The present study enrolled 928 patients who underwent coronary arteriography (CAG) for coronary atherosclerosis at the Cardiology Department of the Second Affiliated Hospital of Nanjing Medical University between January 2013–December 2017. Of these, 478 patients enrolled between January 2013–December 2015 were assessed during the discovery phases of the study, while 450 subjects enrolled between January 2016–December 2017 were evaluated during the validation phase of the study.
The inclusion criteria were patients with suspected CAD who underwent coronary angiography, the exclusion criteria stipulated that patients have been previously diagnosed with congenital heart disease, cardiomyopathy, valvar heart disease, thrombotic disease, diffuse intravascular coagulation, liver disease, kidney failure, and/or have undergone a recent surgery or injury, and/or have a history of malignant tumors or other infectious diseases (such as sepsis or pneumonia)
All coronary angiograms were conducted by inserting a Judkins catheter into the right radial artery, and the produced results were independently reviewed by two experienced angiographers, both of whom were blinded to corresponding genotyping data. Patients were categorized according to their degree of coronary stenosis, such that they were determined to have ‘no CAD’, ‘nonobstructive CAD’, or ‘significant CAD’ if they exhibited 0%, 1–49%, or ≥50% luminal narrowing in any epicardial coronary artery, respectively, as reported in the CSBC Registry . In our study, ‘CAD’ is composed of ‘nonobstructive CAD’ and ‘significant CAD’.
In the vessel scoring, stenosis <50% in any major coronary artery was defined as 0-vessel disease; having ≥50% stenosis in the luminal diameter of one or more coronary arteries was defined as a significant coronary lesion. Major coronary artery disease included left main coronary artery disease (considered a two-vessel disease characterized by stenosis ≥50%), left anterior descending artery disease, left circumflex artery disease, and right coronary artery disease .
The severity and extent of coronary atherosclerosis was assessed via angiographic Gensini scoring , such that 0–24%, 25–49%, 50–74%, 75–89%, and 90–99% angiographic stenosis was indicated by a score of 1, 2, 4, 8, and 16 points, respectively, while total occlusion was indicated by a score of 32 points. Each stenosed segment was then assessed and assigned a score between 0.5 and 5, dependent upon the functional significance of the area supplied by that segment. These scores were multiplied by the coefficient defined for each coronary artery and segment, and the results then summed. Finally, the patients were divided into four groups, comprising those that scored a total of 0, 0.5–19.5, 20–39.5, and ≥40 points.
Patients with a systolic blood pressure ≥140 mmHg, and/or a diastolic blood pressure ≥90 mmHg, and/or who were currently using antihypertensive drugs were defined as being hypertensive. Those that exhibited a fasting plasma glucose level ≥7.0 mmol/l, and/or that were currently using anti-diabetic drugs were defined as being diabetic. Finally, patients who exhibited high serum total cholesterol (TC) levels (TC ≥5.17 mmol/l), and/or high triglyceride (TG) levels (TG ≥1.70 mmol/l), and/or that were currently using lipid-lowering drugs were defined as having hyperlipemia.
The blood pressure, height, and body mass of each subject was measured upon admission. Prior to CAG assessment, blood samples were drawn into an inertia separation gel coagulation tube immediately after vascular puncture. TC, TG, high-density lipoprotein-cholesterol (HDL-C) and low-density lipoprotein-cholesterol (LDL-C) concentrations were determined by the Clinical Laboratory of the Second Affiliated Hospital of Nanjing Medical University, using routine clinical methods. Serum CD121a, IL-8, IL-11 and IL-1β were measured using the CBA Human Soluble Protein Detection Kit (BD Biosciences, New Jersey, USA).
Genomic DNA was isolated from patient venous blood leukocyte pellets using a genomic DNA isolation kit (Applied Biomiga, San Diego, USA). Primers were designed using primer premier5 (Applied Premier, Vancouver, British Columbia, Canada) software to target rs2287048 (forward, 5’-actaggcctcagtttcccactctata-3’; reverse, 5’-gtccaagactggaaatgtgtaatgg-3’), and rs956730, rs2234650, and rs2234651 (forward, 5’-caggttgcaagacagcttttagg-3’; reverse, 5’-cttatgcccaccagctcacttt-3’). The thermal cycling conditions were as follows: denaturation at 94°C for 3 min; followed by 35 cycles of 94°C for 30 s, 64°C for 30 s, and 72°C for 1 min; and a final extension at 72°C for 7 min. Generated products were (570bp for rs2287048; 484bp for rs956730, rs2234650, and rs2234651) stored at 4°C. Finally, the polymerase chain reaction products were identified via direct sequencing using an ABI 3730XL DNA Sequencer (Applied Biosystems, New York, USA).
All statistical analyses were performed using Statistical Package for the Social Sciences version 22.0(SPSS, Inc., Chicago, IL, USA) software. Continuous variables with normal distribution were expressed as the mean ± standard deviation, and compared by unpaired Student’s t-test. Continuous variables with abnormal distribution were analyzed by Mann-Whitney test. Categorical variables were presented as frequency (n) and proportion (%) which were analyzed by Chi-square test.
Genotype distributions were tested at each polymorphic locus for deviation from Hardy-Weinberg equilibrium, and Haploview software (version 4.2, Broad Institute, Cambridge, MA) was used to assess linkage disequilibrium (LD). The chromatogram of direct sequencing was prepared using BioEdit 220.127.116.11 software. To minimize the effect of age and gender differences between patients with and without CAD, Propensity Score Matching (PSM) was performed for the population.
Association between genotypes and CAD severity (i.e. the degree of exhibited vascular stenosis, Gensini scoring and the number of affected vessels) was analyzed by Mann-Whitney or Kruskal-Wallis test. Logistic regression model was used to estimate the association between genetic variants and the risk of CAD, and summarized as odds ratio (OR) and 95% confidence interval (95%CI), factors associated with the dependent variable at P <0.1 on univariate analysis were then entered into the multivariate model. P <0.05 is considered to indicate statistical significance.