Association of G-protein coupled purinergic receptor P2Y2 with ischemic stroke in a Han Chinese population of North China

Li-Ying Yuan, Zhi-Yi He, Lei Li, Yan-Zhe Wang

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

AbstractThe G-protein-coupled purinergic receptor P2Y2 (P2RY2) plays an important role in the mechanism of atherosclerosis, which is relevant to ischemic stroke. This retrospective case-control study aimed to assess the relationship between P2RY2 gene polymorphisms and ischemic stroke risk in the northern Han Chinese population. In this study, clinical data and peripheral blood specimens were collected from 378 ischemic stroke patients and 344 controls. The ischemic stroke participants were recruited from the First Affiiliated Hospital of China Medical University and the First Affiiliated Hospital of Liaoning Medical University. The controls were recruited from the Health Check Center at the First Affiiliated Hospital of China Medical University. Ischemic stroke patients were divided into two subgroups according to the Trial of ORG 10172 in Acute Stroke Treatment (TOAST) classification: large-artery atherosclerosis (n = 178) and small-artery occlusion (n =200) strokes. All subjects were genotyped for three single nucleotide polymorphisms (rs4944831, rs1783596, and rs4944832) in the P2RY2 gene using peripheral venous blood samples. The distribution of the dominant rs4944832 phenotype (GG vs. GA+AA) differed significantly between small-artery occlusion patients and control subjects (odds ratio (OR) = 1.720, 95% confidence interval (CI): 1.203-2.458, P ＜ 0.01).Multivariable logistic regression analysis revealed that the GG genotype of rs4944832 was significantly more prevalent in small-artery occlusion patients than in control subjects (OR = 1.807, 95% CI: 1.215-2.687, P ＜ 0.01). The overall distribution of the haplotype established by rs4944831-rs1783596-rs4944832 was significantly different between ischemic stroke patients and controls (P ＜ 0.01). In ischemic stroke patients, the frequency of the G-C-G haplotype was significantly higher than in control subjects (P = 0.028), whereas the frequency of the T-C-A haplotype was lower than in control subjects (P = 0.047). These results indicate that the G-C-G haplotype of P2RY2 is a susceptibility haplotype for ischemic stroke. In addition, the GG genotype of rs4944832 may be associated with the development of small-artery occlusion in the northern Han Chinese population. The study protocol was approved by the Ethics Committee of the First Affiiliated Hospital of China Medical University on February 20, 2012 (No. 2012-38-1) and the First Affiiliated Hospital of Liaoning Medical University,China, on March 1, 2013 (No. 2013-03-1). All participants gave their informed consent. This trial was registered with the ISRCTN Registry(ISRCTN11439124) on October 24, 2018. Protocol version (1.0).

Key Words: nerve regeneration; P2RY2 gene; ischemic stroke; single nucleotide polymorphism; case-control study; haplotype; northern Han Chinese population; large-artery atherosclerosis; small-artery occlusion; hypertension; candidate gene; neural regeneration

Introduction

Ischemic stroke is a leading cause of disability and death,both in China and worldwide. The incidence of ischemic stroke is higher in northern China (486 per 100,000 person-years) than elsewhere (Wu et al., 2013). The Han ethnicity is the main ethnic group in northern China. Many previous studies have confirmed a relationship between various genetic polymorphisms and the incidence of ischemic stroke in northern Han Chinese (Li et al., 2018b; Ye et al., 2018).Some studies have demonstrated that many polymorphisms in candidate genes are associated with atherosclerosis and hypertension in certain populations, and these studies have contributed to the identification of ischemic stroke susceptibility loci (Kaneko et al., 2006; Naganuma et al., 2009; Wang et al., 2009, 2010; Stepanyan et al., 2018).

