(HIF1A) gene in elite endurance athletes - Journal of Applied Physiology

J Appl Physiol 108: 1497–1500, 2010. First published March 18, 2010; doi:10.1152/japplphysiol.01165.2009.

A common haplotype and the Pro582Ser polymorphism of the hypoxia-inducible factor-1␣ (HIF1A) gene in elite endurance athletes Frank Döring,1 Simone Onur,1 Alexandra Fischer,1 Marcel R. Boulay,2 Louis Pérusse,2 Tuomo Rankinen,4 Rainer Rauramaa,4 Bernd Wolfarth,5 and Claude Bouchard3 1

Department of Molecular Prevention, Institute of Human Nutrition and Food Science, Christian-Albrechts University, Kiel, Germany; 2Physical Activity Sciences Laboratory, Laval University, Ste-Foy, Quebec, Canada; 3Human Genomics Laboratory, Pennington Biomedical Research Center, Baton Rouge, Louisiana; 4Kuopio Research Institute of Exercise Medicine, Department of Physiology and Department of Clinical Physiology, University of Kuopio, Kuopio, Finland; and 5 Department of Preventive and Rehabilitative Sports Medicine, Technical University Munich, Munich, Germany Submitted 21 October 2009; accepted in final form 12 February 2010

Döring F, Onur S, Fischer A, Boulay MR, Pérusse L, Rankinen T, Rauramaa R, Wolfarth B, Bouchard C. A common haplotype and the Pro582Ser polymorphism of the hypoxia-inducible factor-1␣ (HIF1A) gene in elite endurance athletes. J Appl Physiol 108: 1497–1500, 2010. First published March 18, 2010; doi:10.1152/japplphysiol.01165.2009.— Hypoxia-inducible factor-1␣ (HIF1A) is a transcription factor regulating several genes in response to hypoxic stimuli. HIF1A target genes code for proteins involved in oxygen transport, glycolytic enzymes, and glucose transporters. We investigated whether single-nucleotide polymorphisms and haplotypes in the HIF1A gene are associated with endurance performance in the Genathlete cohort, which includes 316 Caucasian male elite endurance athletes (EEA) with a maximal oxygen uptake of 79.0 ⫾ 3.5 ml·kg⫺1·min⫺1 (mean ⫾ SD) and 304 Caucasian male sedentary controls with a maximal oxygen uptake of 40.1 ⫾ 7.0 ml·kg⫺1·min⫺1. Six single-nucleotide polymorphisms (rs1951795, rs11158358, rs2301113, rs11549465, rs115494657, rs17099207) were genotyped with the TaqMan system. We found a nominal significant tendency for a difference between the two groups for HIF1A Pro582Ser (rs11549465) genotype distributions (P ␹2 ⫽ 0.017). Homozygotes of the Pro genotype were slightly more frequent in athletes than in controls (84 vs. 75%). Compared with Ser carriers, the odds ratio (OR) of being an EEA in Pro/Pro homozygotes was 1.77 [95% confidence interval (CI): 1.18 –2.67, P ⫽ 0.006] compared with the other genotypes. A common HIF1A haplotype (frequency: 15%), including the rs11549465 Pro allele and the minor A allele of rs17099207 in the 3= flanking region of the gene, showed a significant association with EEA status (OR: 2.37, 95% CI: 1.21– 4.66, P ⫽ 0.012), whereas the most prevalent haplotype (frequency: 59%) comprising the rs11549465 Pro allele and the major G allele of rs1709920 showed no association with EEA status (OR: 0.93, 95% CI: 0.58 – 1.50, P ⫽ 0.769). We found preliminary evidence that the HIF1A Pro582Ser polymorphism and a common haplotype of the HIF1A gene may be associated with EEA status in Caucasian men.

