This study found that ischemic stroke and other vascular events were common in subjects with the high-risk stroke population during the 4.7-year follow-up. SNPs in TNF rs3093662, IL6Rrs4845625, and TLR4 rs752998 were associated with outcomes. Furthermore, we observed a significant interaction among IL6R rs4845625, TLR4 rs1927911, and HABP2 rs932650. The high-risk interactive genotypes in the three SNPs were independently associated with a higher risk for ischemic stroke and a composite of vascular events during the 4.7-year follow-up.
The incidence of stroke in China increased steadily in the past decade, and the conventional risk factors for stroke demonstrated substantial contributions to the risk of stroke [1, 3, 16]. Chinese stroke or recurrence risk factors include high sodium intake, low fruit and vegetable intake in diets, smoking, diabetes, dyslipidemia, hypertension, physical inactivity, and overweight [3, 16]. Developing evidence-based intervention strategies for these risk factors could help reduce the incidence of stroke. The administration of anti-hypertensives, lipid-lowering drugs, hypoglycemics, and anti-thrombotic agents in managing stroke has reduced the incidence of stroke and other vascular events [16]. However, treatment and standard-reaching rates for these risk factors are lower in China than in high-income countries [20]. This study revealed that a previous history of stroke was found to be independently correlated with an increased chance of experiencing new ischemic stroke and a combination of vascular events. This finding is consistent with prior research [16, 19]. However, the study did not uncover any correlations between other conventional risk variables and the occurrence of new ischemic stroke or composite vascular events. The reason may be due to differences in study design. This study focused on the high-risk stroke population with at least three of the eight risk factors mentioned above.
Although genetic etiologies for stroke remain unclear, few gene variants have been identified as associations with stroke [8, 9], and genetic predisposition may play a critical role in the pathogenesis of stroke [21]. Atherosclerosis, a chronic inflammatory disease, may increase the risk of stroke and other cardiovascular events because of luminal stenosis or plaque rupture [11, 12]. Inflammation and endothelial injury play essential roles in the pathogenesis of atherosclerosis. Gardener et al. [13] were the first to reveal the association of inflammation-related gene variants and endothelial function with carotid plaque in the Northern Manhattan populations. Subsequently, our studies also demonstrated that specific variants in genes involved in inflammation and endothelial function were associated with carotid plaque and carotid stenosis in high-risk stroke populations [14, 15]. Nevertheless, in high-risk stroke populations, relatively few studies examine the relationship between SNPs in these genes and stroke and other vascular events during follow-up.
Several case-control studies explored the effect of SNPs in inflammatory and endothelial function-relevant genes on ischemic stroke. They found that SNPs in genes related to inflammation (CRP rs1205, NOS3 rs1800779, and rs2257073) and 1 SNP in gene-related to endothelial function (HABP2rs11196288) were strongly associated with ischemic stroke [22, 23]. In this study, we observed significant differences in the genotype distribution of TNF rs3093662, IL6Rrs4845625, and TLR4 rs752998 between subjects with and without outcomes by univariate analyses.
Tumor necrosis factor (TNF) in plasma may serve as a risk factor for ischemic stroke and the biomarker for the patient survival rate [24]. The TNF gene is responsible for encoding a cytokine belonging to the TNF ligand family, which is crucial in regulating the adherence of activated T lymphocytes to endothelial cells. The findings of a meta-analysis revealed that TNF-α polymorphism was correlated with elevated levels of TNF-α in the plasma, indicating an increased susceptibility to ischemic stroke [25]. Interleukin-6 (IL-6) as a cytokine plays a vital role in the "response to injury" model of endothelial cells[26]. The IL-6 receptor (IL6R) gene is located in chromosome 1q21 and regulates IL-6 level. Huang et al. [27] found an association between SNPs in IL6R and ischemic stroke in patients with metabolic syndrome. A study from Korea demonstrated polymorphism of IL-6R was associated with the neurologic status of patients with ischemic stroke [28]. Toll-like receptor 4 (TLR4) is essential in oxidative stress, inflammation, and cardiovascular diseases [29]. The TLR4 gene contributes to the innate immune response. The association between TLR4 polymorphism and myocardial infarction risk has been reported, but no association with ischemic stroke has been observed [30]. This study discovered an association between TLR4 polymorphism and outcome during follow-up. Previous studies investigating associations between variants in genes related to inflammation and endothelial function and ischemic stroke reported inconsistent results [23, 31]. Cheng et al. [23] reported polymorphism of HABP2 and PPARA were significantly associated with ischemic stroke, and polymorphism of C807T in ITGA2 increased the risk of ischemic stroke [31]. However, our current and other studies did not find the associations [5–7, 30]. The differences in findings may be attributable to the diverse genetic backgrounds of the subject populations, as genetic polymorphisms vary considerably across ethnic groups.
