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SHORT COMMUNICATION - DETECTION AND ANALYSIS OF POLYMORPHISM IN THE PROMOTER REGION OF EQUINE PPARCG1A GENE.

Byline: D. Polasik, B. Rogers, A. Bobrowska-Chwat and R. Pikula

ABSTRACT

The protein encoded by PPARCG1a gene is a key regulator of genes involved in energy metabolism. Numerous studies indicate that PPARGC1a gene may be a good marker for athletic performance in horses. Therefore the aim of this study was to detect polymorphisms in the promoter region of PPARGC1a gene and to evaluate its presence in horses belonging to nine different breeds (n = 268) as well as in elite and non-elite Thoroughbred racehorses with known racing outcomes (n = 276). By sequencing regulatory region of equine PPARGC1a gene one novel SNP (g.100.784.525 C>G) was detected which changes transcription factors binding sites. To genotype polymorphism PCR-RFLP method was applied. Frequency of alleles and genotypes showed considerable differentiation in analyzed breeds. GG genotype was found only in Standardbred, Polish Heavy Draft horses and Purebred Arabian (0.04-0.13). The distribution of PPARGC1A genotypes was in Hardy-Weinberg equilibrium except Polish Heavy Draft horses (P[?]0.05).

Moreover disproportionate presence of the GC genotype in elite Thoroughbred racehorses over the CC genotype in the non-elite population (OR=1.69) was noticed. Similarly GC genotype was overrepresented in distance runners in relation to sprinters analyzing all Thoroughbreds (OR=2.00) as well as only elite horses (OR=1.31). While there are multiple genes involved in athletic performance, given the association of PPARGC1A to mitochondrial biogenesis and conversion slow-twitch type I muscle fibers, this novel SNP may explain adaption in aerobic metabolism. The relationship between genotypes and gene expression should be performed next to evaluate its functional role.

Key words: PPARCG1a gene, SNP, Thoroughbred, horses.

INTRODUCTION

Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1a) is a protein encoded by the PPARGC1a gene, which is involved in mitochondrial biogenesis, fatty acid oxidation, glucose utilization, thermogenesis, angiogenesis and muscle fiber-type conversion toward slow-twitch type I fibers (Ahmetov and Rogozkin, 2009). Different variants of this gene were associated with milk-fat composition, carcass and growth traits in cattle (Schennink et al. 2009, Shin and Chung, 2013, Li et al. 2014), muscle fiber characteristics and meat quality in pigs (Kim et al. 2012) and chicken skeletal muscle fiber types (Shu et al. 2014). PPARGC1a gene polymorphism in human was investigated only in relation to physical performance (Maciejowska et al. 2012, He et al. 2015). The PPARGC1a gene was proposed as a candidate for physical performance in the horses by Schroder et al. (2011).

The Equine PPARGC1A gene is localized on chromosome 3. (ECA3) and consists of 13 exons. The transcript length is 2814 bp, however the protein is - 796 aa (http://www.ensembl.org). Investigations of oxidative gene expression in equine skeletal muscle showed significant (P<0.05) difference in PPARGC1A transcripts levels 4h after exercise. Moreover velocity at maximum heart rate (VHR-max) and peak post-exercise plasma lactate concentration ([LA]T1) were also associated with PPARGC1A expression (PG). Sequence polymorphism is submitted in Genbank with the accession number JX948085. Analysis of transcription factors binding sites showed that the G allele introduces site for v-ErbA and AML-1a transcription factors that are abolished by the C allele.

Detected SNP was genotyped in 9 equine breeds and Thoroughbred racehorses by PCR-RFLP based on the following restriction fragments lengths: C allele - 327, 202, 183 bp, G allele - 529, 183 bp. Genotypes and alleles frequency with other population statistics are given in Table 1.

Analysis of genotypes frequency showed that CC genotype appeared with highest frequency in all horse breeds (0.53-1.00) except Purebred Arabian (0.27). GG genotype was present only in Standardbred, Polish Heavy Draft and Purebred Arabian horses (0.04-0.13). The distribution of PPARGC1A genotypes deviated from Hardy-Weinberg equilibrium only in Polish Heavy Draft horses (P[?]0.05). Highest observed and expected heterozygosity was noticed for Purebred Arabian (0.60, 0.49 respectively) whereas lowest in Polish Konik (0.06, 0.06). In Hutsul horses only CC genotype was observed. Analysis of alleles frequency indicated that C allele occurred more often (0.57-1.00) in relation to G allele, which highest frequency was observed in Purebred Arabian (0.43).

