Apolipoprotein E gene polymorphism: effects on plasma lipids and risk of type 2 diabetes and coronary artery disease

Type 2 diabetes mellitus (T2DM) is one of the most common diseases with a high incidence and prevalence throughout the world. It affects nearly 4% of the world's population and this percentage will supposedly be increasing up to 5.4% by year 2025 [1]. Prevalence of diabetes in Thai adults as shown by the previous study on Thai population was 9.6% (2.4 million population) and the impaired fasting glucose was 5.4% (1.4 million people). Mean fasting plasma glucose level by age, sex, and area of residence was found to be substantially higher in urban population group than rural [2]. T2DM is also known as a major independent risk factor for coronary artery disease (CAD) and is the major cause of morbidity and mortality affecting people with diabetes. To date, several mechanisms such as dyslipoproteinemia, obesity, oxidative stress, smoking, exercise, alcohol intake, and genetic factors have been identified as risk factors of both T2DM and CAD. Lack of apolipoprotein E (apoE) gene has been clearly demonstrated as a leading cause of severe hyperlipidemia and spontaneous development of atherosclerosis in mammals [3, 4]. However, few studies have been able to demonstrate an association between T2DM and various single nucleotide polymorphisms (SNPs) [5]. Recently, Zeljko et al. 2011 [6] indicated that apoE gene polymorphism is also associated with obesity in normal Croatian Roma population. Adipocytes in an obese person which are the central and causal components in T2DM can generate high amount of biologically active molecules called adipokines or adipocytokines such as plasminogen activator inhibitor-1 (PAI-1), resistin, leptin, interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α) [7]. These inflammatory cytokines inhibit insulin-stimulated glucose metabolism in skeletal muscles and stimulate gluconeogenesis in hepatocytes causing hyperglycemia [8]. Hyperglycemia-induced oxidative stress results in reducing glucose uptake from blood by muscle cells and develops into insulin resistance by decreasing insulin secretion from pancreatic β-cells [9]. Taken these together, increased oxidative stress in hyperglycemia and reduced lipid clearance because of apoE gene polymorphism are effective in developing insulin resistance and T2DM. In addition, high oxidative stress can also cause vascular inflammation leading to atherosclerosis through several cytokines such as NF-κB, TNF-α, IL-1β, and other proinflammatory cytokines [10]. All of these factors are one of the underlying causes of metabolic syndrome [7]. Grundy reported that subjects suffering from the metabolic syndrome are at 2-fold higher risk of developing CAD and at 5-fold higher risk of developing T2DM; however, persons suffering from T2DM are at 3-fold higher risk of developing CAD [11].

ApoE gene is one of the most studied genes which is responsible for stabilizing and solubilizing circulating lipoproteins in our body and also responsible for the development of CAD [10, 11]. ApoE acts as a high affinity ligand for several hepatic lipoprotein receptors such as low-density lipoprotein receptor (LDLR) and LDL-related protein (LRP) and is involved in the process of cellular incorporation of several lipoproteins for transport and digestion [12]. ApoE is a plasma glycoprotein of 34 kDa with 299-amino acids associated with several other plasma glycoproteins, such as high density lipoprotein (HDL), very low density lipoprotein (VLDL), and chylomicrons [13]. In humans, apoE gene located on the chromosome at position 19q13.2 has been known to be polymorphic. SNPs at positions 112 (rs 429358) and 158 (rs 7412) determine three major alleles: ε2 (T to C substitution at position 158), the most common ε3, and ε4 (C to T substitution at position 112); 3 isoforms: ApoE2 (Cys112, 158Cys), ApoE3 (Cys112, 158Arg), and ApoE4 (Arg112, 158Arg); and 6 genotypes having 3 homozygous: E2/E2, E3/E3, E4/E4, and 3 heterozygous: E2/E3, E2/E4, E3/E4 [13]. Previous studies have shown that apoE alleles have influence on the lipid clearance and metabolism in humans. ApoE ε2 allele has been reported to be associated with higher plasma levels of apoE, decreased plasma levels of LDL cholesterol (LDL-C) and lower risk of CAD [14] while apoE ε4 is associated with lower plasma level of apoE, increased plasma levels of total cholesterol (TC), LDL-C, VLDL cholesterol (VLDL-C), and greater risk of CAD when compared to apoE3 homozygotes [15]. One reason for this impaired clearance by apoE ε4 leading to pathogenesis of CAD might be that apoE ε4 binds strongly to LDLR compared to other genotypes. The resulting high amount of lipid can suppress the synthesis of LDLR leading to lower clearance of lipoprotein from our body through LDLR [15]. Other studies have also supported that apoE ε4 allele is associated with the risk of CAD [16]. However, according to a recent meta-analysis, the cardiovascular role of apoE2 is uncertain [17] because of its tendency to increase triglyceride (TG) level [18]. In addition, apo ε2 homozygote in combination with certain additional disorders may develop type III familial hyperlipidemia and premature atherosclerosis [12]. Consequently, with their ability to affect lipid levels, the apoE gene polymorphism could be one of the factors influencing development of both T2DM and CAD. The prevalence of T2DM and CAD is increasing in Thai population according to the cross-sectional ECG survey of 1991 in Thai population, which found that the age-standardized prevalence rate of CAD was 9.9/1000 subjects (men 9.2/1000, women 10.7/1000) [19]. World Health Organization (WHO) global prevalence of diabetes report estimated that by the year 2030, 366 million people, particularly in developing countries, will be affected by diabetes [20]. Genetic factors like apoE are also considered to be genetic determinants of plasma lipoprotein levels and play a central role in the development of CAD. However, it is unclear whether apoE gene is associated with T2DM. The primary aim of this study was to demonstrate the influence of the apoE gene polymorphism on plasma lipids is notable and is an important determinant of T2DM and CAD. Secondarily, this genetic study might also add more information about a Thai population beyond traditional risk factors.

