Importance of NOS3 Genetic Polymorphisms in the Risk of Development of Ischemic Stroke in the Turkish Population

Abstract

In the present study, we aimed to investigate the relationship between endothelial nitric oxide synthase 3 (NOS3) G894T, T-786C, and intron 4 variable number of tandem repeat (VNTR) variants, alone or in combination, and the risk of incidence of ischemic stroke in the Turkish population. The genotypes for all polymorphisms were determined by polymerase chain reaction/restriction fragment length polymorphism techniques on 245 ischemic stroke patients and 145 controls. In the case-control analysis, no significant difference was observed between stroke patients and controls with respect to NOS3 G894T, T-786C, and intron 4 VNTR polymorphisms genotype and allele frequency distribution. However, the copresence of G894T and intron 4 VNTR risk-elevating genotypes in the same individual increased the risk of stroke seven times (odds ratio=7.083, 95% confidence interval=0.866-57.963, p=0.029).

Introduction

Stroke is a sudden interruption in the blood supply of the brain. Most strokes are caused by an abrupt blockage of arteries leading to the brain known as ischemic stroke. Carotid stenosis, resulting from atherosclerosis, is a risk factor for stroke (Nagai et al., 2001). Oxidative stress, the condition occurring when the physiological balance between oxidants and antioxidants is disrupted, plays an important role in the pathogenesis of atherosclerosis, cancer, and diabetes. Free radical production is increased in ischemic stroke, leading to oxidative stress that contributes to brain damage (Alexandrova et al., 2004; Crack and Taylor, 2005).

In the atherosclerotic process, the vascular endothelium has an important effect by releasing of a mediator such as nitric oxide (NO), which plays a key role in the relaxation of vascular smooth muscle cells, inhibits adhesion of platelets and monocytes to the endothelium, reduces the migration and proliferation of vascular smooth muscle cells, and slows down atherogenesis (Draijer et al., 1995; Myers and Tanner, 1998). On the other hand, excess NO may be harmful because of its oxidative role (Schmidt and Walter, 1994).

NO is an uncharged diatomic gas and a potent regulator of vasomotor tone and peripheral resistance. Studies have shown that NO has various effects such as vasodilatation, inhibition of platelet adherence and aggregation, suppression of smooth muscle proliferation, and reduction of adherence of leukocytes to the endothelium (Moncada and Higgs, 1993; Radomski and Salas, 1995; Cooke and Dzau, 1997). Nitric oxide synthase (NOS) produces endogenous NO from the amino acid L-arginine. In mammals, there are 3 distinct genes that encode NOS isoenzymes: neuronal (nNOS or NOS1), cytokine-inducible (iNOS or NOS2), and endothelial (eNOS or NOS3). Wilcox et al. (1997) reported that reduction in the activity of the vascular NOS3 enzyme impairs endothelium-dependent vasodilatation in atherosclerotic vessels. According to a study conducted by Willmot et al. (2005), extended ischemic area and reduction of blood flow in the penumbra were observed in NOS3-deficient transgenic mice. Because of these characteristics, varieties in the NOS3 gene have been considered to contribute to the development of ischemic stroke.

Several SNPs have been identified in the promoter, exons, and introns of the NOS3 gene. G894T in exon 7 of the NOS3 gene leads to a change of Glu to Asp at site 298. It has been demonstrated that this polymorphism alters the NOS3 activity and may be associated with a reduction in the basal NO production (Tesauro et al., 2000). The mutant T allele was reported to be associated with hypertension, coronary artery disease (CAD) (Hingorani et al., 1999), stroke (Hassan et al., 2004), and a number of stenotic vessels. Another NOS3 gene polymorphism, T-786C located in the promoter region of the NOS3 gene, results from replacement of a thymidine by a cytosine at nucleotide -786. It was reported that the T-786C polymorphism may change NOS3 gene expression and result in a significant reduction in the NOS3 gene promoter activity (Nakayama et al., 1999). The T-786C polymorphism was also demonstrated to be associated with an increased predisposition to CAD in Chileans and Koreans (Kim et al., 2007; Jaramillo et al., 2008), to ischemic stroke in young black women (Howard et al., 2005), to coronary spasm in Japanese, and to myocardial infarction (MI) in Koreans (Nakayama et al., 1999; Jo et al., 2006), but not to increase the predisposition to MI and CAD in other Caucasians (Poirier et al., 1999; Granath et al., 2001). The other NOS3 gene polymorphism is the variable number of the tandem repeat (VNTR) polymorphism located in intron 4 of NOS3 (eNOS4b/a polymorphism), which is significantly associated with plasma NO concentration. In repeats of a 27-bp consensus sequence, there are two alleles, a common large allele and a smaller allele. The larger allele (eNOS4b allele), designated b-insertion, has five tandem repeats, and the smaller allele (eNOS4a allele), a-deletion, has four repeats. It has been reported that eNOS4 VNTR polymorphism may be responsible for plasma NO levels (Tsukada et al., 1998).

