Endothelial nitric oxide synthase gene

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Nov 15, 2018 - candidate genes for CAD (Hartiala et al., 2017). eNOS gene (OMIM. 163729) is located on chromosome 7q35–36 region and possesses 26.
Meta Gene 19 (2019) 111–116

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Endothelial nitric oxide synthase gene -922A > G, -786 T > C, 4b/4a, and 894 G > T variants and premature coronary artery disease: An association study with haplotype analysis

T

Amirsaeed Sabeti Aghabozorgia,b, Hossein Farshidic, Zahra Farbooda, Najmeh Ahangarid, ⁎ Tasnim Eghbal Eftekhaaric,f, Amirhossein Bahreynie, Azim Nejatizadehf, a

Department of Human Genetics, Faculty of Medicine, Hormozgan University of Medical Sciences, Bandar Abbas, Iran Student Research Committee, Hormozgan University of Medical Sciences, Bandar Abbas, Iran Cardiovascular Disease Research Center, Hormozgan University of Medical Sciences, Bandar Abbas, Iran d Department of Modern Sciences & Technology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran e Pharmaceutical Research Center, pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran f Molecular Medicine Research Center, Hormozgan University of Medical Sciences, Bandar Abbas, Iran b c

A R T I C LE I N FO

A B S T R A C T

Keywords: Cardiovascular disease Endothelium nitric oxide Haplotype Polymorphism Premature coronary artery disease

Background: Endothelium nitric oxide (eNO) deficiency may lead to premature coronary artery disease (pCAD). This defect could be due to the effect of some eNOS gene variants on its gene expression. The aim of the study: The aim of this study is to investigate the association between four eNOS gene variants, independently and as four-locus haplotypes, with pCAD in the southern population of Iran. Methods: One hundred fifty pCAD patients and 150 age and sex-matched controls were enrolled in this study. Polymerase chain reaction and PCR restriction fragment length polymorphism methods were used for 4a/4b variable number tandem repeat and “–922A/G, –786T/C, 894G/T” single nucleotide polymorphisms, respectively. Results: The results indicated that genotype frequencies of four studied variants between case and control groups were different significantly (p < .05) except for GG (−922A/G), CC (−786 T/C), GT and TT (894G/T) genotypes (p > .05). Likewise, all of the studied four mutant alleles (−922G, e786C, 894 T, and 4a) were associated with increased risk of pCAD (p < .05, OR = 1.966, OR = 3.107, OR = 2.21, OR = 1.650, respectively). H4 (bGCT) and H7 (aGCG) haplotypes were significantly associated with pCAD which introduced susceptible haplotypes (p < .01, p < .0001, respectively). However, H1 (bATG) as a protective haplotype, was in a significant negative association with pCAD (P < .0001). Conclusions: In conclusion, this study suggested three novel four-locus haplotypes of eNOS gene variants that could be in association with pCAD in the southern population of Iran.

1. Introduction Coronary artery disease (CAD) is the most prevalent form of cardiovascular diseases (CVDs), considering as the main cause of morbidity and mortality in both developed and developing countries (Lilly, n.d.). It is estimated that since 2015, approximately one-third of all deaths around the world, are caused by CVD (Mahmood et al., 2014). In various studies, early onset of CAD is characterized as demonstrating manifestations of the disease between 30 and 56 years old and is called premature coronary artery disease (pCAD) (Mohammad et al., 2015). pCAD occurs in around 10% of all CAD patients (Roberts et al.,

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2007). In 2013, almost 5.9 million people died because of pCAD and it is predicted that the world will face around 7.8 million pCAD deaths in 2025 (Eslami et al., 2017). It has been demonstrated that endothelium nitric oxide (eNO) is the most prominent vasodilator component which contributes to the regulation of cardiovascular homeostasis and function of endothelial cells (Metzger et al., 2011). Endothelial nitric oxide synthase (eNOS), which produces eNO from L arginine, regulates vascular functions by controlling eNO levels in vascular endothelium (Pan, 2009). Because of eNOS enzyme role in synthesis and determination of eNO bioavailability, eNOS (or NOS3) gene is one of the best-known

Corresponding author at: Ostandary Street, Bandar Abbas City, Hormozgan Province, Iran. E-mail address: [email protected] (A. Nejatizadeh).

https://doi.org/10.1016/j.mgene.2018.11.008 Received 29 August 2018; Received in revised form 22 October 2018; Accepted 14 November 2018 Available online 15 November 2018 2214-5400/ © 2018 Published by Elsevier B.V.

