eNOS G894T Polymorphism and Abdominal Aortic Aneurysms

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Angiology

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eNOS G894T Polymorphism and Abdominal Aortic Aneurysms Fahri Hayri Atli, Sinasi Manduz, Nurkay Katrancioglu, Unal Ozum, Olcay Murat Disli, Erhan Atahan, Ozturk Ozdemir, Kasim Dogan and Ocal Berkan ANGIOLOGY 2010 61: 125 originally published online 27 July 2009 DOI: 10.1177/0003319709339589 The online version of this article can be found at: http://ang.sagepub.com/content/61/2/125

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Abdominal Aortic Aneurysm

eNOS G894T Polymorphism and Abdominal Aortic Aneurysms

Angiology Volume 61 Number 2 February 2010 125-130 # 2010 The Author(s) 10.1177/0003319709339589 http://ang.sagepub.com

Fahri Hayri Atli, MD, Sinasi Manduz, MD, Nurkay Katrancioglu, MD, Unal Ozum, MD, Olcay Murat Disli, MD, Erhan Atahan, MD, Ozturk Ozdemir, PhD, Kasim Dogan, MD, and Ocal Berkan, MD Background: The genetic risk factors that contribute to the risk of developing abdominal aortic aneurysm (AAA) are poorly understood. We assessed the association of endothelial nitric oxide synthase (eNOS) gene polymorphism with AAA. Methods: eNOS gene polymorphism of 61 patients with AAA and 62 control participants were analyzed by polymerase chain reaction (PCR)-restriction technique. Results: eNOS G894 homozygote T/T genotype polymorphism and 894T allele frequency in patients with AAA were significantly

Introduction Aortic aneurysm (AA) is known as an irreversible segmental enlargement of aorta in age- and body surface–dependent manner relatively. Focal dilation of the aorta, at least 50% larger than the expected normal diameter in transversal measurement, is accepted as an aneurysm.1 The pathogenesis of AAs is a complex phenomenon that caused by genetics and other factors.2 Histologically, AAs are characterized by signs of chronic inflammation, destructive remodeling of the extracellular matrix, cell degeneration, connective tissue dissection and/or degeneration, trauma, aortitis, and other congenital abnormalities that contribute to aneurysm formation in humans. First-degree relatives of patients with abdominal aortic aneurysm (AAA) are commonly From the Department of Cardiovascular Surgery, Urfa State Hospital, Urfa (FHA); and Departments of Cardiovascular Surgery (SM, NK, MD, EA, KD, OB), Neurosurgery (UO), and Medical Biology and Genetics (OO), Cumhuriyet University School of Medicine, 58140 Sivas, Turkey. Address correspondence to: Nurkay Katrancioglu, Department of Cardiovascular Surgery, Cumhuriyet University School of Medicine, 58140 Sivas, Turkey; e-mail: [email protected]

higher than those of the control participants (P ¼ .01, P ¼ .03). Among patients with AAA, the eNOS G894 T/T polymorphism and 894T allele frequency were associated with larger AAAs. Conclusion: The current study, in a small group of participants, showed a relationship between eNOS G894T polymorphism and AAA. Keywords: aortic aneurysm; abdominal; eNOS gene polymorphism; human; nitric oxide

accepted to have a 10% to 15% greater risk for an AAA compared with the general population.3 Abdominal aortic aneurysm is an important vascular disease, but the genetic and environmental risk factors, which contribute to individual’s risk to develop an AAA, are poorly understood. Many reports in literature addressed different associations in genetic basis of AAA.4 Nitric oxide (NO) is synthesized from l-arginine by nitric oxide synthase (NOS). Three isoforms of NOS have been described: endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS), and inducible nitric oxide synthase (iNOS). Nitric oxide synthases use l-arginine as substrate, converting a terminal guanidino nitrogen to produce NO.5 Genetic polymorphisms of the eNOS gene may have a potential role in cardiovascular diseases, stroke, hypertension, and renal dysfunction in human.6-9 Polymorphisms of eNOS gene may influence the expression and functional activity of the enzyme. In this study, it was aimed to investigate the potential role of the eNOS 894G>T gene polymorphism in patients with AAA versus controls in the general Turkish population. 125

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Materials and Methods Participants The current study was approved by local ethics committee and all participants gave their informed consent. Our study group consisted of 61 patients (40 males, 21 females) who were admitted between January 2005 and April 2007, with a diagnosis of AAA by computed tomographic (CT) scan of thorax and abdomen, defined as a focal dilation of the aorta at least 50% larger than the expected normal diameter.1 Patients with chronic inflammatory diseases were excluded from the study. Mean age of patient group was 63.6 + 7.1 years. Our control group consisted of 62 (43 males, 19 females) persons who were selected randomly from age-matched adult Turkish volunteers who have no focal dilation of the abdominal aorta more than 50% of the expected normal diameter of the aorta in CT scans of thorax and abdomen taken with other clinical reasons 2 and who living in Sivas, Turkey. Mean age of control group was 62.1 + 8.0 years.

