Association of NOS3-c.894G>T transversion with susceptibility to metabolic syndrome in Azar-cohort population: A case-control study and in silico analysis of the SNP molecular effects

Document Type : Original Article

Authors

1 Tuberculosis and Lung Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

2 Liver and Gastrointestinal Diseases Research Center, Clinical Research Institute, Tabriz University of Medical Sciences, Tabriz, Iran

3 Department of Statistics and Epidemiology, Faculty of Health, Tabriz University of Medical Sciences, Tabriz, Iran

Abstract

Objective(s): We investigated whether NOS3-c.894G>T transversion (rs1799983), which causes the substitution of glutamate with aspartate (E298D) in the oxygenase domain of endothelial nitric oxide synthase (eNOS), is associated with susceptibility to metabolic syndrome (MetS) risk in Iranian-Azerbaijanis.
Materials and Methods: The frequencies of the alleles and genotypes were compared in the 300 cases and 300 controls using PCR-RFLP assay. Also, higher-order MetS interaction with the genotypes, gender, age, and body mass index (BMI) was evaluated by classification and regression tree (CART) analysis. In silico analysis was done to introduce a hypothesis describing the molecular effects of NOS3-c.894G>T.
Results: The T allele (OR:1.46; CI:1.054-2.04; P=0.02), GT genotype (OR:1.44; CI:1.02-2.03; P=0.03), and dominant model (TT+GT vs GG, OR:1.48; CI:1.06-2.06; P=0.01) were found to be associated with increased risk of MetS. In the male subpopulation TT genotype (OR:7.19; CI:1.53-33.70; P=0.01) was discovered to be associated with increased odds of MetS. CART analysis showed that NOS3-c.894G>T genotypes and BMI significantly contribute to modulating MetS risk. Furthermore, in silico investigation revealed that c.894G>T may alter eNOS function through affecting interactions of its oxygenase domain with proteins such as B2R, b-actin, CALM1, CAV1, GIT1, HSP90AA1, NOSIP, and NOSTRIN.
Conclusion: We showed that NOS3-c.894G>T was associated with an increased risk of MetS in Iranian-Azerbaijanis, and BMI modulates the effects of NOS3-c.894G>T genotypes on MetS risk. Also, in silico analysis found that NOS3-c.894G>T may affect the interaction of the eNOS oxygenase domain with its several functional partners.

