2010, Vol. 5 No. 1, Article 49

Highly Polymorphic Bovine Leptin Gene – A Review

Anjan Dandapat*¹, Dibyendu Chakraborty² and Dipak Banerjee³

^1,2Ph.D. Scholar (Animal Genetics and Breeding)
³Ph.D. Scholar (Animal Physiology)
National Dairy Research Institute,
Karnal (Haryana), India-132001

*Corresponding Author; e-mail address: [email protected]

ABSTRACT

Leptin is a 167-amino acid protein produced by the leptin gene. Mal production of leptin may cause severe hereditary obesity in animals. Regulating through a neuro endocrine pathway.it performs an important role in the control of body weight, feed intake, energy expenditure, immune function and reproduction.

KEY WORDS

Leptin gene, bovine, buffalo.

INTRODUCTION

Leptin is a 167-amino acid protein produced by the leptin gene (LEP), whose name is derived from the Greek word "leptos," which means "thin. It is a 16-kD protein that plays a critical role in the regulation of body weight by inhibiting food intake and stimulating energy expenditure. Defects in leptin production cause severe hereditary obesity in animals. It has an important role in regulation of hematopoiesis, angiogenesis, wound healing, and the immune and inflammatory response. The LEP gene is the human homolog of the gene (ob) mutant in the mouse 'obese' phenotype (Zhang et al., 1997).
Since the bovine leptin gene has been identified on chromosome 4, several SNPs have been previously identified in introns and exons of leptin among different breeds of cattle. The physiological role and biology of leptin is well reviewed (Hossner, 1998 and Houseknechcht et al., 1998). The polymorphic studies on bovine leptin gene have been reported (Pomp et al., 1997; Haegeman et al., 2000; Lien et al., 1997 and Wilkins and Davey, 1997).

