Associations between thromboxane A synthase 1 gene polymorphisms and the risk of ischemic stroke in a Chinese Han population

Lei Li, Zhi-yi He, Yan-zhe Wang, Xu Liu, Li-ying Yuan

Department of Neurology, First Affiliated Hospital of China Medical University, Shenyang, Liaoning Province, China

Introduction

Worldwide, stroke is the main cause of disability and death(Ikram et al., 2009; Malik et al., 2014). Unlike in western countries, stroke is the leading cause of death in China (Wu et al., 2001; Yang et al., 2013). In China, ischemic stroke accounts for approximately 70% of all stroke cases (Wang et al., 2012), with the incidence exhibiting substantial geographic variation. Northern China has the highest incidence(486 per 100,000 person-years), whereas Southern China has a much lower incidence (136 per 100,000 person-years) (Wu et al., 2013). Although age, hypertension, diabetes mellitus,and environmental factors are well-known risk factors for ischemic stroke, it is a multifactorial and multigenic disorder (Matarin et al., 2009; Hachiya et al., 2017). Identifying gene variants that are associated with the risk of ischemic stroke could elucidate the pathogenesis of stroke and lead to new approaches to the prevention and management of this complicated disease. Although several genome-wide association studies have assessed various polymorphisms that may contribute to ischemic stroke (Ikram et al., 2009; Yamada et al., 2009; Bellenguez et al., 2012; Holliday et al., 2012; Lee et al., 2016), the genetic variants associated with predisposition to ischemic stroke have not been unequivocally determined in Chinese individuals.Thromboxane A synthase 1 (TBXAS1) is a downstream enzyme of arachidonic acid metabolism and is the obligate enzyme required to synthesize thromboxane A2 (TXA2) (Hsu et al., 2000; Iñiguez et al., 2008). rs2267682 and rs10487667 are two intronic single-nucleotide polymorphism (SNP)variations in the TBXAS1 gene (Miyata et al., 1994; Chevalier et al., 2001) that may influence the structure and stability of TBXAS1 mRNA, thus affecting the metabolite production of TXA2 (Wang et al., 2010). It is thought that TXA2, a prothrombotic lipid mediator, is implicated in the development and thrombogenicity of atherosclerotic lesions(Ishizuka et al., 1998; Wang et al., 2001; Sellers and Stallone,2008; Calder, 2009; Kim et al., 2010). Considering that atherosclerosis is a well-known risk factor for ischemic stroke,we hypothesized there may be a relationship between these TBXAS1 SNPs and ischemic stroke.

Several studies have revealed the associations the rs2267682 and rs10487667 TBXAS1 SNPs and the risk of cardiovascular diseases in Caucasians, a Korean population and a Uyghur population in Xinjiang (Lemaitre et al., 2009;Park et al., 2009; Wang et al., 2010). However, the results are inconsistent in different diseases and ethnic populations.Currently, the association between TBXAS1 and ischemic stroke in the Chinese Han population is not known.

This study investigated the associations rs2267682 and rs10487667 and susceptibility to ischemic stroke in a Northern Chinese Han population, which has a high incidence of ischemic stroke (Liu et al., 2007).

Participants and Methods

Participants

Ischemic stroke patients were enrolled from the First Affiliated Hospital of China Medical University in Shenyang city and the First Affiliated Hospital of Liaoning Medical University in Jinzhou city of China between October 2010 and May 2011. Healthy control participants were selected from the Health Check Center at the First Affiliated Hospital of China Medical University in China during the same period.All participants were unrelated members of the Chinese Han population in northern China. This study consecutively enrolled 404 ischemic stroke patients and 340 healthy controls. The study protocol was approved by the Animal Ethics Committee of the First Affiliated Hospital of China Medical University in Shenyang city and the First Affiliated Hospital of Liaoning Medical University in Jinzhou city. Written informed consent was provided by all participants. The protocol has been registered with the Chinese Clinical Trial Registry (registration number: Chi-CTRCOC-17013559).

