Genetic Variation of ACTN3 and MSTN Genes in a Ccohort of Endurance Arabian Horses

S. Almarzook¹*, A. Said Ahmed¹, M. Reissmann¹, G. A. Brockmann¹

¹Humboldt-University of Berlin, Faculty of Life Sciences, Albrecht Daniel Thaer-Institute for Agricultural and Horticultural Sciences, Department of Animal Breeding Biology and Molecular Genetics, Invalidenstrasse 42, 10115 Berlin, Germany

The corresponding author:

- Dr. Saria Almarzook: almarzos@hu-berlin.de

ABSTRACT

Arabian horses are readily distinguishable in form and features, and they are widely known for endurance capability. This study is the first examining of endurance-related genes in Arabian horses born and raised in Syria. The major objective was to identify genetic variation in candidate genes that could potentially affect endurance traits and to associate them with endurance phenotypes. The two genes Alpha-actinin skeletal muscle isoform 3 (ACTN3) and Myostatin (MSTN) were sequenced. Performance traits were available for 42 recorded Arabian horses from Syria performed endurance racing over 40, 80, and 120 km distances. Based on the recorded mean speeds, horses were grouped according to their performance index into low and high performers.

The comparative sequencing revealed a total of 13 variants in both studied genes, 12 variants in ACTN3 and one variant in MSTN. General linear model analyses showed that none of the analyzed variants has significant effect on any of the studied traits. However, for ACTN3, we found a 5’ UTR variant (12:26511704G>A) that predicted to cause a gain of an E4BP4 transcription factor binding site, and a variant in the 3´ UTR 12:26524930T>C that predicted to cause the abrogation of two predicted miRNA target sites (eca-miR-1296 and eca-miR-326) and thereby affect gene expression. For MSTN, a 5’ UTR variant 18:66495696A>G is predicted to cause the substitution of the transcription factor binding sites for HFH-1 and Sox-5 by binding sites for HFH-3 and E4BP4.

Keywords: Endurance, candidate genes, transcription factor binding site, Arabian horse, Syria.

INTRODUCTION

Endurance played a vital role in the evolutionary history of human and other species, because it enabled them to survive and preserve their lives under different conditions (Maffetone 2010). In horses, endurance is a trait of great economic value. Humans invested in endurance to improve labor capability and athletic performance of horses. Nowadays, endurance performance is very important in the equestrian competitions. According to Bergero et al., (2005), the endurance performance of horses is identified as a low-intensity long-term trial. Most of the international equine endurance distances range between 30 and 500 km that can be run in one to five days. Different parameters were used to measure endurance performance. Endurance horses can achieve an average speed in endurance races exceeds 25 km/h in the last phase of the race (Nagy et al.,, 2012).

Heritability estimates for mean speed in eight endurance horse breeds (including Arabian horses) vary considerably between 0.16 and 0.40 at distances of 90 km and ≥ 120 km, respectively (Ricard & Touvais 2007). This makes the mean speed as one selection parameter for endurance performance. Studies demonstrated differences in biomarkers related to endurance performance including heart rate, lactate and uric acid (Adamu et al., 2012), physiology and metabolic performance (Bergero et al., 2005; Castejon et al., 2006), morphological factors and gaiting variability (Metayer et al., 2004; Cottin et al., 2010), skeletal muscle fiber types (fast/low twitch fibers), and fiber composition (Rivero et al., 1993; Rivero & Barrey 2001). Pathways involved in endurance as a complex quantitative trait provide a list of candidate genes to be tested for association with endurance trait. There are approximately 230 candidate genes suggested for athletic performance in humans (Bray et al., 2009; Schröder et al., 2011). Recently, studies have tried to identify a DNA profile specific to endurance performance in humans, but it is still under consideration (Rankinen et al., 2016). In contrast to humans, in horses, only a small set of genes related to racing performance has been genotyped until today (Hill et al., 2010a; Silva et al., 2015)

Genes of Interest

The Alpha-actinin skeletal muscle isoform 3 (ACTN3) gene (ENSECAG00000018961) encodes the equine α-actinin 3 protein. The gene is located between 26,511,750 and 26,524,992 bp on horse chromosome 12. ACTN3 is composed of 21 exons. ACTN3 is expressed mainly in the fast twitch muscle fibers (type 2 muscle fibers) which are responsible for high speed and important for the maintenance of muscle contraction (Yang et al., 2003; MacArthur & North 2004; Sjöblom et al., 2008). The ACTN3 gene is highly conserved and its mutation rate is lower than average (North 2008; Fattahi & Najmabadi 2012) which reflects the importance of its function. In humans, the homozygosity for a nonsense polymorphism (R577X), which converts Arginine at position 577 of the protein into a stop codon, causes complete deficiency of the fast skeletal muscle fiber protein α-actinin-3 (Mata et al., 2012; Orysiak et al., 2014). In a study of endurance athletes, the XX genotype was over-represented (Yang et al., 2003). This suggest that ACTN3 variants may contribute to enhancing the endurance performance (Yang et al., 2003; Zanoteli et al., 2003). MacArthur et al., (2008) supported this statement by mouse studies. Their analysis of knockout mouse muscle showed a shift in the properties from fast fibers towards slow fibers, increased activity of the metabolic enzymes and better resistance to fatigue. In horses, ACTN3 is suggested to affect muscle strength and insulin sensitivity which are related to endurance performance in different horse breeds (Gu et al., 2009; Thomas et al., 2014).