oxia, HIF1A accumulates and forms a heterodimer (HIF1) with HIF1B. HIF1A- and/or -B-responsive genes are involved in angiogenesis, glucose metabolism, vasomotor control, and erythropoiesis, many of which are implicated in either the delivery of oxygen and nutrients to cells, or controlling cellular utilization of these substrates (16). Several lines of evidence suggest that the occurrence of local hypoxia in the muscle causes a drop in oxygen pressure within the myocyte during exercise (13, 14). This causes an induction of the HIF1A-mediated signaling pathway in human skeletal muscle, thereby provoking a shift toward increased use of oxidative pathways for energy production (3, 8). In Caucasians, a nonsynonymous coding single-nucleotide polymorphism (SNP) of the HIF1A gene (Pro582Ser, rs11549465 C ⬎ T) has been shown to be associated with changes of maximal oxygen consumption with exercise training in elderly persons (11). Given this evidence and the impact of HIF1A on oxygen metabolism in endurance activities, we hypothesized that variants of the HIF1A gene might be associated with endurance performance status. We examined this hypothesis in the Genathlete study, which includes 316 Caucasian male elite endurance athletes (EEA) and 304 Caucasian male sedentary controls (SC). METHODS

Participants and maximal oxygen uptake test. The Genathlete cohort was established in the late 1990s (15) and has been described in detail before (19). Briefly, this cohort consists of 316 male endur˙ O2 max) of at least 75 ance athletes with a maximal oxygen uptake (V ml·kg⫺1·min⫺1 (mean ⫾ SD 79.04 ⫾ 3.45 ml·kg⫺1·min⫺1, range

single nucleotide polymorphism; exercise genetics

homeostasis is regulated by the transcription factor hypoxia-inducible factor-1␣ (HIF1A). In normoxia (21% O2), HIF1A is rapidly ubiquitinylated and degraded by hydroxylation of proline residues within the degradation domain of HIF1A, whereas, at hypoxia (1% O2), the protein is stabilized. The degradation of newly synthesized HIF1A under normoxic conditions is regulated by a family of oxygen-activated HIF1 prolyl-hydroxylases (4). During hypIN MAMMALIAN CELLS, OXYGEN

Address for reprint requests and other correspondence: F. Doering, Dept. of Molecular Prevention, Institute of Human Nutrition and Food Science, ChristianAlbrechts-Univ., Heinrich-Hecht-Platz 10, 24118 Kiel, Germany (e-mail: [email protected]). http://www.jap.org

Table 1. Descriptive characteristics of endurance athletes and sedentary controls Total number Age, yr Height, cm Weight, kg ˙ O2max, ml 䡠 kg⫺1 䡠 min⫺1 V ˙ O2max, l/min V Country of origin, n (%) Germany USA/Canada Finland

EEA

SC

P

316 21.5 ⫾ 5.4 178.7 ⫾ 6.2 69.3 ⫾ 7.1 79.04 ⫾ 3.45 5.48 ⫾ 0.615

304 28.8 ⫾ 12.0 178.1 ⫾ 7.4 75.4 ⫾ 9.4 40.23 ⫾ 6.97 3.03 ⫾ 0.625

⬍.0001 0.09 ⬍.0001 ⬍.0001 ⬍.0001

187 (59.2) 79 (25.0) 50 (15.8)

169 (55.6) 89 (29.3) 46 (15.1)

Values are given as means ⫾ SD or n (%). EEA, elite endurance athletes; ˙ O2max, maximum oxygen uptake. SC, sedentary controls; V

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HIF1A POLYMORPHISMS AND ELITE ENDURANCE ATHLETE STATUS

Table 2. Positions and minor allele frequencies of HIF1A single-nucleotide polymorphisms in elite endurance athletes and sedentary controls Position

rs1951795 rs11158358 rs2301113 rs11549465 rs11549467 rs17099207

intron 1 intron 6 intron 10 exon 12 exon 12 3= gene region

AA Change

Pro582Ser Ala588Thr

Common Allele

Minor Allele

P HWEEEA

P HWESC

MAFEEA

MAFSC

C C C C G G

A G G T A A

0.35 0.15 0.32 0.91 0.77 0.68

0.18 0.31 0.09 0.83 0.88 0.32

0.163 0.140 0.204 0.084 0.016 0.299

0.210 0.178 0.249 0.137 0.008 0.248

HIF1A, hypoxia-inducible factor-1␣; AA, amino acid; MAF, minor allele frequency; HWE, Hardy-Weinberg equilibrium.