This study aimed to elucidate the risk of three important NOS3 polymorphisms, alone or in combination, on the development of ischemic stroke in a Turkish population.

Materials and Methods

Study population

A total of 245 blood samples were obtained from consecutive unrelated adult Caucasian patients with acute hemispheric ischemic stroke, and 145 blood samples were obtained from symptom-free Caucasian controls from central Anatolia, Turkey, with the collaboration of the Gülhane Military Medical Academy, Department of Neurology, Ankara. Informed consent was obtained from all participants before study entry. The study was approved by the Ethics Committee of the Medical Academy and was carried out according to the principles of the Declaration of Helsinki. The details of inclusion and exclusion criteria were as described before (Can Demirdöğen et al., 2008; Türkanoğlu et al., 2010).

Control subjects were selected randomly from neurology outpatient clinics and did not have a stroke or transient ischemic attack at any time. All exclusion criteria were applied to the controls exactly, plus not having carotid stenosis (lumen narrowing) >70% or ulcerated carotid plaque.

DNA isolation and genotyping

DNA was extracted from peripheral blood leukocytes by salt extraction (Lahiri and Schanabel, 1993) and resuspended in the Tris-EDTA (pH 8.0) buffer.

Two genetic polymorphisms of the NOS3 gene, G894T at exon 7 and T-786C in the 5′-flanking region, were determined by using the polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) technique. Primer pairs used for PCR amplification of the NOS3 G894T region were 5′-AAGGCAGGAGACAGTGGATGGA-3′ and 5′-CCCAGTCAATCCCTTTGGTGCTCA-3′ (Guldiken et al., 2009), and those for the NOS3 T-786C region were 5′-ATGCTCCCAGGGCATCA-3′ and 5′-GTCCTTGAGTCTGACATTAGGG-3′ (Colomba et al., 2008). PCRs of two polymorphic regions were carried out in 50 μL of reaction mixture in the presence of dNTPs, PCR buffer, MgCl₂, and Taq polymerase. Genotyping of two polymorphic sites was carried out by digesting PCR product with BanII for G894T polymorphism and with PdiI for T-786C polymorphism. Results of digestions were analyzed on 2.5% agarose gel.

To determine the intron 4 VNTR polymorphism, the PCR technique was used (Kunnas et al., 2002). DNA was amplified in a reaction mixture containing the following primers: 5′-TTATCAGGCCCTATGGTAGT-3′ and 5′-AACTCCGCTCAGCTGTCCT-3′ in the presence of dNTPs, PCR buffer, MgCl₂, and Taq polymerase. The amplified PCR products of 167 and 194 bp were separated on 3% agarose gels stained with ethidium bromide.

Statistical analyses

Continuous variables are expressed as mean±SD. Normality of the sample distribution of each continuous variable was tested with the Kolmogorov-Smirnov test. Differences of continuous variables were evaluated by the independent samples t or Mann-Whitney U test. Categorical variables were expressed as proportions and compared using the χ² test. When the expected values in any of the cells of a contingency table were below 5, the Fisher exact test was used to calculate p-value. Allele distributions were compared using the χ² test. The associations of the effects of vascular risk factors and genotypes in ischemic stroke cases and controls were assessed by odds ratios (ORs) and 95% confidence intervals (CIs). p-Values <0.05 were considered statistically significant. These statistical analyses were performed with SPSS 20.0 software (SPSS, Inc., Chicago, IL).