Analysis of Thoroughbreds showed that GC genotype was overrepresented (0.23) in elite racehorses when compared to non-elite (0.15) (OR=1.69; p=0.129). In case of race distance in all Thoroughbreds statistically significant differences were found between distance runners and sprinters (OR=2.00; p=0.029). GC genotype appeared with higher frequency in distance runners (0.26) in relation to sprinters (0.15). When comparing elite sprinters against elite distance runners the similar tendency was observed (OR=1.31; p=0.522). Due to small number of none-elite distance horses these subgroups were not compared.

Many studies have focused on searching for polymorphisms in equine genes among different breeds and utility types; myostatin (MSTN), alpha amylases (AMY1, AMY2) and actin alpha 1 (ACTA1) genes may serve as good examples (Baron et al. 2012, Coizet et al. 2014, Polasik and Pikula, 2014).

In our study we observed presence of GG genotype only in three breeds. While it is present in other closely related breeds to the Thoroughbred, in the Arabian and Standardbred, there was an absence of the GG genotype in the racehorses samples studied. This may be due to the low average competing distance range of the horses sampled (1507m), so it is possible that the GG variant exists in outlier sub-populations of the Thoroughbred breed including but not limited to National Hunt performers who regularly run distances of 4000m and beyond which are similar distances to those that the Arabian breed compete at with the latter breed having the highest frequency of the GG genotype.

Presence of GG genotype in Polish Heavy Draft may be explained by high strength and endurance of this breed.

Previous studies in Thoroughbreds have found polymorphisms within exercise relevant genes associated with elite racetrack performance. Gu et al. (2010) studied polymorphism in candidate genes in relation to racing performance in Thoroughbred horses including the PPARGC1A gene. Analyzed horses in that study were divided into elite (n=150) and non elite (n=80) in a similar fashion to our study. An investigated polymorphism (A>G) was detected in noncoding region of PPARGC1A gene - intron 10 however statistical analysis showed that it was not associated with elite racing performance (OR = 0.884, p = 0.621) and therefore not considered for further analysis. In that study 3 SNPs in creatine kinase, muscle (CKM), pyruvate dehydrogenase lipoamide kinase isozyme 4 (PDK4) and cytochrome c oxidase, subunit 4, isoform 2 (COX4I2) genotype frequency distributions were significantly (P<0.05) different between elite and non-elite thoroughbred racehorses.

A subsequent study by Pereira et al. (2015), in Quarter Horses, a breed noted for their sprinting speed and high percentage of type II muscle fiber, indicated that the SNPs in PDK4 and COX4I2 showed no significant associations with a Quarter Horse speed index. Pereira et al. suggested that the alleles of the PDK4 and COX4I2 genes are probably associated with beneficial adaptations in aerobic metabolism and therefore play secondary roles in sprint racing performance in Quarter Horses, which is mainly anaerobic.

Given the association of peroxisome proliferator-activated receptor gamma coactivator 1-alpha to mitochondrial biogenesis and conversion slow-twitch type I muscle fibers, it is reasonable to suggest that GG genotype, more readily associated with distance breeds in this study, may also explain adaptions in aerobic metabolism and similar to the other SNPs in the study by Pereira et al (2015) be not present in breeds generally associated with sprinting.

This novel polymorphism in the regulatory part of PPARCG1a gene is one of many polymorphisms found in equine genome. While it showed variability in different breeds and an association with elite racehorse performance, the relationship between genotypes and gene expression should be performed next to evaluate its functional role. Moreover investigations on a larger Thoroughbred racehorse population including those excelling over longer distance ranges could confirm obtained results and allow to application of PPARCG1a gene as a marker for racing performance.

Table 1. Population statistics in analyzed horses based on PPARGC1A gene polymorphism.

###Genotype frequency###Allele frequency

Breed###He###Z2###p

###GG###GC###CC###G###C

Purebred Arabian###0.49###0.74###0.39###0.13###0.60###0.27###0.43###0.57

Holstein Breed###0.24###0.86###0.35###-###0.28###0.72###0.14###0.86

Malopolski Horse###0.26###0.98###0.32###-###0.31###0.69###0.16###0.84

Wielkopolski Horse###0.20###0.58###0.44###-###0.23###0.77###0.11###0.89

Standardbred###0.38###0.80###0.37###0.04###0.43###0.53###0.26###0.74

Deutsche Reitpony###0.14###0.14###0.70###-###0.16###0.84###0.14###0.86

Polish Cold-blooded###0.24###4.00###0.04###0.06###0.16###0.78###0.14###0.86

Polish Konik###0.06###0.03###0.85###-###0.06###0.94###0.03###0.97

Hutsul###-###-###-###-###-###1.00###-###1.00

Thoroughbred###0.19###3.66###0.06###-###0.21###0.79###0.10###0.90

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Publication:Journal of Animal and Plant Sciences
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Date:Apr 30, 2017
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