Aim: To investigate incidence of ischemic stroke and other vascular events during a 4.7-year follow-up in the high-risk stroke population, and to identify the associations of the 19 single nucleotide polymorphisms (SNPs) in genes related to inflammation and endothelial function and interaction among these SNPs with outcomes.  

Methods: According to the China National Stroke Screening Survey programme, we performed this multi-center community-based sectional survey and prospective cohort study in the Sichuan of southwestern China from May 2015 to January 2020. Eight communities were randomly selected in Sichuan, and the residents in each community volunteered to participate in a face-to-face survey. The 19 SNPs in genes related to endothelial function and inflammation were measured in the high-risk stroke population. All subjects in high-risk stroke populations were followed-up for 4.7 years after the face-to-face survey. The primary outcome was a new ischemic stroke; the secondary outcome was a composite of new vascular events. 

Results: A total of 2893 high-risk stroke population, 2698 (93.3%) completed a 4.7-year follow-up. Outcomes occurred in 192 (7.1%) subjects (118 [4.4%] new ischemic stroke, 24 [0.9%] hemorrhagic stroke, 53 [2.0%] myocardial infarction, and 33[1.2%] death) in the 2698 high-risk stroke population. There were significant differences in genotype distribution of TNF rs3093662, IL6R rs4845625 and TLR4 rs752998 between subjects with and without outcomes by univariate analyses. Generalized multifactor dimensionality reduction (GMDR) analysis showed that there was a significant gene-gene interaction among the 19 SNPs, the best model for outcomes was interaction among IL6R rs4845625, TLR4 rs1927911 and HABP2 rs932650 (P = 0.004). The high-risk interactive genotypes among the 3 SNPs were independently associated with a higher risk for new ischemic stroke  (OR = 2.187, 95% CI: 1.256–5.374, P＜0.001) and total vascular events (OR = 2.382, 95% CI: 1.423–5.894, P＜0.001) after adjustment with covariates.

Conclusion: The incidence of ischemic stroke and other vascular events was found to be very high in the high-risk stroke population. There were associations of specific SNPs in genes related to inflammation and endothelial function with outcomes. The high-risk interactive genotypes among IL6R rs4845625, TLR4 rs1927911 and HABP2 rs932650 were independently associated with a higher risk for new ischemic stroke and other vascular events. These findings are expected to provide new strategies for prevention of ischemic stroke and other vascular events.

Study design and participants

 According to the CNSSS programme, we performed this multi-center community-based cross-sectional survey and prospective cohort study in Sichuan of southwestern China from May 2015 to January 2020. The survey protocol was reviewed and approved by the Ethics Committee of participating hospitals and informed consent (in the Chinese language) was obtained from all participants before enrollment. The community-based cross-sectional survey was conducted in 8 communities of Sichuan between May 2015 and September 2015. The detailed procedures for recruitment of participants and evaluation of risk factors were described in our previous articles. In brief, the 8 communities were selected using cluster randomization method in Sichuan. The residents aged ≥ 40 years in each community were screened using a structured face-to-face questionnaire by interviewers. The questionnaire included demographic characteristics, behavioural factors, history of chronic diseases, personal and family medical history of stroke, and physical examination.

Evaluation of risk factors and definition of high-risk population for stroke

Information on demographic characteristics was collected, and the eight conventional risk factors for stroke and history of stroke were evaluated. The eight risk factors included overweight/obesity, smoking, physical inactivity, family history of stroke, hypertension, diabetes mellitus, dyslipidemia, and atrial fibrillation.  The detailed diagnostic criteria for the eight risk factors and stroke were described in our previous article. Subjects with at least three of the aforementioned eight risk factors or a history of stroke were defined as the high-risk stroke population. The exclusion criteria were as follows: (1) residents declined to participate in the study; (2) severe cardiovascular, liver or renal disease, blood system disease, acute and chronic inflammation, malignant tumors, and immune system diseases;  (3) history of carotid artery stenting or endarterectomy.

Genotyping

The 19 SNPs in 10 genes related to inflammation and endothelial function were selected from the NCBI database (http://www.ncbi.nlm.nih.gov/SNP), according to the following criteria: (1) the SNPs have been evaluated in previous studies ;  (2) the SNPs have a minor allele frequency > 0.05; (3) the SNPs may lead to amino acid changes. For the high-risk stroke population, peripheral blood (3 mL) was drawn from an arm vein and was stored in cryogenic refrigerator at -80 °C until genotype analysis was performed.  Genomic DNA was extracted by a modified phenol/chloroform method and purified using a UNIQ-10 kit (Sangon Biotech Co., Ltd., Shanghai, China). Genotypes of the 19 SNPs were evaluated using a matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) method by investigators, as were previously described.  The investigators were blinded to the data of participants.

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