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participants were unrelated individuals. According to world health organization (WHO) criteria, we included patients who underwent angiography procedure showing at least a stenosis > 50% in one of the arteries on a coronary angiogram. Patients demonstrating < 50% documented coronary obstruction, were excluded from the study. Control group were individuals from the same population who underwent angiography due to unrelated reasons for CAD such as indeterminate noninvasive test results, electrophysiological studies and/ or preoperative risk assessment, etc. Control subjects were proved by coronary angiography along with 0% of atheromatous plaques. In addition, all of the subjects had normal electrocardiogram (ECG) results. Exclusion criteria for the control group were any documented clinical history of atherosclerosis and/or coronary artery bypass grafting (CABG), cardiac medications, myocardial infarction (MI), heart failure, peripheral vascular disease, stroke, and dialysis. Moreover, all of the 300 participants were free of diseases including liver failure, renal failure, chronic obstructive pulmonary disease (COPD) and different types of tumors and cancers. Demographic and clinical variables such as hypertension, body mass index (BMI), family history and smoking status, were filled after a thorough interview and clinical examination. Hypertension is defined as systolic pressure of > 140 mmHg and/or diastolic pressure > 90 mmHg in more than one occasion and/or use of hypertension medications. BMI was calculated as weight divided by the square of height (kg/m2) (Smith et al., 2001). Positive family history is defined as the first-degree relative with CAD or sudden death up to the age of 55 for men and 65 for women. Smoking status is divided into two groups: smokers who have a daily consumer of > 5 cigarettes and nonsmokers as a person who never smoked or had stopped smoking for one year or more.

candidate genes for CAD (Hartiala et al., 2017). eNOS gene (OMIM 163729) is located on chromosome 7q35–36 region and possesses 26 exons and 25 introns which constitutes 21 k bases of DNA (Marsden et al., 1993). There are several significant polymorphisms (genetic variants) in promoter, introns and exons regions of eNOS gene which is evaluated to increase the risk of CAD and pCAD. Several genetic NOS3 variants such as -922A/G (rs1800779), −786 T/C (rs2070744), 4a/4b (rs61722009) and 894G/T (rs1799983, Glu298Asp), may affect both expression and activity as well as eNO production, increasing the risk of CAD (Khera and Kathiresan, 2017). -922 A/G and − 786 T/C polymorphisms in promoter area, 4a/4b VNTR polymorphism in intron 4 and 894 G/T polymorphism in exon 7 of eNOS gene exhibit a high relevance to CAD and pCAD (Ekmekci et al., 2013; Zhu et al., 2017; Jia et al., 2007; Nakayama et al., 1999). However, there are conflicting results regarding each variant. The reason could be due to the fact that CAD is a multifactorial disease. Therefore, many genetic variants collaborating environmental factors may have an additive or synergic effects, indicating the importance of investigating similar studies in different geographically and ethnically regions (Nejatizadeh et al., 2008). pCAD is established to be a genetic disease with 0.63 heritability, suggesting that genetic factors are more efficient than environmental factors (Roberts et al., 2007). Because of afflicted individuals are young, pCAD could have catastrophic outcomes for both families and societies (Klein and Nathan, 2003). Likewise, it is commonly accepted that haplotype analysis is more informative and practical than a single polymorphism analysis (Sandrim and Tanus-Santos, 2007). Therefore, the aim of this study is to evaluate four genetic markers separately and as haplotypes, in predisposition to pCAD.