Results

In the present study, three NOS3 genetic polymorphisms were investigated in a sample of 245 ischemic stroke patients and 145 healthy controls in a Turkish population. The results of clinical laboratory tests and some risk factors of acute ischemic stroke patients and control subjects are given in Table 1. The age status among case and control populations was not significantly different. The risk factors of ischemic stroke, such as hypertension, diabetes, obesity, smoking, and levels of total cholesterol, triglyceride, and LDL-cholesterol, were found to be higher in the patient group. On the other hand, the HDL-cholesterol level was significantly lower in ischemic stroke patients (1.1±0.3 mM) when compared with the control group (1.2±0.3 mM, p=0.001).

Table 1.

Clinical Laboratory Data and Conventional Risk Factors of Acute Ischemic Stroke Patients and Controls

Parameter	Patients (n=245)	Controls (n=145)	p	OR (95% CI)
Age (years)	64.5±13.3	62.1±14.1	0.061^a
Male, n (%)	141 (57.6)	73 (50.3)	0.167^b	1.337 (0.885-2.020)
Hypertension, n (%)	163 (66.5)	55 (37.9)	<0.01^b	3.253 (2.121-4.989)
Diabetes mellitus, n (%)	84 (34.3)	25 (17.2)	<0.01^b	2.504 (1.511-4.151)
Obesity, n (%)	56 (22.9)	9 (6.2)	<0.01^b	4.477 (2.141-9.361)
Smokers, n (%)	69 (28.2)	21 (14.5)	0.001^b	2.315 (1.349-3.972)
Total cholesterol (mM)	4.8±1.3	4.6±1.2	0.112^c
Triglycerides (mM)	1.4±0.2	1.3±0.2	0.174^c
HDL-cholesterol (mM)	1.1±0.3	1.2±0.3	0.001^c
LDL-cholesterol (mM)	2.9±1.0	2.7±1.0	0.007^c

Values are either number of subjects, percentage, or mean±SD.

Mann-Whitney U test is applied.

χ² test is applied.

Independent samples t-test is applied.

CI, confidence interval; OR, odds ratio.

Table 2 summarizes the genotype and allele distributions of NOS3 G894T, T-786C, and intron 4 VNTR polymorphisms in ischemic stroke patients and control subjects. Among 245 patients, 156 (63.7%) were heterozygous and 7 (2.8%) were homozygous for the G894T polymorphism. In the control population, 80 (55.2%) individuals were heterozygous and 10 (6.9%) individuals were homozygous for the mutation. The variant T allele frequency was found to be almost the same in patients and controls. With respect to the T-786C polymorphism, 93 (38%) patients were found to be heterozygous with 6 (2.4%) homozygous individuals, while 57 (39.3%) subjects in the control sample were heterozygous and 6 (4.1%) were homozygous. The C allele was found in 21.4% of patients and 23.8% of controls. For the intron 4 VNTR polymorphism, 71 (29%) patients had the heterozygous ab genotype and 2 (0.8%) had the homozygous aa genotype. In the controls, there were 33 (22.7%) heterozygous ab genotyped and 3 (2.1%) homozygous aa genotyped individuals. The 4a allele frequency was 15.3% in stroke patients and 13.4% in the control group.

Table 2.

Frequencies of NOS3 Genotypes and Alleles in Stroke Patients and Controls

	Cases		Controls
SNP	n	%	n	%
G894T
GG	82	33.5	55	37.9
GT	156	63.7	80	55.2
TT	7	2.8	10	6.9
894T allele frequency	170	34.7	100	34.5
NOS3 T-786C
TT	146	59.6	82	56.6
TC	93	38.0	57	39.3
CC	6	2.4	6	4.1
-786C allele frequency	105	21.4	69	23.8
NOS3 Intron 4 VNTR
bb	172	70.2	109	75.2
ab	71	29.0	33	22.7
aa	2	0.8	3	2.1
4a allele frequency	75	15.3	39	13.4

NOS3, nitric oxide synthase 3; VNTR, variable number of tandem repeat.

Results of single-locus analysis of the effect of NOS3 G894T, T-786C, and intron 4 VNTR polymorphisms on the risk of development of ischemic stroke are summarized in Table 3.

Table 3.

