The endothelial nitric oxide synthase gene variant rs2070744 in turkish elite athletes

Authors

Abstract

AimGenetic variations have been associated with physical performance. The Endothelial Nitric Oxide Synthase (eNOS) gene variants have been widely studied in this context. The aim of the present study is to compare the T-786C variant of the eNOS gene in Turkish elite athletes and control groups.
MethodsDNA samples were obtained from 52 elite athletes (45 male, 7 female) and 60 controlsubjects (49 male, 11 female). The T-786C variant of the eNOS gene was genotyped by polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) method.
ResultsTT, TC, CC genotypes of the T-786C variant of eNOS gene were observed in 40.0%, 48.3%, and 11.6% ofcontrol subjects and in 55.7%, 30.7% and 13.4% of elite athletes, respectively. There was not any statistically significant difference in genotype and allele frequencies of T-786C of the eNOS between the elite athlete and the control groups (p>0.05).
ConclusionThe present study demonstrated that the T-786C variant of the eNOS gene is not associated withstudy population but larger sample analyses are needed in different groups of elite athletes in order to substantiate these findings.

Keywords

Introduction

An elite athlete is defined as the person who has competed at a national or international level in a given sport.¹ In the past decade, the idea that genetic traits bear a strong association with physical performance has been widely accepted. Researchers are now focusing on investigation of the exact genetic profiles that contribute to sport performance and they are trying to determine the underlying mechanisms that play a role in specific fields of elite athletic performance. Nitric oxide (NO) affects the control of skeletal muscle function, increases skeletal muscle glucose uptake during exercise and enhances mitochondrial ATP production. All of these processes modulate muscle strength.² NO is synthesized from L-arginine by the nitric oxide synthase (NOS) gene.^3,4 NOS family has three distinct isoforms: neuronal NOS (nNOS/NOS1), inducible NOS (iNOS/NOS2), and endothelial NOS (eNOS/NOS3).⁴ (Higashibata T). eNOS gene is one of the candidate genes to clarify human variations in health and exercise-related phenotypes. Human eNOS gene is localized on chromosome 7 (7q35-36) and contains 26 exons.⁵ The T-786C variant (rs2070744), a thymidine to cytosine transition mutation, is present in the 5’ flanking region of eNOS gene and decreases the promoter activity of eNOS, resulting in decrease of endothelial NO production.⁶ It was reported that eNOS T-786C variant is related with resting blood pressure.⁷ and the blood pressure response to acute event of maximal aerobic exercise.⁸ The C allele affects eNOS transcription, which is consistent with reduced NO production.⁹ Because of the crucial role of NO in muscle adaptation to exercise, we compared the T-786C variant of eNOS gene in Turkish elite athletes and control groups in this study.

Materials and Methods

PatientsThe study population consisted of 52 Turkish elite athletes (45 males and 7 females; aged between 14-30) and 60 unrelated controls (49 males and 11 females; aged between 18-34) who had no competitive sport experience. Subjects had similar ethnic backgrounds and they were all from the same geographic area. A written-informed consent was obtained from each participant before blood sampling. The study involving human subjects was approved by the Ethics Committee in Clinical Research of Gaziosmanpaşa University (11-BADK-095) and the study was conducted in accord with the Helsinki Declaration.
Genetic analysisGenomic DNA was isolated from 2 mL venous blood according to kit procedure (Sigma, USA) and stored at - 20oC. The eNOS T-786C variant was analyzed by polymerase chain reaction-restriction-restriction fragment lenght polymorphism (PCR-RFLP) methods using the following primers: 5’-TGGAGAGTGCTGGTGTACCCCA-3’ (forward) and 5’-GCCTCCACCCCCACCCTGTC-3’ (reverse). Method was carried out as described previously by Ordenez et al..¹⁰ Amplified products were digested by MspI enzyme. Two sets of digested products were formed as a result of allelic variation. One of these products was of 140 and 40 bp (-786T allele) and another set was of the 90, 50 and 40 bp (-786C allele) in length. Digested products were examined on a 2.5 % agarose gel stained with ethidium bromide.
Statistical AnalysisAll statistical analyses were performed using computer SPSS Statistical Program Version 20.0 and Openepi 3.01 software package program. Continuous data were given as mean±SD (standart deviation) and minmax. Chi-square test was used to determine the significance of differences in the allele frequency and genotype distribution between the two study groups. Hardy-Weinberg equilibrium test was performed for both study groups. Odds ratio (OR) and 95% confidence intervals (CIs) were calculated. A p value < 0.5 was considered statistically significant.