DNA Extraction and Polymerase Chain Reaction The ultrapure genomic DNA was isolated from EDTA-anticoagulate peripheral blood leukocytes with DNA isolating kit (Nucleospin Blood, MachereyNagel, Germany). A set of forward and reverse primers, F-50 -AAGGCAGGAGACACTGATGGA-30 and R-50 CCCAGTCAATCCCTTTGGTGCT-30 , were used for the amplification of the 248-bp fragment including the G894T variant site of exon 7. The polymerase chain reaction (PCR) mixture in a 20-mL final volume consisted of 12.5 mL PCR master mix (Fermantas, Canada), 3.5 mL buffers, 1 mL of each primer, and 2 mL DNA. The target gene region amplified for 35 cycles and each cycle comprised of denaturation at 94 C for 30 seconds, annealing at 58 C for 30 seconds, extension at 72 C 40 seconds, and final extension at 72 C for 5 minutes. Initial denaturation stage was applied at 94 C for 2 minutes. The amplified products were used for agarose and restriction fragment length polymorphism (RFLP) analyses.

Restriction Fragment Length Polymorphisms Amplified PCR products were digested with restriction enzyme BanII (Fermantas, Canada) at 37 C for

16 hours. The digested fragments were analyzed by 2.5% agarose gel electrophoresis technique. The BanII specific homozygous (Asp/Asp and/or Glu/ Glu) and heterozygous (Asp/Glu) alleles were identified and compared to control group statistically.

Statistics SPSS for Windows version 16.0 (SPSS Inc, Chicago, IL) was used for summarizing and analyzing data of this study. Data were expressed as mean + standard deviation (SD) or percentage of column totals. Demographic variables were analyzed by using t test. Chi-square test was used to compare the proportions of genotype distribution from Hardy-Weinberg equilibrium. Odd ratios (ORs) with 95% confidence interval were determined with the effect of T (mutant) allele assumed to be an additive (abnormal T allele vs. normal G allele), a recessive (abnormal homozygote eNOS T/T vs. abnormal heterozygote eNOS T/G and normal homozygote G/G combined), and dominant (normal homozygote eNOS G/G vs. abnormal heterozygote eNOS T/G and abnormal homozygote eNOS T/T combined) model of inheritance. The relationship between genetic variants and AAA was determined by univariate regression analysis. Variables that were significant in univariate analysis were introduced in a multivariate model. Significance was accepted at P < .05.

Results The demographic and clinical characteristics of patients and control participants are shown in Table 1. Hypertension was significantly higher in the patient group than in the controls (P < .001); however, no significant differences in demographic data, smoking status, dyslipidemia, coronary heart disease, and diabetes mellitus were detected between the study groups (Table 1). Genotype distribution and allele frequencies in each group are shown in Table 2. eNOS G894 homozygote T/T genotype polymorphism was more frequently observed in the patient group than in the controls (P ¼ .01), just as 894T allele frequency (P ¼ .03), whereas there were no significant differences between the patient group and control participants for eNOS G894 T/G and eNOS G894 G/G polymorphisms. Of the 61 patients, 25 had AAA diameter 5 cm and 36 had AAA diameter >5 cm according to CT

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eNOS Polymorphism and Abdominal Aortic Aneurysm / Atli et al 127

Table 1. Demographic and Clinical Parameters in Patient and Control Groups Patients (n ¼ 61)

Variable Age (year) Males (%) Current smoker (%) Exsmoker (%) Hypertension (%) Dyslipidemia (%) Diabetes mellitus (%) Coronary heart disease (%) Aortic diameter (cm)

63.65 40 19 8 37 13 5 17 6.21

+ 7.06 (65.6) (31.1) (13.1) (60.7) (21.3) (8.2) (27.8) + 1.42

Allele

Patients (n ¼ 61)

Controls (n ¼ 62)

894T

24 (39) 21 (34) 16 (26) 0.57

12 (19) 29 (47) 21 (34) 0.44

TT (%) TG (%) GG (%)

P .01 .16 .35 .03

NOTES: TT ¼ homozygous carrier of eNOS gene G894T variant; TG ¼ heterozygous carrier of eNOS gene G894T variant; GG ¼ homozygous normal; 894T ¼ variant allele.

