BACKGROUND
maximal oxygen uptake  refers to the highest rate at which oxygen can be consumed by the body during intense exercise  <cit> , and is, among other factors, an important predictor of elite endurance performance  <cit> .
studies have shown that the changes in vo2max following exercise training vary markedly between individuals and that ~ 50% of the variance can be explained by genetic factors  <cit> .
over the last two decades, many single nucleotide polymorphisms  have been suggested to influence elite performance and the variability in vo2max increase following exercise training  <cit> .
while most of these snps were discovered using the candidate gene approach  <cit> , a more comprehensive, genome-wide linkage approach has identified a genomic region on chromosome  <dig> that is associated with the vo2max training response.
the endothelial pas domain protein  <dig>  gene was one of the four genes in this region responsible for this linkage  <cit> .
epas <dig> is a transcription factor playing a key role in the hypoxia inducible factor  pathway in blood, which is responsible for activating gene expression in response to hypoxia  <cit> .
in normal oxygen conditions, epas <dig> is quickly degraded in the cytoplasm.
however, when oxygen levels drop, epas <dig> becomes stabilized, translocates to the nucleus and activates genes that are involved in erythropoiesis , and angiogenesis , thus favoring a better delivery of oxygen to the tissues  <cit> .
delivery of oxygen to skeletal muscles during endurance exercise is viewed a factor limiting vo2max <cit> .
therefore, as a hypoxia detector and as an activator of improved oxygen delivery to the active tissues, epas <dig> is a plausible candidate to influence endurance performance.
epas <dig> snps have been previously associated with blood parameters, such as alterations in erythropoietin, hemoglobin and hematocrit , that are important for success in athletic performance.
for instance, tibetans with the tt genotype in registered snp   <dig> had lower hemoglobin concentration compared with their tc counterparts  <cit> .
however, to date, only one study has examined a possible link between snps within epas <dig> and elite athletic performance  <cit> .
this study looked at snps and haplotypes within epas <dig> in elite australian athletes, stratified to two groups participating in middle-distance , and long-distance  events.
these groups were compared to a non-athletic control group.
the t allele in rs <dig> and the g allele in rs <dig>  two snps located in the first intron of epas <dig>  were overrepresented in the group of endurance athletes compared with controls  <cit> .
furthermore, in the same study, two haplotypes involving rs <dig>  rs <dig>  rs <dig> and rs <dig> were associated with elite endurance performance.
while haplotype g  was overrepresented in elite endurance athletes, haplotype f  was underrepresented in elite endurance athletes compared to controls  <cit> .
despite these positive findings, and the strong biological rational behind investigating epas <dig> in relation to elite endurance performance, this is the only genetic association study showing that epas <dig> snps impact performance, and the sprinters consisting primarily of 100–400 m track runners and sprint cyclists were excluded from the analysis.
replication studies are therefore needed to confirm this association, particularly in different populations, and with a larger sample size  <cit> .
therefore, the aim of this study was to compare genotype distributions and interactions of the epas <dig> snps rs <dig>  rs <dig>  rs <dig>  rs <dig> and rs <dig> between sprint/power athletes, endurance athletes, and controls in polish and russian cohorts.
we also examined the association between the epas <dig> snps and athletic status according to the athletes’ level of competition .
in light of the relationship previously observed between endurance-related phenotypes and snps in the epas <dig>  we hypothesized that epas <dig> snps would be associated with elite endurance performance compared to controls and sprint/power athletes.
we did not have specific directional hypotheses for rs <dig>  rs <dig> and rs <dig> as these snps have not previously been associated with any performance and/or endurance-related phenotypes.
we did not have any directional hypothesis for rs <dig> either, due to the conflicting results reported for this snp   <cit> .
however, in line with the findings of henderson et al.
<cit>  we did expect the rs <dig> g allele to be associated with elite endurance performance.
finally, we did not have any directional hypotheses for any of the snps in relation to sprint/power performance.
methods
the study was approved by the pomeranian medical university ethics committee, poland, and the ural state university of physical culture, russia, and written informed consent was obtained from each participant.
the study complied with the guidelines set out in the declaration of helsinki and the ethics policy of the szczecin university  <cit> .
participants
the athletes and controls were all european caucasians.
the athletes were categorized as either endurance athletes or sprint/power athletes as determined by the distance, duration and energy requirements of their event/sport.
all athletes were ranked in the top  <dig> nationally in their sport discipline and grouped as being either ‘elite-level’ or ‘sub-elite’ based on their best personal performance.
those in the elite group had participated in international competitions such as world and european championships, and/or olympic games, whereas those in the sub-elite group had participated in national competitions only.
details on the number of participants in the elite and sub-elite group are presented in table  <dig> table  <dig>
athletes’ description
polish sample
the sample comprised  <dig> polish athletes , including  <dig> elite and sub-elite sprint/power athletes and  <dig> elite and sub-elite endurance athletes, as well as  <dig> healthy, unrelated, sedentary controls .
russian sample
the russian sample comprised  <dig> athletes , including  <dig> elite and sub-elite sprint/power athletes and  <dig> elite and sub-elite endurance athletes, as well as  <dig> healthy unrelated sedentary controls .
the description of the polish and the russian athletes according to their event/sport is summarized in table  <dig>
genotyping
polish sample
genomic dna was isolated from buccal epithelium using genelute mammalian genomic dna miniprep kit  according to the manufacturer’s instructions.
all genetic analyses were performed at the molecular biology and biotechnology center, faculty of biology, university of szczecin.
all samples were genotyped in duplicate using allelic discrimination assays with taqman® probes  on a cfx <dig> touch™ real-time pcr detection system .
to discriminate epas <dig> rs <dig>  rs <dig>  rs <dig>  rs <dig> and rs <dig> alleles, taqman® pre-designed snp genotyping assays were used , including appropriate primers and fluorescently labeled  mgb™ probes to detect the alleles.
genotypes were assigned using all of the data from the study simultaneously.
russian sample
genomic dna was isolated from buccal epithelium or peripheral blood, during the years 2011– <dig>  using the diatom™ dna prep kit .
genotyping of five selected snps was performed by single-base extension  method.
the sequence surrounding each snp was obtained from the genome reference consortium human genome build  <dig> assembly from the ensembl project  <cit> .
the primer3web software v.  <dig> .
