Polymorphism of the ovine β₃-adrenergic receptor gene

Abstract

The β₃-adrenergic receptors (ARs) appear to play a pivotal role in the regulation of energy balance under sympathetic neural control. These receptors are predominantly found on the surface of adipocytes and are the major mediators of the lipolytic and thermogenic effects of high catecholamine concentrations. Variation within the ovine β₃-AR gene may impact on the expression, tissue distribution and pharmacology of the receptor, which may in turn lead to a spectrum of thermogenic and lipolytic capabilities. In the human β₃-AR an amino acid substitution (Trp64->Arg) has been associated with obesity and insulin resistance in Pima Indians, Japanese, Swedish, Finnish and French individuals.

Previously, β₃-AR genes have been cloned and characterised from human, mouse, rat, and dog genomic libraries. This accumulation of sequence information allowed a polymerase chain reaction (PCR)-based cloning strategy to be employed for the isolation of the bovine, caprine and ovine β₃-AR genes from genomic DNA. The ruminant gene sequences were found to share the same exon-intron structure as the human and dog β₃-AR genes, in which a large exon (~1.4 kb) encodes the entire polypeptide except for the six carboxy-terminal residues which are encoded by a second exon (~0.7 kb). Intron length varies with species. The introns within the bovine, caprine and ovine genes were found to be 724 bp, 730 bp and 724 bp, respectively, and are comparable in size to that of the dog β₃-AR gene (704 bp), but not the human gene which is 1025 bp. The homologies of the putative amino acid sequences of the β₃-ARs from human and dog with the ruminant genes are 85% and 86%, respectively.

Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis of part of the ovine β₃-AR intron was used to screen 12 large Merino half-sib families for sequence variation. Six different alleles that segregated in a Mendelian fashion were observed. The genetic basis for the allelic differences were identified by sequencing the β₃-AR gene (coding and non-coding regions) from animals that were homozygous for each of the alleles. The alleles were designated A through F, with GenBank Accession numbers AΒ14200 through AΒ14205, respectively. This sequence information allowed a new primer set to be designed to facilitate the development of a second, more rapid allele-typing system using PCR-SSCP.

Twenty-seven sire-lines (13 Merino x Merino, 3 Merino x Coopworth, 4 Borderdale x Borderdale, 2 Dorset Down x Coopworth, 5 Coopworth x Coopworth) were used to provide phenotypic and genotypic data to ascertain the affects of allelic variation at the β₃-AR locus on birth weight, weaning weight, growth rate (up until weaning), carcass composition at 63 days post-weaning and cold survival.

Eleven of the twenty-seven sire-lines had greater than 10% cold deaths. Of the 11 sire-lines, the Fisher's exact test revealed an association in one sire-line, M14, in which the E allele was associated with cold survival and the D allele with cold-related mortality. Pooled analysis using the data from all twenty-seven sire-lines (including those animals that genotyped the same as their sire) to determine the association between β₃-AR alleles (inherited from either parent) and the relative risk of cold-related mortality found allele E to be associated with a low relative risk of cold-related mortality (P = 0.008), and allele F to be associated with a high risk of cold related mortality (P < 0.001). None of the other β₃-AR alleles had a significant association with either a high or low relative risk of cold-related mortality.

Analyses of variance within each half-sib family showed that the inheritance of a particular allele was associated with increased birth weights in six sire-lines (M8, M10, M12, M14, B1, and D1). However, in all but one of these sire-lines, these associations occurred in the form of an interaction with either birth rank or gender (M8, multiples only P = 0.050; M10, ewes only P = 0.050; M12, singles only P = 0.034; M14, multiples only P = 0.034; B1, sire allele main effect P = 0.018; D1, rams only P = 0.001). Significant growth rate differences associated with the inheritance of a particular sire allele were detected in sire-lines M7 (ANOVA, P = 0.032), M15 (ANOVA, P =0.024), B2 (ANOVA, P = 0.001), and D1 (ANOVA, P = 0.030). In sire-lines M15 and B2, these differences in growth rate were dependent on both birth rank and gender, with the association being limited to single-born, ewe lambs.

Twenty animals (10 ewes and 10 rams) of birth rank 2 (twin) or 3 (triplet) and between 35 to 40 kg from sire-lines M14, M15, M16, D1, and D2 (i.e. 100 lambs) were randomly chosen at 63 days post-weaning for computer tomography (CT) scanning to predict carcass composition. For each animal the carcass tissue weights (bone, fat and muscle) were predicted, and muscle to bone and muscle to fat ratios were calculated. The inheritance of a particular sire allele was found to be associated with carcass composition in sire-line M16 (sire genotyped as DE) only. In this sire-line those progeny inheriting the D allele from the sire had a significantly higher mean muscle to fat ratio to those inheriting the E allele (ANOVA P = 0.037; l.84 ± 0.096 and l.56 ± 0.070, respectively). Consistent with the above finding, those progeny inheriting the E allele tended to have more fat than those inheriting the D allele (ANOVA P = 0.054; mean total carcass fat: allele D = 4128 ± 282 g, allele E = 4874 ± 206 g) while muscle mass was not significantly different (ANOVA P = 0.863). Total carcass weight was also not significantly different for each of the sire allele groups (ANOVA P = 0.122). However, those progeny inheriting the E allele tended to have a heavier total carcass weight compared to those inheriting the D allele (allele E = 15.2 ± 0.31, allele D = 14.3 ± 0.43).

Such associations support the hypothesis that β₃-ARs are involved in energy homeostasis. With more research, the variation at the β₃-AR locus may assist in the genetic selection for desirable animal production traits.