A total of eight P2Y receptor subtypes (P2Y1, P2Y2,P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14) have been identified (Webb et al., 1996; Von et al., 2000). Dasari et al. (1996) first mapped the human P2RY2 gene to chromosome 11q13.5-q14.1. P2RY2 plays an important role in the progression of hypertension and atherosclerosis, and it is involved in platelet aggregation, coagulation, and inflammation (Gachet et al., 2006). Furthermore, P2RY2 has been implicated in the development of vascular diseases (Ralevic et al., 1991; Di et al., 2002; Dissmore et al., 2016). The role of P2RY2 in endothelial dysfunction has been reported in many studies, which have also shown that this receptor plays an important role in the mechanism of atherosclerosis,which is related to ischemic stroke (Seye et al., 2004, 2006;Guns et al., 2006). Apolipoprotein E null (ApoE-/-) mice deficient in P2Y2R exhibit low VCAM-1 levels in endothelial cells and have a delayed onset of atherosclerosis; the results of this study suggest that targeting this nucleotide receptor may be a new therapeutic approach for atherosclerosis (Qian et al., 2016).

Ischemic stroke is known to be a multifactorial and multigenic disorder (Gao et al., 2017; Hachiya et al., 2017; Li et al., 2018a, c). Many studies have attempted to identify gene variants that are associated with the risk of ischemic stroke(Wang et al., 2009; Matsushita et al., 2010). Although a previous study revealed some P2RY2 polymorphisms that may contribute to ischemic stroke (Wang et al., 2009), no studies have yet examined the association between human P2RY2 gene polymorphisms and ischemic stroke in the northern Han Chinese population. We performed this case-control study to investigate the association between P2RY2 single nucleotide polymorphisms (SNPs: rs4944831, rs1783596,and rs4944832) and the incidence of ischemic stroke in the northern Han Chinese population. In addition, we divided the ischemic stroke subjects into two subgroups, large-artery atherosclerosis (LAA) and small-artery occlusion (SAO),according to the Trial of ORG 10172 in Acute Stroke Treatment (TOAST) classification. This study aimed to confirm the relationships between ischemic stroke of different etiologies and gene polymorphisms in the northern Han Chinese population.

Participants and Methods

Participants

This retrospective case-control study was carried out using 378 ischemic stroke patients (178 with LAA, 200 with SAO)and 344 unrelated healthy controls. All subjects were Han Chinese individuals from Liaoning Province, China. The ischemic stroke patients were enrolled from the First Affiiliated Hospital of China Medical University, Shenyang, and the First Affiliated Hospital of Liaoning Medical University, Jinzhou, from September 1, 2010 to May 30, 2011. The healthy controls were enrolled from the Health Check Center of the First Affiliated Hospital during the same period.The study protocol was approved by the Ethics Committee of the First Affiiliated Hospital of China Medical University on February 20, 2012 (No. 2012-38-1) and the First Affiliated Hospital of Liaoning Medical University in China on March 1, 2013 (No. 2013-03-1) (Additional file 1). This study was performed in strict accordance with the Declaration of Helsinki developed by the World Medical Association. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement (Additional file 2). All participants provided written informed consent (Additional file 3).

Inclusion criteria for the ischemic stroke group

According to the TOAST classification, patients in the ischemic stroke group were diagnosed depending on clinical features and neuroimaging criteria that included the sudden onset of a global or focal neurological deficit, with corresponding brain infarction as seen on computed tomography(CT) imaging or magnetic resonance imaging (MRI) (Adams et al., 1993). Examinations of cerebral blood vessels, such as computed tomography angiography and magnetic resonance angiography, were performed when necessary.

Exclusion criteria for the ischemic stroke group

Patients were excluded if they had cancer, hemorrhagic stroke, transient ischemic attack, or severe cardiac, hepatic,or renal diseases.

Inclusion criteria for the control group

Unrelated healthy controls presented no clinical or radiological evidence of stroke or cerebrovascular diseases and were matched with ischemic stroke patients in sex, age, ethnic origin, and area of residence. Some healthy controls had vascular risk factors such as hypertension, diabetes, smoking,drinking, and hypercholesterolemia.

Exclusion criteria for control subjects

Control subjects were excluded if they had cancer, autoimmune disease, chronic inflammation, renal insufficiency,liver insuffiiciency, or hematopathy.

Data collection

All participant interviews were administered by our investigators, who followed a Chinese structured questionnaire of patients (Additional file 4). Any history of vascular risk factors, such as smoking, drinking, hypertension, and diabetes, was recorded. Body weight, height, blood pressure, fasting plasma glucose, total plasma cholesterol, triglycerides,low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) measures were also measured. To obtain body mass index (BMI) scores, body weight (kg) was divided by height squared (m2).