<dig>  <cit>  was used for designing the pcr primers.
pcr product range was 109–173 bp.
sbe primers to detect rs <dig>  rs <dig>  rs <dig>  and rs <dig> were designed to anneal on the positive strand immediately adjacent to the single nucleotide variation sites.
sbe primer for detection of rs <dig> was designed to anneal on the negative strand.
to avoid any non-specific amplification and extension products, all primers were blasted against human genome reference sequence.
sets of preselected pcr primer pairs and sbe primers were screened for potential cross-reactivity by using autodimer software.
multiplex pcr was performed in a volume of 15 μl containing 1 × pcr buffer,  <dig>  mm mgcl <dig>   <dig>  mm dntps,  <dig>  μm of each primer ,  <dig> unit smartaq dna polymerase  and 5 ng of template dna.
thermal cycler conditions were: 95°c for 30 s, 30 cycles of 95°c for 45 s, 60°c for 45 s, 72°c for 60 sec and finally 10 min at 72°c in geneamp® pcr system  <dig> .
multiplex pcr products were checked for quality and yield by running 3 μl in 2% agarose-tbe gels.
5 μl of pcr products were cleaned with  <dig> unit of fastap thermosensitive alkaline phosphatase  and  <dig> units of exonuclease i .
multiplex sbe reaction was performed by using snapshot® multiplex kit  in 5 μl final volume, including  <dig>  μl of snapshot multiplex ready reaction mix,  <dig>  μl pooled sbe primers and  <dig>  μl of cleaned pcr product .
the cycling conditions were 96°c 10 s, 50°c 5 s and 60°c 30 s, during 25 cycles in geneamp® pcr system  <dig> .
to remove the unincorporated ddntps, the final product was incubated with  <dig> unit of tap .
snapshot products with genescan™ -  <dig> liz™ size standard  were diluted in hi-di™ formamide , denatured and separated using an abi prism  <dig> genetic analyzer  with a 47 cm length capillary and pop-4™ polymer .
the snapshot® primer focus® kit  was used to analyze individual sbe primers for their approximate sizing locations prior to performing the multiplex sbe reaction.
final data were analyzed using the genemapper® software v.  <dig>  from applied biosystems following the software manual.
k <dig> dna high molecular weight from promega corp.  served as positive control sample.
genetic profile of k <dig> dna was following: rs <dig> – g/g, rs <dig> – c/c, rs <dig> – t/t , rs <dig> – g/g, rs <dig> – c/c.
each of the five pcr products  was formed from five different individuals, and was sequenced in separate reaction using bigdye® terminator v <dig>  cycle sequencing kit  with full coincidence of expected and observed sequences.
sequencing was performed in a second laboratory , according to latest recommendations  <cit> .
genotyping reliability across two laboratories
genotyping was performed in duplicate in the same laboratory for accuracy.
two independent investigators have called the genotyping score in each laboratory-100% of the genotypes could be called.
for the purpose of results reliability across two laboratories in two different countries , different dna samples  were shipped from russia to poland and were genotyped by taqman assays.
the results of the genotyping were in 100% agreement across the two laboratories.
statistical analysis
the genotype frequencies of all individual snps are presented in additional file  <dig>  before looking at both the individual effects of the five epas <dig> snps and their interactions, we selected the best genetic model for each snp.
then, snp main effects, as well as snp-snp interactions, were investigated using multivariate adaptive regression splines , a nonparametric regression method  <cit>  that has been successfully applied for detecting snp-snp interaction in several studies .
finally, the odds ratios  of being either a sprint/power athlete or an endurance athlete were calculated for each significant snp and significant interacting pairs of snps using the best genetic model for each snp.
details on the steps that have been taken in the statistical analysis are shown in figure  <dig> figure  <dig>
the five steps that were followed in the statistical analysis.
mars: multivariate adaptive regression splines, bif: bootstrap inclusion fraction.
firstly, we selected the best genetic model for each snp by testing three inheritance models  for each snp in the entire cohort of sprint/power athletes.
secondly, mars was used to detect snp main effects and snp-snp interactions .
thirdly, the covariates selected by mars were input into a logistic regression model to determine their significance, and all covariates with p-value >  <dig>  were excluded.
fourthly, to validate the selected covariates, we repeated steps  <dig> and  <dig> on  <dig> random samples with replacement from the original dataset and calculated the how many times the selected covariates were significant in the  <dig> random samples .
all covariates with a bif < 50% were excluded.
fifthly, we calculated the odds ratio of the genotype combinations for each selected covariate to give a clear biological interpretation.
hardy–weinberg equilibrium
χ <dig> analysis was used to confirm if the control group, in each of the two samples, met hwe expectations.
hwe was tested separately for each snp.
selection of the best genetic model for each snp
three inheritance models  were assessed in the pooled cohorts of sprint/power and endurance athletes  using the wald test in logistic regression models, and the best model was selected based on the minimum p-value for each snp.
the athletic status was divided into two categories and encoded as a dummy variable: athlete  and control .
to adjust for multiple comparisons, the false discovery rate was controlled using the benjamini and hochberg correction  <cit> .
for consistency, the same genetic model was applied separately in the russian and polish groups.
all of the following steps were performed once when comparing sprint/power athletes with controls, and once again when comparing endurance athletes with controls.
analysis of snp main effects and snp-snp interactions
variable importance ranking for snps with strong linkage disequilibrium  has been shown to be biased in previous studies  <cit> .
therefore, ld in the  <dig> snps was examined using r <dig> <cit> , and the pairs of snps displaying a strong ld  were identified .
only rs <dig> and rs <dig> were in strong ld .
consequently, two mars models, all corrected for sex, were developed: one including rs <dig>  rs <dig>  rs <dig> and rs <dig>  and another independent mars model including rs <dig>  rs <dig>  rs <dig> and rs <dig>  for simplicity, we have only reported the mars model excluding rs <dig> in the results section.