2. Materials and methods 2.1. Subjects and study protocol

2.2. Sample collection and genotyping

The present case-control study, which performed in Bandar Abbas, Iran, was approved by Hormozgan University of medical science ethics committee. Due to heterogeneity in Bandar Abbas population, no notable isolated ethnic groups are exhibited. Therefore, according to Prado-Lima et al. (do Prado-Lima et al., 2004), we assumed all of the subjects belonged to a single population, with no social stratification. Written informed consent was signed by every participant in this study. Three hundred Iranian individuals (men ≤45 and women ≤55 years old) consist of 150 patients as case group and 150 healthy subjects as control group were consecutively recruited between January and July 2017 from the department of cardiology in Shahid Mohammadi hospital. All subjects underwent a coronary angiography procedure by recommendation of a relevant cardiologist, before recruitment in this study. For all of the enrolled subjects, a cardiologist, who was blind to genotypes, diagnosed CAD based on angiogram results. Moreover, in order to avoid genetic frequency bias, all

In order to DNA preparation, whole blood samples were obtained from all subjects and stored at −20 °C at ethylenediaminetetraacetic acid (EDTA) anticoagulated tubes. Genomic DNA was extracted using a DNA extraction kit (Genet bio, Korea) according to its manufacture protocol. Amplification of eNOS gene regions containing T-786C, G894T, A-922G, 4a/4b variants, carried out by polymerase chain reaction (PCR) standard method. PCR procedure was sufficient in order to determine aa, ab and bb genotypes (4a/4b variant) on an agarose gel. Whereas, in order to genotype the other three polymorphisms, PCR products must be digested through their specific restriction enzymes and then restriction fragments were electrophoresed (Jeerooburkhan et al., 2001). The primers and restricted enzymes used in this study are implicated in Table 1.

Table 1 Primer pair sequences used for PCR amplification and restriction enzymes of the investigated polymorphic sites of the NOS3 gene. Variant

Primer sequence

Ta1 (°C)

Product size (bp2)

Enzyme

Digested products

-922A/G

Forward primer: 5′-CAGCTAGTGGCCTTTCTCC-3′ Reverse primer: 5′-AGAGCTTGATGCCCTGGTG-3′ Forward primer: 5′-AGTTTCCCTAGTCCCCCATGC-3′ Reverse primer: 5′-CCACACCCCCATGCTCAAGT-3′ Forward primer: 5′-AGG CCCTATGGTAGTGCCTT-3′ Reverse primer: 5′-TCTCTTAGTGCTGTGGTCAC-3′ Forward primer: 5′-TCCCTGAGGAGGGCATGAGGCT-3′ Reverse primer: 5′- TGAGGGTCACACAGGTTCCT-3′