The Myostatin (MSTN) gene (ENSECAG00000021373) is located between 66,490,208 and 66,495,180 bp on horse chromosome 18. MSTN comprises three exons. MSTN encodes the growth differentiation factor 8 (GDF-8) which belongs to the TGF-β protein family affecting growth, differentiation and regulation of muscle proliferation as well as controlling the muscle fiber’s growth (Carnac et al., 2006). Additionally, MSTN is involved in performance relevant functions such as regeneration of skeletal muscles, bone formation, glucose metabolisms and adipocyte proliferation. In different species, mutations which result in an inhibition of MSTN cause increased muscle mass, for instance in Bully Whippet dogs and Belgian blue cattle (Mosher et al., 2007). In horses, findings implied that MSTN variants can be potential predictors of racing performance and morphological traits (Gu et al., 2009; Hill et al., 2010b; Tozaki et al., 2011; Petersen et al., 2014; François et al., 2016).

Horses, in general, vary in their ability to perform endurance due to variability of genetic background and morphological differences, health conditions, and training programs. Arabian and Arabian-cross horses possess morphological and physiological characters making them very suitable for endurance performance and long distance riding under harsh conditions (Wood & Jackson 1989; Metzger et al., 2015; USEF 2016). It is strongly thought that adaptation to extreme endurance exercise is influenced by genetic factors in this breed (Ricard et al., 2017).

Therefore, the ultimate purpose of studying the racing genetics was to provide genetic predictors of the horse’s potential for high racing ability. In this study, we focus on Syrian Arabian horses which provide a valuable model to investigate genetic variants in candidate genes for endurance in horses.

MATERIALS AND METHODS

Animals and endurance data

For this study, we sampled blood or hair from 42 endurance Arabian horses born in Syria between 1994 and 2007. Samples were collected by state veterinarian according to the animal welfare regulations set by the Syrian Ministry of Agriculture and Agrarian Reform and the Syrian Arabian horse official authorities. The studied horses performed endurance in official national events carried out in Syria between 2001 and 2010. Mean speeds for short and medium endurance distances based on international rules (Ricard & Touvais 2007) have been recorded. Based on mean speeds, 24 individuals were indicated as high endurance performers (18-25 km/h) and 18 were indicated as low endurance performers (6-16 km/h). Individuals who were excluded from race due to lameness were included in our study as low performance horses (Table 1). Weights (jockeys and their kits including lead weights) were optimized to 75 Kg. 78.5% of the horses were born in the south of Syria (Damascus and Dara) which suggests the homogeneity in geographical affiliation of the studied group (supplementary Table S1).

Table 1: Arabian horses reported for endurance performance (high and low) for three distances

	40 km		80 km		120 km
	High performance	Low performance	High performance	Low performance	High performance	Low performance
	7	5	7	11	10	2
SUM	12		18		12

Table S1: Origins and sexes of 42 Syrian Arabian horses reported for endurance performance

Region in Syria	Number of individuals
	Males	Females
Al Hasakah	1	6
Damascus	8	23
Dara	-	2
Hama	1	-
Hims	1	-
Total	11	31

Genotyping of the Candidate Genes

Promoters, exons and 3´ UTRs of two autosomal genes (ACTN3 and MSTN) were amplified using primers designed by using Primer3 online tool (Untergasser et al., 2012). If the intron was smaller than 350 bp, two exons were amplified using one primer pair was designed based on the reference equine genome assembly EquCab2. Primers information with PCR product sizes are listed in the supplementary Table S2.

Table S2: Information of gene specific sequences, PCR product sizes and annealing temperature for the analyzed fragments

Primers ID	Sequence	Product (bp)	Annealing T˚
ACTN3 prom up	AGG TTG AGC AGC TGG AAG G	633 633 635 802 642 489 363 400 645 482 462 417 774 547 1052	59 59 59 59 59 59 59 59 59 59 59 59 59 59 59
ACTN3 prom low	CTG TTC CAT ATA CTC GCC GC
ACTN3 Exon1 up	CTT TCC CAA GGT CAC ACA GC
ACTN3 Exon1 low	TCC CCT TGT CAC CCT AAA CC
ACTN3 Exon2-3 up	ACT AGA GCT CAG GGA GGG AA
ACTN3 Exon2-3 low	TGT GAG GCA TGG GTG GTT AT
ACTN3 Exon4-5 up	GAT CTG AAC CCG TGA AGC TG
ACTN3 Exon4-5 low	CAT TAC CAG ACT TGC GCC AT
ACTN3 Exon6-7 up	TGG TAA TGA AGG GCC TCA CA
ACTN3 Exon6-7 low	GGG ACC AAT ATG CTC CCA GA
ACTN3 Exon8 up	CAG GGA AGA AGA CAC TGG GT
ACTN3 Exon8 low	CTC CCT GTG TGA TGC CCT TA
ACTN3 Exon9 up	CTT TGC ATG GGT CCA GGT TT
ACTN3 Exon9 low	GAG CTT GGA TGG GCA GAA AG
ACTN3 Exon10 up	GAG ATG GGT GGA TGA GGT GA
ACTN3 Exon10 low	CCA TCA CGG TTC ACC CAT TG
ACTN3 Exon11 up	ATC AAC TTC AAC ACG CTG CA
ACTN3 Exon11 low	CCT TTG GAC ACC TGC TAT GC
ACTN3 Exon12 up	TAT CAC ACT AGC GCC TCA GG
ACTN3 Exon12 low	GGG ACA AGT GAT GAT GGG GA
ACTN3 Exon13-14 up	GCA GGC AAG GAG GAA ATC TG
ACTN3 Exon13-14low	AGC TTC CCT GTC ATC CCA TC
ACTN3 Exon15 up	AAA GCG CCA GTT CTT GAG TG
ACTN3 Exon15 low	TGA GGT TTC AGG GTG GCT AG
ACTN3 Exon16-17 up	GTA AAT GGT GCA CTG ACC CC
ACTN3 Exon16-17low	TTA GAC TGC TCT GTG ACC GG
ACTN3 Exon18-19 up	AAC CTC CAG ATG CGG ACA G
ACTN3 Exon18-19low	GCG TGA TGA GGA GGA AGT GA
ACTN3 Exon20-21 up	TCT GTG TGA CTC CAA AGC CT
ACTN3 Exon20-21low	TGT TCC CTT CCA CGG TGT AA
MSTN prom up	TGC CCT GGT AAT AAC AAT GAA GA	1200 682 878 801	58 58 59 59
MSTN prom low	TGC CTG TAC AGT CTG AGA GA
MSTN Exon1 up	CTG GTG TGG CAA GTT GTC TC
MSTN Exon1 low	TGC AGC AGA TTT CAG TCT CA
MSTN Exon2 up	GTT CCT CCA CGG TGT CTC TT
MSTN Exon2 low	TTA TTG GGT ACA GGG CTG CC
MSTN Exon3 up	AAC AAG CGT GAA GAG AGG GA
MSTN Exon3 low	AAT TGT GAG GGG AAG GCC TT