in mars, the maximum number of basis functions was set at  <dig>  and the maximum degree of interaction was set at  <dig>  as mars does not provide variable significance using p-values, each covariate selected by mars was used as input into a logistic regression model to determine its significance.
all non-significant covariates  were excluded.
to confirm the significance of the covariates identified by the logistic regression, and to rank their importance, we used the bootstrap inclusion fraction  criterion  <cit> .
we obtained  <dig> mars models using  <dig> random bootstrap samples with replacement from the original data set.
then, we calculated the proportion of times that a significant variable appeared in the  <dig> mars models and called this number the bif.
a variable which is approximately uncorrelated with others, and is only significant at the chosen nominal α level in the mars model, is selected in about 50% of bootstrap samples.
as the p-value diminishes, the bif tends toward 100%.
thus, we only included covariates with a bif > 50%.
for each significant covariate, the odds ratio  of being either a sprint/power or an endurance athlete, depending on the genotype, was calculated.
the 95% confidence intervals  were calculated by unconditional maximum likelihood estimation.
RESULTS
in the pooled cohort of russian and polish controls, genotype distributions for each of the five snps was in agreement with hwe .
endurance athletes vs. controls
in the russian sample, the mars model excluding rs <dig> yielded only one significant covariate, with a bif > 50%.
an interaction between sex and rs <dig> was observed: the ga and gg genotypes in rs <dig> were underrepresented in women endurance athletes .table  <dig>
covariates identified in the mars model excluding rs11689011
1p-value obtained by logistic regression.
2bootstrap inclusion fraction calculated after running  <dig> mars models on  <dig> bootstrap samples.
a bif of  <dig>  indicates that the covariate of interest was selected in  <dig> % of the mars models.
*denotes an interaction.
in the polish sample, no covariate was selected in the mars model excluding rs <dig>  when combining the russian and the polish groups, no covariate was selected in the mars model excluding rs <dig>
using the mars model excluding rs <dig>  the results were almost identical to those of the mars model excluding rs <dig> .
however, in the polish sample one significant covariate was retained with a bif > 50%.
the tt genotype in rs <dig> was underrepresented in the cohort of endurance athletes , especially in the elite-level cohort compared to their sub-elite counterparts .table  <dig>
genotype frequencies of the three single nucleotide polymorphisms  significantly associated with athletic performance
sprint/power athletes vs. controls
in the russian sample, the mars model excluding rs <dig> yielded two significant covariates ; only one covariate, however, had a bif > 50% .
the aa genotype in rs <dig> was underrepresented in sprint/power athletes , especially in the elite sprint/power athletes compared to their sub-elite counterparts .
in the polish sample, no covariates were selected in the mars model .figure  <dig>
genotype distributions of rs <dig> in the different groups.
c: controls, e: endurance athletes, s/p: sprint/power athletes, **: p <  <dig>  in linear regression, ***: p <  <dig>  in linear regression.
in the russian and polish samples combined, the mars model excluding rs <dig> yielded three significant covariates, two had a bif > 50% .
the trend already observed for rs <dig> in the russian sprint/power group was even stronger when combined with the polish group ; the aa genotype in rs <dig> was underrepresented in sprint/power athletes , especially in elite sprint/power athletes compared to their sub-elite counterparts .
furthermore, an interaction between rs <dig> and rs <dig> was found; the combination of the aa genotype in rs <dig> and the ga or gg genotype in rs <dig> was underrepresented in sprint/power athletes .
we note that the genotype distribution of individual snps does not provide information regarding the genotype distribution of their combinations.
for example, snpa and snpb are two snps with alleles a/a and b/b, respectively.
in this example allele a is advantageous to performance, and allele b is also advantageous to performance but only when combined with allele a. from this pattern, one would expect allele a to be overrepresented in athletes, but the a + b combination to be underrepresented in athletes.figure  <dig>
interaction between rs <dig>  rs <dig> and athletic status in the different groups.
c: controls, e: endurance athletes, s/p: sprint/power, ***: p <  <dig>  in linear regression.
using the mars model excluding rs <dig>  the results were almost identical to those of the mars model excluding rs <dig> .
however, the interaction between rs <dig> and rs <dig> in the russian and polish sample combined was not significant .
discussion
we have examined the genotype distribution and snp-snp interaction of five snps in the first intron of the epas <dig> gene in european sprint/power and endurance athletes.
we initially hypothesised that these snps would be associated with endurance athletic status.
however, contrary to our hypotheses our main findings were as follow: in the pooled cohort of polish and russian athletes, 1) rs <dig> was associated with sprint/power athletic status; the aa genotype in rs <dig> was underrepresented in sprint/power athletes, 2) rs <dig> was also associated with sprint/power athletic status; the tt genotype in rs <dig> was underrepresented in sprint/power athletes, and 3) the interaction between rs1867785/rs <dig>  and rs <dig> was associated with sprint/power athletic performance; the combinations of the aa genotype in rs <dig> with either the ga or gg genotypes in rs <dig>  or with the ct or cc genotypes in rs <dig>  were underrepresented in two cohorts of sprint/power athletes.
compared with a previous study, the results of the present study were unexpected.
henderson et al.
<cit>  have reported that the g allele in rs <dig> and the t allele in rs <dig> were overrepresented in endurance athletes, whereas we have observed that the g allele in rs <dig> was underrepresented  in female russian endurance athletes.
furthermore, in our cohort, the t allele in rs <dig> was overrepresented in female russian endurance athletes, but underrepresented in male polish endurance athletes.
a possible explanation for the discrepancies between our study and the study by henderson et al.
