Study of the association of 17 lipid-related gene polymorphisms with coronary heart disease

Objective Blood lipids are well-known risk factors for coronary heart disease (CHD). The aim of this study was to explore the association between 17 lipid-related gene polymorphisms and CHD. Methods The current study examined with 784 CHD cases and 739 non-CHD controls. Genotyping was performed on the MassARRAY iPLEX® assay platform. Results Our analyses revealed a significant association of APOE rs7259620 with CHD (genotype: χ2=6.353, df=2, p=0.042; allele: χ2=5.05, df=1, p=0.025; recessive model: χ2=5.57, df=1, p=0.018). A further gender-based subgroup analysis revealed significant associations of APOE rs7259620 and PPAP2B rs72664392 with CHD in males (genotype: χ2=8.379, df=2, p=0.015; allele: χ2=5.190, df=1, p=0.023; recessive model: χ2=19.3, df=1, p<0.0001) and females (genotype: χ2=9.878, df=2, p=0.007), respectively. Subsequent breakdown analysis by age showed that CETP rs4783961, MLXIPL rs35493868, and PON2 rs12704796 were significantly associated with CHD among individuals younger than 55 years of age (CETP rs4783961: χ2=8.966, df=1, p=0.011 by genotype; MLXIPL rs35493868: χ2=4.87, df=1, p=0.027 by allele; χ2=4.88, df=1, p=0.027 by dominant model; PON2 rs12704796: χ2=6.511, df=2, p=0.039 by genotype; χ2=6.210, df=1, p=0.013 by allele; χ2=5.03, df=1, p=0.025 by dominant model). Significant allelic association was observed between LEPR rs656451 and CHD among individuals older than 65 years of age (χ2=4.410, df=1, p=0.036). Conclusion Our study revealed significant associations of APOE, PPAP2B, CETP, MLXIPL, PON2, and LEPR gene polymorphisms with CHD among the Han Chinese.


Introduction
Coronary heart disease (CHD) is characterized by atherosclerosis, which leads to vascular stenosis and occlusion. Dyslipidemia is known as a risk factor for CHD (1). Blood lipids have been reported to predict the risk of CHD (2,3), which encouraged us to examine the association of lipid-related gene polymorphisms with CHD (4).
On the basis of previous studies, the aim of this study was to assess the association of 17 lipid-related gene polymorphisms with CHD in the Han Chinese.

Sample collection
A total of 784 CHD cases and 739 non-CHD controls were enrolled in the current study. The enrolled cases were classified on the basis of the findings of standardized coronary angiography according to the Seldinger's method (18). The classification details have been reported in our previous studies (19)(20)(21). The study protocol was approved by the ethical committees of Ningbo First Hospital and Ningbo University. Written informed consent was obtained from all the participants.
Single nucleotide polymorphism (SNP) genotyping DNA extraction and quantification were performed as previously described (22,23). Genotyping was performed on the Mas-sARRAY iPLEX ® assay platform (Sequenom, San Diego, CA, USA). The primer sequences and the details of the selected SNPs are presented in Table 1.

Statistical analysis
Genotype and allele distributions were compared between the two groups using the Chi-squared test. Differences were

Results
As shown in the Table 2, 17 SNPs were detected in the upstream regions of lipid-related genes. LEPR rs6656451 was located in the upstream region of a transcript isoform. Among the tested SNPs, APOE rs7259620 was significantly associated with CHD [genotype p=0.042 (df=2), allele p=0.025 (df=1), OR (95% ADIPOQ rs266729, VLDLR rs7852409, and PPAP2B rs72664392 were excluded from further analyses since their genotypic distributions did not meet HWE in the controls (data not shown). In addition, the association of the remaining 12 SNPs with CHD could not be evaluated in the total samples (p>0.05).
Age-based subgroup analyses revealed that CETP rs4783961, MLXIPL rs35493868, and PON2 rs12704796 were significantly as-  Table 4). In addition, LEPR rs656451 was associated with CHD in participants older than 65 years of age (χ 2 =4.410, df=1, p=0.036 by allele, Table 4). No other SNPs were associated with CHD in the age-based subgroup analyses.

