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Association of Sequence Variants in CKM (Creatine Kinase, Muscle) And

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Association of Sequence Variants in CKM (Creatine Kinase, Muscle) And EQUINE VETERINARY JOURNAL 569 Equine vet. J. (2010) 42 (Suppl. 38) 569-575 doi: 10.1111/j.2042-3306.2010.00181.x Association of sequence variants in CKM (creatine kinase, muscle) and COX4I2 (cytochrome c oxidase, subunit 4, isoform 2) genes with racing performance in Thoroughbred horses † J. GU, D. E. MACHUGH, B. A. McGIVNEY, S. D. E. PARK, L. M. KATZ and E. W. HILL* Animal Genomics Laboratory, UCD School of Agriculture, Food Science and Veterinary Medicine, †University Veterinary Hospital, UCD School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Ireland. Keywords: horse; SNP; racing performance; Thoroughbred; CKM; COX4I2 Summary Introduction Reasons for performing study: The wild progenitors of the For 3 centuries the natural athleticism of the horse has been domestic horse were subject to natural selection for speed and selected by breeders to produce Thoroughbred racehorses that, stamina for millennia. Uniquely, this process has been aided by intense management, have become athletes with extreme augmented in Thoroughbreds, which have undergone at least exercise-performance phenotypes. While management of exercise 3 centuries of intense artificial selection for athletic conditioning and nutrition have considerable effects on the phenotypes. While the phenotypic adaptations to exercise are development of elite Thoroughbred athletes (approximately 65%), well described, only a small number of the underlying genetic a significant proportion of variation in athletic ability is heritable variants contributing to these phenotypes have been reported. (Gaffney and Cunningham 1988). Genetic contributions to human Objectives: A panel of candidate performance-related genes athletic performance phenotypes are well documented and more was examined for DNA sequence variation in Thoroughbreds than 220 gene loci have been described (Bray et al. 2009). While it and the association with racecourse performance investigated. is likely that Thoroughbred racing performance is also influenced Materials and methods: Eighteen candidate genes were chosen by a large number of genes, only 2 performance-associated for their putative roles in exercise. Re-sequencing in sequence variants in exercise-relevant genes (MSTN and PDK4), Thoroughbred samples was successful for primer sets in 13 of have been reported for the horse (Hill et al. 2010a,b). As well as these genes. SNPs identified in this study and from the these genes, it has been established that genomic regions EquCab2.0 SNP database were genotyped in 2 sets of containing an over-representation of genes responsible for insulin Thoroughbred samples (n = 150 and 148) and a series of signalling, fatty acid metabolism and muscle strength have been population-based case-control investigations were performed selected during the development of the Thoroughbred (Gu et al. by separating the samples into discrete cohorts on the basis of 2009). Additionally, relationships between mitochondrial retrospective racecourse performance. genotypes and athletic performance in the Thoroughbred have been Results: Twenty novel SNPs were detected in 3 genes: ACTN3, reported (Harrison and Turrion-Gomez 2006). CKM and COX4I2. Genotype frequency distributions for 3 A single nucleotide polymorphism (SNP) is a single base SNPs in CKM and COX4I2 were significantly (P<0.05) substitution in a genomic DNA sequence. SNPs are stable, different between elite Thoroughbreds and racehorses that bi-allelic genetic markers that are abundantly distributed had never won a race. These associations were not validated throughout the genome. They may be discovered by aligning when an additional (n = 130) independent set of samples was segments of a genomic region from different individuals following genotyped, but when analyses included all samples (n = 278) re-sequencing of candidate genes. We hypothesised that genomic the significance of association at COX4I2 g.22684390C>Twas sequence variants may be detected in exercise-relevant genes in confirmed (P<0.02). Thoroughbred horses. Such sequence variants may be developed Conclusions: While molecular genetic information has the for future use to distinguish between individuals with greater potential to become a powerful tool to make improved potential for elite racetrack performance and individuals with lesser decisions in horse industries, it is vital that rigour is applied to prospects for success. The prospective identification of genetic studies generating these data and that adequate and potential may improve selection decisions and reduce operating appropriate sample sets, particularly for independent costs and may provide opportunities to individually design