<cit>  is that our results regarding rs <dig> and rs <dig> were sex-specific.
sex-specific effects of snps are common in a wide range of phenotypes such as waist-hip ratio  <cit> , susceptibility to sport-related injury  <cit> , and increased muscle strength in response to training  <cit> .
we have also recently shown that ppargc1a rs <dig> was associated with elite female, but not with male endurance running performance  <cit> .
however, the polish endurance sample only comprised males, while the russian sample included both males and females.
interestingly, the cohort in the study by henderson et al.
included both males and females athletes, but sex was not investigated as a covariate in their analysis  <cit> .
looking at each snp separately, we found that rs <dig> and rs <dig> were associated with sprint/power performance.
however, these snps had very similar genotype distribution and are in strong ld ; the a allele in rs <dig> segregates with the t allele in rs <dig>  since previous studies have suggested that snps with strong ld cannot be considered in the same statistical model  <cit> , and only one of these snps might influence sprint/power performance, we have created two mars models and considered only one of the snps in each model.
as expected, the two mars models yielded almost identical results in all cohorts.
however, only rs <dig> was associated with endurance athletic status in the polish sample, while this was not the case for rs <dig>  this might be explained by the small difference in genotype distribution in rs <dig> and rs <dig> in the polish control sample.
in addition, the bif obtained for rs <dig>  when comparing polish endurance athletes with controls, was not strong enough , indicating a correlation close to non-significance and sensitive to small changes in genotype frequencies.
the relatively large sample size generated from studying two cohorts of athletes in the present study further reinforces the confidence in the results of the present study.
indeed, in the present study two european caucasian cohorts of athletes were grouped to explore the association between snps in epas <dig> and athletic performance.
our previous results indicated that combining two cohorts of caucasian athletes, especially when they are closely-related, would be useful approach to detect an association between snps and athletic status  <cit> .
while previous studies combined athletes from different ancestries  <cit> , here we have studied athletes from closely-related european ancestries .
this is reinforced by the similarity in the genotype distribution in the control groups .
furthermore, studying two cohorts of athletes has increased the sample size , which further strengthened our results and the likelihood that these specific epas <dig> snps show a genuine association with elite sprint/power performance.
an additional novel finding in the present study is that the aa genotype in rs <dig>  and the tt genotype in rs <dig>  is even more underrepresented in elite sprint/power athletes compared with their sub-elite counterparts.
this has previously been demonstrated for the highly-studied actn <dig> r577x snp, as the 577xx genotype was found in a lower frequency in elite sprint/power athletes compared to their national-level counterparts .
this observation indicates that while the epas <dig> snps are associated with the development of sprint/power ability, they might be even more important in the development of world-class sprint/power ability.
this finding, along with all the other findings in the present study, was obtained using the bootstrap inclusion fractions  statistical method, which as far as we are aware of, has never been used in sports genomics.
the bif analysis is a useful technique for investigating variations among selected models in samples drawn at random with replacement.
such samples mimic datasets that are structurally similar to that under study and that could plausibly have arisen instead  <cit> .
initially designed to test the stability of multivariable models, this non-parametric method allowed us to test whether the epas <dig> snps selected by mars were sensitive to small changes in the data, and confirm that they were unlikely to be false positives.
also, this method allowed us to see the relative importance of the different variables; while rs <dig> and rs <dig> showed very strong main effects in sprint/power athletes, their interaction with rs <dig> was of smaller importance.
we have also shown that several snps within epas <dig> are associated with endurance athletic status, in a sex-specific manner.
the tt genotype in rs <dig> was underrepresented in the cohort of polish endurance athletes.
however, this association was demonstrated only when either the polish or the russian groups were analysed separately, and was abolished when the two cohorts were combined.
we argue that we cannot be certain that these positive findings are not false positives, as they might be a limitation of the smaller sample size.
on the other hand, our positive findings regarding the association between the epas <dig> snps and sprint/power athletic status were found in the combined cohort of sprint/power athletes, and the bifs that were calculated for rs <dig> and rs <dig> were extremely high .
furthermore, these associations were more pronounced when the athletes’ level of competition was considered.
this study is not without limitations.
in case–control studies, the relative proportions of controls and cases impacts the sample size required to detect an association with a given power and significance level.
in the present study, with similar genotype distributions in russians and polish, to detect an association with the same effect size, at the same power and significance level, we would require a larger sample size in the russian population.
we acknowledge that the difference in numbers of athletes and controls in our study might therefore contribute to our results and the lack of replication in both athlete groups.
however, in any association study with elite athletes it is a challenge to increase the sample size due to the very low number of elite athletes available to study.
finally, in previous reports epas <dig> snps have demonstrated an association with performance-related blood parameters  , and elite endurance performance  <cit> , in humans.
however, is has also been shown that epas <dig> deficient mice have greater oxidative stress and an impaired response to oxidative stress  <cit> .
a reduction in hematocrit levels and a global decrease in peripheral blood counts have also been observed in epas1-null mice  <cit> .
although no specific snps were tested in the mice model, these studies illustrate the potential importance of the epas <dig> gene in athletic-related phenotypes.
CONCLUSIONS
we found an association between epas <dig> rs <dig> and epas <dig> rs <dig> and sprint/power athletic status, and an interaction between rs1867785/ <dig> and rs <dig> and sprint/power athletic status in two cohorts of closely-related european athletes.
based on the statistical model used either rs <dig> or rs <dig> are related to sprint/power athletic status.
the association between rs <dig> and sprint/power athletic status is in line with a previous study in australian athletes  <cit> .
unlike the vast number of investigations into the genetics of endurance performance, the genetic influence on elite sprint/power performance has received limited attention, and only a few studies have characterized the associations between genetic variants and elite sprint/power performance.
most studies to date have recruited only one cohort of athletes and were therefore hampered by insufficient sample size.
in the present study, we have combined two cohorts of athletes and used innovative statistical methods, which provide confidence in our results.
functional studies directly demonstrating cause and effect, or providing any proposed cellular or molecular mechanisms to explain the association, are needed to extend and validate these findings.
electronic supplementary material
additional file 1: genotype frequencies of the five investigated single nucleotide polymorphisms .
additional file 2: linkage disequilibrium  map of the five investigated snps in
epas <dig>  the upper horizontal line represents the strand of chromosome  <dig> containing epas <dig> and all five investigated snps.
the triangle below indicates the pairwise ld  between the five snps.
each snp corresponds to a diagonal of this triangle, and the intersection of two diagonals contains the value of ld for the corresponding snp pair.
the colour within the squares represents the strength of the linkage between each pair.
of all possible pairs of snps, only rs <dig> and rs <dig> are in strong ld .
additional file 3: table s2: covariates identified in the different mars models.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
sv and ne have made substantial contributions to conception and design, analysis and interpretation of data, and have been involved in drafting the manuscript or revising it critically for important intellectual content.
pc and vpp conceived the study, participated in its design and coordination and helped drafting the manuscript.
daa and ls carried out the genetic studies and participated in its design and its data collection.
bfv, vas, amk and ms carried out the genetic studies and participated in its design.
zj carried out the molecular genetic studies.
djb has been involved in drafting the manuscript and revising it critically for important intellectual content.
all authors read and approved the final manuscript.