It has been reported that dyslipidemia or dyslipoproteinemia might strongly contribute towards aggravating the problems of micro- and macroangiopathic complications in diabetic patients [26, 27]. This implication was mainly characterized by higher prevalence of male gender, older age, smoking habits, and less physical activity. Other differences were duration of diabetes, lower levels of HDL-C and hypertension when compared to those without CAD which were similar to this study. However, some studies have also shown that elevation of TG and non-esterified fatty acid (NEFA) levels accelerated the pathogenesis of T2DM [28–30]. This suggests that dyslipidemia is associated with both T2DM and CAD. Genetic factors which have strong impact on the metabolism of plasma lipid have been studied regarding the potential effect of T2DM and cardiovascular outcomes in various diabetic and non-diabetic subjects. These studies have underscored that apoE gene encoding apolipoprotein E is associated with significant variation in lipid profile in our body [31]. ApoE gene is one of the most widely studied candidate genes for CAD or any other form of cardiovascular disease and/or diabetes. A significant relationship of apo E polymorphism and CAD has been observed in several ethnic groups, including Caucasian in the USA [32], Austrian [33], Finnish [34], Italian [35], Turkish [36], Indian [37] and Chinese [38] populations. Some studies have shown apoE ε4 allele as an independent risk factor after further adjustment of other established risk factors for development of CAD in T2DM [16] and myocardial infraction [37] patients. However, no independent association was observed after adjusting for age, sex, smoking, BMI, HDL-C and TG in African- Americans and Caucasians [39]. Therefore, it is of great interest to study the independent risk factors of this gene polymorphism in a Thai population. The present study also coincides with a previous study regarding the development of CAD in T2DM subjects indicating that the frequency of ε4 allele is significantly higher in CAD compared to controls and can be one of the factors for the progression of CAD disease [40]. The frequencies of apoE allele and genotype vary between different populations [13]. In this study, ε3 allele was the most frequent allele in control, diabetes, and CAD subjects, while ε2 was less frequent. E3/E3 genotype was the most frequent isoform found in all groups compared to the other genotypes which corresponds to previous reports [13, 41]. Additionally, the higher frequency of E3/E4 genotype was observed only in CAD (p = 0.0004), whereas the higher frequency of ε4 allele was observed in both T2DM and CAD (p = 0.047, p = 0.0009, respectively). From this study we can conclude that among these selected apoE alleles (ε2, ε3, and ε4), ε4 allele is one of the predictors of both diseases in Thai subjects. In addition, the PDAY (Pathobiological Determinants of Atherosclerosis in Youth) study reported that individuals with E2/E3 genotype had fewer atherosclerotic lesions, whereas those with the E3/E4 had more lesions in the abdominal aorta [42]. These observations strongly suggest that the ε2 allele has a protective role against atherosclerosis. However, in this study, we found that E2/E3 genotype and ε2 allele were also significantly lower in T2DM group indicating that the protective effect of ε2 allele on the development of hyperlipidemia might be less in T2DM patients. It is probable that other environmental and genetic factors are involved in the pathogenesis of CAD.

This present study is the first report to demonstrate that the ε4 allele containing genotypes is the major predictor of development of both T2DM and CAD. Our study also shows strong association of E3/E4 genotype with development of CAD in T2DM patients (Table 4) as reported in previous studies [16, 42]. After adjusting for age, sex, smoking, BMI, and physical activity, E3/E4 containing subjects showed 2.52-fold higher risk (p = 0.008) while ε4 allele containing genotypes led to a 2.32-fold higher risk (p = 0.016) for developing CAD and ε4 allele containing genotypes led to a 2.04-fold higher risk (p = 0.029) in the development of T2DM as compared to the controls. This signifies that other risk factors have no influence in development or curbing the disease, suggesting that ε4 allele is the independent risk factor for both T2DM and CAD. However, E3/E4 genotype further increases the risk for the development of diabetes and CAD when combined with smoking and/or obesity (Table 6). This confirms the findings that progression and development of diabetes and CAD is the consequence of multifactorial parameters, for example, obesity, smoking, oxidative stress, defect in pancreatic β-cells, alcohol consumption, exercise, hypertension and genetic factors. In addition, incidence of T2DM is also subject to gene-environment interactions, for example, dietary factors, intake of vegetable fat, polyunsaturated fatty acid, dietary fiber (particularly cereal fiber), magnesium, and caffeine were significantly inversely correlated and intakes of trans fat, saturated fatty acid and heme-iron, glycemic index, and glycemic load were significantly positively correlated with the incidence of T2DM [43]. Similarly, regular exercise has been shown to reduce weight, BMI, HDL-C and insulin resistance. This indicates that exercise is also associated with decrease risk of T2DM and CAD. Furthermore, apoE gene polymorphism has been shown to modulate the effects of exercise on lipoprotein concentrations in plasma [44]. In this study, smoking and obesity are shown to be the two major contributing factors that can promote the development of T2DM and CAD or both when placed in joint association (Table 6). The reason for this is that smoking and obesity enhance the oxidative stress which results in decreased insulin secretion from pancreatic β-cell and decreased uptake of blood glucose into the muscle cells [9, 10]. This evidence was supported by the experimental study on atherosclerosis-susceptible B6 (B6.apoE -/-) and atherosclerosis-resistant BALB (BALB.apoE -/-) mice that showed defects in insulin secretion rather than defects in insulin resistance which explains the mark difference in susceptibility to T2DM [45]. Moreover, oxidative stress also increases vascular inflammation leading to CAD and/or any form of cardiovascular disease with or without combination of T2DM [46].