Allele Frequency of the Identified Variants

Genes were initially sequenced in 10 endurance Arabian horses which belong to two sub cohorts of high (n=5) and low performance horses (n=5). PCR products were sequenced using the ABI PRISM 310 sequencer (Applied Biosystems). Sequences were edited using the Sequence Scanner v2.0 (Applied Biosystems 2012, USA) as well as the BioEdit software (Hall 1999). The multiple sequence alignments were done using Clustal Omega package (Sievers & Higgins 2014).

The genomic positions of the identified sequence variants were determined according to the Equus caballus genome assembly EquCab2 (GCA_000002305.1) and the protein sequence that are available in Ensembl, Release 90, 2017.

The Variant Effect Predictor toolset (Ensembl) was used to determine functional consequences and novelty of the identified variants. Furthermore, we checked both of the transcription factor binding sites (TFBSs) within promoter regions using ConSite online toolset (http://consite.genereg.net) as well as miRNA target sites in the 5´ and 3´ UTRs using miRBase Database, Release 21, with filtering for Equus caballus (Griffiths-Jones et al., 2006; Griffiths-Jones et al., 2007).

For determining alleles and genotypes frequencies after identifying the sequence variants and constructing the haplotypes (manually) based on the common variants, we additionally genotyped 32 Arabian horses for the autosomal genes variants with KASP genotyping method. Reagents were obtained from KBioscience (UK), PCR was performed on a StepOnePlus set, (Applied Biosystems, USA) based on a protocol from Kreuzer et al., (2013) (Table S3).

Genotype and allele frequencies were determined by direct counting. A generalized linear model (GLM) was used to estimate the association of the SNPs with endurance performance traits including the mean speeds and the three distances (40, 80, 120 Km) in both performance indices. The GLM was performed using SAS (version 9.3) with the three genotypes of each SNP as independent variables and the endurance traits as the dependent variable.

Table S3: The customized allele-specific PCR assays and primers for the important identified ACTN3 and MSTN in 32 Arabian horses

SNP	Gene	Primer A1	Primer A2	Primer C	Temp
18:66493737	MSTN	TAT TAA GTA ATC AGG TTA TAA TGC ACC AAA	ATT AAG TAA TCA GGT TAT AAT GCA CCA AG	CCA GGA CTA TTT GAT AGC AGA GTC ATA AA	57
18:66495696	MSTN	ATT CTT TCT ATT TCA AAT GTT TGC CTA AAT AAT	CTT TCT ATT TCA AAT GTT TGC CTA AAT AAC	GAA ATG TTA CTT CCT CAG AAA TTA AGA TTT	57
12:26511704	ACTN3	GGG GCC TCG TTA AGT AGC GT	GGG GCC TCG TTA AGT AGC GC	CCC CAT ATT TAG CGC GAA TCC GAT	57
12:26515885	ACTN3	GAC CCC TTG ACC TCT CCT CTT A	GAC CCC TTG ACC TCT CCT CTT T	GAT TTT GTG GAA GCG CAT CTT GCC TT	57
12:26524894	ACTN3	GTT CTC CAC GCA AGT AGG AGC	GGT TCT CCA CGC AAG TAG GAG T	TGG GAT CAG CCA GAG GGA GCA A	57

RESULTS

The comparative sequence analysis of the candidate genes in the 10 endurance Arabian horses led to identify a total of 13 allelic variants in both ACTN3 and MSTN. The five promising SNPs (three SNPs in ACTN3 and two SNPs in MSTN) were genotyped across the 42 horses (Table S3).

ACTN3

By sequencing the promoter and 21 exons with flanking intron regions of ACTN3 (approximately 8.979 bp), we found 12 variants: one 5´ UTR variant, five intronic variants, four exonic synonymous variants, and two 3´ UTR variants (Table 2). No change in the amino acid was detected. Three ACTN3 variants were genotyped further in 32 horses which are 12:26511704G>A, 12:26515885A>T, 12:26524894T>C.