 maximal oxygen uptake refers to the highest rate at which oxygen can be consumed by the body during intense exercise, and is, among other factors, an important predictor of elite endurance performance. studies have shown that the changes in vo2max following exercise training vary markedly between individuals and that ~ 50% of the variance can be explained by genetic factors. over the last two decades, many single nucleotide polymorphisms have been suggested to influence elite performance and the variability in vo2max increase following exercise training. while most of these snps were discovered using the candidate gene approach, a more comprehensive, genome-wide linkage approach has identified a genomic region on chromosome that is associated with the vo2max training response. the endothelial pas domain protein gene was one of the four genes in this region responsible for this linkage. epas is a transcription factor playing a key role in the hypoxia inducible factor pathway in blood, which is responsible for activating gene expression in response to hypoxia. in normal oxygen conditions, epas is quickly degraded in the cytoplasm. however, when oxygen levels drop, epas becomes stabilized, translocates to the nucleus and activates genes that are involved in erythropoiesis, and angiogenesis, thus favoring a better delivery of oxygen to the tissues. delivery of oxygen to skeletal muscles during endurance exercise is viewed a factor limiting vo2max. therefore, as a hypoxia detector and as an activator of improved oxygen delivery to the active tissues, epas is a plausible candidate to influence endurance performance. epas snps have been previously associated with blood parameters, such as alterations in erythropoietin, hemoglobin and hematocrit, that are important for success in athletic performance. for instance, tibetans with the tt genotype in registered snp had lower hemoglobin concentration compared with their tc counterparts. however, to date, only one study has examined a possible link between snps within epas and elite athletic performance. this study looked at snps and haplotypes within epas in elite australian athletes, stratified to two groups participating in middle-distance, and long-distance events. these groups were compared to a non-athletic control group. the t allele in rs and the g allele in rs two snps located in the first intron of epas were overrepresented in the group of endurance athletes compared with controls. furthermore, in the same study, two haplotypes involving rs rs rs and rs were associated with elite endurance performance. while haplotype g was overrepresented in elite endurance athletes, haplotype f was underrepresented in elite endurance athletes compared to controls. despite these positive findings, and the strong biological rational behind investigating epas in relation to elite endurance performance, this is the only genetic association study showing that epas snps impact performance, and the sprinters consisting primarily of 100–400 m track runners and sprint cyclists were excluded from the analysis. replication studies are therefore needed to confirm this association, particularly in different populations, and with a larger sample size. therefore, the aim of this study was to compare genotype distributions and interactions of the epas snps rs rs rs rs and rs between sprint/power athletes, endurance athletes, and controls in polish and russian cohorts. we also examined the association between the epas snps and athletic status according to the athletes’ level of competition. in light of the relationship previously observed between endurance-related phenotypes and snps in the epas we hypothesized that epas snps would be associated with elite endurance performance compared to controls and sprint/power athletes. we did not have specific directional hypotheses for rs rs and rs as these snps have not previously been associated with any performance and/or endurance-related phenotypes. we did not have any directional hypothesis for rs either, due to the conflicting results reported for this snp. however, in line with the findings of henderson et al. we did expect the rs g allele to be associated with elite endurance performance. finally, we did not have any directional hypotheses for any of the snps in relation to sprint/power performance. methods the study was approved by the pomeranian medical university ethics committee, poland, and the ural state university of physical culture, russia, and written informed consent was obtained from each participant. the study complied with the guidelines set out in the declaration of helsinki and the ethics policy of the szczecin university. participants the athletes and controls were all european caucasians. the athletes were categorized as either endurance athletes or sprint/power athletes as determined by the distance, duration and energy requirements of their event/sport. all athletes were ranked in the top nationally in their sport discipline and grouped as being either ‘elite-level’ or ‘sub-elite’ based on their best personal performance. those in the elite group had participated in international competitions such as world and european championships, and/or olympic games, whereas those in the sub-elite group had participated in national competitions only. details on the number of participants in the elite and sub-elite group are presented in table  table athletes’ description polish sample the sample comprised polish athletes, including elite and sub-elite sprint/power athletes and elite and sub-elite endurance athletes, as well as healthy, unrelated, sedentary controls. russian sample the russian sample comprised athletes, including elite and sub-elite sprint/power athletes and elite and sub-elite endurance athletes, as well as healthy unrelated sedentary controls. the description of the polish and the russian athletes according to their event/sport is summarized in table  genotyping polish sample genomic dna was isolated from buccal epithelium using genelute mammalian genomic dna miniprep kit according to the manufacturer’s instructions. all genetic analyses were performed at the molecular biology and biotechnology center, faculty of biology, university of szczecin. all samples were genotyped in duplicate using allelic discrimination assays with taqman® probes on a cfx touch™ real-time pcr detection system. to discriminate epas rs rs rs rs and rs alleles, taqman® pre-designed snp genotyping assays were used, including appropriate primers and fluorescently labeled mgb™ probes to detect the alleles. genotypes were assigned using all of the data from the study simultaneously. russian sample genomic dna was isolated from buccal epithelium or peripheral blood, during the years 2011– using the diatom™ dna prep kit. genotyping of five selected snps was performed by single-base extension method. the sequence surrounding each snp was obtained from the genome reference consortium human genome build assembly from the ensembl project. the primer3web software v.. was used for designing the pcr primers. pcr product range was 109–173 bp. sbe primers to detect rs rs rs and rs were designed to anneal on the positive strand immediately adjacent to the single nucleotide variation sites. sbe primer for detection of rs was designed to anneal on the negative strand. to avoid any non-specific amplification and extension products, all primers were blasted against human genome reference sequence. sets of preselected pcr primer pairs and sbe primers were screened for potential cross-reactivity by using autodimer software. multiplex pcr was performed in a volume of 15 μl containing 1 × pcr buffer,  mm mgcl  mm dntps,  μm of each primer, unit smartaq dna polymerase and 5 ng of template dna. thermal cycler conditions were: 95°c for 30 s, 30 cycles of 95°c for 45 s, 60°c for 45 s, 72°c for 60 sec and finally 