Recent research on Cattle

A restriction fragment length polymorphism was identified within PCR amplification products of 1820bp of bovine leptin gene using Sau3AI restriction enzyme (Pomp et. al., 1997). Frequencies of A allele in various breeds of cattle were 0.7 in Limousin (n=5), 0.79 in Simmental (n=9), 0.82 in Gelbvich (n=17), 0.71 in Holstein (n=14), 0.5 in Hereford (n=16), 0.73 in Angus (n=15), 0 in Brahman (n=4), and 0.6 in Brangus (n=4).
Lien et al. (1997) has identified two polymorphisms in the intron 2 of bovine leptin gene in Norwegian breed. A 522bp fragment was amplified and digestion with HinfI restriction enzyme revealed fragments of 361 and 161bp with C in position whereas fragments 361, 142 and 19bp were produced with T in the same position. BsaAI digestion of the same PCR product generated fragments 441 and 81bp with G in position whereas undigested single fragment of 522bp was obtained with A in the same position.
Wilkins and Davey (1997) reported a polymorphic microsatellite in the 5' UTR region of the bovine leptin gene. The microsatellite was highly polymorphic with 18 alleles.
QTL for milk production traits is present on fourth chromosome (Lindersson et al., 1998). They found QTL for milk, fat and protein yield at 65 and 85 cM, and for fat and protein percentage at 75 and 95 cM, respectively. However, when these authors tested for a direct effect of the obese locus with BM1501 microsatellite, no associations were found with milk production traits.
Fitzsimmons et al. (1998) characterized the BM 1500 microsatellite, near obese gene in 158 purebred beef bulls (Angus, Charolais, Hereford, and Simmental). Lengths of four alleles were approximately 138, 147, 149, and 140bp with genotypic frequencies of 0.47, 0.44, 0.09, and 0.003 respectively. The 149bp allele was found in low numbers, and no homozygote was identified. Hereford and Angus bulls had the greatest frequencies of 138bp alleles (Hereford = 0.57, Angus = 0.59), while Charolais and Simmental had a greater proportion of 147bp alleles (Charolais = 0.54, Simmental = 0.58). The carcass traits such as rib fat (%), rib lean (%), average fat, and grade fat were found to be significantly associated with the different alleles. The presence of the 138bp allele in the genotype of an animal was correlated with higher levels of fat, whereas the 147bp allele had the opposite effect.
A single nucleotide substitution causes an amino acid change in the leptin molecule, which was consistently different between fat and thin animals (Fitzsimmons et al., 1999).
Konfortov et al. (1999) identified twenty single nucleotide polymorphisms by manual scanning of sequence chromatograms and computerized sequence analysis of 1788bp containing introns and exons of the bovine leptin gene giving a frequency of 1 SNP per 89bp.
Tessanne et al. (1999) studied relationship of polymorphisms in bovine leptin gene with differences in beef carcass traits.
A base substitution, C→T, which results in the replacement of alanine by valine in the leptin protein and an A→G mutation that would result in the replacement of glutamine by arginine has been reported (Haegeman et al., 2000). The frequency of the C→T mutant allele had been studied by PCR-RFLP using HphI restriction enzyme and found to be 0.287, 0.616, 0.245, 0.296, 0.125, 0.113, 0.211, 0.482, 0.264 and 0.24 in Belgium Blue, Red Pied, Red Holstein, Black Holstein, Belgium Blue crossbred, Limousin, Blonde d’ Aquitaine, Red West Flanders, Piedmonte and Charolais breeds of cattle, respectively.
Effect of genetic variation at several loci (leptin, growth hormone, kappa casein, beta lactoglobulin, and Pit-1) and their allelic effects on growth and carcass traits have been reported in beef cattle (Zwierzchowski et al., 2001). The leptin gene polymorphism was shown to affect feed intake, conversion as well as some carcass traits.
No differences in the allele frequencies of A, B, and C alleles of leptin gene have been reported in Polish Red and Polish Black and White cattle (Klauzinska et al., 2001).
Glazko et al. (2002) reported the presence of C-variate of the leptin gene at a frequency of 0.022 in White Head cattle and at a frequency of 0.125 in Polish red cattle.
A cytocine (C) to thymine (T) transition that encode an amino acid change of an arginine to a cysteine was identified in exon 2 of the bovine leptin gene. PCR-RFLP was designed and allele frequencies in four beef breeds were correlated with levels of carcass fat (Buchanan et al., 2002). The frequencies of T allele in different breeds of cattle were found to be 0.58 in Angus (n = 60), 0.34 in Charolais (n = 55), 0.55 in Hereford (n = 22) and 0.32 in Simmental (n = 17). The average fat and grade fat were significantly affected by the genotypes. It was suggested that the T allele, which adds an extra cysteine to the protein, imparts a partial loss of biological function and hence could be the causative mutant.
Liefers et al. (2002) genotyped 613 cows of Holstein Friesian breed by using PCR-RFLP technique for studying association between leptin gene polymorphisms and production, live weight, energy balance, feed intake and fertility traits. Significance of the genotype effects was estimated using the approximated F-statistics provided by ASREML. Cows with AB genotype produce 1.32 kg/day more milk and consume 0.73 kg/day more food compared to the AA genotype. Protein yield and lactose yield were also significantly associated with the genotypes.
Buchanan et al. (2003) genotyped 416 Holstein cows by using restriction enzyme Kpn21 and compared lactation performance data using a mixed model. Animals homozygous for the T allele produced more milk and had higher somatic cell count linear scores, without significantly affecting milk fat or protein percent over the entire lactation.
Five short tandem repeats (STRs) and four RFLPs in the leptin gene in 160 females of synthetic beef cattle breed were reported (Almeida et al., 2003). A high level of genetic diversity was observed, STRs being more variable than RFLPs. Heterozygosities ranged from 0.67 to 0.87 in STRs and from 0.12 to 0.49 in RFLPs. Two alleles (IDVGA51*181 and LEPsau3AI*+) were reported to increase calving interval by about 79 and 81 days, respectively. Heterozygotes (LEPsau3AI*+/ LEPsau3AI*-) had higher weight at first calving than the homozygotes (LEPsau3AI*-/ LEPsau3AI*-).
Lagonigro et al. (2003) screened an experimental cattle population for polymorphisms in the leptin gene and five SNPs were found in the regions containing the coding sequences. The results suggest an association between a polymorphism in exon 2 and feed intake.
HphI polymorphism had a significant effect on milk and protein yield (Madeja et al., 2004). Animals with the TT genotype had approximately 2X higher estimated breeding values for milk and protein yields. They did not find associations between the Kpn21 and Sau3AI polymorphisms and production traits, in contrast with the results published by Liefers et al. (2002). Liefers et al. (2002) did not find any effect of the HphI polymorphism, although they pointed to Sau3AI as a possible marker for milk and protein yield.
Barendse et al. (2004) studied the association of single nucleotide polymorphisms (SNP) in the leptin gene with marbling, fatness including backfat thickness, efficiency of production as well as milk and milk protein yield. However, they found no association between SNP and fatness traits.
The association of leptin gene polymorphism with growth traits and reproduction traits was reported in crossbred and indicine cattle (Choudhary, 2004). Leptin genotypes had significant effect on body weight up to one year of age, milk production and age at first calving. Leptin gene fragment of 330bp and 94bp were polymorphic in crossbred and exotic cattle and monomorphic in indicine cattle. However, leptin gene fragment of 522bp was polymorphic in indicine, crossbreds and exotic cattle.
Fifty seven SNPs in leptin gene including thirty six novel and twenty one known SNPs were found in Korean cattle (Yoon et al., 2005). Allelic frequencies were compared among different breeds of cattle.
Schenkel et al. (2005) evaluated the association of SNP in bovine leptin gene with carcass and meat quality traits from a large sample of crossbred beef cattle. Five SNP (UASMS1, UASMS2, UASMS3, E2JW, and E2FB) were genotyped on 1,111 crossbred bulls, heifers, and steers. Association between SNP within the leptin gene with lean yield, fatness, and tenderness were detected.
Liefers et al. (2005) sequenced the leptin promoter and discovered 20 SNP in a 1.6 Kbp region of the bovine leptin promoter. Fourteen of these SNP were genotyped for all animals and these were found to be associated with leptin concentrations during late pregnancy but not during lactation.
Heravi Moussavi et al. (2006) evaluated the association of genetic differences in the bovine leptin gene and milk yield, reproduction, body condition score (BCS), and plasma glucose level in Iranian Holstein cows. In total, two hundred and thirty eight cows were genotyped by using sau3AI restriction enzyme. A significant association was detected between the RFLP-AB genotype and 305-d milk yield. The heterozygous genotype animals had a trend to better reproductive performance than the homozygous. The results also demonstrated that the RFLP B-allele can yield a higher 305-d milk production with a trend to better reproductive performance.
Dandapat et al. (2009) reported that leptin genotypes have significant effect on growth traits, first lactation milk yield, second lactation milk yield, average daily milk yield during first lactation and days in milk in first lactation. HphI is a possible marker for milk production traits.