Single-Locus Analysis of the Effect of G894T, T-786C, and Intron 4 VNTR Polymorphisms on the Risk of Development of Ischemic Stroke

Genotype	Cases (n=245)	Controls (n=145)	OR (95% CI)	p
GG	82	55	1.0
GT and TT	163	90	1.215 (0.792-1.863)	0.372
TT	146	82	1.0
TC and CC	99	63	0.883 (0.582-1.338)	0.555
bb	172	109	1.0
ab and aa	73	36	1.285 (0.807-2.047)	0.290

Homozygous wild-type genotypes were considered as a reference and individuals carrying the heterozygous and homozygous variant genotypes were grouped together and compared to the reference. As shown in Table 3, the presence of the T allele increased the risk for the incidence of ischemic stroke 1.2-fold; however, this result was not statistically significant. On the other hand, the presence of the C allele has a protective effect against ischemic stroke (OR=0.883, 95% CI=0.582-1.338, p=0.555). The individuals carrying the a allele increased the risk of stroke ∼1.3-fold, but this result was not statistically significant.

Table 4 summarizes the results of multiloci analysis for NOS3 G894T, T-786C, and intron 4 VNTR polymorphisms. We observed that if the risky genotypes of NOS3 G894T and intron 4 VNTR polymorphisms are found on the same subjects at the same time, the double or combined risky genotype had a sevenfold significantly increased risk of stroke (OR=7.083, 95% CI=0.866-57.963, p=0.029). Besides, it was found that the presence of three risky genotypes on the same individual increased the risk of stroke 1.6-fold, which was not statistically significant (p=0.216). Furthermore, when the individual carries two or more risky genotypes at the same time, the risk of incidence of stroke increases ∼1.2-fold, but this was not statistically significant (p=0.560).

Table 4.

Multiloci Analysis of the Effect of G894T, T-786C, and Intron 4 VNTR Polymorphisms on the Risk of Development of Ischemic Stroke

	G894T	T-786C	Intron 4 VNTR	Cases	Controls	OR (95% CI)	p
Total				245	145
No loci at risk	GG	TT	bb	48	34	Reference
One locus at risk	GT/TT	TT	bb	73	38	1.361 (0.755-2.452)	0.304
	GG	TC/CC	bb	3	0	NA
	GG	TT	ab/aa	15	9	1.181 (0.463-3.01)	0.727
Two loci at risk	GT/TT	TC/CC	bb	48	37	0.919 (0.497-1.698)	0.787
	GG	TC/CC	ab/aa	16	12	0.944 (0.397-2.25)	1.000
	GT/TT	TT	ab/aa	10	1	7.083 (0.866-57.963)	0.029^*
Three loci at risk	GT/TT	TC/CC	ab/aa	32	14	1.619 (0.752-3.484)	0.216
Two or more loci at risk				106	64	1.173 (0.685-2.009)	0.560

Comparisons are by χ² test except for those marked with “^*” for which Fisher's exact test was used.

Discussion

NOS3 catalyzes the generation of NO, which mediates vascular relaxation in response to vasoactive substances and shear stress. In addition, it mediates inhibition of platelet adherence and aggregation, suppression of smooth muscle proliferation, and reduction of adherence of leukocytes to the endothelium. These properties of NOS3 make it a biologically reasonable candidate to study as a susceptibility gene in ischemic stroke. Different polymorphisms of NOS3 have been identified and the most studied one is NOS3 G894T polymorphism. However, the results of association of G894T polymorphism with ischemic stroke are quite contradictory. Recently, a positive association has been reported between NOS3 G894T polymorphism and ischemic stroke risk (Elbaz et al., 2000; Berger et al., 2007; Luka et al., 2011). On the other hand, in some studies no association between NOS3 G894T polymorphism and stroke was found (Markus et al., 1998; MacLeod et al., 1999; Szolnoki et al., 2005; Guldiken et al., 2009; Majumdar et al., 2010). In this study, we also observed no significant association between NOS3 G894T polymorphism and ischemic stroke risk.