10 min at 72°c in geneamp® pcr system. multiplex pcr products were checked for quality and yield by running 3 μl in 2% agarose-tbe gels. 5 μl of pcr products were cleaned with unit of fastap thermosensitive alkaline phosphatase and units of exonuclease i. multiplex sbe reaction was performed by using snapshot® multiplex kit in 5 μl final volume, including  μl of snapshot multiplex ready reaction mix,  μl pooled sbe primers and  μl of cleaned pcr product. the cycling conditions were 96°c 10 s, 50°c 5 s and 60°c 30 s, during 25 cycles in geneamp® pcr system. to remove the unincorporated ddntps, the final product was incubated with unit of tap. snapshot products with genescan™ - liz™ size standard were diluted in hi-di™ formamide, denatured and separated using an abi prism genetic analyzer with a 47 cm length capillary and pop-4™ polymer. the snapshot® primer focus® kit was used to analyze individual sbe primers for their approximate sizing locations prior to performing the multiplex sbe reaction. final data were analyzed using the genemapper® software v. from applied biosystems following the software manual. k dna high molecular weight from promega corp. served as positive control sample. genetic profile of k dna was following: rs – g/g, rs – c/c, rs – t/t, rs – g/g, rs – c/c. each of the five pcr products was formed from five different individuals, and was sequenced in separate reaction using bigdye® terminator v cycle sequencing kit with full coincidence of expected and observed sequences. sequencing was performed in a second laboratory, according to latest recommendations. genotyping reliability across two laboratories genotyping was performed in duplicate in the same laboratory for accuracy. two independent investigators have called the genotyping score in each laboratory-100% of the genotypes could be called. for the purpose of results reliability across two laboratories in two different countries, different dna samples were shipped from russia to poland and were genotyped by taqman assays. the results of the genotyping were in 100% agreement across the two laboratories. statistical analysis the genotype frequencies of all individual snps are presented in additional file before looking at both the individual effects of the five epas snps and their interactions, we selected the best genetic model for each snp. then, snp main effects, as well as snp-snp interactions, were investigated using multivariate adaptive regression splines, a nonparametric regression method that has been successfully applied for detecting snp-snp interaction in several studies. finally, the odds ratios of being either a sprint/power athlete or an endurance athlete were calculated for each significant snp and significant interacting pairs of snps using the best genetic model for each snp. details on the steps that have been taken in the statistical analysis are shown in figure  figure the five steps that were followed in the statistical analysis. mars: multivariate adaptive regression splines, bif: bootstrap inclusion fraction. firstly, we selected the best genetic model for each snp by testing three inheritance models for each snp in the entire cohort of sprint/power athletes. secondly, mars was used to detect snp main effects and snp-snp interactions. thirdly, the covariates selected by mars were input into a logistic regression model to determine their significance, and all covariates with p-value >  were excluded. fourthly, to validate the selected covariates, we repeated steps and on random samples with replacement from the original dataset and calculated the how many times the selected covariates were significant in the random samples. all covariates with a bif < 50% were excluded. fifthly, we calculated the odds ratio of the genotype combinations for each selected covariate to give a clear biological interpretation. hardy–weinberg equilibrium χ analysis was used to confirm if the control group, in each of the two samples, met hwe expectations. hwe was tested separately for each snp. selection of the best genetic model for each snp three inheritance models were assessed in the pooled cohorts of sprint/power and endurance athletes using the wald test in logistic regression models, and the best model was selected based on the minimum p-value for each snp. the athletic status was divided into two categories and encoded as a dummy variable: athlete and control. to adjust for multiple comparisons, the false discovery rate was controlled using the benjamini and hochberg correction. for consistency, the same genetic model was applied separately in the russian and polish groups. all of the following steps were performed once when comparing sprint/power athletes with controls, and once again when comparing endurance athletes with controls. analysis of snp main effects and snp-snp interactions variable importance ranking for snps with strong linkage disequilibrium has been shown to be biased in previous studies. therefore, ld in the snps was examined using r, and the pairs of snps displaying a strong ld were identified. only rs and rs were in strong ld. consequently, two mars models, all corrected for sex, were developed: one including rs rs rs and rs and another independent mars model including rs rs rs and rs for simplicity, we have only reported the mars model excluding rs in the results section. in mars, the maximum number of basis functions was set at and the maximum degree of interaction was set at as mars does not provide variable significance using p-values, each covariate selected by mars was used as input into a logistic regression model to determine its significance. all non-significant covariates were excluded. to confirm the significance of the covariates identified by the logistic regression, and to rank their importance, we used the bootstrap inclusion fraction criterion. we obtained mars models using random bootstrap samples with replacement from the original data set. then, we calculated the proportion of times that a significant variable appeared in the mars models and called this number the bif. a variable which is approximately uncorrelated with others, and is only significant at the chosen nominal α level in the mars model, is selected in about 50% of bootstrap samples. as the p-value diminishes, the bif tends toward 100%. thus, we only included covariates with a bif > 50%. for each significant covariate, the odds ratio of being either a sprint/power or an endurance athlete, depending on the genotype, was calculated. the 95% confidence intervals were calculated by unconditional maximum likelihood estimation.  in the pooled cohort of russian and polish controls, genotype distributions for each of the five snps was in agreement with hwe. endurance athletes vs. controls in the russian sample, the mars model excluding rs yielded only one significant covariate, with a bif > 50%. an interaction between sex and rs was observed: the ga and gg genotypes in rs were underrepresented in women endurance athletes.table covariates identified in the mars model excluding rs11689011 1p-value obtained by logistic regression. 