Recent research on Buffalo

Kumar et al. (2003) reported the absence of polymorphism within 522bp PCR product of leptin gene in buffalo digested with HinfI restriction enzyme. Vallinoto et al. (2004) amplified promoter and exon 1 with primers designed from the bovine leptin gene. Three SNPs and one microsatellite were identified. No polymorphisms were detected in exon 2.

REFERENCES

1. Almeida SEM, Almeida, EA, Moraes, JCF, Weimer, TA. Molecular markers in the LEP gene and reproductive performance of beef cattle. J Anim Breeding Genet 2003; 120: 106-113.

2. Barendse W, Bunch RJ, Harrison BE. The leptin C73T missense mutation is not associated with marbling and fatness traits in a large gene mapping experiment in Australian cattle. Anim Genet 2004; 36: 86-88.

3. Buchanan FC, Fitzsimmons CJ, Van Kessel AG, Thue TD, Winkelman Sim DC, Schmutz SM. Association of a missense mutation in the bovine leptin gene with carcass fat content and leptin mRNA levels. Genet Sel Evol 2002; 34: 105-116.

4. Buchanan FC, Van Kessel AG, Waldner C, Christensen DA, Laarveld B, Schmutz SM. Hot topic: an association between a leptin single nucleotide polymorphism and milk and protein yield. J Dairy Sci 2003; 86: 3164-3166.

5. Choudhary V. Molecular studies on leptin and insulin-like growth factor binding protein-3 (IGFBP-3) genes in cattle. Ph.D. thesis submitted to Indian Veterinary Research Institute, Bareily, India, 2004.

6. Dandapat Anjan, Kumar D, Ghosh AK, Banerjee Dipak. Association of leptin polymorphism with growth, milk production and reproduction traits in Sahiwal and crossbred cattle. Indian Journal of Animal Sciences 2009; Volume 79 No. 9, in press.

7. Fitzsimmons CJ, Schmutz SM. A SNP in the leptin gene leads to a change in the amino acid sequence of the mature protein in cattle. In the proceeding of Plant & Animal Genome VII conference, Town & Country Hotel, San Diego, CA, January 17-21, 1999.

8. Fitzsimmons CJ, Schmutz SM, Bergen RD, McKinnon JJ. A potential association between the BM 1500 microsatellite and fat deposition in beef cattle. Mamm Genome 1998; 9: 432-434.

9. Gelderman H. Investigations on inheritance of quantitative characters in animals by gene markers 1. Methods. Theor Appl Gen 1997; 46: 319-330.

10. Glazko VI, Oblap R, Tryapitcina NV, Zwierzchowski L. Analysis of gene polymorphism at locus of myostatin, leptin, beta-globulin and kappa-kaesin genes at some Ukrainian breeds of cattle. In Proceedings of the 7th World Congress on Genetics Applied to Livestock Production, Montpellier, France, 2002, pp. 1-3.

11. Haegeman A, Van Zeveren A, Peelman LJ. New mutation in exon 2 of the bovine leptin gene. Anim Genet 2000; 31:79.

12. Heravi Moussavi A, Ahouei M, Nassiry MR, Javadmanesh A. Association of leptin polymorphism with production, reproduction and plasma glucose level in Iranian Holstein Cows. Asian Aust J Anim Sci 2006; 19: 627-631.

