EDNRA Gene rs1878406 Polymorphism is Associated With Susceptibility to Large Artery Atherosclerotic Stroke

Objective: We performed this study to investigate whether the EDNRA gene rs1878406 C > T polymorphism is associated with risk of large artery atherosclerosis (LAA) stroke in the Chinese Han population. Methods: Genotyping of rs1878406 was performed in 1,112 LAA stroke patients and 1,192 healthy controls. Multivariate logistic regression analyses were applied to assess the effect of the rs1878406 C > T polymorphism on susceptibility to LAA stroke. Results: A significant increase of LAA stroke risk was found in the recessive model (TT vs. CC/TC, OR = 1.74, 95% CI = 1.23–2.48, p = 0.002) and co-dominant model (TC vs. CC, OR = 1.06, 95% CI = 0.89–1.27, TT vs. CC, OR = 1.79, 95% CI = 1.25–2.55, p = 0.006). However, the interaction between age and genotypes of rs1878406 was not statistically significant, and no significant interactive effect was observed between the rs1878406 C > T polymorphism and sex (p > 0.05). Conclusion: The rs1878406 C > T polymorphism is associated with increased risk of LAA stroke in the Chinese Han population.


INTRODUCTION
Stroke has become the second leading cause of death and the third cause of disability in humans. Among them, ischemic stroke accounts for about 80% of all strokes (GBD 2015 DALYs andHALE Collaborators, 2016), and it is a major cause of mortality and disability worldwide (Mendis et al., 2015), loading heavy economic burden especially in low-and middle-income countries (Benjamin et al., 2017). The occurrence and development of stroke are related to endothelial injury, atherosclerosis (Hankey, 2017), and traditional risk factors including age, gender, hypertension, diabetes mellitus, hyperlipidemia, and smoking. Besides traditional risk factors, some specific gene locus polymorphisms have also been confirmed to be associated with LAA stroke (Bellenguez et al., 2012;Malik et al., 2018). In recent years, genome-wide association studies (GWAS) have become an important research method for finding complex disease-related susceptibility genes, and many single-nucleotide polymorphisms (SNPs) related to LAA stroke have been discovered (Traylor et al., 2012). The interaction between gene and environmental factors has also received increasing attention.
Endothelin type A receptor (EDNRA) is a receptor for endothelin-1 (ET-1), a potent vasoconstrictor. EDNRA is expressed in vascular smooth muscle cells (Yu et al., 1995). ET-1, encoded by the EDN1 gene located in chromosome 6p21-24, is a potent vasoconstrictor in the body. ET-1 is expressed in several tissues, including endothelial cells and cardiomyocytes (Brunner et al., 2006). After ET-1 being bounded firmly to EDNRA, it activates calcium channels and phospholipase activation pathway to play a long-lasting vasoconstriction effect (Kampoli et al., 2011). It can also promote the proliferation and migration of vascular smooth muscle cells, increasing the ratio of neointima and media, stimulating the production of cytokines and growth factors, and inducing the formation of extracellular matrix and fibers. Hyperplasia is involved in the occurrence and development of atherosclerosis through many ways (Dai and Dai, 2010;Li et al., 2015a). GWAS have identified that SNP rs1878406 C > T polymorphism in EDNRA gene is related to carotid plaque (Bis et al., 2011). A following study also associated rs1878406 with coronary artery disease (Hemerich et al., 2015). Therefore, we performed this study to investigate the association between the rs1878406 C > T polymorphism and LAA stroke risk in the Chinese Han population and test whether age or gender interacts rs1878406 to influence LAA stroke risk.

Study Subjects
We consecutively recruited first-ever LAA stroke patients between January 2015 and December 2019 from the first hospital of Hangzhou Neurology Department. Ischemic stroke was defined as sudden focal neurological deficits lasting ≥24 h, with evidence of cerebral infarction in clinically relevant areas of the brain confirmed by computed tomography (CT) and/or magnetic resonance imaging (MRI). Patients were enrolled if they (1) were 18 years or older; (2) were of Chinese Han ethnicity; (3) had available blood samples; and (4) diagnosed with LAA stroke according to TOAST classification (Adams et al., 1993). Exclusion criteria included severe liver or kidney dysfunction, hematological diseases, malignancies, and autoimmune disorders. The non-stroke controls were recruited from local inhabitants during the same period and the inclusion criteria were as follows: (1) aged 18 years or older; (2) Chinese Han ethnicity; (3) had regular physical examination; (4) no history of transient ischemic attacks, cerebrovascular, cardiovascular, and atherosclerotic diseases; and (5) no history of severe liver or kidney dysfunction, hematological diseases, malignancies, and autoimmune disorders. The study was approved by the Ethical Review Board of the First Hospital of Hangzhou (Hangzhou, China).