In general, the more frequent variants were the intronic variant 12:26515885A>T (splice region variant), and the exonic variant 12:26524717A>G (synonymous variant) but no significant frequency differences have been detected between the high and low performance groups. One variant in the 3´ UTR (12:26524930T>C) was analyzed for its potential effect on miRNA binding (eca-miR-1296 and eca-miR-326). Additionally, the TFBSs analysis of the ACTN3 5’ UTR 12:26511704G>A was predicted to cause gaining of an E4BP4 binding site (Table 3).

MSTN

By sequencing the promoter and three exons with their flanking intron of MSTN in 10 horses, one transition was detected at 18:66495696A>G within the promoter region. The MSTN promoter polymorphism 18:66495696A>G was genotyped in further 32 horses. The alternative allele G frequencies of the variant 18:66495696A>G are listed in Table 2.

The analysis of TFBSs of the MSTN promoter substitution of A to G at the position 18:66495696 is predicted to cause substitution of two binding sites for HFH-1 and Sox-5 by two binding sites for HFH-3 and E4BP4 (Table 3).

Table 2. The detected variants in the autosomal genes ACTN3 and MSTN, their locations and the mutated allele’s frequencies

Gene	SNP position	Reference> mutated allele	Amino acid change and positions	Variant effect	SNP ID	Frequency of the mutated allele in Arabian horses
						Sequenced individuals N=10	Total N=42	High performers N=24	Low performers N=18
ACTN3	12:26511704	G>A	-	5´ UTR	I1 (Thomas et al., 2014)	0.50	0.42	0.33	0.53
	12:26515793	C>G	-	Intronic (I12)	rs68947239	0.45	0.43	0.46	0.39
	12:26515795	C>T	-	Intronic (I12)	rs68947240	0.45	0.43	0.46	0.39
	12:26515807	T>C	-	Intronic (I12)	I4 (Thomas et al., 2014)	0.45	0.43	0.46	0.39
	12:26515885	A>T	-	splice region (I12)	rs68947241	0.45	0.43	0.46	0.39
	12:26515942	C>T	Ile105Ile	Synonymous (E3)	rs68947242	0.45	0.43	0.46	0.39
	12:26516020	G>C	-	splice region(I3)	rs68947243	0.45	0.43	0.46	0.39
	12:26519406	A>G	Pro366Pro	synonymous (E10)	E3 (Thomas et al., 2014)	0.60	-	-	-
	12:26524504	T>C	Ala814Ala	synonymous (E20)	rs394353570	0.95	-	-	-
	12:26524717	A>G	Leu858Leu	synonymous (E21)	E6 (Thomas et al., 2014)	0.30	0.61	0.58	0.64
	12:26524894	T>C	917	3´ UTR	rs396350893	0.70	0.39	0.42	0.36
	12:26524930	T>C	929	3´ UTR	rs396948497	0.30	0.61	0.58	0.64
MSTN	18:66495696	A>G	-	Upstream	(Stefaniuk et al., 2016)	0.15	0.09	0.10	0.08

E: Exon, I: Intron

Frequencies were calculated after aligning the variants, and constructing the haplotypes of ACTN3 common variants.

Table 3:. Potential effects of variants within the untranslated and promoter regions of ACTN3 and MSTN genes on miRNA and transcription factor binding sites (TFBSs)

Gene	Variant position	Location of variant	Effect of the allelic change
	miRNA gain/loss of binding	TFBSs* gain/loss of binding
ACTN3	12:26511704G>A	5 ´ UTR	-	E4BP4 [+]
ACTN3	12:26524930T>C	3 ´ UTR	eca-miR-1296 [-] eca-miR-326 [-]	-
MSTN	18:66495696A>G	Promoter	-	HFH-1 [-] Sox-5 [-] HFH-3[+] E4BP4[+]

*TFBSs: transcription factor binding sites, [+]=gain, [-]=loss

DISCUSSION

Although ACTN3 is an important functional gene previous studies found variants which infer possible functional changes (Thomas et al., 2014). If we underlay one mutation every 644 to 891 bp in horses (Orlando et al., 2013), we would expect 10 to 14 variants in the ACTN3 gene. In the current study in Syrian Arabian horses, we detected 12 variants, which is consistent with this expectation assuming an average mutation rate. The ACTN3 5’ UTR variant 12:26511704G>A showed significant frequencies differences between four equine phenotypes including endurance, sprint, pace, and strength. The A-allele is overrepresented in the strength (Clydesdale and Shire breeds, frequency=77%) and pace horses (Standardbred breed, frequency=69%), compared to sprint (Thoroughbreds, frequency=17%) and endurance horses (American Arabian, 38%) (Thomas et al., 2014). The A-allele frequency in our current study of Syrian Arabian horses (frequency=42%) is consistent with their findings. The variant 12:26511704G>A is predicted to cause gain of a binding site for the E4 promoter–binding protein 4 (E4BP4), a basic leucine zipper transcription factor. E4BP4 regulates circadian rhythm by competing for DNA binding with a member of the related PAR family of basic leucine zipper transcription factors. E4BP4, also known as nuclear factor interleukin 3 (NFIL3) is thought to affect exercise in the skeletal muscles (Bottinelli & Reggiani 2007). Based on findings by Thomas et al., (2014), the ACTN3 exonic variants 12:26515942C>T (Exon3), 12:26519406A>G (Exon10), and 12:26524717A>G (Exon21) are assigned to three conserved domains (Calpomin homology, Spectrin repeats, and the two EF-hands, respectively), which have an important role in calcium ion binding supporting the protein structure (Djinovic-Carugo et al., 2002; Parry & Squire 2005). The 3´ UTR variants 12:26524894T>C and 12:26524930T>C seems to have no direct effect on the gene function. No clear ACTN3 allele frequency differences were detected in our study between high and low endurance performance horses. However, all variants in the 5´ and 3´ UTR were analyzed for their potential effects on miRNA binding. Interestingly, one variant in the 3´ UTR of ACTN3 (12:26524930T>C) is located within two predicted miRNA target sites (eca-miR-1296 and eca-miR-326). The variant C is responsible for abrogation of these miRNA sites, which might affect their post-transcriptional regulation and consequently the gene expression.