60

138

65

194

(Bsl I) Fermentas Biotechnology company (NgoMIV) New England Biolabs

56

420, 393

61.5

457

A: 138 G: 110 + 28 T: 194 C: 143 + 51 b: 420 a: 393 A: 457 G: 320 + 137

-786T/C 4a/4b 894G/T

1 2

Annealing temperature. Base pair. 112

(Ban II) Fermentas Biotechnology company

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(p < .05). Former studies revealed that -922A/G variant is correlated with MI in Korean population (Jo et al., 2006). In addition, Arnett et al. suggested that A-922G polymorphism may influence the transcription rate and enzyme efficiency through regulation of gene expression. They showed that A-922G variant leads to a higher hazard ratio (HR) in mutant allele carriers for heart failure (AA = 1.00, AG + GG =1.10%95 CI 1.00–1.21, p = .046) (Zhang et al., 2012). Moreover, Ferguson et al. investigated the relationship between eNOS variants with circulating lipids and demonstrated that -922A/G minor allele carriers (−922G) were associated with increased total cholesterol and/ or LDL cholesterol (Ferguson et al., 2010). However, further functional and association studies are required in order to better comprehension of molecular mechanisms involved in A-922G variant. Among four studied variants, −786 T/C SNP had the most significant association at both allelic (C allele, p < .05, OR = 3.107) and genotypic (TC, p < .05, OR = 5.626) levels with pCAD, in our study. Nakayama et al. for the first time discovered three variants involving T786C, A-922G, and T-1468A in the 5′ flanking region of the eNOS gene, which was linked with each other. Consistent with Nakayama et al., our study also revealed that T-786C and A-922G polymorphisms were in close genetic linkage (D′ = 0.30874, p < .00001). Moreover, several pieces of evidence revealed that this variant might be in association with MI and/or CAD (Jo et al., 2006; Colombo et al., 2003). Miyamoto et al., demonstrated that transcription of the eNOS gene containing e786C mutant allele is diminished through binding of replication protein A1 to the e786C nucleotide, but not -786 T wild allele, in the promoter region, which results in significant decrease in serum nitrate levels among -786 T/C carriers (Miyamoto et al., 2000). Therefore, to some extent, the precise molecular pathology of -786 T/C polymorphism has been explained. Parallel with our results, Alvarez et al. demonstrated that -786 T/C variant confers an increased risk of pCAD in Spanish population (Álvarez et al., 2001). Likewise, Zigra et al. demonstrated that both T-786C and G894 T variants are associated with increased risk of MI among patients under 35 years old (Zigra et al., 2013). In contrast, Nassar et al. indicated no significant correlation between T-786CA polymorphism and pCAD patients in Canadian (Nassar et al., 2006). Also, Gluba et al. could not find any association between 894G/T and -786 T/C variants with increased risk of MI patients under 45 years old (Gluba et al., 2009). Our results also demonstrated that 4a/4b variant is correlated with pCAD significantly (p < .05). Furthermore, 4a/4b and -922A/G variants were in strong genetic linkage (D′ = 0.30936, p < .00001). Wang et al., for the first time suggested that the aa genotype is a smokingdependent marker in the pathogenesis of CAD (Wang et al., 1996). Nevertheless, Yokota et al. demonstrated that this polymorphism could behave as an independent risk factor for MI patients in the Japanese population (Ichihara et al., 1998). Recently, Zhang et al. concluded that 27 nucleotides (nt) repeat sequences in eNOS intron 4, produce 27-nt short intronic repeat RNAs (siRNAs) through the eNOS pre mRNA splicing (Zhang et al., 2008a). They also demonstrated that this 27 nt siRNAs could suppress eNOS gene expression through altering histone acetylation and DNA methylation in the promoter region (Zhang et al., 2008b). Since 4a allele possesses one 27 nt repeat sequence less than 4b allele, produces lower levels of 27 nt siRNAs and therefore higher levels of eNOS mRNA exist in 4a allele carrier endothelial cells (Zhang et al., 2008a). Consistent with our findings, Ekmekçi et al. demonstrated that 4a allele of this polymorphism has a significant correlation with MI patients under the 35 years old (EkmekçI et al., 2013). In contrast, Tang et al. presented that neither 4a/4b variant nor 894G/T variant was an associated with pCAD significantly (Wu et al., 2003). Moreover, Gardemann et al. investigated the role of Glu298Asp and 4a/4b polymorphisms in CAD and pCAD German patients. Although 894G/T variant was not associated with increased risk of CAD, limitation of CAD patients to under 61 years old (pCAD group), resulted in a significant association between pCAD with 894G/T variant. However, they did not found any association between 4a/4b polymorphism with CAD

Table 2 Clinical Characteristics of study groups. Parameters

Case ( ± SD1)

Controls ( ± SD)

P-Value2

Age (years) Sex

43.3 ± 7.7 69 (46%) 81 (54%) 26.75 ± 4.8 52 (34.7%) 83 (55.3%) 132.54 ± 23 82.57 ± 14.6 65 (43.3%)

42 ± 7.4 67 (44.7%) 83 (55.3%) 26 ± 4.7 29 (19.3%) 64 (42.7%) 127.24 ± 18 81 ± 13.1 38 (25.3%)

0.394 0.817

Male Female Body mass index Family history Hypertension Systolic blood pressure Diastolic blood pressure Smoking 1 2

0.159 0.003 0.028 0.028 0.332 0.001

Standard deviation. p < .05 is significant.

