Association of Dimethylarginine Dimethylaminohydrolase 2 (DDAH-2 1154 C > A) Gene Polymorphism with Coronary Artery Disease

INTRODUCTION

Coronary artery disease is one of the most common causes of death all over the world. Endothelial dysfunction is one of the initial events in atherosclerosis and is associated with risk factors like smoking, obesity, hypertension, hypercholesterolemia, hypertension, type 1 and 2 diabetes, hyperhomocysteinemia, and chronic renal failure.^1–3 Many factors have been suggested for the impairment of endothelial function and some of them are reduction of the nitric oxide bioactivity, altered endothelial nitric oxide synthase expression, and increased generation of reactive oxygen species. Nitric oxide is the most potent vasodilator synthesized by the endothelial cells from L-arginine by the action of endothelial nitric oxide synthase. Nitric oxide is a gas with a half-life of few seconds. Nitric oxide maintains the vascular endothelial function in many ways and is an important mediator of vasodilation, antithrombotic processes, as well as growth inhibition and anti-inflammation.

The nitric oxide synthase inhibitor, viz. asymmetric dimethylarginine (ADMA) has been identified in the 1990s by Vallance et al.⁴ Asymmetric dimethylarginine is generated by a continuous turnover of proteins in almost every cell of the body. Some arginine residues of proteins are post-translationally dimethylated asymmetrically by protein arginine methyltransferase-1.⁵ The ADMA is a methylated arginine and it competitively inhibits the endothelial and neuronal nitric oxide synthase isoforms.^4,6 It is a less potent inhibitor of the inducible nitric oxide synthase isoform.^4,6 Asymmetric dimethylarginine is eliminated from the body by both metabolic degradations by dimethylarginine dimethylaminohydrolases and also by renal excretion.

Leiper and Vallance in 1999 showed that dimethylarginine dimethylaminohydrolase (DDAH) has two isoforms namely DDAH-1 and DDAH-2, which are expressed in bacteria, rats, mice, and humans.^7,8 Dimethylarginine dimethylaminohydrolase enzyme catalyzes the metabolism of one molecule of ADMA to dimethylamine and L-citrulline (Fig. 1). There are no cofactors needed for the above catalytic reaction. Knipp et al. found that DDAH-1 is a zinc-containing protein.^9,10 However, zinc is not required for the catalytic process, but it is needed to stabilize the DDAH enzyme in an active form.¹⁰ Leiper et al. showed that DDAH-2 isoform was found to be expressed in endothelial cells where it determined the nitric oxide bioactivity.^8,11 Tran et al. mapped the human DDAH-1 gene to the chromosome 1p 22 and DDAH-2 gene to the major histocompatibility complex III region of the chromosome 6p 21.3.⁸

The gene knockout in rats has shown that DDAH-1 plays a vital role in the regulation of serum levels of ADMA, whereas DDAH-2 plays an important role in the control of nitric oxide-mediated functions of the endothelium.¹¹ The DDAH-2 isoform is expressed mainly in the site of eNOS expression, i.e., mainly the endothelium.¹² The gene silencing studies in rats have shown that the DDAH enzyme through the degradation of ADMA, regulates the function of nitric oxide synthase. There are a few studies concerning the association of the DDAH gene polymorphism with risk factors for cardiovascular disease. Gad et al. revealed that a possible association between cardiovascular disease and the A allele/AA genotype for DDAH2 SNP1 (_1151 C/A, rs805304) and G allele/GG genotype for SNP2 (_449 C/G, rs805305) in male 35- to 50-year-old Egyptian patients was observed.¹³ Maas et al. have shown that 449 G/C and 1151 A/C gene polymorphisms in the promoter region of the DDAH-2 gene are associated with an increased incidence of hypertension.¹⁴ Therefore, this study was undertaken to find the association of DDAH-2 gene 1151 C > A polymorphisms with coronary artery disease.

AIMS AND OBJECTIVES

The objective of the study is to find the association of DDAH-2 gene 1151 C > A polymorphism with coronary artery disease. To estimate the serum nitric oxide levels and find its relationship with the above gene polymorphism.