In the MSTN gene, the promoter variant 18:66495696A>G has been previously reported in different horse breeds including Arabian, Thoroughbred, Polish Konik and Hucul horses (Binns et al., 2010; Dall'Olio et al., 2010; Hill et al., 2010c; Tozaki et al., 2012; Stefaniuk et al., 2014). The G-allele has a frequency of 0.23 in Arabian horses in Poland (Stefaniuk et al., 2014). Furthermore, 18:66495696A>G is suggested to be associated with height at withers in Arabian horses and Uruguayan Creolo horses (Dall’Olio et al., 2012; Stefaniuk et al., 2016). In the current study, the frequency of the alternative allele G was 0.09, and it did not differ considerably between high and low performing groups.

Different studies reported promoter variants of the equine MSTN gene. Among those promoter variants, 18:66495826A>G was also found in Hucul, Polish Heavy Draft, and Thoroughbred horses (Stefaniuk et al., (2014). Studies in Thoroughbred horses showed an association between racing performance phenotypes and promoter insertion polymorphisms (227 bp SINE insertion located at -373/-147 bp upstream of the translation start codon ATG). Animals homozygous for the SINE insertion allele were most frequent short distance racing, heterozygous allele carriers were more frequent in horses performing at middle-distance, while homozygous carriers for the wild type allele were most often found in long-distance endurance races (Hill et al., 2010c; Dall’Olio et al., 2014). The SINE insertion is suggested to affect the MSTN gene expression (Santagostino et al., 2015). None of the above promoter variants are observed in the Syrian Arabian horses. Interestingly, the intronic variant 18:66493737T>C has been widely known for its strong positive association with short racing distance, speed and body composition in different racehorse breeds (e.g. Thoroughbred) (Binns et al., 2010; Hill et al., 2010a; Hill et al., 2010c; Tozaki et al., 2011; Hill et al., 2012; Tozaki et al., 2012). In all examined Syrian Arabian horses of the current study, this locus was homozygous for the reference genotype (TT). Homozygosity for the T allele was also found in Arabian horses sampled in Poland (Stefaniuk et al., 2016). In another study, a an unique haplotype was found in the second exon (EU241341) present in 12 of 19 Arabian horses (Baron et al., 2012). In our Syrian Arabian horses, the three MSTN exons were conserved and identical to the reference. As shown in our study, MSTN exons were conserved in 96 Arabian horses from Poland (Stefaniuk et al., 2016).

The TFBSs analysis of the MSTN promoter substitution of A to G at the position 18:66495696 predicted to cause a loss of the Helix factor hepatocyte nuclear factor-3 homologue 1 (HFH-1) and the sex-determining region SRY-box 5 (Sox-5) transcription factor binding sites (UniProt), as well as, gaining of both: Hepatocyte Nuclear Factor 3 Forkhead Homolog 3 (HFH-3) and E4 promoter–binding protein 4 (E4BP4). HFH-1 is one of the nuclear factors that share a conserved DNA-binding domain (winged helix domain). It is thought to have an impact on the nerve and skeletal systems in mammals, particularly during the early stages of the embryo development (Altaba et al., 1993; Hong et al., 1999; Hoggatt et al., 2000). HFH-1 can act as inhibitor for the abundant protein found in smooth muscle-specific promoters (Hoggatt et al., 2000). Smooth muscles are not directly involved in the athletic performance, but their contractions are regulating the internal organs (e.g. blood vessels).

The Sox family of transcription factors is involved in regulating cell development and tissue regeneration (Sarkar & Hochedlinger 2013). The transcription factor Sox-5 (sex-determining region SRY-box 5) is considered to be an enhancer of chondrogenesis; as such it controls the correct development of the skeleton (Smits et al., 2001). Hepatocyte Nuclear Factor 3 Forkhead Homolog 3 (HFH-3 or FoxO1) is a member of the forkhead domain transcription factors family, which are all expressed in skeletal muscle (Sanchez et al., 2014) and involved in a wide range of cellular functions including energy metabolism (Ogg et al., 1997). HFH-3 has a vital role in development the sense of balance, mediating the formation of fatty acids and glycerol to be consumed by muscle cells under exercise, regulating of glucose metabolism in skeletal muscle, as well as, muscle energy homeostasis (Furuyama et al., 2003; Sanchez et al., 2014).

The correlation between endurance traits and ACTN3 and MSTN genotypes was tested by GLM analysis, and we found that none of the analyzed SNPs has significant effect on the endurance traits. Only SNP 12:26511704G>A (located in the 5´UTR of ACTN3) has marginally significant effect (0.04) on the mean speed traits in both performance indices.