To demonstrate that apoE gene polymorphism is involved in the lipid clearance process and it has great influence on the lipid level in our body [47, 48]. Although previous study has shown that non-HDL-C concentration is similar to or better than LDL-C alone in predicting cardiovascular disease (CVD) incidence [49], in this study, the results showed that the decrease in HDL-C and elevation of TG, VLDL-C and LDL-C levels (Table 1) are also the landmarks of diabetes and CAD development as found in an earlier study [50]. However, the association of apoE ε4 allele and lipid profile is still controversial [51]. The ε4 allele has been shown to be associated with high concentration of serum TC and LDL-C in Chinese population [38]. Some studies also showed a significant relationship between E3/E4 genotype with lower HDL-C and higher LDL-C concentrations in CAD patients [52] and with higher TG levels in T2DM patients [48] as compared to the healthy controls. However, a study in the Tunisian population has suggested that HDL-C concentration and other lipid profiles are not associated with apoE gene polymorphism in the total population studied, but has proven that ε4 allele increased LDL-C in type 2 diabetic men [53]. Association of ε4 allele with higher LDL-C and lower HDL-C levels was only found in type 2 diabetic women in Spanish population [54] indicating that gender affects the effect of apoE gene polymorphism. In this study, there is a mean comparison between E4 carriers and E3/E3 genotype in the T2DM and total population studied which showed a significantly higher VLDL-C, TG and lower HDL-C levels in E4 carriers (Table 7) similar to the report of Knouff et al 1999 [15]. Nevertheless, the differences of lipid profiles were not observed in controls and T2DM with CAD. This may be due to dietary restrictions in the controls and lipid-lowering aggressive treatment in T2DM with CAD. Kolovou et al. 2006 [55] reported that the ε4 allele can increase LDL-C concentrations in the presence of an atherogenic diet, but a lower fat diet can suppress this effect. On the other hand, aerobically trained individuals have high HDL and display enhanced glucose tolerance [56]. A lipid-lowering-drug which raises HDL levels and decrease TG levels delays the onset of T2DM and reduces the development of atherosclerosis [57]. In addition, loss of caspase-1 activity which involves the inflammatory process in human atherosclerotic vessel has shown to reduce atherosclerosis lesion formation in Casp1^-/- Apo E^-/- mice [58]. Another reason for these variations of lipid profiles might be because of the differences in genetic background and the prevalence of high oxidative stress in the studied population. When considering the basis of gene-gene and gene-environment interactions, as described above, the polymorphism study at the genome level might not provide a real picture for development of diseases in respect to lipid profile. A more robust parallel study at the protein level, or of other candidate genes, may be necessary to evaluate the mechanism of T2DM and CAD development. However, this study will provide a good starting point for the screening of large populations before proceeding to the protein level study of apoE and other candidate genes in various races and, additionally, including pharmacogenomics study in the future.

This study tentatively supports the fact that variability in apoE gene locus is associated with diabetes with and without CAD complication by influencing the plasma lipid levels that are important risk factors for both T2DM and CAD. No single factor can give a satisfactory explanation regarding development of diabetes and CAD as both diseases are complex diseases. Focusing, therefore, on only one risk factor may be less than optimal in enabling researchers to predict who will develop untoward events in complex diseases like diabetes and CAD. Nonetheless, our study strongly supported that the apoE ε4 allele is an independent risk factor for development of both T2DM and CAD. Moreover, obesity and/or smoking, conditions associated with high oxidative stress, can aggravate the progression of both diseases. Genetic studies can thus provide information that may help to improve the ability to identify individuals, families and populations at increased risk, as well as to improve the clinical management of patients with T2DM and CAD. Such information may be useful in developing public health programs reinforcing primary and secondary prevention for T2DM and CAD. Furthermore, T2DM and CAD patients identified to carry high risk genotype or allele should be treated aggressively to prevent the progression of disease.