2bootstrap inclusion fraction calculated after running mars models on bootstrap samples. a bif of indicates that the covariate of interest was selected in % of the mars models. *denotes an interaction. in the polish sample, no covariate was selected in the mars model excluding rs when combining the russian and the polish groups, no covariate was selected in the mars model excluding rs using the mars model excluding rs the results were almost identical to those of the mars model excluding rs. however, in the polish sample one significant covariate was retained with a bif > 50%. the tt genotype in rs was underrepresented in the cohort of endurance athletes, especially in the elite-level cohort compared to their sub-elite counterparts.table genotype frequencies of the three single nucleotide polymorphisms significantly associated with athletic performance sprint/power athletes vs. controls in the russian sample, the mars model excluding rs yielded two significant covariates; only one covariate, however, had a bif > 50%. the aa genotype in rs was underrepresented in sprint/power athletes, especially in the elite sprint/power athletes compared to their sub-elite counterparts. in the polish sample, no covariates were selected in the mars model.figure genotype distributions of rs in the different groups. c: controls, e: endurance athletes, s/p: sprint/power athletes, **: p <  in linear regression, ***: p <  in linear regression. in the russian and polish samples combined, the mars model excluding rs yielded three significant covariates, two had a bif > 50%. the trend already observed for rs in the russian sprint/power group was even stronger when combined with the polish group; the aa genotype in rs was underrepresented in sprint/power athletes, especially in elite sprint/power athletes compared to their sub-elite counterparts. furthermore, an interaction between rs and rs was found; the combination of the aa genotype in rs and the ga or gg genotype in rs was underrepresented in sprint/power athletes. we note that the genotype distribution of individual snps does not provide information regarding the genotype distribution of their combinations. for example, snpa and snpb are two snps with alleles a/a and b/b, respectively. in this example allele a is advantageous to performance, and allele b is also advantageous to performance but only when combined with allele a. from this pattern, one would expect allele a to be overrepresented in athletes, but the a + b combination to be underrepresented in athletes.figure interaction between rs rs and athletic status in the different groups. c: controls, e: endurance athletes, s/p: sprint/power, ***: p <  in linear regression. using the mars model excluding rs the results were almost identical to those of the mars model excluding rs. however, the interaction between rs and rs in the russian and polish sample combined was not significant. discussion we have examined the genotype distribution and snp-snp interaction of five snps in the first intron of the epas gene in european sprint/power and endurance athletes. we initially hypothesised that these snps would be associated with endurance athletic status. however, contrary to our hypotheses our main findings were as follow: in the pooled cohort of polish and russian athletes, 1) rs was associated with sprint/power athletic status; the aa genotype in rs was underrepresented in sprint/power athletes, 2) rs was also associated with sprint/power athletic status; the tt genotype in rs was underrepresented in sprint/power athletes, and 3) the interaction between rs1867785/rs and rs was associated with sprint/power athletic performance; the combinations of the aa genotype in rs with either the ga or gg genotypes in rs or with the ct or cc genotypes in rs were underrepresented in two cohorts of sprint/power athletes. compared with a previous study, the results of the present study were unexpected. henderson et al. have reported that the g allele in rs and the t allele in rs were overrepresented in endurance athletes, whereas we have observed that the g allele in rs was underrepresented in female russian endurance athletes. furthermore, in our cohort, the t allele in rs was overrepresented in female russian endurance athletes, but underrepresented in male polish endurance athletes. a possible explanation for the discrepancies between our study and the study by henderson et al. is that our results regarding rs and rs were sex-specific. sex-specific effects of snps are common in a wide range of phenotypes such as waist-hip ratio, susceptibility to sport-related injury, and increased muscle strength in response to training. we have also recently shown that ppargc1a rs was associated with elite female, but not with male endurance running performance. however, the polish endurance sample only comprised males, while the russian sample included both males and females. interestingly, the cohort in the study by henderson et al. included both males and females athletes, but sex was not investigated as a covariate in their analysis. looking at each snp separately, we found that rs and rs were associated with sprint/power performance. however, these snps had very similar genotype distribution and are in strong ld; the a allele in rs segregates with the t allele in rs since previous studies have suggested that snps with strong ld cannot be considered in the same statistical model, and only one of these snps might influence sprint/power performance, we have created two mars models and considered only one of the snps in each model. as expected, the two mars models yielded almost identical results in all cohorts. however, only rs was associated with endurance athletic status in the polish sample, while this was not the case for rs this might be explained by the small difference in genotype distribution in rs and rs in the polish control sample. in addition, the bif obtained for rs when comparing polish endurance athletes with controls, was not strong enough, indicating a correlation close to non-significance and sensitive to small changes in genotype frequencies. the relatively large sample size generated from studying two cohorts of athletes in the present study further reinforces the confidence in the results of the present study. indeed, in the present study two european caucasian cohorts of athletes were grouped to explore the association between snps in epas and athletic performance. our previous results indicated that combining two cohorts of caucasian athletes, especially when they are closely-related, would be useful approach to detect an association between snps and athletic status. while previous studies combined athletes from different ancestries, here we have studied athletes from closely-related european ancestries. this is reinforced by the similarity in the genotype distribution in the control groups. furthermore, studying two cohorts of athletes has increased the sample size, which further strengthened our results and the likelihood that these specific epas snps show a genuine association with elite sprint/power performance. an additional novel finding in the present study is that the aa genotype in rs and the tt genotype in rs is even more underrepresented in elite sprint/power athletes compared with their sub-elite counterparts. this has previously been demonstrated for the highly-studied actn r577x snp, as the 577xx genotype was found in a lower frequency in elite sprint/power athletes compared to their national-level counterparts. this observation indicates that while the epas snps are associated with the development of sprint/power ability, they might be even more important in the development of world-class sprint/power ability. this finding, along with all the other findings in the present study, was obtained using the bootstrap inclusion fractions statistical method, which as far as we are aware of, has never been used in sports genomics. the bif analysis is a useful technique for investigating variations among selected models in samples drawn at random with replacement. such samples mimic datasets that are structurally similar to that under study and that could plausibly have arisen instead. initially designed to test the stability of multivariable models, this non-parametric method allowed us to test whether the epas snps selected by mars were sensitive to small changes in the data, and confirm that they were unlikely to be false positives. also, this method allowed us to see the relative importance of the different variables; while rs and rs showed very strong main effects in sprint/power athletes, their interaction with rs