CONCLUSION

In the current study, we could not detect any significant difference in allele frequencies of the 13 variants in both genes ACTN3 and MSTN, therefore no differentiation was detected between the high and low performance groups. But these variants could influence the transcription factor binding sites (TFBSs) and the miRNAs. Results could not show to which extent the possible effects of the changed transcription binding factor sites have a direct influence on the endurance performance of the Arabian horses.

However, this study contributes to the knowledge of candidate genes that are related to endurance performance in Syrian Arabian horses. Detection of the genetic background of the endurance-related genes in Arabian horses is still a question to be answered. This can be achieved if we test the association between polymorphisms of a multi-gene panel in a larger group of endurance Arabian horses using more endurance data. The information of the candidate genes variants could be beneficial in improving selecting criteria and breeding programs in the future.

ACKNOWLEDGEMENTS

We thank the Arabian horse office, the Syrian Ministry of Agriculture and Agrarian Reform, the National commission for biotechnology in Syria, all Arabian horse authorities and owners in Syria and abroad for their support and collaboration on horse sampling and pedigrees identification. Samples were collected by state veterinarian according to the animal welfare regulations set by the Syrian Ministry of Agriculture and Agrarian Reform. This study was financially supported by the IDB scholarship (MERIT).

REFERENCES

Adamu L., Adzahan N.M., Abdullah R. & Ahmad B. (2012) Effects of speed, heart rate, lactate and uric acid on the performance of arabian horses during a 120-km endurance race. IOSR Journal of Agriculture and Veterinary Science 1, 1-4.

Altaba A.R., Prezioso V., Darnell J. & Jessell T. (1993) Sequential expression of HNF-3β and HNF-3α by embryonic organizing centers: the dorsal lip/node, notochord and floor plate. Mechanisms of development 44, 91-108.

Baron E., Lopes M., Mendonça D. & da Câmara Machado A. (2012) SNP identification and polymorphism analysis in exon 2 of the horse myostatin gene. Animal genetics 43, 229-32.

Bergero D., Assenza A. & Caola G. (2005) Contribution to our knowledge of the physiology and metabolism of endurance horses. Livestock Production Science 92, 167-76.

Binns M., Boehler D. & Lambert D. (2010) Identification of the myostatin locus (MSTN) as having a major effect on optimum racing distance in the Thoroughbred horse in the USA. Animal genetics 41, 154-8.

Bottinelli R. & Reggiani C. (2007) Skeletal muscle plasticity in health and disease: from genes to whole muscle. Springer.

Bray M., Hagberg J., Perusse L., Rankinen T., Roth S., Wolfarth B. & Bouchard C. (2009) The human gene map for performance and health-related fitness phenotypes: the 2006-2007 update. Medicine+ Science in Sports+ Exercise 41, 35.

Carnac G., Ricaud S., Vernus B. & Bonnieu A. (2006) Myostatin: biology and clinical relevance. Mini reviews in medicinal chemistry 6, 765-70.

Castejon F., Trigo P., Muñoz A. & Riber C. (2006) Uric acid responses to endurance racing and relationships with performance, plasma biochemistry and metabolic alterations. Equine Veterinary Journal 38, 70-3.

Cottin F., Metayer N., Goachet A., Julliand V., Slawinski J., Billat V. & Barrey E. (2010) Oxygen consumption and gait variables of Arabian endurance horses measured during a field exercise test. Equine Veterinary Journal 42, 1-5.

Dall'Olio S., Fontanesi L., Nanni Costa L., Tassinari M., Minieri L. & Falaschini A. (2010) Analysis of horse myostatin gene and identification of single nucleotide polymorphisms in breeds of different morphological types. BioMed Research International 2010.

Dall’Olio S., Fontanesi L., Antonelli C., Nanni Costa L., Tassinari M. & Falaschini A. (2012) Association study between a SNP of the myostatin gene promoter and morphological traits in Uruguayan Creole horse. Proc. Soc. Ital. Sci. Vet 66, 412-4.

Dall’Olio S., Scotti E., Fontanesi L. & Tassinari M. (2014) Analysis of the 227 bp short interspersed nuclear element (SINE) insertion of the promoter of the myostatin (MSTN) gene in different horse breeds. Veterinaria italiana 50, 193-7.

Djinovic-Carugo K., Gautel M., Ylänne J. & Young P. (2002) The spectrin repeat: a structural platform for cytoskeletal protein assemblies. FEBS letters 513, 119-23.

Fattahi Z. & Najmabadi H. (2012) Prevalence of ACTN3 (the athlete gene) R577X polymorphism in Iranian population. Iranian Red Crescent Medical Journal 14, 617.

François L., Fegraeus K.J., Eriksson S., Andersson L.S., Tesfayonas Y.G., Viluma A., Imsland F., Buys N., Mikko S. & Lindgren G. (2016) Conformation traits and gaits in the Icelandic horse are associated with genetic variants in Myostatin (MSTN). Journal of Heredity, esw031.

Furuyama T., Kitayama K., Yamashita H. & Nozomu M. (2003) Forkhead transcription factor FOXO1 (FKHR)-dependent induction of PDK4 gene expression in skeletal muscle during energy deprivation. Biochemical Journal 375, 365-71.

Griffiths-Jones S., Grocock R.J., Van Dongen S., Bateman A. & Enright A.J. (2006) miRBase: microRNA sequences, targets and gene nomenclature. Nucleic acids research 34, D140-D4.

Griffiths-Jones S., Saini H.K., van Dongen S. & Enright A.J. (2007) miRBase: tools for microRNA genomics. Nucleic acids research 36, D154-D8.