The inclusion criteria for the ischemic stroke group were:patients with ischemic stroke diagnosed according to clinical features and neuroimaging criteria that included the sudden onset of a non-conclusive and focal neurological de ficit with corresponding infarction on brain imaging with computed tomography or magnetic resonance imaging, echocardiography, transcranial Doppler ultrasound, or carotid duplex imaging. Magnetic resonance angiography and computed tomography angiography were performed when necessary.

The exclusion criteria for the ischemic stroke group were:patients with severe cardiac, renal or hepatic diseases and those with cancer.

The inclusion criteria for the control group were: unrelated healthy controls with no clinical or radiological evidence of stroke or cerebrovascular diseases matched with the ischemic stroke patients in terms of area of residence, ethnic origin, gender, and age.

The exclusion criteria for the control group were: patients with cancer, autoimmune disease, chronic inflammation,renal or liver insufficiency, and hematopathy.

Data collection

Demographic and risk factor information was collected using a structured questionnaire. Measurements of body weight, height, blood pressure, plasma glucose, total plasma cholesterol, triglycerides, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol are obtained.The body mass index was calculated as the body weight (kg)divided by the squared height (m2). Blood pressure measurements were acquired at least twice with the patient in the supine position after 15 minutes of rest. Hypertension was de fined by a systolic blood pressure of ≥ 140 mmHg, a diastolic blood pressure of ≥ 90 mmHg (1 mmHg = 0.133 kPa),or the use of antihypertensive medications. Diabetes mellitus was de fined by a fasting plasma glucose concentration of≥ 7.00 mM, a hemoglobin A1c content of ≥ 6.5%, or the use of antidiabetes medication. Hyperlipidemia was de fined by a total plasma cholesterol level ≥ 5.72 mM and/or a plasma triglyceride level ≥ 1.7 mM, or current use of lipid-lowering drugs.

After overall evaluation of the clinical data, patients with cardioembolic stroke, stroke of other determined aetiology,or stroke of undetermined aetiology according to the Trial of ORG 10172 in Acute Stroke Treatment (TOAST) classi fication were excluded from the analysis (Adams et al., 1993).Ischemic strokes were classi fied as either the large-artery atherosclerosis (LAA) or small-artery occlusion (SAO) subtypes.

Genotype determination

SNP ID numbers and detailed sequence information for rs2267682 and rs10487667 were obtained from the public dbSNP database (http://www.ncbi.nlm.nih.gov/SNP/). These SNPs have minor allele frequencies of at least 5% in Han Chinese.

Peripheral blood (10 mL) samples were collected from each participant, and genomic DNA was extracted using the Wizard Genomic DNA Puri fication Kit (Promega, Sunnyvale, CA, USA) according to the manufacturer’s instructions. DNA purity and quantity were assessed using absorbance values obtained using a spectrophotometer (Thermo Scientific, Waltham, MA, USA). The DNA samples were stored at −20°C until use.

Genotyping was performed using the Multiplex SNaPshot sequencing method (Bujalkova et al., 2008; Di Cristofaro et al., 2010). Genomic DNA was first amplified by multiplex polymerase chain reaction (PCR) using speci fic primers. The sequences of the primers are shown inTable 1.

The PCR reactions (10 μL/tube) consisted of 10 ng of ge-nomic template, 1 μM of each primer, 10 μL 1× GC buffer I(Takara, Otsu, Shiga, Japan), 3.0 mM Mg2+(Takara), 0.3 mM dNTPs (Generay Biotech, Shanghai, China), and 1 U Hot-Star Taq polymerase (Qiagen, Hilden, Germany). PCR was performed in duplicate at 95°C for 15 minutes and subjected to 11 cycles of 94°C for 20 seconds, 67.5°C for 40 seconds,and 72°C for 1.5 minutes. Subsequently, PCR was performed using 24 cycles of 94°C for 20 seconds, 63°C for 30 seconds,and 72°C for 110 seconds, followed by an extension at 72°C for 2 minutes. The PCR products were then characterized using SNaPshot Multiplex sequencing and GeneMapper 4.0(Applied Biosystems, Princeton, NJ, USA). Additionally, we randomly selected 10% of the positive samples for repeated genotyping to assess experimental quality, and obtained the same results.