Keywords

References

1. Grundy SM. Metabolic syndrome: Connecting and reconciling cardiovascular and diabetes worlds. J Am Coll Cardiol 2006; 21:1093-1100.
2. Alberti KGMM, Eckel RH, Grundy SM, Zimmet PZ, Cleeman JI, Donato KA, et al. Harmonizing the metabolic syndrome: A joint interim statement of the international diabetes federation task force on epidemiology and prevention; National heart, lung, and blood institute; American heart association; World heart federation; International. Circulation 2009; 120:1640–1645.
3. Galmés S, Cifre M, Palou A, Oliver P, and Serra F. A genetic score of predisposition to low-grade inflammation associated with obesity may contribute to discern population at risk for metabolic syndrome. Nutrients 2019; 11:298-317.
4. McCracken E, Monaghan M, and Sreenivasan S. Pathophysiology of the metabolic syndrome. Clin Dermatol 2018; 36:14–20.
5. Pacher P, Beckman JS, and Liaudet L. Nitric oxide and peroxynitrite in health and disease. Physiol Rev 2007; 87:315–424.
6. Treuer AV. and Gonzalez DR. Nitric oxide synthases, S-nitrosylation and cardiovascular health: From molecular mechanisms to therapeutic opportunities (Review). Mol Med Rep 2015; 11:1555–1565.
7. Siragusa M and Fleming I. The eNOS signalosome and its link to endothelial dysfunction. Pflugers Arch Eur J Physiol 2016; 468:1125–1137.
8. Chernyshova K, Bondar V, Kobeleva H, and Maznichenko I. P6566Association of eNOS gene polymorphisms with features of dyslipidemia in patients with arterial hypertension and metabolic syndrome. Eur Heart J 2018;  39:ehy566-P6566.
9. Nunes RAB, Lima LB, Tanaka NI, da Costa Pereira A, Krieger JE, Mansur AJ. Genetic associations of bradykinin type 2 receptor, alpha-adrenoceptors and endothelial nitric oxide synthase with blood pressure and left ventricular mass in outpatients without overt heart disease. IJC Hear Vasc 2018; 21:45–49.
10. Saleh A, Stathopoulou MG, Dadé S, Ndiaye NC, Azimi-Nezhad M, Murray H, et al. Angiogenesis related genes NOS3, CD14, MMP3 and IL4R are associated to VEGF gene expression and circulating levels in healthy adults. BMC Med Genet 2015; 16:90-99.
11. Dellamea BS, Pinto LCF, Leitão CB, Santos KG, Canani LHS. Endothelial nitric oxide synthase gene polymorphisms and risk of diabetic nephropathy: A systematic review and meta-analysis. BMC Med Genet 2014; 15:1–13.
12. El-Lebedy D. Interaction between endothelial nitric oxide synthase rs1799983, cholesteryl ester-transfer protein rs708272 and angiopoietin-like protein 8 rs2278426 gene variants highly elevates the risk of type 2 diabetes mellitus and cardiovascular disease. Cardiovasc Diabetol 2018; 17:97.
13. Vecoli C. Endothelial nitric oxide synthase gene polymorphisms in cardiovascular disease. Vitam Horm 2014; 96:387–406.
14. Dalvand S, Niksima SH, Meshkani R, Ghanei Gheshlagh R, Sadegh-Nejadi S, Kooti W, et al. Prevalence of metabolic syndrome among Iranian population: A systematic review and meta-analysis. Iran J Public Health 2017; 46:456–467.
15. Farhang S, Faramarzi E, Sani NA, Poustchi H, Ostadrahimi A, Alizadeh BZ, et al. Cohort profile: The AZAR cohort, a health-oriented research model in areas of major environmental change in Central Asia. Int J Epidemiol 2019; 48:1-9.
16. Aftabi Y, Colagar AH, and Mehrnejad F. An in silico approach to investigate the source of the controversial interpretations about the phenotypic results of the human AhR-gene G1661A polymorphism. J Theor Biol 2016; 393:1–15.
17. Stata Press. Stata power and sample-size reference manual release. Texas Stata Press, Version. 2013;13:1-50.
18. Bairaktari ET, Seferiadis KI, Elisaf MS. Evaluation of methods for the measurement of low-density lipoprotein cholesterol. J Cardiovasc Pharmacol Ther 2005; 10:45–54.
19. Poustchi H, Eghtesad S, Kamangar F, Etemadi A, Keshtkar AA, Hekmatdoost A, et al. Prospective epidemiological research studies in Iran (the PERSIAN Cohort Study): Rationale, objectives, and design. Am J Epidemiol 2018; 187:647–655.
20. Grundy SM, Brewer HB, Cleeman JI, Smith SC, Lenfant C. Definition of metabolic syndrome: Report of the national heart, lung, and blood institute/american heart association conference on scientific issues related to definition. Circulation 2004; 109:433–438.
21. Green MR, Hughes H, Sambrook J, MacCallum P. Molecular cloning: A laboratory manual. 4th ed. New York, Cold Spring Harbor, 2012.
