Acyl Coenzyme A Synthetase Long-Chain 1 (ACSL1) Gene Polymorphism (rs6552828) and Elite Endurance Athletic Status: A Replication Study

The aim of this study was to determine the association between the rs6552828 polymorphism in acyl coenzyme A synthetase (ACSL1) and elite endurance athletic status. We studied 82 Caucasian (Spanish) World/Olympic-class endurance male athletes, and a group of sex and ethnically matched healthy young adults (controls, n = 197). The analyses were replicated in a cohort of a different ethnic origin (Chinese of the Han ethnic group), composed of elite endurance athletes (runners) [cases, n = 241 (128 male)] and healthy sedentary adults [controls, n = 504 (267 male)]. In the Spanish cohort, genotype (P = 0.591) and minor allele (A) frequencies were similar in cases and controls (P = 0.978). In the Chinese cohort, genotype (P = 0.973) and minor allele (G) frequencies were comparable in female endurance athletes and sedentary controls (P = 0.881), whereas in males the frequency of the G allele was higher in endurance athletes (0.40) compared with their controls (0.32, P = 0.040). The odds ratio (95%CI) for an elite endurance Chinese athlete to carry the G allele compared with ethnically matched controls was 1.381 (1.015–1.880) (P-value = 0.04). Our findings suggest that the ACSL1 gene polymorphism rs6552828 is not associated with elite endurance athletic status in Caucasians, yet a marginal association seems to exist for the Chinese (Han) male population.


Introduction
Elite athletic status is a complex phenotype, with several genetic polymorphisms, many of which remain to be identified, contributing to athletic success, whether individually or in combination with other polymorphisms [1]. Genome-wide association (GWA) can help identifying novel candidate polymorphisms associated with elite endurance status. This type of studies evaluates association of genetic variation with outcomes or traits of interest by using 100,000 to 1 million or more markers across the genome without any previous hypotheses about potential mechanisms [2]. A GWA study was recently conducted by Bouchard et al. [3] in sedentary Caucasians to study the association of 324,611 singlenucleotide polymorphisms (SNPs) and the trainability of one of the main phenotype traits indicative of human endurance performance, i.e. maximal oxygen uptake (VO 2 max). The strongest association with the training response of VO 2 max was found to acyl coenzyme A synthetase long-chain 1 (ACSL1) gene polymor-phism (rs6552828). The ACSL1 gene is a candidate to explain individual variability in endurance performance, as well as in some health-related phenotypes, owing to its potential role in aerobic metabolism at the adypocite, cardiomyocite, liver and skeletal muscle fiber level [4,5,6,7,8,9].
The findings of GWA studies should be further explored in genetic association studies focused on those SNPs showing the highest level of association [2]. Thus, the purpose of the present case:control study was to determine the association between the ACSL1 rs6552828 polymorphism and elite endurance athletic status. To this end, we studied a cohort that comprised Caucasian (Spanish) World/Olympic-class endurance male athletes (cases), and sex and ethnically-matched healthy young adults (controls). We also studied a replication cohort of a different ethnic origin (Chinese of the Han ethnic group), composed of elite endurance athletes (cases), and healthy sedentary adults (controls). Owing to the important putative role of ACSL1 in aerobic-related phenotypes [4,5,6,7,8,9] we hypothesized that the ACSL1 rs6552828 polymorphism is associated with elite endurance athletic status.

Participants
The research project was in accordance with the Declaration of Helsinki, it was approved by the corresponding University Review Boards [Universidad Europea de Madrid (UEM), Spain and China Institute of Sport Science (Beijing, China)]. Written consent was obtained from each participant. Our study adhered to most of the recent guidelines for STrengthening the REporting of Genetic Association studies (STREGA), including issues such as replication, selection of participants, rationale for choice of genes and variants, statistical methods or relatedness [10].
Spanish cohort.  21 ). All were undergraduate Physical Education students from the same university (UEM). Inclusion and exclusion criteria for this group were to be free of any diagnosed cardiorespiratory disease and not to be engaged in competitive sports or in formal, supervised exercise training (i.e. performing less than 3 structured weekly sessions of strenuous exercise as running, swimming, bicycling, and weight lifting) or to have a family history of competitive sports participation.
The VO 2 max values of runners, cyclists and rowers were obtained using a breath-by-breath system (Oxycon Pro System, Jaeger, Wuerzburg, Germany) in laboratory treadmill, cycleergometer or rower-ergometer tests performed until volitional exhaustion. The VO 2max of controls was estimated from the time to complete 2,000 meter tests [11]; the tests were performed inside a 400-meter outdoor track under similar environmental conditions (temperature, ,23-24uC; relative humidity, 45-55%; barometric pressure, ,720 mmHg).