Table 1 Primer sequences for rs2267682 and rs10487667 ampli fication

Outcome measures

Primary outcome measures

The primary outcome measures were the genotype frequencies of the two TBXAS1 SNPs, rs2267682 and rs10487667, in the patients and controls.

Secondary outcome measures

The secondary outcome measures were the allele frequencies of rs2267682 and rs10487667 in patients and controls, multivariate logistic analysis for risk factors, and haplotype analysis.

Statistical analysis

Means ± standard deviations (SD) and percentages were used to assess continuous and categorical variables, respectively. Statistical analyses were performed using SPSS 13.0 software (IBM Corporation, Armonk, NY, USA).

The allele frequencies were calculated based on the genotypes of all participants. The genotype distributions were checked for Hardy-Weinberg equilibrium using chi-square tests. The distributions of the demographic variables were examined, and the differences in risk factors between cases and controls were reassessed using Pearson’s chi-square tests and Student’s t tests. Differences in the allele and genotype frequencies of TBXAS1 SNPs between the patients and controls were evaluated with chi-square tests and with odds ratios (ORs) and 95% con fidence intervals (95% CIs), respectively, using the most common genotype as the reference group. Multivariable logistic regression analysis was used to evaluate the relationships between TBXAS1 polymorphisms and ischemic stroke by adjusting for confounding variables.

Figure 1 Test intervention flow chart.

The linkage disequilibrium index (D-prime andr2) and the inferred haplotypes of these two SNPs were performed using the SHEsis analysis platform as described previously(Shi and He, 2005; Li et al., 2009). A P value ＜ 0.05 was considered statistically signi ficant.

Results

Clinical characteristics of the participants

To determine the potential associations of TBXAS1 SNPs with the development of ischemic stroke, 404 ischemic stroke patients and 340 controls were recruited for this study at the beginning, but only 370 patients continued into the complete study (Figure 1). Twenty-six patients were excluded for cardiogenic cerebral embolisms. One patient was excluded for cerebral arteritis. Five patients were excluded for ischemic stroke caused by unknown factors. Two individuals were excluded for decline to participate in the study. This study population finally consisted of 370 patients with ischemic stroke (220 males and 150 females) and 340 healthy controls (193 males and 147 females). The general characteristics and biochemical parameters of the patients and controls are summarized inTable 2.

There were no significant differences in mean age, body mass index, or serum total cholesterol, high-density lipoprotein cholesterol, or total triglyceride levels between the patients and controls (Table 2). The risk factor pro file revealed that hypertension, diabetes, smoking, and alcohol use were common risk factors in the patients. The ischemic stroke patients also exhibited signi ficantly higher systolic and diastolic blood pressure and higher serum levels of low-density lipoprotein cholesterol and fasting plasma glucose (P ＜ 0.05).

Ischemic stroke subtype stratification revealed that the percentages of patients with hypertension and type 2 diabetes mellitus, smokers, and elevated systolic and diastolic blood pressure and serum low-density lipoprotein cholesterol and fasting plasma glucose concentrations were markedly higher in the LAA and SAO patients than in the controls. However,the percentage of alcohol consumers was only markedly increased in the SAO patients compared with that in the controls.

Table 2 Clinical characteristics of the study participants

Table 3 Genotype and allele distributions in patients with ischemic stroke and controls

Table 4 Multivariable logistic regression analysis of ischemic stroke

Table 6 Haplotype analysis of the TBXAS1 gene in ischemic stroke patients and controls

Genotype analysis

Table 3presents the genotype and allele frequencies of the TBXAS1 SNPs in the patients and controls. The genotype distributions of the two SNPs were in Hardy—Weinberg equilibrium in both patients and controls (P ＞ 0.05). As presented inTable 3, there were no signi ficant differences in the rs10487667 genotype or the allele distributions between ischemic stroke patients and controls. In contrast, signi ficant differences were observed in the distributions of the TT genotype and the T allele of rs2267682 between ischemic stroke patients and controls (P ＜ 0.01 and P = 0.02, respectively).Further analyses of the LAA or SAO subgroups revealed that the rs2267682 TT genotype frequency and T allele frequency in patients with LAA were signi ficantly greater than those in controls (P ＜ 0.01 and P = 0.02, respectively), but this difference was not observed in patients with SAO.

