Abstract

Background: This study aims to investigate the relationship between the development of aortic dissections and aneurysms with the polymorphisms of angiotensin converting enzyme gene, methylenetetrahydrofolate reductase gene, plasminogen activator inhibitor-1 gene, and nitric oxide synthase gene.

Methods: Between April 2009 and July 2014, 38 patients with aortic dissections (28 males, 10 females; mean age 55.1±10.7 years; range, 30 to 78 years) and 67 patients with aortic aneurysms (57 males, 10 females; mean age 63.0±11.4 years; range, 31 to 82 years) were included in this cross-sectional study. The control group consisted of 60 healthy volunteers (41 males, 19 females; mean age 56.3±11.2 years; range, 30 to 82 years) without an aortic aneurysm or dissection, as assessed by thoracoabdominal computed tomography. The prespecified four genes were genotyped with competitive allelespecific polymerase chain reaction.

Results: The aortic dissection group had higher nitric oxide synthase-3 (4b/4b) expression levels, compared to the control group. The aortic aneurysm group had also higher nitric oxide synthase-3 (4b/4a) expression levels, compared to the control group. Compared to the control group, a higher rate of angiotensin converting enzyme I/D gene polymorphism was detected in the aneurysm group, while higher D/D polymorphism rates were found in the dissection group; although not statistically significant.

Conclusion: Our study results suggest that the nitric oxide synthase-3 intron 4b/4b and nitric oxide synthase-3 intron 4b/4a gene polymorphisms can be used as a predictor of aortic dissection and aneurysm development.

Aortic dissections and aneurysms are life-threatening conditions associated with high morbidity and mortality. Aortic dissection is an aortic pathology characterized by a tear in the intima layer which separates the media layer from the bloodstream, leading to the development of a false lumen between the intima and media layers of the aorta.[1] Aneurysm is an abnormal and irreversible dilatation (two times greater than normal diameter) which can affect any segment of the aorta. Aortic aneurysms are often caused by degenerations which can be also related with dissections, connective tissue diseases, blunt traumas, aortitis, mycotic infections, or congenital anomalies.[2]

Aortic aneurysms and dissections are important health issues in terms of mortality rates and other health services all over the world.[1] The prevalence of aortic aneurysms is between 1.7 and 12.7%.[2] However, it is difficult to determine the true incidence, as aortic dissections usually remain undiagnosed. The prevalence of aortic dissections is reported to be between 0.2 and 0.8%.[2] In a study where 12-year retrospective data was evaluated, the incidence of both diseases was found to increase over the years. Of note, aortic dissections and aortic aneurysms are more common in men than in women.[3,4]

In the literature, there are several studies examining the relationship between the development of aortic dissections and aneurysms and genetic factors. In the present study, we aimed to investigate the relationship between the development of aortic dissections and aneurysms and the polymorphisms of angiotensin converting enzyme (ACE) gene, methylenetetrahydrofolate reductase (MTHFR) gene, plasminogen activator inhibitor-1 (PAI-1) gene, and nitric oxide synthase (NOS) gene.

Methods

This cross-sectional study included a total of 38 patients with aortic dissections (28 males, 10 females; mean age 55.0±10.7 years; range, 30 to 78 years) and 67 patients with aortic aneurysms (57 males, 10 females; mean age 63.0±11.4 years; range, 31 to 82 years) at Erciyes University, Faculty of Medicine, Department of Cardiovascular Surgery between April 2009 and July 2014. The patients were divided into three groups as the control, aneurysm, and dissection group. Patients with aortic dissections were referred to as the dissection group and patients with aortic aneurysms were referred to as the aneurysm group. The dissection group was further divided into two groups according to the Stanford classification: Stanford type A group (n=33) and Stanford type B group (n=5). Aortic dissections and aneurysms were diagnosed with computed tomography and echocardiography (Figures 1-2). The patients with a detectable dissection flap and those with an enlarged aortic diameter (twotimes greater than normal diameter) in any segment of the aorta in the transverse measurements were included in the study group.