2.3. Statistical and haplotype analysis All statistical analyses were conducted using SPSS software version 16.0 (SPSS Inc., Chicago, IL, USA). Continuous variables were analyzed as mean ± SD. Student t-test and Chi-square test were used to compare demographic and clinical data. Deviations of genotype frequencies from Hardy-Weinberg equilibrium (HWE) and differences in genotype distributions and allele frequencies, were analyzed using Chi-square test; odds ratios (OR) with 95% confidence intervals (CI) were calculated. P values less than 0.05 were considered statistically significant. Haplotype analysis and pairwise linkage disequilibrium (LD) between various genotypes of studied polymorphisms were analyzed using SNP analyzer version 2 software (V.2). 3. Results 3.1. Demographic characteristics of the study groups The demographic and clinical characteristics of 150 case subjects and 150 age and gender-matched (p > .05) healthy subjects are categorized in Table 2. Moreover, there was no significant difference between the two groups concerning BMI (p > .05). However, the prevalence of the other risk factors (i.e. hypertension, family history and smoking status) were higher in the case group and the differences were significant (p < .05). 3.2. Single marker association analysis Effects of four candidate variants within eNOS locus were determined by comparing alleles and genotypes frequencies of these variants between two groups (Table 3). Single-locus analysis demonstrated that the selected variants associated with pCAD significantly (p < .05). Likewise, p-value and OR with 95% CI measurements for every allele and genotype of the variants, were listed in Table 3. 3.3. Haplotype association analysis The -922A/G and -786 T/C (D′ = 0.30874, p < .00001) as well as 4a/4b and -922A/G (D′ = 0.30936, p < .00001) were in significant LD in control and case groups. Furthermore, genotype frequencies of the –922A/G and 4a/4b were in HWE (p = .16 for –922A/G and p = .21 for 4a/4b), whereas the 894G/T and –786 T/C polymorphisms were not in HWE (p = .0007 for –786 T/C and p = .000 for 894G/T). In addition, among 16 four-locus haplotypes, three of them (H1, H4, H7, p < .05) were associated with pCAD (Table 4). 4. Discussion To the best of our knowledge, the present study for the first time indicated that -922A/G variant is an independent risk factor for pCAD 113

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Table 3 Genotype and allele frequency of investigated polymorphisms. polymorphism

Parameters

-922A/G

Alleles Genotypes

-786T/C

Alleles Genotypes

4a/4b VNTR

Alleles Genotypes

894G/T SNP

Alleles Genotypes

1 2 3

Study participants

A allele G allele AA genotype AG genotype GG genotype Carrier (AG + GG) T allele C allele TT genotype TC genotype CC genotype Carrier (TC + CC) T allele C allele bb genotype ab genotype aa genotype Carrier (ab + aa) G allele T allele GG genotype GT genotype TT genotype Carrier (GT + TT)

Statistics

Cases (n = 150)

Controls (n = 150)

P value1

OR2 (95% CI3)

189(63%) 111(37%) 52 (34.7%) 85 (56.7%) 13 (8.6%) 98 (65%) 180 (60%) 120 (40%) 38 (25.3%) 104 (69.3%) 8 (5.4%) 112 (74.6%) 205 (68.3%) 95 (31.7%) 72 (48%) 61 (40.7%) 17 (11.3%) 78 (52%) 146 (48.7%) 154 (51.3%) 20 (13.3%) 106 (70.7%) 24 (16%) 130 (86.7%)

231(77%) 69 (23%) 90 (60%) 51 (34%) 9 (6%) 60 (40%) 247 (82%) 53 (18%) 102 (68%) 43 (28.7%) 5 (3.3%) 48 (32%) 248 (82.7%) 52 (17.3%) 103 (68.7%) 42(28%) 5 (3.3%) 47 (31.3%) 183 (61%) 117 (39%) 46 (30.7%) 91 (60.6%) 13 (8.7%) 104 (69.3%)

– 0 0 0 0.376 0 – 0 0 0 0.395 0 – 0 0 0.021 0.008 0 – 0.002 0 0.053 0.068 0

– 1.966 (1.376–2.809) 0.354 (0.221–0.565) 2.538 (1.591–4.051) 1.487 (0.616–3.591) 2.827 (1.769–4.518) – 3.107 (2.133–4.525) 0.16 (0.97–0.264) 5.626 (3.427–9.235) 1.634 (0.522–5.114) 6.263 (3.787–10.358) – 2.21 (1.503–3.249) 0.421 (0.263–0.674) 1.762 (1.087–2.856) 3.707 (1.331–10.326) 2.374 (1.483–3.802) – 1.65 (1.193–2.281) 0.384 (0.194–0.624) 2.007 (0.980–4.112) 1.562 (0.966–2.525) 2.875 (1.602–5.16)

