Genome-wide association study identifies three novel genetic markers associated with elite endurance performance

To investigate the association between multiple single-nucleotide polymorphisms (SNPs), aerobic performance and elite endurance athlete status in Russians. By using GWAS approach, we examined the association between 1,140,419 SNPs and relative maximal oxygen consumption rate (V.O2max) in 80 international-level Russian endurance athletes (46 males and 34 females). To validate obtained results, we further performed case-control studies by comparing the frequencies of the most significant SNPs (with P < 10−5-10−8) between 218 endurance athletes and opposite cohorts (192 Russian controls, 1367 European controls, and 230 Russian power athletes). Initially, six ‘endurance alleles’ were identified showing discrete associations with V.O2max both in males and females. Next, case-control studies resulted in remaining three SNPs (NFIA-AS2 rs1572312, TSHR rs7144481, RBFOX1 rs7191721) associated with endurance athlete status. The C allele of the most significant SNP, rs1572312, was associated with high values of V.O2max (males: P = 0.0051; females: P = 0.0005). Furthermore, the frequency of the rs1572312 C allele was significantly higher in elite endurance athletes (95.5%) in comparison with non-elite endurance athletes (89.8%, P = 0.0257), Russian (88.8%, P = 0.007) and European (90.6%, P = 0.0197) controls and power athletes (86.2%, P = 0.0005). The rs1572312 SNP is located on the nuclear factor I A antisense RNA 2 (NFIA-AS2) gene which is supposed to regulate the expression of the NFIA gene (encodes transcription factor involved in activation of erythropoiesis and repression of the granulopoiesis). Our data show that the NFIA-AS2 rs1572312, TSHR rs7144481 and RBFOX1 rs7191721 polymorphisms are associated with aerobic performance and elite endurance athlete status.


INTRODUCTION
It has long been recognized that the interindividual variability of aerobic performance and the ability to become an elite endurance athlete have a strong genetic basis. A large body of evidence suggests that genetic markers may explain, in part, an interindividual variability of aerobic performance characteristics in response to endurance training [1][2][3][4]. With genotyping becoming widely available, a large number of genetic studies evaluating candidate gene variants have been published with largely unconfirmed associations with elite athlete status [5,6].
Case-control studies remain the most common study design in genomics of aerobic capacity and endurance and generally involve determining whether one allele of a DNA sequence (gene or noncoding region of DNA) is more common in a group of elite endurance athletes than it is in the general population, thus implying that the allele boosts performance [7][8][9][10]. To avoid false-positive results casecontrol studies should have at least one replication with additional athletic and non-athletic cohorts from different populations [6,11,12].
Genome-wide association study identifies three novel genetic markers associated with elite endurance performance AUTHORS: Ahmetov II 1,2,3 , Kulemin NA 3,4 , Popov DV 5 , Naumov VA 3 , Akimov EB 6  Since endurance and power are located at opposite extremes of the muscle performance continuum, the comparison of allelic frequencies between endurance and power athletes is also the way to identify endurance/power markers [13,14].

Study participants
The study involved 449 Russian athletes (267 males and 182 females)

Calculation of total genotype score
To quantify the combined influence of polymorphisms associated with endurance athlete status, an algorithm [22] was used to incorporate all favourable genotype scores for any given individual in a simple additive model. The total score was then transformed mathematically to lie within the range 0-100 and labelled the 'total genotype score' (TGS). A TGS of 100 represents a 'perfect' polygenic profile for endurance and a TGS of 0 represents the 'worst' possible profile for endurance.

Statistical analysis
Linear regression analysis of a quantitative trait ( · VO 2max ) with GWAS data of five discovery cohorts of athletes (all athletes, female athletes, male athletes, long endurance athletes, middle and short endurance athletes) was performed using PLINK software. The squared correlation coefficient R 2 was used as a measure of explained variance.
Differences in phenotypes between groups were analysed using ANOVA or unpaired t tests (when variables of two genotypes were compared). All data are presented as mean (standard deviation).
Genotype distribution and allele frequencies between athletes and controls were compared using χ 2 tests. P values < 0.05 were considered statistically significant. It has been estimated that the sample size required in this study to obtain a statistical power of 80% was sufficient. Statistical analyses were conducted using GraphPad InStat software (GraphPad Software, Inc., USA).  Table 2).

GWAS for
The C allele of one of the most significant SNPs, NFIA-AS2 rs1572312 (discovery P = 1.66 · 10 -6 ), was associated with high

Case-control studies
To validate obtained results, we further performed case-control studies by comparing the frequencies of the six most significant SNPs between 218 endurance athletes (or 100 elite endurance athletes) and opposite cohorts (192 Russian controls, 1367 European controls, and 230 Russian power athletes). We assumed that 'endurance allele' should be under-represented in at least one opposite cohort (Russian controls or Russian power athletes) when compared to endurance athletes. This approach resulted in remaining three SNPs (NFIA-AS2 rs1572312 C, TSHR rs7144481 C, RBFOX1 rs7191721 G) associ-rs7191721 G) associated with endurance athlete status (Table 3). In particular, the frequency of the NFIA-AS2 rs1572312 C allele was significantly higher

NFIA-AS2 rs1572312 genotype and hematological parameters
Since NFIA-AS2 gene may be involved in the regulation of erythropoiesis and granulopoiesis, we hypothesized that variation in this gene had lower number of neutrophils (3.17 ± 1.1 · 10 9 vs. 5.1 ± 1.8 · 10 9 , P = 0.0029) and lower leukocyte/erythrocyte ratio (P = 0.0332) than A allele carriers. The same interrelationship was observed in female athletes, but only as a tendency due to small sample size.
Overall, these results indicate that NFIA-AS2 rs1572312 C allele is associated with activation of erythropoiesis, while A allele with activated granulopoiesis.