Table 3. Genotype Distribution and Allele Frequency for eNOS Gene G894T Polymorphism According to the Aortic Diameter in Patient Group Aortic Diameter Genotype

Allele

5 cm (n ¼ 25)

>5 cm (n ¼ 36)

894T

5 (20) 10 (40) 10 (40) 0.40

19 (53) 11 (31) 6 (16) 0.68

TT (%) TG (%) GG (%)

P

62.11 + 7.99 43 (69.4) 26 (41.9) 4 (6.5) 11 (17.7) 9 (14.5) 4 (6.5) 13 (20.9) 1.95 + 0.79

.26 .65 .21 .35 .00 .33 .71 .49 .00

Table 4. Odds Ratios Associated With eNOS Gene Polymorphisms for Abdominal Aortic Aneurysm According to Univariate Analyses

Table 2. Genotype Distribution and Allele Frequency for eNOS gene G894T Polymorphism in Patient and Control Groups Genotype

Controls (n ¼ 62)

P .02 .44 .08 .002

NOTES: TT ¼ homozygous carrier of eNOS gene G894T variant; TG ¼ heterozygous carrier of eNOS gene G894T variant; GG ¼ homozygous normal; 894T ¼ variant allele.

scan results. Those patients having AAA >5 cm in diameter were found to have eNOS G894 T/T polymorphism more frequently than those with AAA 5 cm in diameter (P ¼ .02). 894T allele was more frequently observed among those with AAA >5 cm in diameter compared to those with AAA 5 cm in diameter (P ¼ .002). No significant difference was observed between the 2 subgroups concerning eNOS G894 T/G and G/G polymorphism (Table 3). Of note, these differences were noted despite similar age and sex distribution between the 2 subgroups.

Univariate Analysis Gender (female vs. male) Hypertension Diabetes Smoking Dyslipidemia eNOS 894T recessive model (TT vs. TG þ GG) eNOS 894T dominant model (GG vs. TGþTT) Multivariate analysis Hypertension eNOS 894T recessive model (TT vs. TGþGG)

OR (95% CI)

P

0.36 (0.13-1.01) 20.48 (6.30-66.54) 0.37 (0.06-2.23) 0.34 (0.11-1.07) 1.15 (0.33-3.96) 2.7 (1.19-6.09)

.06 .00 .28 .07 .82 .01

0.49 (0.24-1.02)

.59

20.11 (6.02-67.13) .00 3.34 (1.18-9.42) .02

NOTES: CI ¼ confidence interval; eNOS ¼ endothelial nitric oxide synthase; OR ¼ odds ratio; TT ¼ homozygous carrier of eNOS gene G894T variant; TG ¼ heterozygous carrier of eNOS gene G894T variant; GG ¼ homozygous normal.

There was significant relationship between both the eNOS recessive model and hypertension and the presence of AAA (eNOS 894T recessive model OR: 2.7, 95% CI: 1.19-6.09, P ¼ .01; hypertension OR: 20.48, 95% CI: 6.30-66.54, P ¼ .000); however, there was no significant relationship between the eNOS dominant model and the presence of AAA and eNOS considered (OR: 0.49, 95% CI: 0.24-1.02, P ¼ .59) in univariate logistic regression analysis (Table 4). In addition, there was significant relationship between both the eNOS recessive model and hypertension and the presence of AAA (eNOS 894T recessive model OR: 3.34, 95% CI: 1.189.42, P ¼ .02; hypertension OR: 20.11, 95% CI: 6.02-67.13, P ¼ .000) in multivariate regression analysis (Table 4).