MATERIALS AND METHODS

The study protocol was approved by the Institutional Ethics Committee of Madras Medical College, Chennai (No. 21032012). One hundred cases of coronary artery disease in the age group between 35 years and 60 years old verified by coronary angiogram having >50% stenosis of at least one of the major coronary arteries were randomly recruited. Cases may be with or without risk factors like smoking, alcohol, and hypertension. Age, sex, and risk factors matched 100 controls who had no clinical evidence of coronary artery disease were selected. Patients with any other acute or chronic illness were excluded. Assessment of DDAH-2 (1154 C > A) gene polymorphism was done by TaqMan assay using real-time PCR and confirmation of SNP genotypes was done by Sanger sequencing.

GENOTYPE ANALYSIS

STATISTICAL ANALYSIS

The statistical analysis was performed using SPSS software version 16. p < 0.05 was considered to be statistically significant. Hardy–Weinberg equilibrium, genotype frequency distribution, and allele frequency distribution between cases and controls were compared with a χ² test. Age, body mass index (BMI), waist/hip ratio, and serum lipid levels were compared between cases and controls by Student’s t-test. One-way ANOVA was used to compare the NO levels between CC, CA, and AA genotypes. Multiple logistic regression analysis was performed to evaluate the interaction between human DDAH 2 CC/AA genotypes and other variables (age, sex smoking, alcohol, serum levels of HDL and LDL) in relation to the prevalence of coronary artery disease.

RESULTS

Table 1 shows the genotype distribution of human DDAH-2 (1151 C/A) gene polymorphism in the study group (both cases and controls). This was found to be in Hardy–Weinberg equilibrium as evidenced by the chi-square test (χ² = 1.6446, p = 0.19).

Table 2 shows the data of age, sex, BMI, waist/hip ratio, HDL-c and LDL-c levels, and risk factor distribution like smoking, alcohol, and hypertension among cases and control subjects. An insignificant p value was obtained with respect to variables like age, sex, BMI, history of smoking, alcoholism, and hypertension by Student’s t-test. There was a significant difference in HDL-c (low in cases 41.77 ± 3.52, high in controls 47.30 ± 3.90) and LDL-c levels (high in cases 117 ± 14.64, low in controls 101.31 ± 8.77) between cases and controls.

Table 3 shows the genotype distribution of human DDAH-2 (1151 C/A) gene polymorphism in patients with coronary artery disease and control subjects. AA genotype was more frequent among cases (29%) when compared with controls (14%). In contrast, CC was more common among controls (31%) when compared with cases (17%). The distribution of CA genotype between cases and controls was 54 and 55%, respectively. p value is 0.009.

Table 4 shows the allele frequency distribution and odds ratio calculation of human DDAH-2 (1151 C/A) gene polymorphism in patients with coronary artery disease and control subjects. A⁺ allele was more frequent among cases (83%) when compared with controls (69%). In contrast, A⁻ allele was more frequent among controls (31%) when compared with cases (17%). p value is 0.02. Odds ratio analysis showed that the A⁺ allele carries a 2.19 times higher risk for coronary artery disease when compared with the A⁻ allele.

Table 5 shows the difference in serum nitric oxide between CC, CA, and AA genotypes of human DDAH-2 (1151 C/A) gene polymorphism. The serum nitric oxide was lower among AA genotype (30.02 ± 6.96) when compared with CA genotype (33.04 ± 7.46) and CC genotype (34.56 ± 6.35) by one-way ANOVA with a p value of <0.009.

After controlling variables namely age, sex, HDL-c, LDL-c, and waist-hip ratio, the AA genotype of human DDAH-2 (1151 C/A) gene polymorphism frequency still remains significantly higher in patients with CAD when compared with controls with a p value of 0.009. Table 6 shows the multiple logistic regression analysis.

DISCUSSION

Various genetic and environmental factors are involved in combination for the development of coronary artery disease. An endothelial dysfunction is an initial event in the development of atherosclerosis. Many factors have been suggested for the impairment of endothelial function and it is mainly due to reduction in the nitric oxide bioavailability, altered endothelial nitric oxide synthase expression, and increased generation of oxygen-derived free radicals.