replication, are used. conditioning programmes to reduce injury risk. evj_181 569..575 *Corresponding email: [email protected] [Paper received for publication 08.01.10; Accepted 01.06.10] © 2010 EVJ Ltd 570 SNP association with elite racing performance Therefore, we investigated a panel of candidate athletic RPR = 115) and 62 nonelite (mean RPR = 59) performing performance genes with functions in muscle development and Thoroughbreds. The elite performer group contained a subset of metabolism, many of which were included in the human gene animals that competed successfully in short distance (Յ8 f) and map for performance (Rankinen et al. 2006). The aim of this long distance (>8 f) races. study was to identify sequence variation in a panel of candidate Validation Sample Set: A set of 130 (97 TBE and 33 TBO) athletic performance genes and to evaluate SNP association with additional Thoroughbred samples was selected from the repository racing performance phenotypes. As the Thoroughbred population for validation of the SNP associations, and criteria for inclusion has been subjected to recent and strong selection, we were as for Sample Set II. hypothesised that adaptation to exercise performance may have resulted in advantageous sequence variants in genes that NonThoroughbreds: Samples from 3 non-Thoroughbred contribute to an athletic phenotype and that these variants may be populations were included as diverse samples in a panel for SNP found at higher frequencies in successful subgroups of the discovery by re-sequencing: Akhal-Teke (AH; Turkmenistan, population. Central Asia), Connemara (CON; Ireland, Western Europe) and Tuva (TU; Republic of Tuva, Southern Siberian Steppes). Materials and methods Genomic DNA was extracted from either fresh whole blood or hair samples using a modified version of a standard phenol/ Ethics chloroform method (Sambrook and Russell 2001). This work has been approved by University College Dublin, Retrieval of equine genomic sequence for SNP discovery Animal Research Ethics Committee. Twenty-three candidate athletic performance genes (Table 1) were Horse populations and DNA samples selected for SNP discovery on the basis that their key functions were relevant to exercise physiology and on the availability of Thoroughbreds: More than 1400 registered Thoroughbred horse equine-specific genomic sequence at the time of PCR assay design. samples (hair or fresh blood) were collected from stud farms, An overview of the study genes including gene symbol, gene racing yards and sales establishments in Ireland and New Zealand name, chromosome location and functional ontology is given in between 1997 and 2009. To minimise confounding effects of racing Table 1. over obstacles, only horses with performance records in Flat races Primers for PCR were selected and designed from 3 different were considered for inclusion in the study cohorts. The highest sources based on the availability of equine-specific genomic standard and most valuable elite Flat races are known as Group sequence for each candidate gene. Before the availability of the first races. Horses were categorised based on retrospective racecourse assembly (EquCab1.0) of the horse (Equus caballus) genome performance records as ‘Elite Thoroughbreds’ (TBE) or ‘Other (February 2007), PCR primers were either: 1) chosen from Thoroughbreds’ (TBO). Elite Thoroughbreds were Flat racehorses published papers; or 2) designed from available sequences in that had won at least one Group race (Group 1, Group 2 or Group GenBank and the Ensembl Trace Archive (Wheeler et al. 2008). In 3). Other Thoroughbreds had competed in at least one race, but had instances where no horse sequence was available, a comparative never won a race and had handicap ratings (Racing Post Rating, genomics approach was taken and PCR primers were designed RPR) <80. Race records were derived from 3 sources - Europe race based on the conserved regions between human (Homo sapiens) records: The Racing Post online database (www.racingpost.co.uk); and cattle (Bos taurus) sequences. Australasia and South East Asia race records: Arion Pedigrees (www.arion.co.nz) and North America race records: Pedigree Online Thoroughbred database (www.pedigreequery.com). In all PCR primer design, amplification and purification cases pedigree information was used to control for genetic and DNA re-sequencing background by attempting to exclude samples sharing sires. No dams were shared. Also, overrepresentation of popular sire lines To amplify candidate gene target regions, PCR primers were (e.g. Northern Dancer, etc.) within the pedigrees was avoided designed using the online tool Primer3 (Rozen and Skaletsky 2000) where possible. and synthesised by Invitrogen1. Assays that were successfully optimised (Table S2) were used Sample Set I: Sample Set I (Table S1) comprised 150 elite (TBE, n to amplify
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