75.0 –92.9 ml·kg⫺1·min⫺1) and 304 sedentary male controls with ˙ O2 max of 50 ml·kg⫺1·min⫺1 or less (mean ⫾ SD 40.23 ⫾ maximal V 6.97 ml·kg⫺1·min⫺1, range 17.2–50.0 ml·kg⫺1·min⫺1) (Table 1). The endurance athletes were recruited in Germany (n ⫽ 187), North America (n ⫽ 79), and Finland (n ⫽ 50) and represented crosscountry skiing (n ⫽ 105), biathlon (n ⫽ 86), cycling (n ⫽ 72), and running (n ⫽ 39). Fourteen athletes were engaged in other endurance sports (n ⫽ 14). All athletes had been competing at the national or international level for several years. The control group comprised healthy sedentary subjects from the same geographical areas as the athletes. The study has been approved by the Ethics Committee of the participating institutes, and all subjects provided written, informed ˙ O2 max values of SC subjects and athletes consent for participation. V are reported here only to indicate the approximate magnitude of the difference in cardio-respiratory fitness between the two groups. These data are not used in any of the genetic analyses reported in this paper. ˙ O2 max of SC subjects were all measured or predicted from cycle V ˙ O2 max of the athletes was determined in ergometer incremental tests. V the course of incremental exercise tests on cycle ergometers or motor-driven treadmills when they were at the peak of their physical performance. Gas exchanges were measured with a variety of commercially available open calorimetry systems. DNA preparation and SNP analysis. Genomic DNA was isolated from white blood cells following a standard protocol, according to the manufacturers instructions (Qiagen, Genomic DNA isolation kit, tip 500). Genomic DNA was amplified using ␾-polymerase, according to the manufacturer’s instructions (Amersham Bio-Science, Uppsala, SE). Six HIF1A polymorphisms were selected: four tagging (tag) SNPs with minor allele frequencies ⬎ 10% (rs1951795, rs11549465, rs11158358, and rs2301113), one nonsynonymous coding SNP located in exon 12 (rs11549467), and one SNP located 3= of the gene (rs17099207). TagSNPs were selected using TAMAL software (7). TAMAL identifies haplotype tagging SNPs from current versions of online resources i.e., HapMap, Perlegen, Affymetrix, dbSNP, and the University of California Santa Cruz (UCSC) genome browser. Genotyping was performed with predesigned 5= nuclease assays (TaqMan SNP Genotyping Assay, Applied Biosystems, Foster City, CA). Fluorescence was measured with the ABI Prism 7900 HT sequence detection system.

Statistical analysis. Pearson ␹2 and/or Fisher exact tests were used to compare genotype or allele frequencies between EEAs and controls, and to assess Hardy-Weinberg equilibrium. Odds ratios (ORs) were calculated using unconditional logistic regression model. Haplotype frequencies were estimated with the expectation-maximization algorithm (5), and individual haplotypes were imputed by the method described by Zaykin et al. (21). Lewontin’s D prime (D=) and the correlation coefficient (r2) were calculated to assess linkage disequilibrium between polymorphisms among SCs. Statistics were computed with the Statistics Package for the Social Sciences 12.0 (SPSS, Chicago, IL) and SAS version 9.3 (SAS Institute, Cary, NC). Twosided nominal P values are reported. RESULTS

The physical positions within the gene and minor allele frequencies of the HIF1A SNPs are shown in Table 2. Genotype distributions were in Hardy-Weinberg equilibrium among athletes (EEAs) and controls (SCs). Allele frequencies did not differ between specific sports or places of origin within the endurance athlete and control group (data not shown). The linkage correlation coefficients (r2) between SNPs ranged from ⬍0.001 and 0.82, and Lewontin’s D= ranged between 0.14 and 1.0. Pearson ␹2 tests showed a strong tendency for a difference (P ⫽ 0.017) in the genotype distribution of the Pro582Ser (rs11549465 C ⬎ T) polymorphism between athletes and controls (Table 3). Among EEAs, 84% were homozygous for the Pro-coding C allele, whereas, in the control group, 75% carried the Pro/Pro genotype. The resulting OR for EEA status in Pro/Pro homozygotes vs. Ser allele carriers (11 vs. 12 ⫹ 22) was 1.77 [95% confidence interval (CI): 1.18 –2.67, P ⫽ 0.006]. After exclusion of rare haplotypes (frequency ⬍ 5%), four common HIF1A haplotypes were defined by rs1951795 (C ⬎ A), rs11158358 (C ⬎ G), rs2301113 (A ⬎ C), rs11549465 (C ⬎ T), rs11549467 (G ⬎ A), and rs17099207 (G ⬎ A), explaining 84% of the haplotypic diversity of the gene (Table 4).