The endogenous inhibitor of the nitric oxide synthase namely ADMA has been identified in the 1990s by Vallance et al.⁴ Although ADMA is excreted partly by the kidneys, the major fate of ADMA in humans is degradation by the DDAH enzymes.⁴ The DDAH-2 isoform is expressed mainly in tissues expressing eNOS, i.e., mainly the endothelium.¹² Thus, the DDAH enzyme through the catabolism of ADMA, regulates the function of nitric oxide synthase. Some studies have focused on the association of the DDAH2 gene polymorphisms as an inheritable risk for coronary artery disease. Jones et al. have identified six common polymorphisms within the promoter region of the human gene for DDAH-2.¹⁶ Maas et al. have shown that 1151 A/C and 449 G/C gene polymorphisms in the DDAH-2 promoter region are associated with an increased incidence of hypertension.¹⁴

This study was conducted to determine the association of DDAH-2 (−1151C/A) gene polymorphism with coronary artery disease. The AA genotype of DDAH-2 (1151 C/A) gene polymorphism was significantly higher among cases (29%) when compared with controls (14%). The odds ratio calculation showed that the A⁺ allele carries a 2.19 times higher risk for coronary artery disease when compared with the A⁻ allele. This is in accordance with Gad et al. possible association between cardiovascular disease and the A allele/AA genotype for DDAH-2 SNP (_1151 C/A, rs805304) in males 35–50-year-old Egyptian patients.¹³ In the present study, significant differences in genotype and allele distributions were observed between cases and controls. This raises the speculation of the susceptibility of having coronary insufficiency with the increase in the frequency of DDAH-2 A allele/AA genotype for DDAH-2 (1151 C/A) gene polymorphism.

Subjects with AA genotype having A allele at 1151 position in the promoter region of DDAH-2 gene, decrease the expression of DDAH-2 gene and in turn its activity. The decreased DDAH activity reduces the metabolism of ADMA to citrulline and dimethylamine. Thereby ADMA increases which competitively inhibits nitric oxide synthase and the bioavailable nitric oxide is decreased leading to endothelial dysfunction, inflammation, and atherosclerosis. Schnabel et al. reported that plasma ADMA is an independent predictor of future cardiovascular events in patients with coronary artery disease.¹⁷ The elevated plasma levels of ADMA in conditions of increased cardiovascular risk have been linked experimentally to changes in DDAH expression or activity.¹⁸ Zoccali showed that the ADMA levels are an important independent risk factor for atherosclerotic cardiovascular disease, the prognosis of chronic kidney disease, and the formation of cardiovascular disease in chronic kidney disease patients.¹⁹

A decrease in DDAH expression or activity is emerging as a potential mediator of cardiovascular disease.^20,21 Thus, DDAH emerges as an important regulator of NO bioavailability. Subjects with CC genotype with C allele at 1151 position in the promoter region of DDAH-2 gene will have a normal expression of DDAH-2 gene and in turn, normal DDAH activity, which metabolizes ADMA to citrulline and dimethylamine, thereby inhibition of nitric oxide synthase by ADMA is avoided and the bioavailability of nitric oxide is not affected.

CONCLUSION

In this study, the AA genotype of DDAH-2 (1151 C/A) gene polymorphism was shown to be an independent predictor of coronary artery disease. The A⁺ allele carries a 2.19 times higher risk for coronary artery disease when compared with the A⁻ allele. Low levels of nitric oxide could have been a contributing factor in the higher incidence of coronary artery disease in AA genotype patients.

LIMITATION OF THE STUDY

This study has certain limitations. Serum levels of ADMA were not measured and its correlation with DDAH-2 gene polymorphism and nitric oxide levels was not done.

INFORMED CONSENT

Informed consent was obtained from all participants included in the study.

REFERENCES

1. Chowienczyk PJ, Watts GF, Cockcroft JR, et al. Impaired endothelium-dependent vasodilation of forearm resistance vessels in hypercholesterolaemia. Lancet 1992;340(8833):1430–1432. DOI: 10.1016/0140-6736(92)92621-L.

2. Panza JA, Quyyumi AA, Brush JE,Jr et al. Abnormal endothelium-dependent vascular relaxation in patients with essential hypertension. N Eng J Med 1990;323(1):22–27. DOI: 10.1056/NEJM199007053230105.

3. Williams SB, Cusco JA, Roddy MA, et al. Impaired nitric oxide-mediated vasodilation in patients with non-insulin-dependent diabetes mellitus. J Am Colle Cardiol 1996;27(3):567–574. DOI: 10.1016/0735-1097(95)00522-6.

4. Leone A, Moncada S, Vallance P, et al. Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure. Lancet 1992;339(8793):572–575. DOI: 10.1016/0140-6736(92)90865-Z.

5. Vallance P, Leone A, Calver A, et al. Endogenous dimethylarginine as an inhibitor of nitric oxide synthesis. J Cardiovascu Pharmacol 1992;20:S60–S62. DOI: 10.1097/00005344-199204002-00018.