The second polymorphism studied in the present study was NOS3 T-786C. Cheng et al. (2008) reported that the -786CC genotype was more prone to develop stroke in the Chinese population. On the other hand, some studies showed that the -786TT genotype prevalence was higher in ischemic stroke patients when compared with controls (Howard et al., 2005; Majumdar et al., 2010). We could not find any association between T-786C polymorphism and ischemic stroke risk (Table 3).

The last polymorphism analyzed in this study was NOS3 intron 4 VNTR polymorphism. Again, there were controversial results about this polymorphism and stroke association in the literature. The intron 4bb genotype was shown as an important genetic risk factor for early-onset ischemic stroke in the Chinese population (Shi et al., 2008). On the contrary, according to some studies, the 4a allele was found to be a risk factor for stroke (Hou et al., 2001; Majumdar et al., 2010). According to the present study, there was no relevance between intron 4 VNTR polymorphism and ischemic stroke risk. In this study, the effect of the combination of three different NOS3 polymorphisms, namely G894T, T-786C, and intron 4 VNTR, on the risk of stroke was analyzed. It was observed that 10 stroke patients heterozygous for the G894T were also heterozygous for the intron 4 VNTR polymorphism. When both polymorphisms were analyzed with the presence of a single risk-elevating genotype, neither the G894T nor the intron 4 VNTR risky genotypes had a significant effect on the development of ischemic stroke risk. However, the presence of G894T and intron 4 VNTR risky genotypes on the same individual increased the risk of stroke sevenfold, which was statistically significant (OR=7.083, 95% CI=0.866-57.963, p=0.029). This result suggests that G894T and intron 4 VNTR polymorphisms have a combined effect on the risk of stroke.

The comparison of NOS3 G894T, T-786C, and intron 4 VNTR polymorphisms in the Turkish population and various ethnic populations is represented in Table 5. In the present study, the frequency of the 894T allele in 145 subjects was found as 0.345, which was higher when compared to other Turkish studies in the literature (Bayazit et al., 2009; Guldiken et al., 2009). On the other hand, higher mutant 894T allele frequency was obtained from a study performed by Yemişçi et al. (2009). The mutant 894T allele frequency of our population was found to be highly similar with those of the French (Elbaz et al., 2000), Caucasian (Tanus-Santos et al., 2002), and Greek people (Andrikopoulos et al., 2008; Vasilakou et al., 2008; Kitsios and Zintzaras, 2010) (Table 5). Mutant allele frequencies in Asian populations (Cheng et al., 2008; Moe et al., 2008; Tamemoto et al., 2008; Shin et al., 2010) were found to be considerably lower than that determined in our study. Moreover, this allele frequency was found to be lower in American populations than Turkish populations.

Table 5.

Comparison of the Genotype and Allele Frequencies Distributions of NOS3 G894T, T-786C, and Intron 4 VNTR Polymorphisms in Turkish Population Control Groups and Previously Published Data in Other Population Control Groups

NOS3 G894T SNP
Population	n	GG	GT	TT	G	T	Reference
Turkish (this study)	145	37.9	55.2	6.9	0.655	0.345
Turkish	81	27.2	58	14.8	0.562	0.438	Yemişçi et al. (2009)
Turkish	133	49.3	45.8	4.9	0.722	0.278	Guldiken et al. (2009)
Turkish	159	56.6	33.3	10.1	0.733	0.268	Bayazit et al. (2009)
Asian
Chinese	309	78.6	19.7	1.6	0.885	0.115	Cheng et al. (2008)
Japanese	283	88	11.3	0.7	0.937	0.064	Tamemoto et al. (2008)
Korean	115	89.6	10.4	0	0.948	0.052	Shin et al. (2010)
Singaporean	207	77.3	20.3	2.4	0.875	0.126	Moe et al. (2008)
European
Caucasian	47	36.2	55.3	8.5	0.639	0.362	Tanus-Santos et al. (2002)
French	460	35.4	50.4	14.1	0.606	0.393	Elbaz et al. (2000)
Greek	727	48.4	40.9	10.7	0.689	0.312	Andrikopoulos et al. (2008)
Greek	161	47	46	7	0.700	0.300	Vasilakou et al. (2008)
Greek	302	44.7	43.1	12.2	0.663	0.338	Kitsios and Zintzaras (2010)
Italian	67	28	61	11	0.585	0.415	Colomba et al. (2008)
American
Brazilian	102	52	44	4	0.740	0.260	Sandrim et al. (2006)
Chilean	112	66	32	2	0.820	0.180	Jaramillo et al. (2010)