After the overall collection and evaluation of the clinical data, ischemic stroke patients with cardioembolic stroke,stroke of other determined etiology, or stroke of undetermined etiology according to TOAST classification, were excluded from the analysis (Adams et al., 1993). In our study,ischemic stroke patients included two subgroups: LAA and SAO. LAA patients had clinical and brain imaging findings of either significant (＞ 50%) stenosis or occlusion of a major brain artery or branch cortical artery, presumably due to atherosclerosis. The SAO subgroup included patients whose strokes were often labeled lacunar infarcts in other classifications. The patients had a normal CT/MRI examination or a relevant brainstem or subcortical hemispheric lesion with a diameter of less than 1.5 cm.

Polymorphism genotyping

Three P2RY2 SNPs (rs4944831, rs1783596, and rs4944832)were obtained from the public dbSNP database (http://www.ncbi.nlm.nih.gov/SNP). All three SNPs represent minor allele frequencies of at least 5% in the Han Chinese population. SNP1 (rs4944831) is located in intron 1, SNP2(rs1783596) is located in the coding regions of exon 3, and SNP3 (rs4944832) is located in the 3 flanking region of the P2RY2 gene.

Peripheral blood samples were collected from the median cubital vein of each subject. Genomic DNA was extracted from peripheral blood leukocytes using a DNA extraction kit(Wizard Genomic DNA purification kit; Promega, Sunnyvale, CA, USA) and quantified with a spectrophotometer(Thermo Scientific, Waltham, MA, USA). All DNA samples were stored at -20°C (Nakayama et al., 2001).

Genotyping of P2RY2 SNPs was performed using Multiplex SNaPshot sequencing. Specifically, genomic DNA was first amplified by multiplex polymerase chain reaction (PCR)using the primers shown in Table 1.

Table 1 Primer sequences for P2RY2 gene variants rs4944831,rs1783596, and rs4944832 amplification

PCR samples (10 μL/tube) consisted of 10 ng genomic template, 1 μM forward primer, 1 μM reverse primer, 10 μL of 1× GC buffer I (Takara, Otsu, Shiga, Japan), 3 mM Mg2+(Takara), 0.3 mM dNTPs (Generay Biotech, Shanghai, China), and 1 U of HotStar Taq polymerase (Qiagen, Hilden,Germany). PCR was performed in duplicate. The cycling conditions consisted of 95°C for 15 minutes; 11 cycles of 94°C for 20 seconds, 67.5°C for 40 seconds, and 72°C for 90 seconds; 24 cycles of 94°C for 20 seconds, 63°C for 30 seconds, and 72°C for 110 seconds; and an extension step at 72°C for 120 seconds.

PCR products were characterized via SNaPshot Multiplex sequencing and GeneMapper 4.0 (Applied Biosystems,Princeton, NJ, USA). Following characterization, 10% of positive samples were randomly selected for repeated genotyping to assess the experimental quality, and the same results were obtained.

Primary outcome measures

The primary outcome measures of this study were the genotype frequencies of the three P2RY2 SNPs (rs4944831,rs1783596, and rs4944832) in ischemic stroke patients and controls.

Secondary outcome measures

Secondary outcome measures were as follows:

(1) Allele frequencies of the three P2RY2 SNPs (rs4944831,rs1783596, and rs4944832) in ischemic stroke patients and controls.

(2) Inferred haplotypes of these three P2RY2 SNPs(rs4944831, rs1783596, and rs4944832).

Statistical analysis

Statistical analyses were performed using SPSS 19.0 software(IBM Corporation, Armonk, NY, USA). Continuous and categorical data are presented as the mean ± SD and percentages, respectively. Differences in vascular risk factors between ischemic stroke patients and healthy controls were assessed using Pearson's chi-squared test and Student's t test. The allele frequencies of the P2RY2 SNPs were calculated based on the genotypes of all participants. Differences in the genotype and allele frequencies of P2RY2 SNPs between ischemic stroke patients and control subjects were evaluated with the chi-squared test and reported as odds ratios (ORs)and 95% confidence intervals (95% CIs). The Hardy-Weinberg equilibrium was used to examine genotype distributions. Multivariable logistic regression analysis was selected to assess the relationships between P2RY2 polymorphisms and ischemic stroke by adjusting for confounding variables.