22. Questier F, Put R, Coomans D, Walczak B, and Heyden Y Vander. The use of CART and multivariate regression trees for supervised and unsupervised feature selection. Chemom Intell Lab Syst 2005; 76:45–54.
23. Trevethan R. Sensitivity, specificity, and predictive values: Foundations, pliabilities, and pitfalls in research and practice. Front Public Heal 2017; 5:307-314.
24. Reva B, Antipin Y, and Sander C. Predicting the functional impact of protein mutations: Application to cancer genomics. Nucleic Acids Res 2011; 39:e118.
25. Capriotti E and Fariselli P. PhD-SNPg: A webserver and lightweight tool for scoring single nucleotide variants. Nucleic Acids Res 2017; 45:W247–W252.
26. Adzhubei I, Jordan DM, and Sunyaev SR. Predicting functional effect of human missense mutations using PolyPhen-2. Curr Protoc Hum Genet 2013; 76:7-20.
27. Bendl J, Stourac J, Salanda O, Pavelka A, Wieben ED, Zendulka J, et al. PredictSNP: Robust and accurate consensus classifier for prediction of disease-related mutations. Gardner PP, editor. PLoS Comput Biol 2014; 10:1–17.
28. Choi Y and Chan AP. PROVEAN web server: A tool to predict the functional effect of amino acid substitutions and indels. Bioinformatics 2015; 31:2745–2747.
29. Kumar P, Henikoff S, and Ng PC. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat Protoc 2009; 4:1073–1082.
30. Johnson AD, Handsaker RE, Pulit SL, Nizzari MM, O’Donnell CJ, and De Bakker PIW. SNAP: A web-based tool for identification and annotation of proxy SNPs using HapMap. Bioinformatics 2008; 24:2938–2939.
31. Hecht M, Bromberg Y, and Rost B. Better prediction of functional effects for sequence variants. BMC Genomics 2015; 16:1-12.
32. De Baets G, Van Durme J, Reumers J, Maurer-Stroh S, Vanhee P, Dopazo J, et al. SNPeffect 4.0: On-line prediction of molecular and structural effects of protein-coding variants. Nucleic Acids Res 2012; 40:D935–D939.
33. El-Gebali S, Mistry J, Bateman A, Eddy SR, Luciani A, Potter SC, et al. The Pfam protein families database in 2019. Nucleic Acids Res 2019; 47:D427–D432.
34. Yachdav G, Kloppmann E, Kajan L, Hecht M, Goldberg T, Hamp T, et al. PredictProtein - An open resource for online prediction of protein structural and functional features. Nucleic Acids Res 2014; 42:337–343.
35. Szklarczyk D, Gable AL, Lyon D, Junge A, Wyder S, Huerta-Cepas J, et al. STRING v11: Protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nucleic Acids Res 2019; 47:D607–D613.
36. Yan Y, Tao H, He J, and Huang SY. The HDOCK server for integrated protein–protein docking. Nat Protoc 2020; 15:1829–1852.
37. Ju H, Zou R, Venema VJ, and Venema RC. Direct interaction of endothelial nitric-oxide synthase and caveolin-1 inhibits synthase activity. J Biol Chem 1997; 272:18522–18525.
38. Fontana J, Fulton D, Chen Y, Fairchild TA, McCabe TJ, Fujita N, et al. Domain mapping studies reveal that the M domain of hsp90 serves as a molecular scaffold to regulate Akt-dependent phosphorylation of endothelial nitric oxide synthase and NO release. Circ Res 2002; 90:866–873.
39. Hellermann GR and Solomonson LP. Calmodulin promotes dimerization of the oxygenase domain of human endothelial nitric-oxide synthase. J Biol Chem 1997; 272:12030–12034.
40. Icking A. NOSTRIN functions as a homotrimeric adaptor protein facilitating internalization of eNOS. J Cell Sci 2005; 5059–5070.
41. Golser R, Gorren ACF, Leber A, Andrew P, Habisch HJ, Werner ER, et al. Interaction of endothelial and neuronal nitric-oxide synthases with the bradykinin B2 receptor. Binding of an inhibitory peptide to the oxygenase domain blocks uncoupled NADPH oxidation. J Biol Chem 2000; 275:5291–5296.
42. Liu S, Premont RT, Rockey DC. Endothelial nitric-oxide synthase (eNOS) is activated through G-protein-coupled receptor kinase-interacting protein 1 (GIT1) tyrosine phosphorylation and Src protein. J Biol Chem 2014; 289:18163–18174.
43. Dedio J, König P, Wohlfart P, Schroeder C, Kummer W,  Müller-Esterl W. NOSIP, a novel modulator of endothelial nitric oxide synthase activity. FASEB J 2001; 15:79–89.
44. Kondrikov D, Fonseca F V., Elms S, Fulton D, Black SM, Block ER, et al. Β-Actin association with endothelial nitric-oxide synthase modulates nitric oxide and superoxide generation from the enzyme. J Biol Chem 2010; 285:4319–4327.
45. Sadati SM, Radfar M, Hamidi AK, Abdollahi M, Qorbani M, Esfahani EN, et al. Association between the polymorphism of Glu298Asp in Exon 7 of the eNOS gene with foot ulcer and oxidative stress in adult patients with type 2 diabetes. Can J Diabetes 2018; 42:18–22.