Discussion
In the present study, we examined the association of 17 lipidrelated SNPs with CHD among 784 CHD cases and 739 non-CHD controls. We identified a male-specific association of APOE rs7259620 with CHD. Meanwhile, we also found a significant association of PON2 rs12704796 with CHD among participants younger than 55 years of age. On the genotypic level, we identify a significant association of CHD with PPAP2B rs72664392 in females and CETP rs4783961 in participants younger than 55 years of age. On the allelic level, we identified a significant association of CHD with MLXIPL rs35493868 in participants younger than 55 years of age and LEPR rs6656451 in participants older than 65 years of age.
Previous studies have indicated that APOE is significantly associated with CHD. APOE ε2 was shown to reduce the risk of CHD by 20% (24), whereas ε4 was shown to increase the risk of CHD by approximately 42% compared with ε3/ε3 genotype (25). Epidemiological evidence has shown that males are at a higher risk of CHD than females worldwide (26). Gender disparity has been found in APOE-related cardiovascular disease (27). In the previous studies, we have shown that CHD risk was gender-dependent in the Han Chinese and that APOE rs4420638 polymorphism was significantly associated with increased CHD risk in male Han Chinese (28). This observation might be explained by the differences of hormonal profiles, smoking status, alcohol-drinking, occupation, and dietary habits between males and females (29,30). In the present study, we also identified a novel genetic variant of APOE associated with CHD in males.
PPAR2B is a negative regulator of inflammatory cytokines, leucocyte adhesion, cell survival, and migration in human primary aortic endothelial cells (31), suggesting that PPAR2B can protect blood vessel against inflammation (32). Mechanosensitive PPAP2B plays a critical role in promoting anti-inflammatory phenotype and maintaining the vascular integrity of endothelial monolayer under atheroprotective flow (33). However, discrepancies exist regarding the association of PPAP2B with CHD (34). PPAP2B rs1759752 is associated with increased CHD risk in males, while PPAP2B rs12566304 is associated with a decreased CHD risk in females (34). Other studies have shown that PPAP2B rs17114036-A is associated with CHD (35,36). In contrast, PPAP2B rs17114036 is not associated with CHD after adjustments for gender (16,35). Here, we identified a novel   polymorphism (rs72664392) in PPAP2B promoter associated with CHD in females. This finding could be partly explained by the particular genetic background. Aging is a pivotal risk factor for CHD (37,38). The incidence of CHD in people younger than 40 years of age is 0.6%, and it increases two-fold or more with every 10-year increase in age (39). High adiponectin concentration has been shown to be associated with a lower risk of CHD in people younger 65 years of age (40). In people younger than 55 years of age, PON2 rs12704796-A has been shown to increase the risk of CHD by 43.1%, whereas MLXIPL rs35493868-G has been shown to reduce the risk of CHD by 38.1%. In addition, LEPR rs6656451-T has been reported to reduce the risk of CHD by 45.5% among people older than 65 years of age.

Study limitations
Our results did not demonstrate a significant association of 11 of the tested SNPs with CHD. A power analysis revealed that these SNPs showed a minimal or moderate power to detect a significant association in the current study (power=0.074-0.425). In addition, several SNPs did not present reliable association results in genderand age-based subgroup analyses since their genotype distributions did not meet HWE in the controls. Future association study of these SNPs with CHD is warranted in other cohorts.

Conclusion
Our study demonstrated the gender-or age-dependent association of six SNPs (APOE rs7259620, PPAP2B rs72664392, CETP rs4783961, PON2 rs12704796, MLXIPL rs35493868, and LEPR rs6656451) CHD in Han Chinese population. However, future replication is required to validate our findings.