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Discussion eNOS gene is located on chromosome 7q35-36.10 Genetic variation, referred to as ‘‘functional polymorphisms’’ including the eNOS gene intron-4 27base pair variable number of tandem repeat (eNOS 27 VNTR), eNOS gene promoter single-nucleotide polymorphism (eNOS T786C), and eNOS gene exon-7 G894T polymorphism were investigated in different clinical pathologies.11,12 eNOS gene is proposed as the susceptible gene for some vascular diseases including coronary artery disease, carotid atherosclerosis, hypertension, and AAA.11,13 An experimental study model demonstrated that the eNOS deficiency is able to change the disease pattern of atherosclerosis, thus determining peripheral arterial disease, myocardial ischemia, and vascular complications, such as aortic dissection and AAA formation.13,14 Abdominal aortic aneurysm is a morphological vascular disease with multifactorial pathogenesis including genetic and environmental factors. Nitric oxide is an important mediator of endothelial function. Nitric oxide synthesis is performed by cells expressing eNOS, nNOS, or iNOS. Nitric oxide produced by eNOS is regarded as vasculoprotective and it has a role in maintaining vessel wall geometry in addition to its role as a vasodilator.11,15 Nitric oxide produced by iNOS in vascular smooth cells occurs primarily in inflammatory media resulting in high level of NO and its toxic product that degrade elastin and disturb extracellular matrix.16,17 Dosenko et al18 suggested that uncontrolled NO produced by iNOS or unstable NO produced by eNOS may destroy integrity of vessel wall.19,20 It has been suggested that polymorphisms in the eNOS gene are related to impaired expression and functional activity of the eNOS enzyme.21,22 A positive association between plasma NO levels and G894T polymorphisms in eNOS gene has been found in different populations.23,24 Wattanapitayakul et al12 concluded that eNOS gene polymorphism might alter interaction with other components of eNOS gene and/or enzymatic stability. Because of this alteration, polymorphic eNOS gene may affect the expression and functional activity of eNOS enzyme thereby reducing NO availability.21,23 It may be resulted from inactivation by cleavage, possibly because of a tighter turn of the a helix, thus determining a reduction in the capacity for NO production.21,22

There are only 2 reports, which focus on the link between functional polymorphisms of eNOS gene and AAA. Kotani et al25 reported that 4a/4b polymorphism of eNOS gene is not associated with presence of AAA. However, they also found strong association between 4a/4b polymorphism of eNOS gene and elective repair of AAA, reflecting the expansion of AAA. Fatini et al22 reported significant difference in genotype distribution and allele frequency for eNOS G894T but not for T-786C and 4a/4b polymorphisms. In our study, a significant difference was found in genotype distribution for eNOS G894 T/T between patients with AAA and controls. In addition, genetic distribution for eNOS G894 T/T and allele frequency for eNOS G894T were higher in patients with AAA >5 cm compared with those with AAA 5 cm despite no significant differences in age and sex distribution between 2 subgroups. This might reflect the association between disease progression rate and genetic predilection. This finding is novel and should be verified by further studies. Our study has some limitations. First, it only involves 61 patients with AAA. However, we performed a post hoc power analyses with 39% and 19% of homozygozity in patients with AAA and controls, respectively. We calculated a power of 73% with a P < .05 and our case numbers in the study groups. This issue can be solved by an expanded cooperative study providing a higher power. Second, the control group consists of truly healthy participants. Therefore, there are significant differences between control and AAA groups for traditional risk factors. Abdominal aortic aneurysm is a complex disease; the effect of 1 gene on complex diseases might be difficult to explore in a population study exposed to environmental confounding factors known to influence the disease. Smoking is an important initiating factor in the development of AAA. In addition to its established role in the atherosclerotic process, it mainly affects elastin degradation in the vascular wall. Smoking promotes the expression of proteolytic systems while at the same time it attenuates the activity of their inhibitors.26 In our study, smoking status of the study groups were comparable, and it is not possible to evaluate the effect of smoking in the development of AAA. Although we demonstrated that the effect of the eNOS gene is relevant independently from hypertension, the best way to test the potential association between genetic polymorphisms and the disease would be comparable traditional risk factors in both the study and control populations. However, the significant difference

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eNOS Polymorphism and Abdominal Aortic Aneurysm / Atli et al 129

between 2 subgroups (large vs. small aneurysm) provides support for a genetic link. In conclusion, we report a relationship between eNOS G894T polymorphism and the presence and the extent of AAA. Abdominal aortic aneurysm is a complex disease that is affected by changeable and nonchangeable risk factors. Early identification of nonmodifiable risk factors such as eNOS G894T polymorphism may allow physicians to address modifiable risk factors to prevent or delay the development of AAA.

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NO metabolite levels in humans. Biochem Biophysic Res Commun. 1998;245(1):190-193. 25. Kotani K, Shimomura F, Ikawa F, et al. Allele frequency of human endothelial nitric oxide synthase gene

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