Multivariate logistic regression analysis was used to evaluate the relationships between the rs2267682 polymorphism and ischemic stroke. After adjustments for confounding variables, the TT genotype of rs2267682 remained significantly associated with an increased risk of ischemic stroke(OR = 1.94, 95% CI: 1.13—3.33, P = 0.02;Table 4). Similarresults were observed in patients with the LAA subtype (OR= 2.20, 95% CI : 1.16—4.18, P = 0.02;Table 5).

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Table 5 Multivariable logistic regression analysis of the LAA subtypes

Using the SHEsis program platform, our data revealed that these two TBXAS1 SNPs were in linkage disequilibrium in the Chinese population. Four haplotypes with frequencies greater than 3% among both cases and controls were included in the haplotype analysis.Table 6presents the distribution of the individual haplotypes constructed with rs2267682 and rs10487667. The overall haplotype distributions were significantly different between the cases and controls (global test, P = 0.03). The frequency of the T-G haplotype constructed with rs22676821 and rs10487667 in patients with ischemic stroke was signi ficantly greater than that in controls (OR = 1.49, 95% CI: 1.10—2.00, P ＜ 0.01).

Discussion

Ischemic stroke is a multigenic and multifactorial disease.Environmental, cultural, and genetic factors may participate in its development. Identification of the genetic factors for stroke plays an important role in the early recognition of susceptibility to stroke in people at a high risk, before symptoms are found, and early intervention. To our knowledge, this report is the first to reveal the correlation between TBXAS1 and susceptibility to ischemic stroke in a Chinese Han population.

In line with our results, Park et al. (2009) reported that the TT genotype of rs2267682 and a specific haplotype of TBXAS1 exhibit significant associations with increased susceptibility to non-cardiogenic stroke in a Korean population. However, the molecular mechanism underlying the association between this SNP with ischemic stroke remains unclear. One possible reason is the effect on TXA2 synthesis. TXA2 results from the isomerization of prostaglandin H2 (PGH2) by TBXAS1 (Wang and Kulmacz, 2002) and is formed in different cell types in the blood and vascular wall in the presence of various physiological and pathological stimuli (Needleman et al., 1976; Pyo et al., 2007; Gabrielsen et al., 2010; Muzaffar et al., 2011). TXA2 is a potent platelet activator and vasoconstrictor, and may play a key role in acute coronary syndromes and atherosclerosis (Koudstaal et al., 1993; Davi and Patrono, 2007; Calder, 2009; Borow et al.,2015). A previous study suggested that the TBXAS1 binding sites for transcriptional regulatory factors are located in in-tron 1, including the cytosine-cytosine-adenosine-adenosine-thymidine box, polyoma enhancer activator 3, cyclic adenosine monophosphate-response element, and lymphocyte function-associated antigen 1 (Miyata et al., 1994). These observations suggest that SNPs in intron 1 may influence promoter/enhancer activity. The TT genotype and T allele of rs2267682 may enhance promoter activity and lead to up-regulation of the expression of TBXAS1. The observation that the TT genotype of the TBXAS1 rs2267682 SNP increases susceptibility to ischemic stroke might be explained,at least in part, by the role of TBXAS1 in synthesizing TXA2.

In contrast, the TBXAS1 rs2267682 SNP is independent of ischemic stroke incidence, according to a recent study of white participants in North America (Lemaitre et al., 2009).Combined with the results in a Korean population (Park et al., 2009), this phenomenon might be explained at least in part by differences in race and geographical groups, and these associations could be applied to Asian populations.Moreover, the study in an American population placed more emphasis on cardiogenic stroke (Lemaitre et al., 2009).Disparate mechanisms may be critical in the development of acute ischemic coronary and cerebrovascular events (Cheng et al., 2012). This disagreement could be due to differences in study design and sample selection, as well as sample size.