Gu J., Orr N., Park S.D., Katz L.M., Sulimova G., MacHugh D.E. & Hill E.W. (2009) A genome scan for positive selection in thoroughbred horses. PLoS One 4, e5767.

Hall T.A. (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. In: Nucleic acids symposium series, pp. 95-8.

Hill E., Gu J., McGivney B. & MacHugh D. (2010a) Targets of selection in the Thoroughbred genome contain exercise‐relevant gene SNPs associated with elite racecourse performance. Animal genetics 41, 56-63.

Hill E.W., Fonseca R.G., McGivney B.A., Gu J., MacHugh D.E. & Katz L.M. (2012) MSTN genotype (g. 66493737C/T) association with speed indices in Thoroughbred racehorses. Journal of Applied Physiology 112, 86-90.

Hill E.W., Gu J., Eivers S.S., Fonseca R.G., McGivney B.A., Govindarajan P., Orr N., Katz L.M. & MacHugh D. (2010b) A sequence polymorphism in MSTN predicts sprinting ability and racing stamina in thoroughbred horses. PLoS One 5, e8645.

Hill E.W., McGivney B.A., Gu J., Whiston R. & MacHugh D.E. (2010c) A genome-wide SNP-association study confirms a sequence variant (g. 66493737C> T) in the equine myostatin (MSTN) gene as the most powerful predictor of optimum racing distance for Thoroughbred racehorses. BMC genomics 11, 1.

Hoggatt A.M., Kriegel A.M., Smith A.F. & Herring B.P. (2000) Hepatocyte nuclear factor-3 homologue 1 (HFH-1) represses transcription of smooth muscle-specific genes. Journal of Biological Chemistry 275, 31162-70.

Hong H.-K., Lass J.H. & Chakravarti A. (1999) Pleiotropic skeletal and ocular phenotypes of the mouse mutation congenital hydrocephalus (ch/Mf1) arise from a winged helix/forkhead transcription factor gene. Human molecular genetics 8, 625-37.

Kreuzer S., Reissmann M. & Brockmann G.A. (2013) Gene test to elucidate the ETEC F4ab/F4ac receptor status in pigs. Veterinary microbiology 162, 293.

MacArthur D.G. & North K.N. (2004) A gene for speed? The evolution and function of α‐actinin‐3. Bioessays 26, 786-95.

MacArthur D.G., Seto J.T., Chan S., Quinlan K.G., Raftery J.M., Turner N., Nicholson M.D., Kee A.J., Hardeman E.C. & Gunning P.W. (2008) An Actn3 knockout mouse provides mechanistic insights into the association between α-actinin-3 deficiency and human athletic performance. Human molecular genetics 17, 1076-86.

Maffetone P. (2010) The big book of endurance training and racing. Skyhorse Publishing, Inc.

Mata X., Vaiman A., Ducasse A., Diribarne M., Schibler L. & Guerin G. (2012) Genomic structure, polymorphism and expression of the horse alpha-actinin-3 gene. Gene 491, 20-4.

Metayer N., Biau S., Cochet J. & Barrey E. (2004) Stduy of locomotion and morphological factors in the performance of the horse specialized in endurance tests. In: 30ème journée de la recherche équine, 3 mars 2004., pp. 67-76. Les Haras Nationaux Direction du Développement.

Metzger J., Karwath M., Tonda R., Beltran S., Águeda L., Gut M., Gut I.G. & Distl O. (2015) Runs of homozygosity reveal signatures of positive selection for reproduction traits in breed and non-breed horses. BMC genomics 16, 764.

Mosher D.S., Quignon P., Bustamante C.D., Sutter N.B., Mellersh C.S., Parker H.G. & Ostrander E.A. (2007) A mutation in the myostatin gene increases muscle mass and enhances racing performance in heterozygote dogs. PLoS genetics 3, e79.

Nagy A., Dyson S.J. & Murray J.K. (2012) A veterinary review of endurance riding as an international competitive sport. The Veterinary Journal 194, 288-93.

North K. (2008) Why is α-actinin-3 deficiency so common in the general population? The evolution of athletic performance. Twin Research and Human Genetics 11, 384-94.

Ogg S., Paradis S., Gottlieb S., Patterson G.I., Lee L., Tissenbaum H.A. & Ruvkun G. (1997) The Fork head transcription factor DAF-16 transduces insulin-like metabolic and longevity signals in C. elegans. Nature 389, 994-9.

Orlando L., Ginolhac A., Zhang G., Froese D., Albrechtsen A., Stiller M., Schubert M., Cappellini E., Petersen B. & Moltke I. (2013) Recalibrating Equus evolution using the genome sequence of an early Middle Pleistocene horse. Nature 499, 74-8.

Orysiak J., Busko K., Michalski R., Mazur-Różycka J., Gajewski J., Malczewska-Lenczowska J., Sitkowski D. & Pokrywka A. (2014) Relationship between ACTN3 R577X polymorphism and maximal power output in elite Polish athletes. Medicina 50, 303-8.

Parry D.A. & Squire J.M. (2005) Fibrous proteins: Coiled-coils, collagen and elastomers. Gulf Professional Publishing.

Petersen J.L., Valberg S.J., Mickelson J.R. & McCue M.E. (2014) Haplotype diversity in the equine myostatin gene with focus on variants associated with race distance propensity and muscle fiber type proportions. Animal genetics 45, 827-35.