Results

We genotyped 60 controls (average age: 23.12±3.59 years; 49 male, 11 female) and 52 elite athletes (average age: 21.52±4.46 years; 45 male, 7 female) for the T-786C variant of the eNOS gene. The demographic and clinical characteristics are presented Table 1. In result of analysis eNOS T-786C variant, it was determined that there was no any statistical significant differences between Turkish elite athletes and unrelated control who had no competitive sport experience in terms of genotype and allel frequencies. The genotype and allele frequencies of the eNOS T-786C of both group are reported in Table 2
.

Discussion

Elite athletes represent both endurance and power related traits. Sport performance is rather polygenic in nature because of the combined effect of hundreds of factors in genetic variance among individuals. Even though it is difficult to determine the accurate genetic factors of performance, in recent years, various gene variants have been analyzed to evaluate individual differences in elite athletes with phenotype-genotype association studies.eNOS-derived NO is also known as “endothelial-derived relaxing factor” and has crucial functions, including regulation of vascular tone and regional blood flow, inhibition of vascular smooth muscle cell proliferation, modulation of leukocyte–endothelial interactions and thrombosis.¹¹ Furthermore, NO has an impact as a neurotransmitter in the brain by facilitating the conversion of soluble guanylyl cyclase to the second messenger molecule, cyclic guanisine monophosphate (cGMP). cGMP relaxes the blood vessels following exercise, increasing blood flow to muscles following exercise to enhance glucose uptake.¹² Evidence from several studies suggests that NO also plays a role in human skeletal muscle glucose uptake during exercise.¹³ as well as in the regulation of oxygen consumption in the myocardium.¹⁴ and skeletal muscles.¹⁵ eNOS gene has been considered as one of the candidate genes affecting high endurance performance due to the effects of NO on vascular tone. The functions of eNOS gene are confirmed with experimental studies using eNOS-knockout mice. It was reported that eNOS gene deficiency causes increased vascular smooth muscle cell proliferation in
response to vessel injury.¹⁶ hypertension.¹⁷ increased diet-induced atherosclerosis.¹⁸ and decreased bleeding times.¹⁹ eNOS gene has several polymorphic sites. In various studies, it was reported that eNOS T-786C variant to be associated with resting forearm blood flow.²⁰ and the parasympathetic modulation response to aerobic exercise training.²¹ besides the differentiation of elite power from endurance athletes.²² In in-vitro luciferase-based transcription analysis, it was shown that C allele of eNOS has a lower promoter activity than T allele.⁶ In previous studies, it was reported that eNOS G894T variant was associated with physical performance.²³ however in another study, it wasn’t found difference three variants of eNOS compared with controls.²⁴ When distribution of alleles is investigated, there are studies reporting that T allele is more abundant in athletes.^25,26,27 There are also studies suggesting that C allele is abundant.²⁸ In present study, we compared eNOS T-786C genotypes in elite athletes and control groups. The genotype and allele frequencies of eNOS T-786C variant showed no significant differences between athletes and control groups (p>0.05).

Conclusion

Although the present study does not imply any difference between the groups, larger sample analyses are needed in different groups of elite athletes to substantiate these findings.