Genotyping
Our study followed recent recommendations for replicating genotype-phenotype association studies [12]: genotyping was performed specifically for research purposes, and the researchers in charge of genotyping were totally blinded to the participants' identities [DNA samples were tracked solely with bar-coding and personal identities were only made available to the main study researcher (in Spain or China) who was not involved in actual genotyping].
Spanish cohort. We obtained DNA from participants' blood or saliva samples over years 2004-2008 and used the classical phenol-chloroform DNA extraction protocol with alcoholic precipitation. Genomic DNA was resuspended in 50 ml milli-Q H 2 O and stored at 220uC. Genotyping was performed during April-May 2011 in the Genetics laboratory of the UEM. Polymerase chain reaction (PCR) amplification was performed using a StepOne TM Real-Time PCR System (Applied Biosystems, Foster City, CA). Allelic discrimination analysis for the ACSL1 rs6552828 polymorphism was performed with predesigned Applied Biosystems TaqManH SNP Genotyping Assays on demand (Assay ID: C__30469648_10).
Replication cohort. We obtained samples of peripheral whole blood from elite athletes (during years 2003

Statistical analysis
Genotypic and allele frequencies were compared among sedentary controls and endurance athletes using the x 2 test. We used logistic regression analysis to analyse the association of the ACSL1 rs6552828 polymorphism with elite endurance athletic status. The analyses were conducted in Spanish and Chinese separately, and in the case of the Chinese population, the analyses were conducted separately for men and women. All statistical analyses were performed using the PASW (v. 18.0 for WIN-DOWS, Chicago) and the a was set at 0.05.

Spanish cohort
There were no failures in sample collection, DNA acquisition or genotyping procedures, except for 11 athletes, for which the amount of DNA gathered from saliva was insufficient to allow ACSL1 rs6552828 genotype assessment. Genotype distributions met Hardy-Weinberg equilibrium (HWE) in both controls and athletes (Table 1). Genotype (P = 0.591) and minor allele (A) frequencies were similar in sedentary controls and athletes (P = 0.978). The odds ratio (OR) and 95% confidence interval (95%CI) for the association between carriage of the A allele of the ACSL1 rs6552828 polymorphism and athletic status was 0.997 (0.819-1.214).

Replication cohort
There were no failures in sample collection, DNA acquisition or genotyping procedures, except for 1 participant (female) in the control group, and 5 (3 male, 2 female) in the athlete's group. Genotype distributions were in HWE except for male endurance athletes (P = 0.035, Table 2). In females, genotypic (P = 0.973) and allele frequencies (P = 0.881) were similar in endurance and controls, whereas in males both genotypic (P = 0.019) and allelic (P = 0.040) differed in endurance and controls. The odds ratio (95%CI) for an elite endurance Chinese athlete of having the G allele compared with ethnically-matched controls was 0.975 (0.697-1.364, P = 0.881) for females, whereas in males, the odds ratio of having the G allele was 1.381 (1.015-1.880, P = 0.04).