The linkage disequilibrium index (D-prime and r2) and the inferred haplotypes of these three SNPs (rs4944831,rs1783596, and rs4944832) were assessed using the SHEsis analysis platform (http://analysis.bio-x.cn/myAnalysis.php)(Shi and He, 2005). A difference was considered statistically significant if P ＜ 0.05.

Results

Clinical characteristics of study subjects

A total of 378 ischemic stroke patients (227 males and 151 females) and 344 control subjects (208 males and 136 females) were enrolled to determine the potential association of P2RY2 SNPs with ischemic stroke risk. There were no significant differences in age, BMI, or serum HDL-C lev-els between ischemic stroke patients and control subjects.However, the percentages of individuals with hypertension,diabetes, current smoking status, or current alcohol consumption were higher in ischemic stroke patients (LAA:178, SAO: 200) than in controls (Table 2). Levels of serum fasting plasma glucose, total cholesterol, and LDL-C were also higher in ischemic stroke patients than in controls.

Genotype analysis

The genotype distributions of the three SNPs (rs4944831,rs1783596, and rs4944832) were in Hardy-Weinberg equilibrium in both patients and controls (P ＞ 0.05). There was no significant difference in the distributions of each SNP between ischemic stroke patients and controls (P ＞ 0.05).The genotype and allele frequencies of the P2RY2 SNPs in ischemic stroke patients (including in the LAA and SAO subgroups) and controls are shown in Table 3.

There were no significant differences in the rs4944831 and rs1783596 genotypes or allele distributions between ischemic stroke patients and controls. In contrast, there were significant differences in the distributions of the GG genotype and of the G allele of rs4944832 between SAO patients and control subjects (P ＜ 0.01). However, this difference was not observed in patients with LAA. Multivariable logistic regression analysis was used to assess the relationship between the rs4944832 polymorphism and SAO by adjusting for confounding variables. The GG genotype of rs4944832 remained significantly associated with an increased risk of SAO (OR = 1.807, 95% CI: 1.215-2.687, P ＜ 0.01; Table 4).

Using the SHEsis program platform, these P2RY2 SNPs(rs4944831, rs1783596, and rs4944832) were found to be in linkage disequilibrium in the Chinese population. Six haplotypes with frequencies greater than 3% among both ischemic stroke patients and control subjects were selected in the haplotype analysis. Table 5 presents the distribution of the individual haplotypes constructed with rs4944831-rs1783596-rs4944832. The overall haplotype distributions were significantly different between the ischemic stroke patients and controls (global test, P ＜ 0.01). The frequency of the G-C-G haplotype constructed with rs4944831-rs1783596-rs4944832 in ischemic stroke patients was significantly higher than that in controls (OR = 1.772, 95% CI:1.057-2.971, P = 0.028), while the frequency of the T-C-A haplotype in ischemic stroke patients was lower than that in control subjects (OR = 0.754, 95% CI: 0.570-0.997, P =0.047).

Discussion

P2RY2 belongs to a family of G-protein coupled receptors and participates in various physiological responses upon adenosine triphosphate binding and releasing; these responses include shifts in membrane potential, secretion, platelet aggregation, and neurotransmission (Dubyak et al., 1993; Parr et al., 1994; Foresta et al., 1996; Kunapuli et al., 1998; Kunzelmann et al., 2005; Rieg et al., 2007; Orriss et al., 2017).

The P2RY2 gene is considered to be critical in the mechanism of atherosclerosis and hypertension (Seye et al., 2002,2003; Wang et al., 2010; Dissmore et al., 2016). Marrelli et al. (1999) found that P2Y2 receptors have a significant influence on middle cerebral artery dilation after ischemia reperfusion in rats. P2RY2 also has a significant effect on blood pressure regulation in response to adenosine triphosphate binding (Von et al., 2000; Ruppert et al., 2003). In addition,recent studies have reported that endothelial cell-specific P2Y2R deficiency promotes plaque stability in ApoE-/-mice(Chen et al., 2017). The P2RY2 gene may also play a causative role in hypertension (Rieg et al., 2007, 2011; Erlinge et al., 2008; Wang et al., 2015) and myocardial infarction (Wihlborg et al., 2006). In a recent study in Japan, rs4944831 and the T-A-G haplotype of the P2RY2 gene were geneticmarkers for essential hypertension (Wang et al., 2010).These studies show that the P2RY2 gene has a strong association with ischemic stroke.

