–2553. Purnell, J.Q., et al., 2003. Ghrelin levels correlate with insulin levels, insulin resistance, and high-density lipoprotein cholesterol, but not with gender, menopausal status, or cortisol levels in humans. J. Clin. Endocrinol. Metab. 88, 5747–5752. Seoane, L.M., et al., 2000. Ghrelin elicits a marked stimulatory effect on GH secretion in freely-moving rats. Eur. J. Endocrinol. 143, 7–9. Shearman, L.P., et al., 2006. Ghrelin neutralization by a ribonucleic acid-SPM ameliorates obesity in diet-induced obese mice. Endocrinology 147, 1517–1526. Shuto, Y., et al., 2002. Hypothalamic growth hormone secretagogue receptor regulates growth hormone secretion, feeding, and adiposity. J. Clin. Invest. 109, 1429–1436. Steinle, N.I., et al., 2002. Eating behavior in the Old Order Amish: heritability analysis and a genome-wide linkage analysis. Am. J. Clin. Nutr. 75, 1098–1106. Sun, Y., Ahmed, S., Smith, R.G., 2003. Deletion of ghrelin impairs neither growth nor appetite. Mol. Cell. Biol. 23, 7973–7981. Tschop, M., Smiley, D.L., Heiman, M.L., 2000. Ghrelin induces adiposity in rodents. Nature 407, 908–913. Ueda, H., et al., 1999. Genetic analysis of late-onset type 2 diabetes in a mouse model of human complex trait. Diabetes 48, 1168–1174. Vizcarra, J.A., et al., 2007. Active immunization against ghrelin decreases weight gain and alters plasma concentrations of growth hormone in growing pigs. Domest. Anim. Endocrinol. 33, 176–189.
A. Bahrami et al. / Gene 505 (2012) 379–383 Wortley, K.E., et al., 2004. Genetic deletion of ghrelin does not decrease food intake but influences metabolic fuel preference. Proc. Natl. Acad. Sci. U. S. A. 101, 8227–8232. Wortley, K.E., et al., 2005. Absence of ghrelin protects against early-onset obesity. J. Clin. Invest. 115, 3573–3578. Wu, X., et al., 2002. A combined analysis of genomewide linkage scans for body mass index from the National Heart, Lung, and Blood Institute Family Blood Pressure Program. Am. J. Hum. Genet. 70, 1247–1256. Yang, J., et al., 2008. Identification of the acyltransferase that octanoylates ghrelin, an appetite-stimulating peptide hormone. Cell 132, 387–396.
383
Zhang, J.V., et al., 2005. Obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin's effects on food intake. Science 310, 996–999. Zhang, C.L., et al., 2007. Enhance the efficiency of single-strand conformation polymorphism analysis by short polyacrylamide gel and modified silver staining. Anal. Biochem. 365, 286–287. Zhang, B., et al., 2009. Associations of polymorphism within the GHSR gene with growth traits in Nanyang cattle. Mol. Biol. Rep. 36, 2259–2263. Zigman, J.M., et al., 2005. Mice lacking ghrelin receptors resist the development of dietinduced obesity. J. Clin. Invest. 115, 3564–3572.