Rankinen T., Fuku N., Wolfarth B., Wang G., Sarzynski M.A., Alexeev D.G., Ahmetov I.I., Boulay M.R., Cieszczyk P. & Eynon N. (2016) No evidence of a common DNA variant profile specific to world class endurance athletes. PLoS One 11, e0147330.

Ricard A., Robert C., Blouin C., Baste F., Torquet G., Morgenthaler C., Rivière J., Mach N., Mata X. & Schibler L. (2017) Endurance exercise ability in the horse: a trait with complex polygenic determinism. Frontiers in genetics 8, 89.

Ricard A. & Touvais M. (2007) Genetic parameters of performance traits in horse endurance races. Livestock Science 110, 118-25.

Rivero J.-L.L. & Barrey E. (2001) Heritabilities and genetic and phenotypic parameters for gluteus medius muscle fibre type composition, fibre size and capillaries in purebred Spanish horses. Livestock Production Science 72, 233-41.

Rivero J., Serrano A.L., Henckel P. & Aguera E. (1993) Muscle fiber type composition and fiber size in successfully and unsuccessfully endurance-raced horses. Journal of Applied Physiology 75, 1758-66.

Sanchez A.M., Candau R.B. & Bernardi H. (2014) FoxO transcription factors: their roles in the maintenance of skeletal muscle homeostasis. Cellular and molecular life sciences 71, 1657-71.

Santagostino M., Khoriauli L., Gamba R., Bonuglia M., Klipstein O., Piras F.M., Vella F., Russo A., Badiale C. & Mazzagatti A. (2015) Genome-wide evolutionary and functional analysis of the Equine Repetitive Element 1: an insertion in the myostatin promoter affects gene expression. BMC genetics 16, 126.

Sarkar A. & Hochedlinger K. (2013) The sox family of transcription factors: versatile regulators of stem and progenitor cell fate. Cell stem cell 12, 15-30.

Schröder W., Klostermann A. & Distl O. (2011) Candidate genes for physical performance in the horse. The Veterinary Journal 190, 39-48.

Sievers F. & Higgins D.G. (2014) Clustal omega. Current Protocols in Bioinformatics, 3.13. 1-3.. 6.

Silva M.S., Bolani W., Alves C.R., Biagi D.G., Lemos Jr J.R., da Silva J.L., de Oliveira P.A., Alves G.B., de Oliveira E.M. & Negrão C.E. (2015) Elimination of Influences of the ACTN3 R577X Variant on Oxygen Uptake by Endurance Training in Healthy Individuals. IJSPP 10.

Sjöblom B., Salmazo A. & Djinović-Carugo K. (2008) α-Actinin structure and regulation. Cellular and molecular life sciences 65, 2688-701.

Smits P., Li P., Mandel J., Zhang Z., Deng J.M., Behringer R.R., De Crombrugghe B. & Lefebvre V. (2001) The transcription factors L-Sox5 and Sox6 are essential for cartilage formation. Developmental cell 1, 277-90.

Stefaniuk M., Kaczor U., Augustyn R., Gurgul A., Kulisa M. & Podstawski Z. (2014) Identification of a New Haplotype within the Promoter Region of the MSTN Gene in Horses from Five of the most Common Breeds in Poland. Folia biologica 62, 219-22.

Stefaniuk M., Ropka-Molik K., Piórkowska K., Kulisa M. & Podstawski Z. (2016) Analysis of polymorphisms in the equine MSTN gene in Polish populations of horse breeds. Livestock Science 187, 151-7.

Thomas K.C., Hamilton N.A., North K.N. & Houweling P.J. (2014) Sequence analysis of the equine ACTN3 gene in Australian horse breeds. Gene 538, 88-93.

Tozaki T., Hill E., Hirota K., Kakoi H., Gawahara H., Miyake T., Sugita S., Hasegawa T., Ishida N. & Nakano Y. (2012) A cohort study of racing performance in Japanese Thoroughbred racehorses using genome information on ECA18. Animal genetics 43, 42-52.

Tozaki T., Sato F., Hill E.W., Miyake T., Endo Y., Kakoi H., Gawahara H., Hirota K.-i., Nakano Y. & Nambo Y. (2011) Sequence variants at the myostatin gene locus influence the body composition of Thoroughbred horses. Journal of Veterinary Medical Science 73, 1617-24.

UniProt The Universal Protein Resource. The UniProt Consortium, http://www.uniprot.org.

Untergasser A., Cutcutache I., Koressaar T., Ye J., Faircloth B.C., Remm M. & Rozen S.G. (2012) Primer3—new capabilities and interfaces. Nucleic acids research 40, e115-e.

USEF (2016) RULE BOOK. United States Equestrian Federation,, 4047 Iron Works Parkway Lexington, KY 40511

Wood C.H. & Jackson S.G. (1989) Horse Judging Manual. University of Kentucky Cooperative Extension Service.

Yang N., MacArthur D.G., Gulbin J.P., Hahn A.G., Beggs A.H., Easteal S. & North K. (2003) ACTN3 genotype is associated with human elite athletic performance. The American Journal of Human Genetics 73, 627-31.

Zanoteli E., Lotuffo R.M., Oliveira A.S., Beggs A.H., Canovas M., Zatz M. & Vainzof M. (2003) Deficiency of muscle α-actinin-3 is compatible with high muscle performance. Journal of molecular neuroscience 20, 39-42.

Copyright (c) 2019 Global Journal of Animal Scientific Research. All rights reserved.

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.