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

Our haplotype-based case-control study aimed to assess the association between the P2RY2 gene and ischemic stroke in the northern Han Chinese population. The ischemic stroke group was also divided into two subgroups for further analysis. The results revealed that the genotypic distribution of the G allele of rs4944832 and the dominant model (GG vs. GA+AA) of rs4944832 significantly differed between SAO stroke patients and control subjects. Logistic regression analyses showed that SAO stroke patients had a significantly different GG genotype distribution of rs4944832 compared with control subjects. This difference illustrates that the rs4944832 SNP is most likely associated with SAO stroke in the northern Han Chinese population.

Haplotype-based analyses have several well-known advantages over those based on individual SNPs (Morris et al.,2002). In this haplotype-based case-control study, we established P2RY2 haplotypes from different combinations of the selected SNPs (rs4944831, rs1783596, and rs4944832). The haplotype analysis showed that the overall haplotype distri-bution (rs4944831-rs1783596-rs4944832) significantly differed between ischemic stroke patients and control subjects(P ＜ 0.01). Significant differences were also observed in the frequency of the G-C-G haplotype between ischemic stroke patients and control subjects. The G-C-G haplotype can be regarded as a susceptibility haplotype for ischemic stroke in northern Han Chinese individuals, because this haplotype was significantly more common among ischemic stroke patients in our study. Furthermore, the frequency of the T-C-A haplotype was lower for ischemic stroke patients than for control subjects; this was therefore regarded as a resistant haplotype for ischemic stroke in the northern Han Chinese population.

Table 4 Multivariable logistic regression analysis of the small-artery occlusion stroke subtype

In summary, the GG genotype of P2RY2 and the G allele of the rs4944832 SNP increase susceptibility to SAO stroke in the northern Han Chinese population. The G-C-G haplotype of the P2RY2 gene may be a susceptibility haplotype for ischemic stroke in this population. The results of this study also suggest that the T-C-A haplotype is a resistance marker for ischemic stroke.

No previous studies have linked the P2RY2 gene to the risk of SAO; they may be linked via the mechanism by which P2RY2 increases the risk of primary hypertension. However,our study has a relatively small sample size. In the future,further studies in different ethnic groups should be carried out using larger samples to further investigate detailed genotyping and to isolate the functional mutations in the P2RY2 gene that can influence the occurrence of ischemic stroke.

Author contributions:Study design, experiment implement, data analysis, paper drafting and revision: LYY; provision of reagents/materials/analysis tools: LL and YZW; data analysis: LL; paper revision: ZYH. All authors approved the final version of the paper.

Financial support:This study was supported by the National Natural Science Foundation of China, No. 81070913 (to ZYH). The funding body played no role in the study design, in the collection, analysis and interpretation of data, in the writing of the paper, and in the decision to submit the paper for publication.

Conflicts of interest:The authors have no conflicts of interest to declare.

Institutional review board statement:The study protocol was approved by the Ethics Committee of the First Affiiliated Hospital of China Medical University on February 20, 2012 (No. 2012-38-1) and the First Affiiliated Hospital of Liaoning Medical University in China on March 1, 2013 (No.2013-03-1). The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a prior approval by the institution's human research committee.

Table 5 An rs4944831-rs1783596-rs4944832 haplotype analysis[n (%)] of the P2RY2 gene in ischemic stroke patients and controls

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

Reporting statement:This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement.

Biostatistics statement:The statistical methods of this study were reviewed by the biostatistician of First Affiiliated Hospital of China Medical University, China.

Copyright license agreement:The Copyright License Agreement has been signed by all authors before publication.

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

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Peer review:Externally peer reviewed.

Open access statement:This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-Non-Commercial-ShareAlike 4.0 License, which allows others to remix,tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.

Open peer reviewer:Zhongwu Liu, Henry Ford Hospital, USA.

Additional files:

Additional file 1:Ethical examination and approval document (Chinese).

Additional file 2: STROBE checklist.

Additional file 3: Informed consent (Chinese).

Additional file 4: Chinese structured questionnaire of patients (Chinese).

Additional file 5: Open peer review report 1.