Declarations

References

1. Macarthur DG, North KN. Genes and human elite athletic performance. Hum Genet. 2005;116:331-339. doi:10.1007/s00439-005-1261-8
   Article PubMed Google Scholar
2. Gao Y. The multiple actions of NO. Pflugers Arch. 2010;459(6):829-839. doi:10.1007/s00424-009-0773-9
   Article PubMed Google Scholar
3. Ozturk E, Balat O, Pehlivan S, Ugur MG, Ozcan C, Sever T, et al. Endothelial nitric oxide synthase gene polymorphisms in preeclampsia with or without eclampsia in a Turkish population. J Obstet Gynaecol Res. 2011;37(12):1778-1783. doi:10.1111/j.1447-0756.2011.01606.x
   Article PubMed Google Scholar
4. Higashibata T, Hamajima N, Naito M, Kawai S, Yin G, Suzuki S, et al. eNOS genotype modifies the effect of leisure-time physical activity on serum triglyceride levels in a Japanese population. Lipids Health Dis. 2012;11:150. doi:10.1186/1476-511x-11-150
   Article PubMed Google Scholar
5. Kara N, Senturk N, Gunes SO, Bagci H, Yigit S, Turanli AY. Lack of evidence for association between endothelial nitric oxide synthase gene polymorphism (Glu298Asp) with Behçet’s disease in the Turkish population. Arch Dermatol Res. 2006;297(10):468-471. doi:10.1007/s00403-006-0643-7
   Article PubMed Google Scholar
6. Nakayama M, Yasue H, Yoshimura M, Shimasaki Y, Kugiyama K, Ogawa H, et al. T-786→C mutation in the 5′-flanking region of the endothelial nitric oxide synthase gene is associated with coronary spasm. Circulation. 1999;99(22):2864-2870. doi:10.1161/01.cir.99.22.2864
   Article PubMed Google Scholar
7. Augeri AL, Tsongalis GJ, Van Heest JL, Maresh CM, Thompson PD, Pescatello LS. The endothelial nitric oxide synthase −786 T>C polymorphism and the exercise-induced blood pressure and nitric oxide responses among men with elevated blood pressure. Atherosclerosis. 2009;204(2):e28-e34. doi:10.1016/j.atherosclerosis.2008.12.015
   Article PubMed Google Scholar
8. Olson KM, Augeri AL, Seip RL, Tsongalis GJ, Thompson PD, Pescatello LS. Correlates of endothelial function and the peak systolic blood pressure response to a graded maximal exercise test. Atherosclerosis. 2012;222(1):202-207. doi:10.1016/j.atherosclerosis.2012.01.027
   Article PubMed Google Scholar
9. Dengel DR, Brown MD, Ferrell RE, Reynolds TH, Supiano MA. A preliminary study on T-786C endothelial nitric oxide synthase gene and renal hemodynamic and blood pressure responses to dietary sodium. Physiol Res. 2007;56(4):393-401. doi:10.33549/physiolres.931002
   Article PubMed Google Scholar
10. Ordóñez AJ, Carreira JM, Franco AG, Sánchez LM, Alvarez MV, García EC. Two expressive polymorphisms on the endothelial nitric oxide synthase gene (intron 4, 27 bp repeat and −786 T/C) and venous thromboembolism. Thromb Res. 2000;99(6):563-566. doi:10.1016/s0049-3848(00)00288-7
    Article PubMed Google Scholar
11. Huang PL. eNOS, metabolic syndrome and cardiovascular disease. Trends Endocrinol Metab. 2009;20(6):295-302. doi:10.1016/j.tem.2009.03.005
    Article PubMed Google Scholar
12. Pellinger TK, Simmons GH, Maclean DA, Halliwill JR. Local histamine H1- and H2-receptor blockade reduces postexercise skeletal muscle interstitial glucose concentrations in humans. Appl Physiol Nutr Metab. 2010;35(5):617-626. doi:10.1139/h10-055
    Article PubMed Google Scholar
13. McConell GK, Kingwell BA. Does nitric oxide regulate skeletal muscle glucose uptake during exercise? Exerc Sport Sci Rev. 2006;34(1):36-41. doi:10.1097/00003677-200601000-00008
    Article PubMed Google Scholar
14. Loke KE, Laycock SK, Mital S, Wolin MS, Bernstein R, Oz M, et al. Nitric oxide modulates mitochondrial respiration in failing human heart. Circulation. 1999;100(12):1291-1297. doi:10.1161/01.cir.100.12.1291
    Article PubMed Google Scholar
15. Wilkerson DP, Campbell IT, Jones AM. Influence of nitric oxide synthase inhibition on pulmonary O2 uptake kinetics during supra-maximal exercise in humans. J Physiol. 2004;561(Pt 2):623-635. doi:10.1113/jphysiol.2004.071894
    Article PubMed Google Scholar