Table 3. Association of HIF1A single-nucleotide polymorphisms with elite endurance status Genotype nEEA/SC*

rs1951795 rs11158358 rs2301113 rs11549465‡ rs11549467 rs17099207

Dominant Model 2

11

12

22

␹

P†

OR (95% CI)

P†

214/189 224/204 193/173 254/220 298/294 153/165

89/91 80/82 106/100 47/69 10/5 126/117

6/17 3/12 10/24 2/6 0/0 29/15

4.41 3.55 3.24 8.48 1.53 3.65

0.038 0.072 0.064 0.017 0.212 0.045

0.76 (0.54–1.08) 0.80 (0.56–1.15) 0.83 (0.59–1.16) 1.77 (1.18–2.67)

0.122 0.229 0.267 0.006

1.29 (0.93–1.77)

0.128

nEEA/SC, No. of EEA/SC subjects. *Because we excluded rare haplotypes, the sum of genotypes does not fit with the counts of participants. 11, Homozygous major allele; 12, heterozygote; 22, homozygous minor allele. †Nominal P values. Odds ratios (ORs) were calculated for minor allele carriers (11 ⫹ 12 vs. 22; dominant model) with one exception: ‡for rs11549465, ORs were calculated for the homozygous major allele carriers (11 vs. 12 ⫹ 22). CI, confidence interval. J Appl Physiol • VOL

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Table 4. Association of HIF1A haplotypes with elite endurance status Frequency Haplotype*

EEA

SC

␹2

P†

OR

P†

C-C-C-T-G-G C-C-C-T-G-A A-G-G-C-G-G A-G-G-C-G-A

0.588 0.183 0.042 0.042

0.593 0.134 0.076 0.063

0.19 5.76 6.67 2.05

0.661 0.016 0.010 0.152

0.93 (0.58–1.50) 2.37 (1.21–4.66) 0.24 (0.08–0.70) 0.36 (0.11–1.14)

0.769 0.012 0.009 0.082

*Order of bases corresponds to rs1951795 C ⬎ A, rs11158358 C ⬎ G, rs2301113 C ⬎ G, rs11549465 C ⬎ T, rs15549467 G ⬎ A, and rs17099207 G ⬎ A. †Nominal P values.

Haplotype C-C-C-T-G-A comprising the major Pro allele of the Pro582Ser (rs11549465) variant and the rare A allele of rs17099207 was slightly more frequent in athletes than in controls (18.5 vs. 13.4%, P ⫽ 0.016). The resulting OR for EEA status was 2.37 (95% CI: 1.21– 4.66, P ⫽ 0.012). As suspected, the counterpart (A-G-G-C-G-G) of the haplotype C-C-C-T-G-A is also significantly and inversely associated with athlete status (OR: 0.24; 95% CI: 0.08 – 0.70, P ⫽ 0.009). The most common haplotype C-C-C-T-G-G (frequencies, EEA: 58.8%, SC: 59.3%) comprising the major alleles of the six polymorphisms showed no association with EEA status (OR: 0.93, 95% CI: 0.58 –1.50, P ⫽ 0.769). DISCUSSION