13. Hossner KL. Cellular, molecular and physiological aspects of leptin: Potential application in animal production. Canadian J Anim Sci 1998; 78: 463-472.

14. Houseknecht KL, Baile CA, Matteri RL, Spurlock ME. The biology of leptin: a review. J Anim Sci 1998; 76: 1405-1420.

15. Klauzinska M, Siadowska E, Grochowska R, Zwierzchowski L, Zurkowski M. Polymorphism of molecular genetic systems in the Polish Red cattle. Tsitologiya i Genetika 2001; 35: 58-60.

16. Konfortov BA, Licence VE, Miller JR. Re-sequencing of DNA from a diverse panel of cattle reveals a high level of polymorphism in both intron and exon. Mamm Genome 1999; 10: 1142-1145.

17. Kumar P, Choudhary V, Bhattacharya TK, Bhushan B, Sharma A. Studies on HinfI-RFLP on leptin gene in riverine buffaloes. In Proceedings of the 4th Asian Buffalo Congress, New Delhi, India, 25-28 February, 2003.

18. Lagonigro R, Wiener P, Pilla F, Woolliams JA, Williams JL. A new mutation in the coding region of the bovine leptin gene associated with feed intake. Anim Genet 2003; 34: 371-374.

19. Lande R. The minimum number of gene contributing to qualitative variation between and within populations. Gene 1981; 99: 541-553.

20. Liefers SC, te Pas MFW, Veerkamp RF, van der Lende T. Associations between leptin gene polymorphisms and production, live weight, energy balance, feed intake, and fertility in Holstein Heifers. J Dairy Sci 2002; 85: 1633-1638.

21. Liefers SC, Veerkamp RF, te Pas MFW, Delavaud C, Chilliard Y, Platje M, van der Lende T. Leptin promoter mutations affect leptin levels and performance traits in dairy cows. Anim Genet 2005; 36: 111-118.

22. Lien S, Sundvold H, Klungland H, Vage DI. Two novel polymorphisms in the bovine obesity gene (OBS). Anim Genet 1997; 28: 245.

23. Lindersson M, Andersson-Eklund L, De Koning DJ, Lunden A, Maki-Tanila A, Andersson L. Mapping of Serum amylase-1 and Quantative Trait Loci for Milk Production Traits to Cattle Chromosome. J Dairy Sci 1998; 81: 1454-1461.

24. Madeja Z, Adamowicz T, Chmurzynska A, Jankowski T, Melonek J, Switonski M, Strabel T. Effect of Leptin Gene Polymorphisms on Breeding Value for Milk Production Traits. J Dairy Sci 2004; 87: 3925-3927.

25. Pomp D, Zou T, Clutter AC, Barendse W. Mapping of leptin to bovine chromosome 4 by linkage analysis of a PCR based polymorphism. J Anim Sci 1997; 75: 1427.

26. Schenkel FS, Miller SP, Ye X, Moore SS, Nkrumah JD, Li C, Yu J, Mandell IB, Wilton JW, Williams JL. Association of single nucleotide polymorphisms in the leptin gene with carcass and meat quality traits of beef cattle. J Anim Sci 2005; 83: 2009-2020.

27. Tessanne K, Hines HC, Davis ME. Relationship of polymorphisms in the bovine leptin gene with differences in beef carcass traits. Special-circular Ohio Agricultural Research and Development centre 1999; 170: 36-41.

28. Vallinoto M, Schneider MPC, Silva A, Iannuzzi L, Brenig B. Molecular cloning and analysis of the swamp and river buffalo leptin gene. Anim Genet 2004; 35: 462-463.

29. Wilkins RJ, Davey HW. A polymorphic microsatellite in the bovine leptin gene. Anim Genet 1997; 28: 376.

30. Yoon DH, Cho BH, Park BL, Choi YH, Cheong HS, Lee HK, Chung ER, Cheong IC, Shin HD. Highly polymorphic bovine leptin gene. Asian Aust J Anim Sci 2005; 18: 1548-1551.

31. Zhang F, Basinski MB, Beals JM, Briggs SL, Churgay LM, Clawson DK, DiMarchi RD, Furman TC, Hale JE, Hsiung HM, Schoner BE, Smith DP, Zhang XY, Wery JP, Schevitz RW. Crystal structure of the obese protein leptin-E100. Nature 1997; 387: 206-209.

32. Zwierzchowski L, Oprzadek J, Dymnick E, Dzierzbicki P. An association of growth hormone, kappa casein, beta-lactoglobulin, leptin and Pit-1 loci polymorphism with growth rate and carcass traits in beef cattle. Animal science papers and Reports 2001; 19: 65-77.