Data Collection
Baseline characteristics included demographic information (age, gender, height, and weight), medical histories (hypertension, diabetes mellitus, hyperlipidemia, and smoking), and laboratory tests. Smokers were those who had smoked ≥100 cigarettes during their lifetime and currently smoke every day or some days (Loke et al., 2014). Routine laboratory investigations were performed after overnight fasting within 24 h of admission. The definition of hyperlipidemia is serum triglyceride ≥150 mg/dl, low-density lipoprotein cholesterol ≥130 mg/dl, high-density lipoprotein cholesterol ≤40 mg/dl in adult males and ≤50 mg/dl in adult females, any use of lipid-lowering drugs, or any self-reported history of hyperlipidemia (Wang et al., 2015).

Genotyping
All patients took 5 ml of fasting peripheral venous blood in the early morning and placed it in an EDTA anticoagulation tube. After centrifugation, the lower layer of blood cells was removed and stored in a refrigerator at −80°C. Genotyping was conducted by SNPscan technology, supported by the Center for Human Genetics Research, Genesky Biotechnology Co. Ltd. (Shanghai, China). About 5% of the samples were randomly selected and genotyped repeatedly to confirm the genotyping credibility, and the results were 100% consistent.

Statistical Analyses
Statistical analyses were performed with SPSS Statistics Version 22.0 (IBM, Armonk, NY, United States). Student's t-test was conducted for continuous variable. Categorical data were expressed as frequency and percentage. Categorical variables were compared using Chi-square test. The mean age of LAA stroke onset for three polymorphisms was compared using oneway ANOVA. Univariate and multivariate logistic regression analyses were performed to investigate the association of the rs1878406 C > T polymorphism with risk of LAA stroke. Akaike information criterion (AIC) was calculated for selecting the best model for the SNP. All statistical tests were two-sided and p < 0.05 was considered statistically significant.

Baseline Characteristics of the Subjects
A total of 1,112 LAA stroke patients and 1,192 healthy controls were recruited in this study. Baseline clinical and demographic characteristics are shown in Table 1. Compared with the controls, patients were older (p < 0.001) and had a higher proportion of male and traditional risk factors for ischemic stroke, such as history of hypertension, diabetes mellitus, hyperlipidemia, and smoking (p < 0.001).

Association of rs1878406 Polymorphism With Age of Onset of LAA Stroke
We first evaluated the correlation between the rs1878406 polymorphism and the age of onset of LAA stroke. As shown in Figure 1, these three polymorphisms were not significantly related to the age at onset of LAA stroke (p > 0.05).

Association of rs1878406 C > T Polymorphism With the Risk of LAA Stroke
The frequencies of CC, TC, and TT genotypes were 60.1%, 35.3%, and 4.5%, among the LAA stroke patients, respectively, and 56.8%, 35.5%, and 7.6%, among the controls, respectively. According to the AIC values, the recessive model is the bestfitting model. In the recessive model, compared with CC and TC genotype, TT genotype of the rs1878406 was associated with significantly increased risk of LAA   >60 years, however, no significant association was found. In the stratified analysis by sex, the T allele of rs1878406 significantly increased the risk of LAA stroke in males (CC/CT vs. TT, OR 1.89, 95% CI 1.14-3.11, p 0.011; Table 4). In females, however, we did not detect any significant association of rs1878406 with risk of LAA stroke. The results suggested potential interactions between rs1878406 polymorphism, age, and sex in the etiology of LAA stroke. However, the interaction between age and genotypes of rs1878406 was not statistically significant (p interaction 0.442; Figure 2A). Similarly, there was no statistical evidence for interaction between these rs1878406 polymorphism and sex on the risk of LAA stroke (p interaction 0.553; Figure 2B).

Functional Annotation
Bioinformatics analysis by HaploReg v4.1 indicated that rs1878406 could change the binding affinity of regulatory motifs Myf_4 (Table 5).  FIGURE 2 | (A) Adjusted OR of LAA stroke according to rs1878406 and age; the interaction between rs1878406 and age was non-significant (p interaction 0.442).
(B) Adjusted OR of LAA stroke according to rs1878406 and sex; the interaction between rs1878406 and sex was non-significant (p interaction 0.553).