16. Moroi M, Zhang L, Yasuda T, Virmani R, Gold HK, Fishman MC, et al. Interaction of genetic deficiency of endothelial nitric oxide, gender, and pregnancy in vascular response to injury in mice. J Clin Invest. 1998;101(6):1225-1232. doi:10.1172/jci1293
    Article PubMed Google Scholar
17. Huang PL, Huang Z, Mashimo H, Bloch KD, Moskowitz MA, Bevan JA, et al. Hypertension in mice lacking the gene for endothelial nitric oxide synthase. Nature. 1995;377(6546):239-242. doi:10.1038/377239a0
    Article PubMed Google Scholar
18. Kuhlencordt PJ, Gyurko R, Han F, Scherrer-Crosbie M, Aretz TH, Hajjar R, et al. Accelerated atherosclerosis, aortic aneurysm formation, and ischemic heart disease in apolipoprotein E/endothelial nitric oxide synthase double-knockout mice. Circulation. 2001;104(4):448-454. doi:10.1161/hc2901.091399
    Article PubMed Google Scholar
19. Freedman JE, Sauter R, Battinelli EM, Ault K, Knowles C, Huang PL, et al. Deficient platelet-derived nitric oxide and enhanced hemostasis in mice lacking the NOS3 gene. Circ Res. 1999;84(12):1416-1421. doi:10.1161/01.res.84.12.1416
    Article PubMed Google Scholar
20. Data SA, Roltsch MH, Hand B, Ferrell RE, Park JJ, Brown MD. eNOS T-786C genotype, physical activity, and peak forearm blood flow in females. Med Sci Sports Exerc. 2003;35(12):1991-1997. doi:10.1249/01.mss.0000099105.99682.8b
    Article PubMed Google Scholar
21. Silva BM, Neves FJ, Negrão MV, Alves CR, Dias RG, Alves GB, et al. Endothelial nitric oxide synthase polymorphisms and adaptation of parasympathetic modulation to exercise training. Med Sci Sports Exerc. 2011;43(9):1611-1618. doi:10.1249/mss.0b013e3182152197
    Article PubMed Google Scholar
22. Gómez-Gallego F, Ruiz JR, Buxens A, Altmäe S, Artieda M, Santiago C, et al. Are elite endurance athletes genetically predisposed to lower disease risk? Physiol Genomics. 2010;41(1):82-90. doi:10.1152/physiolgenomics.00183.2009
    Article PubMed Google Scholar
23. Saunders CJ, Xenophontos SL, Cariolou MA, Anastassiades LC, Noakes TD, Collins M. The bradykinin beta 2 receptor (BDKRB2) and endothelial nitric oxide synthase 3 (NOS3) genes and endurance performance during Ironman triathlons. Hum Mol Genet. 2006;15(6):979-987. doi:10.1093/hmg/ddl014
    Article PubMed Google Scholar
24. Wolfarth B, Rankinen T, Mühlbauer S, Ducke M, Rauramaa R, Boulay MR, et al. Endothelial nitric oxide synthase gene polymorphism and elite endurance athlete status: the Genathlete study. Scand J Med Sci Sports. 2008;18(4):485-490. doi:10.1111/j.1600-0838.2007.00717.x
    Article PubMed Google Scholar
25. Gómez-Gallego F, Ruiz JR, Buxens A, Artieda M, Arteta D, Santiago C, et al. The −786 T/C polymorphism of the NOS3 gene is associated with elite performance in power sports. Eur J Appl Physiol. 2009;107(5):565-569.
    PubMed Google Scholar
26. Drozdovs’ka SB, Lysenko OM, Dosenko VI, Il’in VM, Moibenko OO. T(-786)→C polymorphism of the endothelial nitric oxide synthase promoter gene (eNOS) and exercise performance in sport. Fiziol Zh. 2013;59(6):63-71.
    PubMed Google Scholar
27. Sessa F, Chetta M, Petito A, Franzetti M, Bafunno V, Pisanelli D, et al. Gene polymorphisms and sport attitude in Italian athletes. Genet Test Mol Biomarkers. 2011;15(4):285-290. doi:10.1089/gtmb.2010.0179
    Article PubMed Google Scholar
28. Eynon N, Ruiz JR, Yvert T, Santiago C, Gómez-Gallego F, Lucia A, et al. The C allele in NOS3 −786 T/C polymorphism is associated with elite soccer player status. Int J Sports Med. 2012;33(7):521-524. doi:10.1055/s-0032-1306337
    Article PubMed Google Scholar

Additional Information

Publisher’s Note
Bayrakol MP remains neutral with regard to jurisdictional and institutional claims.

Rights and Permissions

Creative Commons License

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0). To view a copy of the license, visit https://creativecommons.org/licenses/by-nc/4.0/

View full license details →

About This Article

Received:

April 4, 2016

Accepted:

April 12, 2016

Published Online:

May 1, 2016

Printed:

May 1, 2016