Discussion
The main finding of our study was that the ACSL1 rs6552828 SNP, located in the first intron of the ACSL1 gene, 715 bp and 718 bp upstream of exon 2 and the start codon, respectively, was not associated with elite endurance athletic status in Spanish men, yet a marginal association was found in Chinese men. The ACSL1 gene is a putative candidate to explain individual variability in endurance performance, as well as in some health related phenotypes, owing to its potential role in aerobic metabolic adaptations to regular exercise, at the adypocite, cardiomyocite, liver and skeletal muscle fiber level. Long-chain acyl coenzyme A (acyl-CoA) synthetase (ACSL) isoenzymes, of which ACSL1 is the main and most studied isoenzyme convert free fatty acids (FFA) to acyl coenzyme A (acyl-CoA) in an ATP-dependent manner, simultaneously activating and trapping FFA within cells [13]. Activation of FFA to acyl-CoA is required before FFAs can be oxidized to provide ATP. ACSL1 is highly expressed in major energy-metabolizing tissues such as fat, liver, and skeletal muscles [5,6]. Recent research also supports evidence for an important role of ACSL1 in heart metabolism [14].
The role of ACSL1 in FFA oxidation in different tissues has been shown using transgenic mice models. Mice lacking ACSL1 specifically in adipose tissue have defects in adipose FFA oxidation [9], whereas those unable to express ACSL1 in heart ventricles show compensatory catabolism of glucose and amino acids leading to mTOR activation and cardiac hypertrophy without lipid accumulation or dysfunction [14]. In contrast, mice overexpressing ACSL1 specifically at the heart level show markedly impaired metabolic homeostasis with accumulation of triglycerides and phospholipids [15].  ACSL1 is a candidate to explain individual differences in some several disease and endurance exercise-related phenotypes. Recent research has shown that the ACSL1 rs9997745 polymorphism influences the risk of metabolic disease, most likely via disturbances in FFA metabolism [16]; no individual or combined association was however found for other SNPs of this gene, i.e. rs4862417, rs13120078, rs12503643 and the one we studied here, rs6552828. A GWA study recently conducted by Bouchard et al. [3] on 324,611 SNPs identified a set of 21 SNPs accounting for 49% of the variance in the trainability of VO 2 max [3]. The strongest association with the training response of VO 2 max was found to ACSL1 rs6552828. In the single-SNP analyses, rs6552828 explained 6.1% of the variance in the response of VO 2 max. Homozygotes of the rs6552828 minor allele (AA) had 125 mL/ min (228%) and 63 mL/min (217%) lower VO 2 max response than the common allele homozygotes (GG) and the heterozygotes (AG) respectively. Interestingly, in our study the A allele was less frequent in elite male endurance Chinese athletes compared with their controls. It must be also kept in mind that the A allele was the major allele in the Chinese cohort, which highlights inter-ethnic differences in genotype distributions.
To our knowledge, there is no functional data on the rs6552828 SNP; thus, we can only speculate about mechanisms underlying our findings. The intronic location of this SNP has the potential to affect mRNA stability or to modulate ACSL1 gene transcriptional activity. Indeed, non-coding SNPs could regulate the alternative splicing of mRNA leading to changes in gene expression [17] and phenotype traits [18,19]. Non-coding SNPs can also influence the binding of transcription factors [20]. Another possibility is that the ACSL1 is part of the group of candidate genes, among which are calcineurin genes [21], but many of which are yet to be identified, whose cumulative effect explains, at least partly, individual variations in endurance performance in the Chinese Han population. It could also be possible that the rs6552828 SNP may be a surrogate marker for other functional ACSL1 SNPs in the region.
We believe the results of our study are overall valid, as all the following criteria were met [2]: cases clearly presented the main study phenotype (i.e. being an elite athlete), as we studied some of the best elite endurance athletes world-wide, participants within both cohorts were ethnically-matched, genetic assessment was accurate and unbiased, genotype distributions were in HWE in the control group of the two cohorts, and we used a replication cohort of a different ethnic origin. Current body of knowledge on genetic factors associated with exercise phenotypes and athletic status comes mainly from research performed on Caucasian populations. Further investigations are thus needed with other ethnic groups and populations as the one studied here, i.e. representing an important fraction of the total planet population.
In summary, our findings suggest that the ACSL1 gene polymorphism rs6552828 is marginally associated with male elite endurance status in Chinese (Han) population yet such association was not found in Chinese females or in a different (Caucasian) cohort. Our findings exemplify the need for further genetic association studies in the field of sport sciences to use at least two cohorts of a different ethnic background in order to increase the generalisability of their results.