DISCUSSION
We confirmed that age, blood pressure, diabetes, hypertension, hyperlipidemia, and smoking were independent risk factors for cerebral infarction by comparing the general data of atherosclerotic cerebral infarction with those of normal controls, which were consistent with the results of previous studies. After adjusting for factors such as gender, age, and hypertension under the recessive model, it was shown that TT genotype at rs1878406 was a risk factor for susceptibility to cerebral infarction as compared with the CC/TC genotype. Under the recessive model, the interaction between age or gender and genotypes of rs1878406 C > T polymorphism was not statistically significant. Altogether, it is found that the T allele at EDNRA rs1878406 is a risk factor for cerebral infarction.
The EDNRA gene encodes ET-1 receptor A, which plays an important role in effective and durable vasoconstriction (Dai and Dai, 2010;Li et al., 2015a), and is widely distributed in the cardiovascular and central nervous system. Encoded by EDN1, ET-1 is a potent vasoconstrictor that is expressed in a variety of tissues, including endothelial cells and cardiomyocytes (Brunner et al., 2006). ET-1 is closely related to cardiovascular and cerebrovascular diseases, and endothelial dysfunction can be found in hypertension, atherosclerosis, diabetes, hyperlipidemia, and cerebrovascular spasm, with increased ET release (Balletshofer et al., 2000). In 1992, Zir et al. (Balletshofer et al., 2000) first confirmed the elevated plasma ET-1 level in patients with acute ischemic cerebral infarction. In recent years, the relationship between ET-1 and ischemic cerebrovascular disease has become a hot topic in basic and clinical medicine. ET-1 mediates endothelial dysfunction mainly by increasing fibroblasts and macrophages. Endothelial damage greatly enhances the sensitivity of blood vessels to ET-1, causing durable contraction of local blood vessels, and ischemia and hypoxia of brain tissue. In addition to its effect on blood vessels, ET-1 can also directly act on nerve cells, causing cell death, accelerating the death of neurons in the hypoxia area by promoting the release of excitatory amino acids (Lin et al., 1990). Studies have shown that ET-1 can bind to its receptor and activate the voltage-sensitive L-type calcium channel, causing extracellular calcium influx and intracellular calcium overload, thus aggravating the damage of nerve cells (Marsden et al., 1989). All the above lines of evidence provide us with the theoretical basis that the rs1878406 variant in EDNRA may be related to cerebral infarction.
Multiple previous studies have shown that the rs1878406 variant is associated with atherosclerosis and endothelial dysfunction. For example, in the 2011 meta-analysis by Bis et al. (2011), it was found that the C allele of rs1878406 was associated with lower risk of plaque. However, the T allele was associated with a 22% increased odds of the presence of plaque. These associations may provide important insights into the pathophysiological mechanisms relating the genes to atherosclerosis and subsequent artery disease. Another study (Li et al., 2015b) explored the potential relationship between EDNRA rs1878406 polymorphisms and the carotid intima-media thickness (IMT) levels, but no statistically significant differences were found when this polymorphism was assessed according to carotid IMT values. Instead, they identified a significant interaction of gender with this variant rs1878406 in the EDNRA gene. For gene-gender interaction on common carotid arteries IMT, the adjusted mean for men carrying the GA/GG genotype of EDNRA SNP rs1878406 was 1.18 times higher than that for men carrying the AA genotype. This may be due to the fact that the sample size of their study was relatively small. Zhang et al. (2017) found in their analysis of the Han population that the rs1878406 TT/TC genotype could be a significant risk factor for severe multiple coronary artery lesions. Moreover, our results indicated that rs1878406 could change the binding affinity of regulatory motifs Myf_4. The above results of previous studies are consistent with our studies, suggesting that the C→T mutation in the EDNRA gene rs1878406 may influence the function of ET-1, thereby affecting the occurrence of atherosclerosis.
Our study has some limitations. First of all, we only analyzed the Han population, which is not representative of other ethnic groups. More international institutions should further combine for sample collection, so that possible geographical and ethnic differences can be compared in the future. Secondly, the sample size of this study is relatively small, and further larger sample study is required. In addition, there is a lack of in-depth study on the related mechanism of rs1878406 polymorphism increasing the risk of cerebral infarction, and further functional verification can be conducted, including in vivo and in vitro experiments.
In conclusion, the results of this study suggest that individuals carrying the EDNRA rs1878406 TT genotype may be a risk factor against cerebral infarction. However, to explore the relationship between rs1878406 and pre-cerebral infarction, we need to conduct joint analysis on the basis of the largest sample size as possible, so as to provide a reliable theoretical basis for the early prevention and treatment of cerebral infarction.

DATA AVAILABILITY STATEMENT
The data presented in the study are deposited in the EMBL-EBI repository, accession number PRJEB48922.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the Affiliated Hangzhou ninth People's Hospital, Zhejiang University School of Medicine. The patients/ participants provided their written informed consent to participate in this study.

AUTHOR CONTRIBUTIONS
WW and XX contributed to conception and design of the study. WW, JZ, TC, YF, JD, DJ, and GZ organized the database. BT and XH performed the statistical analysis. WW wrote the first draft of the manuscript. WW and XH wrote the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.