Research paperCharacterisation of single nucleotide polymorphisms identified in the bovine lactoferrin gene sequences across a range of dairy cow breeds
Introduction
Lactoferrin is a bioactive glycoprotein found in the secretions of a wide range of mammalian species, including bovine, human, goat and porcine. The amino acid sequences of the lactoferrin proteins are highly conserved among the species of higher mammals [1]. The level of lactoferrin in milk however, varies enormously between species with the highest concentrations recorded in human milk (1.0–5.0 mg/ml) [1]. However, bovine milk, which is the most common source of commercially produced lactoferrin contains about one-tenth of the lactoferrin found in other mammals with concentrations ranging between 0.02 and 0.2 mg/ml [2]. This can however, dramatically increase during the dry period and during a mastitis incidence, with concentrations increasing by about 100-fold [3]. This suggests that the protein plays important physiological roles, particularly during a mastitis event.
The bovine lactoferrin gene is located on chromosome 22 spanning approximately 34.5 kb of genomic DNA and comprises 17 exons ranging from 82 (exon 1) to 225 bp (exon 17). The 5′-regulatory region of the bovine lactoferrin gene contains the promoter sequence, a non-canonical TATA box and a number of transcription factor binding sites necessary for expression of the gene [4]. The coding sequence of the functional bovine lactoferrin protein defines a single polypeptide chain of 708 amino acids, which is folded into two separate lobes (N- and C-terminals) that each contains two iron-binding and two bicarbonate-binding sites [5]. There are five N-linked glycosylation sites present in bovine lactoferrin located at asparagine (Asn) residues 233, 281, 368, 476 and 545. The significance of glycosylation for lactoferrin is not yet completely understood [6].
Single nucleotide polymorphisms (SNPs) are the most abundant genetic variations in mammalian genomes, occurring in regulatory, exon and intron regions of genes [7]. To date, genetic polymorphisms in the regulatory region of bovine and human [5], [7], [8] lactoferrin genes have been described. However, the significance and associated functional effects of these polymorphisms have yet to be established. Polymorphisms have also been identified in the coding sequence of lactoferrin genes. Mutations in the coding regions of a protein can affect protein structure, which may potentially alter the biological functions of that protein [9]. In addition, polymorphisms that cause amino acid substitutions on the surface of the protein that have no affect on protein structure could alter the surface properties of the protein [10]. Alterations to the N-terminal sequence of the protein are particularly significant as this region is largely responsible for the biological activity of lactoferrin. To date, polymorphisms in the coding region of human and goat lactoferrin that affect the antimicrobial properties of the lactoferrin have been identified [11], [12]. In addition Mohamed et al. identified a unique polymorphism in human lactoferrin genes that leads to increased susceptibility to diarrhoea [13].
Bovine mastitis is defined as inflammation of a cows’ udder and is predominantly caused by bacterial infections. The financial loss to the dairy industry caused by mastitis is significant [14]. A logical approach to reducing mastitis incidence in dairy cattle would be to select for animals in breeding programmes that are genetically resistant or less susceptible to the disease [15]. Lactoferrin plays a vital role in the natural defence mechanism of the mammary gland and therefore, is a potential candidate gene for imparting resistance to mastitis in dairy cows [4], [16]. This study was conducted to establish a baseline of genetic polymorphisms present in the lactoferrin genes of Irish dairy cattle and identify single genetic events which could potentially affect milk lactoferrin concentration or produce lactoferrin protein variants.
Section snippets
Sample population and blood collection
Seventy cows representing six different breeds were used in this study. The group comprised 47 Holstein Friesians, 6 Jersey, 5 Jersey–Friesian crossbreeds, 7 Norwegian Reds, 4 Montbéliard and 1 Norwegian Red–Friesian crossbreed. To provide a diverse genetic pool, the animals selected were largely unrelated being from six different breeds and within each breed they had different sires. Blood samples (7 ml) were collected (in duplicate) by caudal venipuncture from each animal into sample tubes
Results
In total, 2.2 kb of the promoter region of the bovine lactoferrin gene was sequenced in both forward and reverse directions to confirm the presence of polymorphisms. The consensus sequence identified in this study deviated from the published bovine lactoferrin sequence (GenBank Ref. NC_007320) by 29 nucleotides, giving rise to an average of one polymorphism per 76 bases. The SNP loci, percentage homo- and heterozygosity of the SNPs and the major allele frequency determined in this study are
Discussion
Twenty-nine polymorphisms were identified within the 2.2 kb regulatory region of the bovine lactoferrin gene. Among these, 10 were previously reported [5], [8] while others (n = 19) were unique polymorphisms, some of which were found to be associated with putative transcription factor binding sites. For example, the SNP at position −1155 is located on the GATA-1 and SPI transcription factor binding sites. SP1 DNA-binding sites are known enhancer elements in eukaryotic genomes that are considered
Conclusions
Bovine mastitis is a costly disease to the dairy industry. The documented association between lactoferrin concentrations in milk and bovine mastitis incidence makes this gene an obvious candidate to investigate whether variants impart differences in disease resistance. This study has identified a baseline of polymorphisms in lactoferrin gene sequences that are present within the Irish bovine population. Several potentially interesting novel polymorphisms were identified within the coding
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