Figure 1: A standard axial image from a computed tomography scan demonstrating a descending thoracic aortic aneurysm and dissection. A: Ascending aorta; D: Descending aorta; PA: Pulmonary artery.

Figure 2: A standard axial image from a computed tomography scan demonstrating an ascending aortic aneurysm. A: Ascending aorta; D: Descending aorta; PA: Pulmonary artery; VCS: Vena cava superior.

The control group consisted of 60 healthy volunteers (41 males, 19 females; mean age 56.3±11.2 years; range, 30 to 82 years) who did not have an aortic aneurysm or dissection, as assessed by the thoracoabdominal computed tomography and transthoracic echocardiography. Patients with a disease which may cause aneurysms such as Marfan syndrome or Behçet's disease and those with a syndromic phenotype were excluded from the study.

The study protocol was approved by the Erciyes University, Faculty of Medicine, Ethics Committee (E.C. Number: 09/202, Date: 07.04.2009). A written informed consent was obtained from each participant. The study was conducted in accordance with the principles of the Declaration of Helsinki.

Data collection
A 5 mL blood sample was taken with vacutainer from each individual. Blood samples were stored at +4°C in a tube containing 1/100 volume and 0.5 mmol/L sodium ethylenediaminetetraacetic acid (EDTA).

Genetic testing
All of the genetic studies were performed in the Genome and Stem Cell Center at Erciyes University (GENKOK). A 2 mL of blood samples with EDTA were obtained from each participant. Genomic deoxyribonucleic acid (DNA) was extracted from the peripheral blood samples using standard procedures of High Pure PCR Template Preparation Kit (Roche, Germany). The final DNA concentration was determined with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). Amplifications of NOS3 intron 4 polymorphism (rs61722009) were performed with standard polymerase chain reaction (PCR). The PCR products were visualized with 2% agarose gel stained with ethidium bromide. The NOS3 Glu298Asp (894G>T, rs1799983) polymorphism was determined using a PCR-restriction fragment length polymorphism (RFLP)-based protocol. The PCR products were checked on 2.5% agarose gel, 206-bp eNOS product was digested overnight at 37°C with Mbo I restriction endonuclease enzyme.[3]ACE gene I/D polymorphism were amplified with PCR method. Fifty microliters final volume of PCR mixture consisted of 50 ng genomic DNA, 10¥PCR buffer, 0.2 mM of each dNTP, MgCl2 (1.5 mM), Taq DNA polymerase (1 U/mL), and 10 pmol of each primer.[4] The ACE deletion polymorphism is characterized by a 190-bp fragment, whereas the presence of the insertion leads to a 490-bp fragment. PAI-1 4G/5G amplification was performed for 30 cycles with denaturation temperature of 94°C for three min, 94°C for 30 sec, 60°C for 30 sec, and 72°C for 30 sec, the final extension step at 72°C was extended by one min. Amplified 98-bp product was digested overnight with Bsl I at 55°C and subjected to 4% agarose gel electrophoresis.[5] M THFR C 677T p olymorphism (rs1801133) was shown with PCR-RFLP. PCR cycling conditions included an initial denaturation at 94°C for two min, followed by 40 cycles of 30 sec at 94°C, 30 sec at 62°C, and 30 sec at 72°C, and a final extension step at 72°C for seven min. A total of 198 base pairs of PCR products were, then, digested overnight at 37°C with Hinf I restriction enzyme and checked with 3% agarose gel electrophoresis.[6] A ll of t he P CR p rimers of the polymorphisms and annealing temperatures are summarized in Table 1. The genotypes of the related polymorphisms after RFLP are also summarized in Table 2.

