Ethics approval of study protocol
Written informed consent was obtained from each participant after a full explanation of this study. The study protocol was conducted according to the standards of the Declaration of Helsinki, and the study was approved by the Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University.
Study population
All 1022 participations were selected from Xinjiang, the northwestern part of China. From January 2016 to December 2018, 567 CAD patients were recruited from the First Affiliated Hospital of Xinjiang Medical University. This study population consisted of 308 Han patients and 259 Uygur patients with CAD. CAD patients with typical chest pain, electrocardiographic changes (new pathologic Q waves, at least 1 mm ST segment elevation in any two or more contiguous limb leads or a new left bundle branch block, or new persistent ST-T wave changes indicative of a non-Q wave myocardial infarction) and serum creatine kinase-MB isoenzyme (CK-MB) elevations (more than 3-fold higher than the upper reference limit) were examined by coronary angiography according to the guidelines [17]. The diagnostic criteria of CAD were defined as the presence of at least one significant coronary artery stenosis of more than 50% luminal diameter based on coronary angiography. CAD patients who had a congenital hypercoagulable status with proven disease-limiting life expectancy, malignancy, connective tissue disease, impaired renal function, or chronic inflammatory disease were excluded from the study.
The control (non-CAD) participants of 294 Han and 161 Uygur Chinese individuals were selected from the Cardiovascular Risk Survey [18,19]. This study comprised 14,618 subjects and is a multiple-ethnic, community-based, cross-sectional study designed to investigate the prevalence, incidence, and risk factors for cardiovascular diseases in the Han, Uygur, and Kazakh populations of Xinjiang, in the northwestern part of China. This study consisted of interviews, physical examinations, and data from blood sample analyses. These subjects did not have any of the following conditions related to CAD: a positive family history, stable and unstable angina, myocardial infarction, evidence of CAD by electrocardiography and angiography, abnormality of regional wall motion, or relevant valvular abnormalities based on echocardiography. Both CAD patients and control subjects were matched for age and sex.
Data collection
Clinical data and information about the presence of traditional CAD risk factors including essential hypertension, diabetes mellitus (DM), total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), smoking, drinking, height, and weight were obtained from all study participants by reviewing the patients’ medical records. Essential hypertension was defined as a history of hypertension and/or average systolic blood pressure (SBP) ≥ 140 mmHg and/or an average diastolic blood pressure (DBP) ≥ 90 mmHg on at least two separate occasions according to medical examination and history. DM was defined as fasting plasma glucose levels ≥ 7.0 mmol/L (126 mg/dL), glucose levels ≥ 11.1 mmol/L (200 mg/dL) 2h after the administration of a 75g oral glucose load, a history of diabetes, or patients with a history of anti-diabetic medication use. Participants were considered smokers when consuming more than five cigarettes per day or nonsmokers when they had never smoked or had stopped smoking at least 1 year before sample collection. Patients who consumed 20ml or more of alcohol per day in the previous 6 months were considered alcohol users. The height and weight of each individual were recorded to calculate the body mass index (BMI) and determine the risk of obesity. BMI was calculated as weight divided by height squared (kg/m2).
Blood collection and DNA extraction
Blood samples were collected from all subjects using a standard venipuncture technique with ethylene diamine tetraacetic acid (EDTA)-containing tubes and centrifuged at 4000 ´ g for 5 min to separate the plasma for a range of biochemical assays. DNA was extracted from the peripheral vein blood leukocytes using a whole-blood genome extraction kit (Beijing Bioteke Corporation, Beijing, China). DNA samples were stored at −80 ℃ for genotyping.
Biochemical measurements and genotyping
Serum and plasma collected for measurements were immediately frozen at −80 ℃ until use. Plasma concentrations of TG, TC, HDL-C, and LDL-C were measured using standard methods in the Central Laboratory of the First Affiliated Hospital of Xinjiang Medical University. Using the Haploview 4.2 software and the 1000 Genomes database, we obtained two tagging SNPs (rs1892818, rs9693898) for Chinese individuals using a minor allele frequency ≥ 0.05 and linkage disequilibrium patterns with r2 ≥ 0.8 as a cutoff (Figure 1). We titrated the DNA concentration at 50 ng/mL. The rs1892818 and rs9693898 polymorphisms of ADRB3 were detected by TaqMan single nucleotide polymorphism genotyping assays in a 7900 HT Standard real-time polymerase chain reaction (PCR) system, according to the manufacturer’s recommendations (Applied Biosystems). Primers and probes used in the TaqMan SNP Genotyping Assays (Applied Biosystems) were chosen based on information available at the ABI website (www3.appliedbiosys tems.com/AB_Home/index.htm). PCR amplification was performed in a total reaction volume of 6 μL containing 3 μL of TaqMan Universal Master Mix, 0.15 μL probes, 1.85 μL ddH2O, and 1 μL DNA with the following amplification protocol: 95 ℃ for 10 min, 40 cycles of 95 ℃ for 15 s and 60 ℃ for 1 min. All 96-well plates were read using the Sequence Detection Systems (SDS) automation controller software v2.4 (ABI). The SDSv2.4 endpoint typing software package was used for automatic genotyping. The results are shown in Figure 2, and the three colors represent different genotypes. Variants in the SNP (rs1892818) of the ADRB3 gene were classified into two genotypes, namely CC and CT genotypes, whereas rs9693898 of the ADRB3 gene was classified into three genotypes, AA, AG, and GG (Figure 2).
Statistical analyses
The normality of parameters was assessed by a Shapiro–Wilk test. Continuous statistics that met the normality assumption are shown as the mean and standard deviation (mean ± SD), and differences in continuous variables between the CAD patients and control subjects were analyzed using an independent-sample t-test. As some measurement data in this study did not meet the normality assumption, they were described as medians (interquartile range) and compared with the Mann–Whitney U test. The Hardy–Weinberg equilibrium was assessed by Chi-square analysis. Differences in enumeration data between CAD patients and control subjects were analyzed using the Chi-square test, as were differences in distributions of genotypes and alleles between CAD patients and control subjects. Logistic regression analyses with effect ratios (odds ratio [OR] and 95% confidence interval [CI]) were used to assess the contribution of the major risk factors. All statistical analyses were performed using SPSS 22.0 for Windows (SPSS Institute). A two-tailed value of p < 0.05 was considered statistically significant.