Multi-Species Comparative Analysis of the Equine ACE Gene Identifies a Highly Conserved Potential Transcription Factor Binding Site in Intron 16

Multi-Species Comparative Analysis of the Equine ACE Gene Identifies a Highly Conserved Potential Transcription Factor Binding Site in Intron 16 Natasha A. Hamilton1*, Imke Tammen2, Herman W. Raadsma2 1 ReproGen-Animal Bioscience Group, Faculty of Veterinary Science, University of Sydney, Camperdown, New South Wales, Australia, 2 ReproGen-Animal Bioscience Group, Faculty of Veterinary Science, University of Sydney Camden, New South Wales, Australia Abstract Angiotensin converting enzyme (ACE) is essential for control of blood pressure. The human ACE gene contains an intronic Alu indel (I/D) polymorphism that has been associated with variation in serum enzyme levels, although the functional mechanism has not been identified. The polymorphism has also been associated with cardiovascular disease, type II diabetes, renal disease and elite athleticism. We have characterized the ACE gene in horses of breeds selected for differing physical abilities. The equine gene has a similar structure to that of all known mammalian ACE genes. Nine common single nucleotide polymorphisms (SNPs) discovered in pooled DNA were found to be inherited in nine haplotypes. Three of these SNPs were located in intron 16, homologous to that containing the Alu polymorphism in the human. A highly conserved 18 bp sequence, also within that intron, was identified as being a potential binding site for the transcription factors Oct-1, HFH-1 and HNF-3b, and lies within a larger area of higher than normal homology. This putative regulatory element may contribute to regulation of the documented inter-individual variation in human circulating enzyme levels, for which a functional mechanism is yet to be defined. Two equine SNPs occurred within the conserved area in intron 16, although neither of them disrupted the putative binding site. We propose a possible regulatory mechanism of the ACE gene in mammalian species which was previously unknown. This advance will allow further analysis leading to a better understanding of the mechanisms underpinning the associations seen between the human Alu polymorphism and enzyme levels, cardiovascular disease states and elite athleticism. Citation: Hamilton NA, Tammen I, Raadsma HW (2013) Multi-Species Comparative Analysis of the Equine ACE Gene Identifies a Highly Conserved Potential Transcription Factor Binding Site in Intron 16. PLoS ONE 8(2): e55434. doi:10.1371/journal.pone.0055434 Editor: Vincent Laudet, Ecole Normale Supérieure de Lyon, France Received July 6, 2012; Accepted December 23, 2012; Published February 8, 2013 Copyright: ß 2013 Hamilton et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was partly funded by the NSW Racing Research Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding received for this study. Competing Interests: The authors of this paper also hold the inventors rights within the related patent ‘‘Equine performance test’’ International Patent Application no pct/au2007/001379. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: mRNA, or lower stability of the I allele mRNA [3,19,20]. Circulating enzyme levels were found to be influenced by the action of a major codominant gene, with adults homozygous for the I allele having significantly lower circulating enzyme levels than homozygotes for the D allele. Heterozygotes fall in between these levels [3,19]. The suggestion that an unidentified intronic silencer element is eliminated by the deletion variant, and thus increases observed circulating enzyme levels, has been ruled out [21]. Other studies have hypothesised that other polymorphisms are responsible for the effects attributed to the I/D, and instead indicate that potentially two functional variants exist, probably in the 39 region of the gene, accounting for these effects [22–24]. However, these studies were unable to identify the functional variant(s) and only investigated a selection of known ACE polymorphisms. Furthermore, the mode of action of alternate allelic forms of the ACE gene on variation in circulating enzyme levels in addition to performance is yet to be elucidated. The human ACE gene spans 21308 bp [GenBank:NG_011648] of which 4422 bp comprises coding sequence across 26 exons [25]. It encodes two commonly expressed isozymes, the larger of which is membrane bound and primarily found in both epithelial and endothelial cells. In particular, vascular cells from the brain and Introduction Angiotensin converting enzyme (ACE) is an essential component of the renin-angiotensin system and plays an important role in the control of blood pressure, renal function and male fertility [1]. The presence of a 287 bp Alu insertion/deletion (I/D) polymorphism with a high minor allele frequency (0.4–0.47) within the ACE gene [2,3], combined with the intrinsic function of the enzyme, has resulted in over 500 association studies between the human ACE gene and a wide range of disorders, most notably cardiovascular disease, type II diabetes and related renal disease [4–7]. More than 20 studies have explored a possible association with extreme athleticism, with conflicting results. In many studies, the insertion (I) allele has been associated with anabolic response to training and elite endurance performance, whilst the deletion (D) allele was associated with sprint or power performance [8–12], and both variants have been associated with response to strength training [13,14]. However, some studies have found no connection with athletic performance [15–18]. The I/D polymorphism, which is found in intron 16, appears to account for 28–47% of the inter-individual variation in serum ACE levels, either due to increased expression of the D allele PLOS ONE | www.plosone.org 1 February 2013 | Volume 8 | Issue 2 | e55434 Multi-Species Comparison of the Equine ACE Gene lung produce large amounts of ACE, as do the brush border cells of kidney tubules, while all mammalian endothelial cells appear to produce ACE (endothelial or somatic ACE, sACE) [26]. Vascular endothelial cells also release a circulating form of the enzyme by cleaving it from the membrane bound tail [27,28]. This large isozyme is transcribed from exons 1 to 26, excluding exon 13. The smaller ACE variant is only expressed after puberty in the germinal cells of the testes, is encoded by exons 13 to 26 through initiation of a separate promoter in intron 12, and is known as testicular ACE (tACE). A feature of the ACE gene is a high degree of homology between two distinct regions of the gene, namely exons 4 to 11 (region 1) and 17 to 24 (region 2). The exons in regions 1 and 2 have conserved codon phases, are up to 80 (...truncated)