Table 1: Primer sequences and polymerase chain reaction program for genotyping gene polymorphisms

Table 2: Genotypes of related polymorphisms

Statistical analysis The statistical analysis was performed using the IBM SPSS for Windows, version 21.0 software (IBM Corp., Armonk, NY, USA). First, the control, aneurysm, and dissection groups were compared in terms of variables. Subsequently, the Stanford type A and Stanford type B dissection groups were compared within themselves. Statistical data were expressed in the number of units (n), percentage (%), mean ± standard deviation (SD) and median (25th-75th percentile). The normal distribution of numerical variables was assessed with the Shapiro-Wilk test. One-way analysis of variance (ANOVA) was used to compare the means of more than two groups. The Mann-Whitney U test was used to compare the two groups. The chi-square test was used to compare categorical variables. A p value of <0.05 was considered statistically significant.

Results

The age and gender distribution of the groups is shown in Table 3. In terms of age, the mean age of the aneurysm group was statistically higher than the dissection and control groups (p<0.001). However, there were no statistically significant differences among the groups in terms of gender (p>0.05).

Table 3: Comparison of groups in terms of genetic variables

There was a statistically significant difference in the NOS3 intron 4 polymorphism between the dissection group and the control group (p<0.05). In addition, there was a statistically significant difference between the aneurysm group and the control group in terms of NOS34b/4a polymorphism (p<0.05). However, we found no statistically significant difference between the control group and the patient groups in terms of other variables (p>0.05) (Table 3). Also, there were no statistically significant differences between the Stanford type A and Stanford type B groups (p>0.05) (Table 4).

Table 4: Dissection groups according to genetic variables

Discussion

Previous studies have shown that aortic aneurysms and dissections have a strong genetic background.[1,2] To date, a number of genetic analyses has been used to understand the underlying mechanisms of the development of aortic aneurysms and dissections.[2,3] However, these studies are mostly related to syndromic disorders.[2] The g enetic basis of non-syndromic aortic aneurysms and dissections is much more complex. In several studies in which the patients with aortic aneurysms and dissections were included, various non-syndromic mutations were identified in the genetic tests.[2,3] Still, new investigations are needed to determine which gene mutations cause non-syndromic diseases. In this study, we investigated the relationship between the development of aortic dissections and aneurysms and the ACE I/D gene polymorphism, MTHFRC 677T gene polymorphism, PAI-1 4G/5G gene polymorphism, NOS3 intron 4, and NOS3 G894T gene polymorphism.

Many researchers have suggested that genetic components may play a role in the pathogenesis of aortic diseases.[7,8] Researchers have searched for genes encoding key enzymes that play an important role in aneurysms, dissections, and inflammatory responses, and elastin, elastase, collagen, collagenase, metalloproteinase (MMP-1, 8, 13), tissue inhibitors, PAI-1, interleukins, ACE, MTHFR, NOS3, platelet activating factor, human leucocyte antigens, and inflammatory receptors have been investigated.[7,8]

The PAI-1 gene is located on the long arm of chromosome 7 (q21.3-q22). It is an important regulator of plasma activation in the tissue. A polymorphism within the gene sequence of PAI-1 has changed PAI-1 expression and plasminogen activation.[9] The most frequently investigated PAI-1 genetic variation is the 4G/5G insertion/deletion polymorphism. This polymorphism causes the nucleotide sequence of 4 or 5 guanine (4G or 5G), and the emerging different alleles lead to changes in the expression of PAI-1.[10] In their study, Jones et al.[11] found that polymorphism of the PAI-1 4G/5G gene was not related with the development of aortic aneurysms. Also, Rossaak et al.[9] reported t hat t here was no statistically significant difference between the healthy controls and patients with aortic aneurysms in terms of characteristics of the PAI-1 gene distribution. Similarly, in our study, there was no statistically significant difference between the aneurysm and control groups in terms of the PAI-1 4G/5G gene polymorphism.

