Highly Polymorphic Bovine Leptin Gene

The leptin, an anti-obesity protein, is a hormone protein expressed and secreted mainly from adipocyte tissue, and involved in regulation of body weight, food intake and energy metabolism. In an effort to discover polymorphism(s) in genes whose variant(s) might be implicated in phenotypic traits of growth, we have sequenced exons and their boundaries of leptin gene including 1,000 bp upstream of promoter region with twenty-four unrelated Korean cattle. Fifty-seven sequence variants were identified: fourteen in 5’ flanking region, twenty-seven in introns, eight in exons, and eight in 3’ flanking region. By pair-wise linkage analysis among polymorphisms, ten sets of SNPs were in absolute linkage disequilibrium (LD) (|D’| = 1 and r = 1). Among variants identified, thirty-six SNPs were newly identified, and twenty-one SNPs, which were reported in other breeds, were also confirmed in Korean cattle. The allele frequencies of variants were quite different among breeds. The information from SNPs of bovine leptin gene could be useful for further genetic studies of this gene. (Asian-Aust. J. Anim. Sci. 2005. Vol 18, No. 11 : 1548-1551)


INTRODUCTION
Leptin, the product of obese (ob) gene, is a hormone protein expressed and secreted mainly from adipocyte tissue.It is involved in the control of body weight through the effects on food intake and energy expenditure.The mice with mutant ob are obese and diabetic whereas the ob/ob mice and wild-type mice, injected with leptin, exhibit weight loss, decreased food intake and reduced body fat (Halaas et al., 1995;Pelleymounter et al., 1995).It was initially reported that the mutant ob of mouse is closely associated with obesity (Zhang et al., 1994).Several studies have suggested that leptin, as anti-obesity hormone, acts on receptor in the hypothalamus of brain where it upregulate appetite-reducing neuropeptides inhibiting appetiteinducing through orexigenic and anorexic signaling pathways ( Lee et al., 1996;Schwartz et al., 1996;Ahima et al., 2000;Di Marzo et al., 2001).
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.Several studies have revealed that polymorphisms were associated with lean and fat cattle (Stone et al., 1996;Fitzsimmons et al., 1998) and with the fat contents and feed intakes (Konfortov et al., 1999;Buchanan et al., 2002;Lagonigro et al., 2003).These genetic informations for fatty-phenotypic traits are valuable for breeding for high quality meat through breeding by maker-assisted selection (MAS).Important economic factors for animal production may be influenced by leptin include feed conversion efficiency and intra-muscular fat, which is considered to improve meat quality (Wheeler et al., 1994).Although it has been reported that genetic variants of candidate genes such as mitochondrial DNA is related to growth traits in Korean cattle, polymorphism information of leptin in Korean cattle is still obscure (Jeon et al., 2005).
In this study, leptin gene, including -1,000 bp promoter region, has been examined to identify single nucleotide polymorphisms (SNPs) in leptin gene.

Sequencing analysis of the bovine leptin gene
Genomic DNA for sequencing was isolated from twenty-four unrelated Korean cattle.Full gene including -1,000 bp 5' upstream region were PCR-amplified and directly sequenced using ABI PRISM 3700 genetic analyzer (Applied Biosystems, Foster City CA).Seventeen primer sets for the amplification and sequencing analysis were designed based on GenBank sequences (AB070368 and U50365) (Table 1).Sequence variants were verified by chromatograms.

Statistics
The X 2 tests were used to determine whether individual variants were in equilibrium at each locus in the population (Hardy-Weinberg equilibrium).Heterozygosity (H) for each locus with allele frequencies p and q = 1-p was given by H = 1-p2-q2 = 2p(1-p).We examined Lewontin's D' (|D'|) and the linkage disequilibrium coefficient, r 2 , among all biallelic loci (Hedrick 1987;Hedrick and Kumar 2001).Haplotypes for measure of LDs were inferred using the algorithm developed by Stephens et al. (2001).

RESULTS AND DISCUSSION
As one of efforts to identify genetic polymorphisms in potent candidate genes for meat quality, we have sequenced all exons and their boundaries of leptin gene including 1,000 bp upstream of promoter region with twenty-four unrelated Korean cattle (Hanwoo, Bos taurus coreanae).We identified fifty-seven sequence variants; fourteen in 5' flanking region, twenty-seven in introns, eight in exons (3 non-synonymous and 5 synonymous), and eight in 3' flanking region.The locations and allele frequencies of SNPs are shown in Figure 1 and Table 2.By pair-wise linkage analysis among polymorphisms, we have found that ten sets of SNPs were in absolute linkage disequilibrium (LD) (|D'| = 1 and r 2 = 1) (Figure 1).The identifications of SNP for leptin gene have been widely accomplished in a diverse breed of cattle.In the present study, we discovered thirty-six novel SNPs and also confirmed twenty-one SNPs reported previously in Korean cattle (Hanwoo, Bos taurus coreanae).Confirmed SNPs and their frequencies were summarized in Table 2.The

Table 1 .Figure 1 .
Figure 1.Gene map of Bos taurus Leptin.Coding exons are marked by black blocks, and 5' and 3' UTR by white blocks.The first base of the translation start site was denoted as nucleotide +1 and the nomenclature of each polymorphism was referred to the recommendation of Human Genome Variation Society (http://www.hgvs.org/mutnomen/).The polymorphisms in absolute LD (r 2 = 1) are indicated.The frequencies of polymorphisms were based on sequencing data (n = 24).

Table 2 .
Frequencies of Leptin polymorphisms identified in Korean cattle and comparison with other breed The first base of translational start site is denoted as nucleotide +1.The nomenclature of each polymorphism was based on the cDNA reference numbers and referred to the Mutation Nomenclature Homepage at the HGVS website (http://www.hgvs.org/mutnomen/) *