P < .05 is significant. Odds ratio. Confidence interval.

activation of eNOS protein in caveolae, disrupts the native endothelial cell function of eNOS enzyme (Joshi et al., 2007). There are several studies, which replicate our findings. Abdel-Aziz et al. indicated that the TT genotype of this variant is an independent risk factor for pCAD patients (Abdel-Aziz and Mohamed, 2013). In addition, in Mexico, Salas et al. introduced Gu298Asp polymorphism as an independent risk factor for premature ST-elevation myocardial infarction (STEMI) in < 45 patients (Isordia-Salas et al., 2010). However, Nassar et al. conducted a study in Caucasian Canadian which demonstrated no association between Asp298 allele as well as Asp298 homozygotes with pCAD patients (Nassar et al., 2001). Our results of haplotype analysis revealed that among sixteen fourlocus haplotypes, H1 haplotype consists of bATG allele (stands for 4a/

and/or pCAD (Gardemann et al., n.d.). The present study also suggested that the 894G/T variant is correlated with pCAD significantly. Several association studies confirmed that this variant might have a decisive role in the pathogenesis of CAD, coronary spasm, MI and carotid atherosclerosis (Hingorani et al., 1999; Yoshimura et al., 1998; Lembo et al., 2001). 894G/T polymorphism within exon 7 of eNOS gene results in the substitution of glutamate (GAG codon) by aspartate (GAT codon) in the oxygenase domain of the eNOS protein (Zhang et al., 2012). Senthil et al. demonstrated that TT carriers of this polymorphism have high levels of eNOS mRNA but low protein levels (Senthil et al., 2005). Recently, Joshi et al. revealed that impairment interaction of 298asp eNOS protein (TT, GT genotypes) with caveolin-1, which has an important role for localization and Table 4 Haplotype analysis. Haplotype

Associated haplotypes

Cases (%)

Controls (%)

Total frequency (%)

OR1

95% CI2

P-value3

bATG bATT bACG bGCT aATG bGTT aGCG aGTT aGTG bGTG aATT aGCT bGCG aACT bACT aACG

H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14 H15 H16

11.2 28.4 7.7 6.7 7.9 3.5 8.9 2.9 1.4 1.6 3.4 2.7 9.5 4 0 0

40 18 6.3 1.3 3.4 7.6 1.9 3.4 3.7 2.5 3.7 0 2.5 0 4.3 0.8

27.8 21.5 8 6.8 5.7 5.3 4.8 3.02 2.6 2.6 2.5 2.4 2.4 2.4 1.06 0.9

0.391 1.097 0.821 3.696 1.336 0.485 4.69 0.704 0.14 0.495 3.02 10.31 1 4.125 0 0

0.271–0.564 0.752–1.601 0.444–1.521 1.847–7.395 0.683–2.614 0.214–1.097 2.225–9.883 0.308–1.612 0.017–1.145 0.123–1.998 0.312–29.202 1.311–81.057 0.200–4.995 1.152–14.770 0.000–0.000 0.000–0.000

< 0.0001 NS4 NS < 0.01 NS NS < 0.0001 NS NS NS NS NS NS NS NS NS

1 2 3 4

Odds ratio. Confidence interval. p < .05 is significant; P-value adjusted with Bonferroni Correction. Non-significant. 114