6. Leiper J, Vallance P. Biological significance of endogenous methylarginines that inhibit nitric oxide synthases. Cardiovascu Res 1999;43(3):542–548. DOI: 10.1016/S0008-6363(99)00162-5.

7. Leiper JM, Maria JS, Chubb A, et al. Identification of two human dimethylarginine dimethylaminohydrolases with distinct tissue distributions and homology with microbial arginine deiminases. Biochem J 1999;343(1):209–214. DOI: 10.1042/bj3430209.

8. Tran CT, Fox MF, Vallance P, et al. Chromosomal localization, gene structure, and expression pattern of DDAH1: comparison with DDAH2 and implications for evolutionary origins. Genomics 2000;68(1):101–105. DOI: 10.1006/geno.2000.6262.

9. Bogumil R, Knipp M, Fundel SM, et al. Characterization of dimethylargininase from bovine brain: evidence for a zinc binding site. Biochemistry 1998;37(14):4791–4798. DOI: 10.1021/bi972312t.

10. Knipp M, Charnock JM, Garner CD, et al. Structural and functional characterization of the Zn (II) site in dimethylargininase-1 (DDAH-1) from bovine brain Zn (II) release activates DDAH-1. J Biolog Chemis 2001;276(44):40449–40456. DOI: 10.1074/jbc.M104056200.

11. Wang D, Gill PS, Chabrashvili T, et al. Isoform-specific regulation by NG, N G-dimethylarginine dimethylaminohydrolase of rat serum asymmetric dimethylarginine and vascular endothelium-derived relaxing factor/NO. Circulat Res 2007;101(6):627–635. DOI: 10.1161/CIRCRESAHA.107.158915.

12. Jones LC, Hingorani AD. Genetic regulation of endothelial function. Heart 2005;91(10):1275–1277. DOI: 10.1136/hrt.2005.061325.

13. Gad MZ, Hassanein SI, Abdel-Maksoud SM, et al. Association of DDAH2 gene polymorphism with cardiovascular disease in Egyptian patients. J Genet 2011;90(1):161. DOI: 10.1007/s12041-011-0043-4.

14. Maas R, Erdmann J, Lüneburg N, et al. Polymorphisms in the promoter region of the dimethylarginine dimethylaminohydrolase 2 gene are associated with prevalence of hypertension. Pharmacologi Res 2009;60(6):488–493. DOI: 10.1016/j.phrs.2009.07.013.

15. Cortas NK, Wakid NW. Determination of inorganic nitrate in serum and urine by a kinetic cadmium-reduction method. Clin Chem 1990;36(8):1440–1443. DOI: 10.1093/clinchem/36.8.1440.

16. Jones LC, Tran CT, Leiper JM, et al. Common genetic variation in a basal promoter element alters DDAH2 expression in endothelial cells. Biochem Biophysi Res Communicati 2003;310(3):836–843. DOI: 10.1016/j.bbrc.2003.09.097.

17. Schnabel R, Blankenberg S, Lubos E, et al. Asymmetric dimethylarginine and the risk of cardiovascular events and death in patients with coronary artery disease: results from the athero gene study. Circulat Res 2005;97(5):e53–e59. DOI: 10.1161/01.RES.0000181286.44222.61.

18. Bouras G, Deftereos S, Tousoulis D, et al. Dimethylarginine (ADMA): a promising biomarker for cardiovascular disease? Curr Top Med Chemis 2013;13(2):180–200. DOI: 10.2174/1568026611313020007.

19. Zoccali C. Traditional and emerging cardiovascular and renal risk factors: an epidemiologic perspective. Kidney Int 2006;70(1):26–33. DOI: 10.1038/sj.ki.5000417.

20. Xuan C, Xu LQ, Tian QW, et al. Dimethylarginine dimethylaminohydrolase 2 (DDAH 2) gene polymorphism, asymmetric dimethylarginine (ADMA) concentrations, and risk of coronary artery disease: a case-control study. Scienti Rep 2016;6(1):1–6. DOI: 10.1038/srep33934.

21. Mannino GC, Pezzilli S, Averta C, et al. A functional variant of the dimethylarginine dimethylaminohydrolase-2 gene is associated with myocardial infarction in type 2 diabetic patients. Cardiovascu Diabetol 2019;18(1):102. DOI: 10.1186/s12933-019-0906-1.