Today, heart disease is currently the main cause of mortality in the world and approximately takes 17.9 million lives each year [22]. There are several types of heart disease such as Arrhythmia, Atherosclerosis, Cardiomyopathy, Congenital heart defects, heart infections, and CAD and different terms are used for them [23]. CAD is the situation that plaque buildup in the coronary arteries and it's sometimes known as ischemic heart disease. [4]. In the past years, many studies have been done to understand the physiological mechanism of CAD development. Environment and genetics play an important role in the contraction of cardiovascular disease. The understanding of disease-causing factors allows us for better prognosis and determining the treatment strategies of patients in the future. The conventional risk factors for CAD are high LDL-cholesterol, low HDL-cholesterol, high blood pressure, first-degree family history, smoking, diabetes mellitus (DM), etc. CAD is one of the long-time complications of DM and it seems for many reasons, including metabolic factors such as hyperglycemia, dyslipidemia, and insulin resistance, which leads to abnormal function of endothelial cells, vascular smooth muscle, impaired platelet function, and at last abnormal coagulation [24]. It is well-known stress oxidative and enzymes involved in its pathways such as SOD2 and PON1 contribute to endothelial dysfunction and development of cardiovascular disease. PON1 acts as an antioxidant factor, it's associated with HDL that metabolize organophosphates and prevent the onset of coronary artery disease. Also, SOD2 protects cells by catalytically scavenging harmful superoxide radicals [25, 26]. Moreover, clinical and experimental studies indicate that the oxidative stress pathway is associated with susceptibility to T2DM, insulin resistance, impaired glucose tolerance, and related complications through decreased activity of antioxidant enzymes.
Until now, several biological pathways are identified and well-understood that are related to the development of DM and CAD [27, 28]. GWAS and case-control studies have found more than 200 genetic loci that are associated with DM and/or CAD susceptibility in humans [7, 29]. However, causal genes, variants, and molecular pathways have not been completely identified for most loci. Polymorphisms are the most common genetic variants that repeat in more than 1% of the population and are related to multifactorial diseases like T2DM and CAD. Here, we investigated the prevalence of three independent polymorphisms in four separate groups. To our knowledge, there is no previous study on the relationship between polymorphisms CDKN2B-AS, SOD2, and PON1 and risk of T2DM/ CAD in Iranian population.
In this study, we found that rs2891168 is significantly associated with CAD. We used TaqMan allelic discrimination which is one of the most developed genotyping techniques to detect genotypes. Several previous GWASs confirmed that rs2891168 is associated with CAD [7, 30]. The rs2891168 SNP on chromosome 9p21.3 is found in the intronic sequence of the CDKN2B-AS gene, which is thought to be a susceptibility locus for the pathogenesis of CAD. Though the function of CDKN2BAS is not completed known, its level of expression has been associated with CAD and it seems allelic modification, changes CDKN2BAS production in the blood and finally increases the risk for CAD development [31–33]. As mentioned in the result, rs2891168 was significantly associated with CAD but we couldn’t find any association between rs2891168 and T2DM. These findings were consistent with previous reports [8]. After comparing no-T2DM/no-CAD and T2DM/no-CAD̶ groups, the distribution of G-allele was relatively similar in both groups and our result might happen because of the distinct process of causing T2DM.
In the case of PON1 rs662 A > G, the GG, and AG genotypes were more frequent among the T2DM/no-CAD and T2DM/CAD groups compared to no-T2DM/no-CAD group, showing the G-allele may be a risk factor for T2DM susceptibility. As a result, we assume that rs662 polymorphism is not a risk factor for CAD even though it may result in a T2DM predisposing factor. The role of paraoxonase1 (PON1) in the inhibition of atherosclerosis is remarkable. Oxidative stress and inflammation convert LDL to oxidized low-density lipoprotein (ox-LDL) and it can internalize by macrophages. PON1 hydrolyses ox-LDL to LDL and promotes cholesterol efflux from macrophage, and through this pathway inhibits atherosclerosis. According to previous studies, it seems the G allele is less effective than the A allele in oxidation and it can be considered as a risk factor for CAD [34, 35]. Randa et al. [36] reported that individuals with GG genotype show 9-fold risks to develop CAD as compared to an individual with AG genotype that demonstrates 4-fold risks. Studies show a contradictory result about the association of rs662 and CAD, but it might associate due to structural change in protein and subsequently altered activity of PON1 [37]. Based on our study, unlike rs2891168, the allelic distribution of rs662 was notably different in the T2DM/no-CAD group compared to no-T2DM/no-CAD group. It supports previous studies that rs662 reduces PON1 activity and concentration that lead to glucose tolerance and insulin resistance in T2DM patients [38–40]. It is supposed that OS induced by decreased PON1 concentration and/or activity, which in turn causes muscle cells to take in less blood glucose and become insulin resistant.
For the SOD2 rs4880 T > C, the frequency of C-allele was not statistically different in comparison with T-allele between groups. Similarly, we did not find a remarkable difference between the genotypes or genetic models in the four groups. According to our study, it may be rs4880 doesn't have any association with CAD risk, although several previous studies showed that SOD2 polymorphism can increase CAD risk by stress-related lipid abnormalities. SOD2 rs4880 T > C, substitute valine with alanine at position 16 of signal peptide and subsequently lead to more efficient import of SOD2 into the mitochondrial matrix [41]. In 2008, Fujimoto et al showed that the alanine variant could increase the SOD2 ability to neutralize superoxide radicals by its increased activity [42]. It seems SOD2 polymorphism is not directly related to coronary atherosclerosis, although more study is needed to confirm this hypothesis.
We faced some limitations in this study. First, different ethnic groups live in Iran, and each of them has a variety of allele frequencies, but we just could reach individuals from southeast of Iran and it probably affect the results. Second, our study only shows an association between three effective SNPs of CDKN2B-AS, SOD2, and PON1 genes in T2DM and CAD development, not a causal relationship. Additionally, we couldn’t investigate the influence of these SNPs on genes expression or function in this study. Thirdly and lastly, our study design does not consider the presence of other mutations, polymorphisms, and the action of conventional risk factors that could influence the TD2M and/or CAD development. CAD is a multifactorial disease that is influenced by both genetic and environmental factors. Genetic association studies like this create a potential genetic knowledge about the process and pathways involved in CAD. Gene expression is affected by one's genetic background, interaction with other genes, and environmental factors. It sounds like further studies are recommended in this field to discover the secret of multifactorial disease pathophysiology like CAD.