Ischemic stroke is a complicated disease that is classi fied into various subtypes according to different pathogeneses.The TOAST criteria have been widely used in studies to etiologically classify acute ischemic stroke. Jerrard-Dunne et al.(2003) suggested that the strongest genetic in fluences would be detected in strokes attributable to LAA or SAO. Therefore, only patients with these two subtypes were enrolled in our study. Based on the TOAST criteria, this study separately examined the association between the two TBXAS1 SNPs and the LAA and SAO subtypes. The present study found that rs2267682 may contribute to an increased risk for the LAA subtype. This signi ficant finding remained after adjusting for potential confounding risk factors.

Recently, Gabrielsen et al. (2010) found that TBXAS1 mRNA is significantly elevated in atherosclerosis with advanced lesions in rats. TBXAS1 is expressed in human carotid atherosclerotic lesions and is related to increases in inflammatory cells, particularly M2 polarized macrophages.Furthermore, these authors discovered that TBXAS1 mRNA levels are increased in the atherosclerotic plaques of patients with recent cerebral symptoms of plaque thrombus formation compared with those of patients without symptoms (Gabrielsen et al., 2010). This research shows a correlation between TBXAS1 expression in advanced atherosclerotic lesions and plaque instability. Another study revealed that a genetic variant of TBXAS1 was associated with carotid artery or intracranial arterial stenosis, carotid plaque vulnerability, platelet activation, and TXA2 levels (Yi et al., 2016a,b, 2017). It seems reasonable that TBXAS1 and TXA2 play important roles in atherosclerosis and thrombosis. These findings might help explain why the TT genotype and T allele of rs2267682 were associated with the development of LAA but not SAO.

In the haplotype analysis, four major haplotypes of these two SNPs with frequencies greater than 5% were identified.Interestingly, compared with the control group, the ischemic stroke group exhibited a remarkably higher frequency of the T-G haplotype comprising rs22676821-rs10487667. This suggests that this haplotype may be a genetic marker for ischemic stroke in Han Chinese.

However, this study is preliminary due to the relatively small sample size, lack of measurements of TBXAS1 mRNA and protein expression, absence of TXA2 measurements, and the assessment of only two SNPs in the TBXAS1 gene. Therefore, the results require con firmation with a larger sample size and further research including genotyping, expression, and translation. If the correlation is con firmed, it is possible that these two TBXAS1 SNPs could be used to indicate the risk of ischemic stroke in Chinese Han individuals.

In conclusion, the TT genotype of TBXAS1 and the T allele of the rs2267682 SNP increase the susceptibility to ischemic stroke in a Northern Chinese Han population. Furthermore,the T-G haplotype comprising rs22676821-rs10487667 confers a genetic risk factor for ischemic stroke in this Chinese population.

Author contributions:LL conceived and designed the study, performed the experiments, analyzed the data, drafted the paper and revised it critically. LL, LYY and YZW provided reagents/materials/analysis tools. XL analyzed the data. ZYH revised this paper critically. All authors approved the final version of the paper.

Con flicts of interest:None declared.

Research ethics:The study protocol was approved by the Animal Ethics Committee of the First Affiliated Hospital of China Medical University and the First Affiliated Hospital of Liaoning Medical University in China.

Declaration of participant consent:The authors certify that they have obtained all appropriate participant consent forms. In the form, participants have given their consent for their images and other clinical information to be reported in the journal. The participants understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Data sharing statement:The datasets analyzed during the current study are available from the corresponding author on reasonable request.

Plagiarism check:Checked twice by iThenticate.

Peer review:Externally peer reviewed.

Open access statement:This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under identical terms.

Open peer reviewer:Takatoshi Hara, the Jikei University School of Medicine, Japan.

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