The participants in this study were recruited from Wuhan General Hospital of Guangzhou Military Region between 2010 and 2016. Individuals with incomplete information were excluded. A total of 435 CAD patients and 196 non-CAD controls were involved in the study. All the participants were unrelated Chinese Han individuals. This study was approved by the Medical Ethics Committee of Southern Medical University and compliant with the principles set forth by the Declaration of Helsinki Principles.

Subjects were defined as smokers if they smoked more than 100 cigarettes in lifetime. Subjects were diagnosed hypertension as systolic blood pressure (SBP) >140 mmHg or diastolic blood pressure (DBP) >90 mmHg [11]. Diabetes mellitus was defined as either fasting plasma glucose levels of ≥7.0 mmol/L or plasma glucose levels of ≥11.1 mmol/L [12]. Total cholesterol, HDL cholesterol, LDL cholesterol, C-reactive protein (CRP) and triglyceride (TG) levels were measured according to standard laboratory methods in Southern Medical University. The diagnostic criteria for CAD cases were defined as followings: at least one of the major segments of coronary arteries (right coronary artery, left circumflex, or left anterior descending arteries) with more than or equal to 50% organic stenosis based on coronary angiography.

Blood samples (5 ml) were collected from participants. DNA extraction was conducted by TianGen DNA extraction kit (TianGen Ltd., Beijing, China) according to the manufacture instruction. DNA quality was analyzed by Electrophoresis and NanoDrop (NanoDrop Technologies, Houston, TX, USA) quantification. 20 ng DNA was used for PCR amplification. Chimeric specific primers were designed using Oligo 6.0, which contain target sequences and universal sequences. The product sizes of PCR reaction were between 107 and 160 bp, and the primer length was 37–38 bp, with melting temperature (Tm) 55–65 °C and the GC content between 20 and 80%. Multiplex PCR was conducted and followed by adaptor adding. Final products were purified by polyacrylamide gel electrophoresis (PAGE) and then sequenced on MiSeq platform (Illumina, USA).

Sequencing data were separated by index sequences using FASTX-toolkit as each sample group has a unique index sequence. Then, the index and adapter sequences were trimed out with cutadapt. And target sequences were mapped to human genome reference sequence (NCBI, dbSNP bulid 142) using BWA software. SNPs were called by samtools.

Continuous variables between cases and controls are calculated using Student’s t test and presented as mean ± SD. Categorical variables were calculated using chi-square test. Gene frequencies, allele frequencies, and differences in genotype and allele frequencies between different groups were also examined. Hardy-Weinberg equilibrium (HWE) test was performed by SHEsis [13]. P value >0.05 was considered in HWE. Allele distribution between cases and controls were analyzed using chi-square test. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by logistic regression analysis after adjusting for age, hypertension, type 2 diabetes, TG, TC, HDL-C and LDL-C. P value <0.05 was considered as statistically significant. Association analysis were conducted by SPSS version 21.0 (SPSS Inc., Chicago, Illinois, USA).

In this study, 435 CAD patients and 196 controls were included. Clinical characteristics are summarized in Table 1. Samples in case group are more likely to have hypertension, diabetes and smoking. EF, HDLC and APOA1 levels are significantly lower in case group than in control group, while TG, APOA1, GLU and GHb levels are significantly higher in case group than in control group. These data indicate that male, EF, HDLC, TG, ApoA, APOA1, GLU, GHb, hypertension, diabetes and smoking are risk factors of developing CAD in this study.

Seven SNPs were genotyped in 435 cases and 196 controls. Allele frequency distribution was shown in Table 2. All SNPs were accorded with HWE. In these 7 SNPs, rs5882 and rs662799 were associated with CAD (p ≤ 0.05), in which rs662799 was explored by Ye et al. of the association between coronary heart disease (CHD) and the APOA5 rs662799 polymorphism [14]. After adjustment of age, gender, smoking, diabetes and hypertension, only rs662799 was significantly associated with increasing risk of developing CAD (risk allele T, OR = 1.374, 95% CI = 1.032–1.83, P = 0.03) (Table 3). Minor (T) allele frequency of SNP rs662799 in control and case group were 33.9 and 38.2% respectively. The results remained significant when HDLC, LDLC, TC and TG were introduced into the model (data not shown). No significant differences were observed in other polymorphisms after adjustment.

Gender, smoking, diabetes and hypertension status were stratified for further investigation (Table 4). Rs662799 was associated with CAD in male and hypertension group. Rs5882 was associated with CAD in non-hypertension group. Rs1800588 was associated with CAD in smoking group.