was of smaller importance. we have also shown that several snps within epas are associated with endurance athletic status, in a sex-specific manner. the tt genotype in rs was underrepresented in the cohort of polish endurance athletes. however, this association was demonstrated only when either the polish or the russian groups were analysed separately, and was abolished when the two cohorts were combined. we argue that we cannot be certain that these positive findings are not false positives, as they might be a limitation of the smaller sample size. on the other hand, our positive findings regarding the association between the epas snps and sprint/power athletic status were found in the combined cohort of sprint/power athletes, and the bifs that were calculated for rs and rs were extremely high. furthermore, these associations were more pronounced when the athletes’ level of competition was considered. this study is not without limitations. in case–control studies, the relative proportions of controls and cases impacts the sample size required to detect an association with a given power and significance level. in the present study, with similar genotype distributions in russians and polish, to detect an association with the same effect size, at the same power and significance level, we would require a larger sample size in the russian population. we acknowledge that the difference in numbers of athletes and controls in our study might therefore contribute to our results and the lack of replication in both athlete groups. however, in any association study with elite athletes it is a challenge to increase the sample size due to the very low number of elite athletes available to study. finally, in previous reports epas snps have demonstrated an association with performance-related blood parameters, and elite endurance performance, in humans. however, is has also been shown that epas deficient mice have greater oxidative stress and an impaired response to oxidative stress. a reduction in hematocrit levels and a global decrease in peripheral blood counts have also been observed in epas1-null mice. although no specific snps were tested in the mice model, these studies illustrate the potential importance of the epas gene in athletic-related phenotypes.  we found an association between epas rs and epas rs and sprint/power athletic status, and an interaction between rs1867785/ and rs and sprint/power athletic status in two cohorts of closely-related european athletes. based on the statistical model used either rs or rs are related to sprint/power athletic status. the association between rs and sprint/power athletic status is in line with a previous study in australian athletes. unlike the vast number of investigations into the genetics of endurance performance, the genetic influence on elite sprint/power performance has received limited attention, and only a few studies have characterized the associations between genetic variants and elite sprint/power performance. most studies to date have recruited only one cohort of athletes and were therefore hampered by insufficient sample size. in the present study, we have combined two cohorts of athletes and used innovative statistical methods, which provide confidence in our results. functional studies directly demonstrating cause and effect, or providing any proposed cellular or molecular mechanisms to explain the association, are needed to extend and validate these findings. electronic supplementary material additional file 1: genotype frequencies of the five investigated single nucleotide polymorphisms. additional file 2: linkage disequilibrium map of the five investigated snps in epas the upper horizontal line represents the strand of chromosome containing epas and all five investigated snps. the triangle below indicates the pairwise ld between the five snps. each snp corresponds to a diagonal of this triangle, and the intersection of two diagonals contains the value of ld for the corresponding snp pair. the colour within the squares represents the strength of the linkage between each pair. of all possible pairs of snps, only rs and rs are in strong ld. additional file 3: table s2: covariates identified in the different mars models. competing interests the authors declare that they have no competing interests. authors’ contributions sv and ne have made substantial contributions to conception and design, analysis and interpretation of data, and have been involved in drafting the manuscript or revising it critically for important intellectual content. pc and vpp conceived the study, participated in its design and coordination and helped drafting the manuscript. daa and ls carried out the genetic studies and participated in its design and its data collection. bfv, vas, amk and ms carried out the genetic studies and participated in its design. zj carried out the molecular genetic studies. djb has been involved in drafting the manuscript and revising it critically for important intellectual content. all authors read and approved the final manuscript. 
using innovative statistical methods we compared genotype distributions and interactions of epas snps rs rs rs rs and rs between sprint/power athletes, endurance athletes, and controls in polish and russian samples. in the pooled cohort of polish and russian athletes, 1) rs was associated with sprint/power athletic status; the aa genotype in rs was underrepresented in the sprint/power athletes, 2) rs was also associated with sprint/power athletic status; the tt genotype in rs was underrepresented sprint/power athletes, and 3) the interaction between rs rs and rs was associated with sprint/power athletic performance; the combinations of the aa genotype in rs with either the ag or gg genotypes in rs or with the ct or cc genotypes in rs were underrepresented in two cohorts of sprint/power athletes. the endothelial pas domain protein activates genes that are involved in erythropoiesis and angiogenesis, thus favoring a better delivery of oxygen to the tissues and is a plausible candidate to influence athletic performance. based on the unique statistical model rs1867785/rs are strong predictors of sprint/power athletic status, and the interaction between rs rs and rs might contribute to success in sprint/power athletic performance.
 the endothelial pas domain protein activates genes that are involved in erythropoiesis and angiogenesis, thus favoring a better delivery of oxygen to the tissues and is a plausible candidate to influence athletic performance. using innovative statistical methods we compared genotype distributions and interactions of epas snps rs rs rs rs and rs between sprint/power athletes, endurance athletes, and controls in polish and russian samples. we also examined the association between these snps and the athletes’ competition level. genotyping was performed by either real-time pcr or by single-base extension method.  in the pooled cohort of polish and russian athletes, 1) rs was associated with sprint/power athletic status; the aa genotype in rs was underrepresented in the sprint/power athletes, 2) rs was also associated with sprint/power athletic status; the tt genotype in rs was underrepresented sprint/power athletes, and 3) the interaction between rs rs and rs was associated with sprint/power athletic performance; the combinations of the aa genotype in rs with either the ag or gg genotypes in rs or with the ct or cc genotypes in rs were underrepresented in two cohorts of sprint/power athletes.  based on the unique statistical model rs1867785/rs are strong predictors of sprint/power athletic status, and the interaction between rs rs and rs might contribute to success in sprint/power athletic performance. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords athletic performanceepas1genomicsgenesendurancesprintissue-copyright-statement© the author 2014 