DISCUSSION
This is the first study to demonstrate that polymorphisms of NFIA-AS2, TSHR and RBFOX1 genes are associated with aerobic perfor-are associated with aerobic performance and elite endurance athlete status. Importantly, these results were obtained by two-stage approach using GWAS, which has been successfully used to identify genetic markers associated with relevant    [1,2,23,24].
The most significant SNP (rs157231) was found in the nuclear factor I A antisense RNA 2 (NFIA-AS2) gene (localized to chromosome 1). More specifically, the CC genotype was associated with high · VO 2max in three subgroups of athletes (male and female middle and short endurance athletes; female long endurance athletes). Additionally, the frequency of the C allele was significantly higher in elite endurance athletes in comparison with non-elite endurance athletes, Russian and European controls and power athletes.
The NFIA-AS2 gene encodes long non-coding RNA (lnc-RNA) with undescribed function. Genes of antisense long non-coding RNAs are transcribed from either the same genomic site or a site distant from the gene locus where the sense transcript counterpart is produced.
Antisense lnc-RNAs repress -and in some cases can also activate -transcription of the targeted protein coding genes via mechanisms such as DNA methylation and chromatin modification at the genomic loci of the targeted genes [25]. lncRNAs can also act as a sponge for microRNAs that affect differentiation programs [26].
Therefore, one might suggest that NFIA-AS2 is involved in the regulation of expression of the nuclear factor I A (NFIA) gene or erythroid/ myeloid specific RNAs. NFIA, as a transcription factor, induces erythropoiesis, whereas its silencing drives granulopoiesis [27]. The rs7144481 polymorphism seems to be a significant predictor of elite endurance performance too (discovery P value -8.92 · 10 -8 ).
That is, rs7144481 C allele in the present study was associated with greater aerobic capacity in three subgroups of athletes (male long endurance athletes; all male and all female athletes). The significance of this SNP was confirmed in case-control studies (higher frequency of the C allele in elite endurance athletes in comparison with Russian and European controls). The rs7144481 polymorphism is located in the 3'-untranslated region of the thyroid stimulating hormone receptor (TSHR) gene (location: 14q31). The protein encoded by this gene is a membrane receptor for thyrothropin (produces thyroid hormones) and thyrostimulin (activates TSHR), and therefore, a major controller of thyroid cell metabolism. Thyroid hormones are known as determinants of metabolic and contractile phenotype of skeletal muscle [30].
TSHR also mediates the effect of thyrothropin on angiogenesis via cAMP-mammalian target of rapamycin signaling [31]. Although further in vitro functional studies are needed, one might speculate that the rs7144481 C allele is associated with greater expression of TSHR gene and increased angiogenesis and/or metabolic rate -factors that directly influence aerobic performance.
Since RNA-binding proteins are key regulators of gene expression, Rbfox1 knockdown leads to the inhibition of muscle differentiation [33]. RBFOX1 (location: 16p13.3) is implicated in multiple medical conditions, including muscular dystrophies, cancers, neurodevelopmental and neuropsychiatric disorders [33,34]. We therefore speculate that RBFOX1 rs7191721 G allele may be favourable for muscle function of endurance athletes. Indeed, our data show that middle and short endurance athletes of both genders, as well as all female endurance athletes with G allele have greater aerobic capacity. Additionally, the G allele is over-represented in endurance athletes when compared to power athletes.
The limitation of the study is the sample size of our discovery cohort with · VO 2max data (n=80). Relatively small cohort did not allow to reach genome-wide significance level which was set at P = 4.38 · 10 -8 (i.e. 0.05/1,140,419 SNPs). However, we believe that using additional approaches (validation of results in different gender groups, casecontrol study, biochemical study) had minimized false-positive results.
Our current progress towards understanding the molecular basis of athletic performance represents only the first steps. The next decade will be an exciting period for sports genomics, as we apply the new DNA technologies (whole genome sequencing, GWAS, epigenomic, transcriptomic and proteomic profiling etc.) and bioinformatics to further dissect and analyze the genetic effects on human physical ability [6]. Efforts to perform GWAS in the cohorts of athletes are presently underway (at least athletes from Australia, Canada, Ethiopia, Finland, Germany, Greece, Italy, Jamaica, Japan, Kenya, Poland, Russia and USA).

CONCLUSIONS
In conclusion, our data show that the NFIA-AS2 rs1572312, TSHR rs7144481 and RBFOX1 rs7191721 polymorphisms are associated with aerobic performance and elite endurance athlete status, but further replication studies using different ethnic cohorts are warranted. -----