NOS3 T-786C SNP
Population	n	TT	TC	CC	T	C	Reference
Turkish (this study)	145	56.6	39.3	4.1	0.763	0.238
Turkish	71	38	49.3	12.7	0.627	0.374	Sinici et al. (2010)
Turkish	79	44.3	51.9	3.8	0.703	0.298	Yemişçi et al. (2009)
Asian
Chinese	309	80.9	18.1	1	0.900	0.101	Cheng et al. (2008)
Korean	115	80.9	18.3	0.8	0.901	0.100	Shin et al. (2010)
Singaporean	207	73.4	25.1	1.5	0.860	0.141	Moe et al. (2008)
European
Caucasian	47	21.3	63.8	14.9	0.532	0.468	Tanus-Santos et al. (2002)
Greek	289	35	51.2	13.8	0.606	0.394	Kitsios and Zintzaras (2010)
Italian	67	21	50	29	0.460	0.540	Colomba et al. (2008)
Italian	360	32.8	48.9	18.3	0.573	0.428	Venturelli et al. (2005)
American
Brazilian	102	37	51	12	0.625	0.375	Sandrim et al. (2006)
Chilean	112	59	38	4	0.780	0.230	Jaramillo et al. (2010)

NOS3 intron 4 VNTR
Population	n	aa	ab	bb	a	b	Reference
Turkish (this study)	145	2.1	22.7	75.2	0.135	0.866
Turkish	71	2.8	25.4	71.8	0.155	0.845	Sinici et al. (2010)
Turkish	81	16	29.6	54.3	0.308	0.691	Yemişçi et al. (2009)
Turkish	181	2.1	19.9	79	0.121	0.890	Bayazit et al. (2009)
Asian
Korean	115	0.8	20.9	78.3	0.113	0.888	Shin et al. (2010)
European
Caucasian	47	4.3	25.5	70.2	0.171	0.830	Tanus-Santos et al. (2002)
Greek	161	3	24	73	0.150	0.850	Vasilakou et al. (2008)
Greek	303	4	33.3	62.7	0.207	0.794	Kitsios and Zintzaras (2010)
Italian	67	0	23	77	0.115	0.885	Colomba et al. (2008)
American
Brazilian	102	4	19	77	0.135	0.865	Sandrim et al. (2006)
Chilean	112	1	14	85	0.080	0.920	Jaramillo et al. (2008)

In this study, the frequency of the -786C allele in 145 healthy subjects was found as 0.238 (Table 5). This result was almost similar to the -786C allele frequency found in the studies on Turkish populations (Yemişçi et al., 2009; Sinici et al., 2010). The mutant -786C allele frequency of the Turkish population was found to be higher than that of Asian populations (Cheng et al., 2008; Moe et al., 2008; Shin et al., 2010). On the contrary, European populations (Tanus-Santos et al., 2002; Venturelli et al., 2005; Colomba et al., 2008; Kitsios and Zintzaras, 2010) had a high mutant allele frequency when compared with the Turkish population. The allele frequency of -786C found in this study was similar to the ones found in American populations (Sandrim et al., 2006; Jaramillo et al., 2010).

For intron 4 VNTR, the small a allele frequency was reported as 0.135 in this study (Table 5). This allele frequency was found to be very similar with Asian (Shin et al., 2010), European (Tanus-Santos et al., 2002; Vasilakou et al., 2008; Colomba et al., 2008), and American (Sandrim et al., 2006) populations.

In conclusion, to the best of our knowledge, this is the first report to evaluate the possible association between the NOS3 G894T, T-786C, and intron 4 VNTR polymorphisms and ischemic stroke risk in a Turkish population. These polymorphisms, when considered alone, did not have a role in the development of ischemic stroke in the studied Turkish population. However, significant results were obtained when the risky genotypes of G894T and intron 4 VNTR were present together.

Footnotes

Acknowledgment

This work was supported by the Middle East Technical University Research Project Fund (Project number: BAP-08-11-2008-R-06).

Author Disclosure Statement

The authors declare no financial conflicts of interest.

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