Endurance performance is a multifactorial and polygenic trait with weak evidence of associations with individual SNPs (2, 12, 18). In one study, the HIF1A Pro582Ser polymorphism was examined in weight-lifters (1). The incidence of the Ser allele was higher in the power-orientated athletes compared with a control group (1). Interestingly, here we found that homozygote carrier of the Pro genotype was slightly more frequent in endurance athletes than in controls. Moreover, one haplotype carrying the Pro-coding C allele and the minor A allele of polymorphism rs17099207 was found to be slightly more frequent in athletes than in controls. Our finding is in agreement with the results of an intervention study (11), which showed that sequence variation of HIF1A was associated with ˙ O2 max before and after 24 wk of aerobic exercise training in V elderly persons. Homozygous subjects (60 yr and older) carrying the Pro variant of HIF1A Pro582Ser exhibited signifi˙ O2 max after training than those cantly higher changes in V carrying the minor allele. Thus the latter report, together with the present study, suggests that HIF1A Pro582 might be related to a higher responsiveness to endurance training. Studies in cancer cells (6, 17) as well as a promotor gene assay (20) suggested a lower transcriptional activity of HIF1A Pro582 compared with the rare Ser582 variant. Furthermore, tissue-specific loss of function of HIF1A in mice led to a decrease of exercise-induced changes in gene expression of glycolytic enzymes in skeletal muscle (10) and caused reductions in contractility and vascularization of the heart (9). These functional studies actually contradict the higher prevalence of the HIF1A Pro582 variant in EEAs in our study. We speculate that homozygosity for Pro582 is related to a higher and faster induction of HIF1A under hypoxic conditions. This assumption is supported by the study of Fu et al. (6): when hydroxylation of HIF1A was blocked in transfected cells through treatment with 2,2=-dipyridyal (an iron chelator), a significant increase in HIF1A Pro582 protein levels was observed. The increase in J Appl Physiol • VOL

levels of the HIF1A Ser582 mutant protein was markedly lower and less rapid. Presumably, the Pro582 variant is associated with higher induction level, which could influence endurance capacity. Of course, functional studies will be needed to fully understand the mechanisms underlying the putative association between the HIF1A polymorphisms and endurance performance. There is always a major element of uncertainty in genetic association studies, particularly if the statistical evidence is weak, due to lack of statistical power, unknown linkage with other variants, environmental interactions, or epistatic (genegene interaction) effects. We, therefore, cannot exclude the possibility that the association seen in our study is due to a type 1 error or unknown linkage to another functional variation close to HIF1A. In conclusion, we found weak evidence for associations between the HIF1A Pro582Ser polymorphism and a haplotype determined by the HIF1A Pro582 coding C allele and the minor allele of a putative regulatory SNP adjacent to the HIF1A 3= end with elite endurance performance status in Caucasian men. ACKNOWLEDGMENTS Thanks are expressed to the colleagues who kindly provided some of the DNA samples. Furthermore, we thank J. Fischer for technical assistance. GRANTS This work was supported by the German Institute for Sportsscience Grant VF 040 8/03/01/2002–2005 and by prior funding to the Physical Activity Sciences Laboratory at Laval University, and to the Human Genomics Laboratory at Pennington Biomedical Research Center. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). REFERENCES 1. Ahmetov II, Hakimullina AM, Lyubaeva EV, Vinogradova OL, Rogozkin VA. Effect of HIF1A gene polymorphism on human muscle performance. Bull Exp Biol Med 146: 351–353, 2008. 2. Ahmetov II, Williams AG, Popov DV, Lyubaeva EV, Hakimullina AM, Fedotovskaya ON, Mozhayskaya IA, Vinogradova OL, Astratenkova IV, Montgomery HE, Rogozkin VA. The combined impact of metabolic gene polymorphisms on elite endurance athlete status and related phenotypes. Hum Genet 126: 751–761, 2009. 3. Ameln H, Gustafsson T, Sundberg CJ, Okamoto K, Jansson E, Poellinger L, Makino Y. Physiological activation of hypoxia inducible factor-1 in human skeletal muscle. FASEB J 19: 1009 –1011, 2005. 4. Epstein AC, Gleadle JM, McNeill LA, Hewitson KS, O’Rourke J, Mole DR, Mukherji M, Metzen E, Wilson MI, Dhanda A, Tian YM, Masson N, Hamilton DL, Jaakkola P, Barstead R, Hodgkin J, Maxwell PH, Pugh CW, Schofield CJ, Ratcliffe PJ. C elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell 107: 43–54, 2001.