Furthermore, stroke as a complex disease does not follow the Mendelian pattern of inheritance [4]. The potential exists for an interaction between a specific gene variant and other gene variants [8, 9]. Therefore, linkage analysis, commonly employed to assess single-gene disorders, is unsuitable for determining the genetic cause of stroke [10].
The most noteworthy finding in this study was a significant interaction among IL6R rs4845625, TLR4 rs1927911, and HABP2rs932650. The high-risk interactive genotypes in the three SNPs were independently associated with a higher risk for ischemic stroke and a composite of vascular events during the 4.7-year follow-up. The molecular interaction mechanisms of the three SNPs that increase the risk of stroke and vascular events remain unknown. As we all know, atherosclerosis is one of the critical risk factors for stroke. In atherosclerosis and stroke, the activation of inflammatory cells, endothelial injury, influx of lipoproteins, and proliferation of smooth muscle cells play crucial roles [22, 32]. SNPs in HABP2, IL6R, ITGA2, NOS2A, and IL1A have been associated with multiple studies of inflammation and endothelial function [13–15]. Hyaluronan-binding protein 2 (HABP2) affects vascular smooth muscle cell proliferation and plaque vulnerability [33]. The HABP2 gene encodes the hyaluronan-binding protein involved in vascular integrity and cell adhesion. Previous studies revealed that HABP2 SNPs were associated with carotid atherosclerosis [13–15], venous thromboembolic disease [34], and ischemic stroke [22, 23]. IL6RSNPs regulate IL-6 levels and increase ischemic stroke [27]. TLR4 variants are involved in immune response and are associated with myocardial infarction risk [30]. Therefore, the reason for the interaction in the three SNPs' contribution to ischemic stroke risk may be that the three SNPs are involved in encoding and regulating the relevant enzymes that affect the pathogenesis of ischemic stroke, inflammation, and endothelial function, resulting in the occurrence of ischemic stroke or other vascular events. However, further studies are necessary to elucidate the molecular mechanisms of interaction among the three variants in the future.
Strengths of the current study included: (1) multi-center community-based cross-sectional survey and prospective cohort study, focus on the high-risk population for stroke; (2) To the best of our knowledge, this study is the first to investigate the associations of the 19 SNPs and interaction among these SNPs in genes related to inflammation and endothelial function with ischemic stroke and other vascular during the follow-up; (3) GMDR approach was used to analyze gene-gene interaction; (4) Long follow-up period (4.7-year follow-up after face to face survey). However, there were some limitations in this study. First, this was a multi-center, cross-sectional survey and prospective cohort study. There may be recall bias because of self-reported questionnaires and follow-up by telephone. Second, this study was performed in Sichuan, southwestern China. Thus, the results of our study may not represent the full spectrum of the Chinese population. Third, our previous study has demonstrated that the persistence of drug therapy is crucial to decreasing stroke and other vascular events [19]. The main aim of this study was to evaluate the association of the 19 SNPs and the interaction among these SNPs with outcomes. Thus, we did not analyze the association between the persistence of drug therapy and the results in this study. Fourth, we assessed the role of the 19 known SNPs in genes related to inflammation and endothelial function, and other relevant genes were not investigated. Furthermore, although we revealed high-risk interactive genotypes in IL6R rs4845625 TLR4 rs1927911, and HABP2 rs932650 increased the risk of ischemic stroke and other vascular events, the molecular mechanisms were unknown. Therefore, future studies involving larger sample sizes and multi-center and more extensive genetic variants should be performed to identify the full extent of gene-gene interaction effects on outcomes in the high-risk stroke population.