The current study indicated that both the C allele of rs428253 and the G allele of rs2243115 were correlated with increased risk of CHD, but rs4740 or rs568408 were not associated with CHD risk. The rs428253 belongs to EBI3 gene, which located on chromosome 19p13.3. The rs2243115 belongs to IL-12A subunit, which is located on chromosome 3q25.33. Previously some studies have investigated the association between the two genes and inflammatory diseases [11, 12]. So far, there are few studies about the effect of IL-35 polymorphism on cardiovascular disease. Zhang et al confirmed that ebi3-rs428253 polymorphism is related to the risk reduction of chronic rhinosinusitis and allergic rhinitis . So far, only two studies [15, 16] have confirmed the relationship between IL-35 gene and coronary heart disease risk, but the conclusions of these two studies are inconsistent. A study  for Mexico populations suggested that our study suggests a relationship of the EBI3 SNPs with IL-35 levels, and Ebi3-rs428253 and il-12a-rs2243115 gene polymorphisms play an important role in the mechanism of CHD risk reduction. However, a recent case-control study in the Chinese population  suggested that there was a statistical correlation between the EBI3 rs428253 mutation genotype and the risk of CHD, but there was no statistical significance between the mutation genotype of IL-12A-rs2243115 and the risk of CHD. The study also found that there was no significant difference in the level of IL-35 between different genotypes in the healthy control group. Although two case- control studies have been performed previously, but the two studies concluded inconsistent results, and just one study was performed for Chinese Zhuang population, but no study focused on Chinese Han population, which was the largest nationality in China. So the difference in nationality for our study and previous studies may lead to different results. The biological mechanism of the association between IL-35 gene and coronary heart disease is not well established, but previous studies suggested that EBI3-rs428253 may be involved in the modification of LEF1 binding site [15, 22], and play an important role in granulocyte proliferation and differentiation . SNP-rs428253 in EBI33 gene is related to the occurrence and development of CHD, which may be caused by regulating β - Catenin pathway and Treg pathway rather than by influencing the production of IL-35, because in the IL-35 levels was not different among different genotype of rs428253 in controls of study by Lin et al .
The pathogenesis of coronary heart disease is very complex. It is not only affected by genetic factors and environmental factors independently, but also by the synergistic effect between them [24, 25]. We were all known that alcohol drinking and smoking were two main modified risk factors for CHD [26, 27]. In this study, the smoking and alcohol drinking rates were higher in cases than in controls, which indicated that smoking and alcohol drinking were two risk factors for CHD. So we performed a GMDR analysis for gene- smoking or alcohol drinking interaction, and the results suggested a significant interaction between rs428253 and current smoking associated with CHD risk. Current smokers with rs428253 - GC/ CC genotype have the highest CHD risk, compared to subjects with never smokers with rs428253 - GG genotype. Environmental factors can cause phenotypic differences by influencing gene expression regulation. Therefore, the study of IL- 35 gene-environment interaction is helpful to better understand the occurrence of CHD.
There were several limitations in this study. Firstly, the sample size is not large enough, so this is only a preliminary study of the polymorphism of this locus, so the results obtained in current study need to be verified in the study with larger sample size and in different populations. Secondly, we just selected four loci in this study. In the future, we will study multiple loci of IL- 35 gene and CHD, in order to better understand the mechanism of CHD from the perspective of genetics. Lastly, the G allele frequency of rs2243115 was higher than that in gene database, so the selection bias may exist in this study.
The C allele of rs428253 and the G allele of rs2243115 were correlated with increased risk of CHD. We also found a significant interaction between rs428253 and current smoking associated with CHD risk. Although the previous studies have reported the relationship between the gene and CHD risk, but these studies concluded conflicting results, so the results obtained from this study verified this association. In addition, we also found the interaction between rs428253 and current smoking, which added more detailed mechanism foe relationship between gene, environmental risk factors and CHD susceptibility.