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5. Excoffier L, Slatkin M. Maximum-likelihood estimation of molecular haplotype frequencies in a diploid population. Mol Biol Evol 12: 921–927, 1995. 6. Fu XS, Choi E, Bubley GJ, Balk SP. Identification of hypoxia-inducible factor-1 alpha (HIF-1 alpha) polymorphism as a mutation in prostate cancer that prevents normoxia-induced degradation. Prostate 63: 215– 221, 2005. 7. Hemminger BM, Saelim B, Sullivan PF. TAMAL: an integrated approach to choosing SNPs for genetic studies of human complex traits. Bioinformatics 22: 626 –627, 2006. 8. Hoppeler H, Fluck M. Plasticity of skeletal muscle mitochondria: structure and function. Med Sci Sports Exerc 35: 95–104, 2003. 9. Huang Y, Hickey RP, Yeh JL, Liu D, Dadak A, Young LH, Johnson RS, Giordano FJ. Cardiac myocyte-specific HIF-1alpha deletion alters vascularization, energy availability, calcium flux, and contractility in the normoxic heart. FASEB J 18: 1138 –1140, 2004. 10. Mason SD, Howlett RA, Kim MJ, Olfert IM, Hogan MC, McNulty W, Hickey RP, Wagner PD, Kahn CR, Giordano FJ, Johnson RS. Loss of skeletal muscle HIF-1 alpha results in altered exercise endurance. PLoS Biol 2: e288, 2004. 11. Prior SJ, Hagberg JM, Phares DA, Brown MD, Fairfull L, Ferrell RE, Roth SM. Sequence variation in hypoxia-inducible factor 1 alpha (HIF1A): association with maximal oxygen consumption. Physiol Genomics 15: 20 –26, 2003. 12. Rankinen T, Bray MS, Hagberg JM, Perusse L, Roth SM, Wolfarth B, Bouchard C. The human gene map for performance and health-related fitness phenotypes: the 2005 update. Med Sci Sports Exerc 38: 1863–1888, 2006. 13. Richardson RS, Newcomer SC, Noyszewski EA. Skeletal muscle intracellular PO2 assessed by myoglobin desaturation: response to graded exercise. J Appl Physiol 91: 2679 –2685, 2001.

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14. Richardson RS, Noyszewski EA, Kendrick KF, Leigh JS, Wagner PD. Myoglobin O2 desaturation during exercise. Evidence of limited O2 transport. J Clin Invest 96: 1916 –1926, 1995. 15. Rivera MA, Dionne FT, Wolfarth B, Chagnon M, Simoneau JA, Perusse L, Boulay MR, Gagnon J, Song TM, Keul J, Bouchard C. Muscle-specific creatine kinase gene polymorphisms in elite endurance athletes and sedentary controls. Med Sci Sports Exerc 29: 1444 –1447, 1997. 16. Semenza G. Signal transduction to hypoxia-inducible factor 1. Biochem Pharmacol 64: 993–998, 2002. 17. Tanimoto K, Yoshiga K, Eguchi H, Kaneyasu M, Ukon K, Kumazaki T, Oue N, Yasui W, Imai K, Nakachi K, Poellinger L, Nishiyama M. Hypoxia-inducible factor-1 alpha polymorphisms associated with enhanced transactivation capacity, implying clinical significance. Carcinogenesis 24: 1779 –1783, 2003. 18. Williams AG, Folland JP. Similarity of polygenic profiles limits the potential for elite human physical performance. J Physiol 586: 113–121, 2008. 19. Wolfarth B, Rankinen T, Muhlbauer S, Scherr J, Boulay MR, Perusse L, Rauramaa R, Bouchard C. Association between a beta 2-adrenergic receptor polymorphism and elite endurance performance. Metabolism 56: 1649 –1651, 2007. 20. Yamada N, Horikawa Y, Oda N, Iizuka K, Shihara N, Kishi S, Takeda J. Genetic variation in the hypoxia-inducible factor-1 alpha gene is associated with type 2 diabetes in Japanese. J Clin Endocrinol Metab 90: 5841–5847, 2005. 21. Zaykin DV, Westfall PH, Young SS, Karnoub MA, Wagner MJ, Ehm MG. Testing association of statistically inferred haplotypes with discrete and continuous traits in samples of unrelated individuals. Hum Hered 53: 79 –91, 2002.

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