46. Zohreh R, Reza N-R. Association of endothelial nitric oxide synthase gene variant (G894T) with coronary artery disease in Western Iran. Clin Biochem 2011; 44:131-137.
47. Mahmoodi K, Nasehi L, Karami E, Soltanpour MS. Association of nitric oxide levels and endothelial nitric oxide synthase G894T polymorphism with coronary artery disease in the Iranian population. Vasc Spec Int 2016; 32:105–112.
48. Hennekens CH and Andreotti F. Leading avoidable cause of premature deaths worldwide: Case for obesity. Am J Med 2013; 126:97–98.
49. Fattakhov NS, Skuratovskaya DA, Vasilenko MA, Kirienkova E V., Zatolokin PA, Mironyuk NI, et al. Association of Glu298Asp polymorphism of endothelial NO synthase gene with metabolic syndrome development: A pilot study. Bull Exp Biol Med 2017; 162:615–618.
50. Yu AP, Tam BT, Yau WY, Chan KS, Yu SS, Chung TL, et al. Association of endothelin-1 and matrix metallopeptidase-9 with metabolic syndrome in middle-aged and older adults. Diabetol Metab Syndr 2015; 7:1-13.
51. Ianas O, Manda D, Heltianu C, Vladoiu S, Popa O, Rosca R, et al. The G894T polymorphism of endothelial nitric oxide synthase gene and the endocrine-metabolic changes in metabolic syndrome: A romanian case-control study. Acta Endocrinol (Copenh) 2009; 5:447–458.
52. Fairchild TA, Fulton D, Fontana JT, Gratton JP, McCabe TJ, and Sessa WC. Acidic hydrolysis as a mechanism for the cleavage of the Glu298 → Asp variant of human endothelial nitric-oxide synthase. J Biol Chem 2001; 276:26674–26679.
53. Griffith O. Nitric Oxide synthases: Properties and catalytic mechanism. Annu Rev Physiol 1995; 57:707–736.
54. Morris CM, Ballard CG, Allan L, Rowan E, Stephens S, Firbank M, et al. NOS3 gene rs1799983 polymorphism and incident dementia in elderly stroke survivors. Neurobiol Aging 2011; 32:554.e1-554.e6.
55. McDonald DM, Alp NJ, Channon KM. Functional comparison of the endothelial nitric oxide synthase Glu 298Asp polymorphic variants in human endothelial cells. Pharmacogenetics 2004; 14:831–839.
56. Wang XL, Sim AS, Wang MX, Murrell GAC, Trudinger B,  Wang J. Genotype dependent and cigarette specific effects on endothelial nitric oxide synthase gene expression and enzyme activity. FEBS Lett 2000; 471:45–50.
57. Ghasemi A, Zahediasl S, and Azizi F. Nitric oxide and clustering of metabolic syndrome components in pediatrics. Eur J Epidemiol 2010; 25:45–53.
58. Dolgikh OV, Zaitseva NV, Nosov AE, Krivtsov AV, Dianova DG, Kazakova OA, et al. Analysis of the role of carriership of polymorphic genotypes of ESR1, eNOS, and APOE4 genes in the development of arterial hypertension in men. Bull Exp Biol Med 2018; 164:753–756.
59. Aftabi Y, Hosseinzadeh Colagar A, Mehrnejad F, Seyedrezazadeh E, and Moudi E. Aryl hydrocarbon receptor gene transitions (c.-742C>T; c.1661G>A) and idiopathic male infertility: A case-control study with in silico and meta-analysis. Environ Sci Pollut Res 2017; 24:20599–20615.
60. Aftabi Y, Rafei S, Zarredar H, Amiri-Sadeghan A, Akbari-Shahpar M, Khoshkam Z, et al. Refinement of coding SNPs in the human aryl hydrocarbon receptor gene using ISNPranker: An integrative-SNP ranking web-tool. Comput Biol Chem 2021; 90:107416.
61. Aftabi Y, Khoshkam Z, Amiri-Sadeghan A, Khalili Y, Ichihara G. An introduction to EpiPol (Epigenetic affecting Polymorphism) concept with an in silico identification of CpG-affecting SNPs in the upstream regulatory sequences of human AHR gene. Meta Gene 2020; 26:100805.
62. Azani A, Hosseinzadeh A, Azadkhah R, Zonouzi AAP, Zonouzi AP, Aftabi Y, et al. Association of endothelial nitric oxide synthase gene variants (-786 T>C, intron 4 b/a VNTR and 894 G>T) with idiopathic recurrent pregnancy loss: A case-control study with haplotype and in silico analysis. Eur J Obstet Gynecol Reprod Biol 2017; 215:93–100.
63. Kone BC. Protein-protein interactions controlling nitric oxide synthases. Acta Physiol Scand 2000; 168:27–31.
64. Coletta A, Pinney JW, Solís DYW, Marsh J, Pettifer SR, and Attwood TK. Low-complexity regions within protein sequences have position-dependent roles. BMC Syst Biol 2010; 4:1–13.
65. Joshi MS and Bauer JA. Preliminary computational modeling of nitric oxide synthase 3 interactions with caveolin-1: Influence of exon 7 Glu298Asp polymorphism. Acta Biochim Biophys Sin (Shanghai) 2008; 40:47–54.
Iranian Journal of Basic Medical Sciences
Volume 24, Issue 3
March 2021
Pages 408-419

Files

Share

How to cite

Statistics