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4b, −922A/G, −786 T/C and 894G/T variants, respectively) had the most frequency in control group, implying a protective role of H1 allele against pCAD (total frequency: %27.8, OR = 0.391, 95% CI 0.271–0.564, p < .0001). Furthermore, H4 (bGCT) and H7 (aGCG) haplotypes were susceptible alleles which enhanced the risk of pCAD (H4, total frequency: percentage6.8, OR = 3.696, 95% CI 1.847–7.395, p < .01; H7, total frequency: percentage4.8, OR = 4.69, 95% CI 2.225–9.883, p < .0001). Similar to our findings, Wang et al. confirmed that 4a/4b variant in cooperation with -786 T/C variant; regulates the transcriptional activity of eNOS gene in a haplotype-specific manner (Wang et al., 2002). Fatini et al. proved that combined genotypes including “-786(CC + TC)/894(TT + GT)” and “aa+ab/ 894(TT + GT)” are significantly associated with carotid stenosis patients (p = .04, OR = 1.5, 95% CI 1.01–2.1; p = .01, OR = 1.8, 95% CI 1.1–2.9, respectively) (Fatini et al., n.d.). Kumar et al. investigated the correlation between 894G/T, −786 T/C and 4a/4b variants/haplotypes with coronary heart disease (CHD) in young Indians (mean age: 42.60 ± 15.3). There was a significant association between the bb genotype, 894 T allele carriers and e786C allele carriers and CHD. They suggested that 894G/−786 T/4b haplotype was the least prevalent among CAD patients compared to the control group, whereas 894G/786C/4b and 894 T/-786C/4b haplotypes were associated with increased risk in CHD patients (Kumar et al., 2016). In contrast, Piccoli et al. demonstrated that haplotypes consist of -786 T/C, 894G/T and intron 4 a/b polymorphisms did not show a significant association with the acute coronary syndrome in Brazilians (da Costa Escobar Piccoli et al., 2012). These possible opposing findings could be due to different ancestral alleles in various ethnic populations and/or modifying effects of environment on the decisive role of NOS3 polymorphisms in pCAD (Hassan et al., 2004). One limitation of our study is small sample size owing to the young ages of subjects and several including and excluding criteria for sample collection. Another limitation is absent of lipid profile, fast blood sugar (FBS) and hemoglobin A1C (HbA1c) markers for determination of dyslipidemia and diabetes status. However, by lowering the age limit, we minimized the effects of these confounding factors. Lack of functional studies such as assessment of mRNA and protein levels and bioavailability of eNOS enzyme, as well as measurement of NO plasma concentrations, where another limitation in our study. However, previous studies had conducted several functional investigations on eNOS gene variants, which emphasized crucial roles of mentioned polymorphisms in CAD development.

Funding This research was supported by Hormozgan University of Medical Sciences [grant number: 950177]. Declaration of interest The Authors declare that there is no conflict of interest. References Abdel-Aziz, T., Mohamed, R., 2013. Association of endothelial nitric oxide synthase gene polymorphisms with classical risk factors in development of premature coronary artery disease. Mol. Biol. Rep. 40 (4), 3065–3071. Álvarez, R., González, P., Batalla, A., Reguero, J.R., Iglesias-Cubero, G., Hevia, S., et al., 2001. Association between the NOS3 (−786 T/C) and the ACE (I/D) DNA Genotypes and Early Coronary Artery Disease. Nitric Oxide 5 (4), 343–348. Colombo, M.G., Paradossi, U., Andreassi, M.G., Botto, N., Manfredi, S., Masetti, S., et al., 2003. Endothelial nitric oxide synthase gene polymorphisms and risk of coronary artery disease. Clin. Chem. 49 (3), 389–395. da Costa Escobar Piccoli, J., Manfredini, V., Hamester, F.I., Bandinelli, J.B., Turkienicz, I.M., Chies, J.A., et al., 2012. 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5. Conclusion Several studies have focused on the association between eNOS gene polymorphisms and CAD. However, only a few studies enrolled pCAD subjects. The more effects of genetic factors on pCAD individuals, as well as the more limited environmental confounding factors in these subjects, are two main causes of inclusion of young individuals in these genetic studies (Ekmekci et al., 2013; Isordia-Salas et al., 2010). It is well defined that haplotype analysis of several effective polymorphisms may give much greater power than single polymorphism analysis for association studies. In summary, we demonstrated a significant association between four individually single locus polymorphisms in NOS3 gene with pCAD. Besides, we introduced three novel 4 locus haplotypes (H1, H4, H7) which are affirmative than their single-locus variants, in the representation of the association between genetic susceptibility factors with increased or decreased risk of pCAD in a south population of Iran. Acknowledgment We are sincerely thankful to our counselors in Clinical Research Development Center of Shahid Mohammadi Hospital. 115

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