Subcutaneous angiotensin II infusion in mice has been shown to affect the renal-angiotensin system, which leads to aortic aneurysms. However, previous studies have not elucidated whether ACE gene polymorphism is a risk factor for aortic aneurysms. In a study, Fatini et al.[12] reported that ACE D/D polymorphism was an independent risk factor for the development of aortic aneurysms. On the other hand, Hamano et al.[13] found exact opposite results. In a study where the long-term effects of the ACE I/D genotype on aortic aneurysms were examined, Yeung et al.[14] also reported no association between the ACE I/D and aortic aneurysms. Additionally, Korcz et al.[15] found that ACE I /D gene p olymorphism was not a predisposing factor for the development of aortic aneurysms. Consistent with the previous findings, we also showed that the ACE gene polymorphism did not play a role in the development of aortic aneurysms.

In the literature, there is a limited number of data about genetic causes of the aortic dissections. Kalay et al.[16] found that the frequency of Da llele in the ACE gene polymorphism significantly increased in aortic dissections.[16] In our study, there was no difference between the aortic dissection and control group. In addition, there was no difference in the development of aortic dissection between the D/D allele and the I/D allele.

It is well-known that nitric oxide (NO), which is regularly released from the endothelium and synthesized by the NOS3 enzyme, has a vasculoprotective effect by providing a regular vasodilatation.[3] Irregular release of the NO leads to aneurysm development, causing weakness in the vessel wall and subsequent injury.[19-21] In addition, irregular NO synthesis is associated with weakened vessel walls by altering the amount of elastin protein, which is an important component of the extracellular matrix.[17]

The clinical and prognostic importance of NOS 4a/b polymorphism has been shown previously in different clinical conditions such as thrombosis, atherosclerosis, and myocardial infarction.[3,4,22] Therefore, negative effects of nos gene polymorphism may be a risk factor for aneurysm and dissection development. Johanning et al.[18] reported that experimentally-induced a neurysms in rats were inhibited by the administration of NO inhibitors. The gene encoding NOS3 has been proposed as a predisposing gene for aortic aneurysms, and polymorphic variants of this gene affect the release and functional activity of the enzyme. Veldman et al.[19] found that Glu298Asp (G894T) polymorphism in the exon 7 region of the NOS3 gene was associated with decreased basal NO synthesis. Fatini et al.[20] reported that the NOS3 Glu298Asp (G894T) polymorphism predisposed to aortic aneurysms. On the contrary, Moon et al.[21] suggested t hat t here was no association between the Glu298Asp (G894T) polymorphism and plasma NO metabolites. Our study findings are consistent with the findings of the Moon"s study. There was no difference between the patients and healthy controls in terms of the NOS3 (G894T) polymorphism.

To the best of our knowledge, there are no studies which investigate the development of aneurysms and dissections in terms of NOS3 intron 4 polymorphism in the literature. Hence, this is the first study on this subject. In this study, NOS3 4b/4b polymorphism in the dissection group and NOS3 4b/4a polymorphism group in the aneurysm group were found to be statistically significantly higher than the control group. These findings indicate that there is a relationship between the development of aortic aneurysms/dissections and NOS3 intron 4 gene polymorphism.

Furthermore, polymorphic variants of the MTHFR gene have been found to be associated with hyperhomocysteinemia, vascular pathologies, neural tube defects, dementia, perinatal mortality, mental disorders, neurodegenerative disorders, migraine, and cancer.[22] Frosst et al.[23] reported an increased risk of aortic aneurysms in MTHFR 677T allele carriers. LaMorte et al.[24] also reported that the MTHFR gene polymorphism was highly correlated with aortic aneurysms in Caucasian males. However, in our study, we found no statistically significant difference between the control and patient groups. It can be attributed to the fact that the ethnicity is different from the reported cohorts. In a previous study, it was reported that individuals with T allele in the Turkish population are fewer than the other races.[25]

The limitations of our study are; it is a single centered study and the number of patients are relatively low.

In conclusion, our study results showed that there was an association between the NOS3 (4b/b, 4b/a) and aortic aneurysms or dissections. Nonetheless, further studies on genetic factors which play an important role in underlying mechanisms of the development of aortic aneurysms and dissections are needed.

Declaration of conflicting interests
The authors declared no conflicts of interest with respect to the authorship and/or publication of this article.

Funding
The authors received no financial support for the research and/or authorship of this article.

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