Apolipoprotein E as Risk Factor for Coronary Heart Disease

Allelic variation of apolipoprotein E (apo E) has been shown to infl uence the concentrations of total cholesterol and low density lipoprotein cholesterol (LDL-C) and considered to play a role as one of risk factors for coronary heart disease (CHD). The aim of this study was to examine the relationship between Apo E polymorphism and the risk of CHD. Blood samples were collected from 33 CHD patients in Dr. Sardjito Hospital Yogyakarta, and 38 apparently healthy control individuals in a cross sectional study. The common allelic variants of ApoE were screened employing polymerase chain reaction and restriction fragment length polymorphism. The results obtained were analyzed by t-test and signifi cantly different if p <0.05 and risk factor was calculated by odd ratio. Frequency of ApoE ε2, ε2 and ε4 alleles in CHD patients were 12.1%, 69.7% and 18.2% while in controls were 18.4%, 72.4% and 9.2% respectively. Dyslipidemia condition was a strong risk factor for CHD. By controlling lipid profi le and applying multifactorial statistic analysis, it was shown that ε4 gene carrier was the risk factor for CHD, but not in triglyceride level, whereas ε2 carrier gene was not the risk factor for CHD. Dislipidemia was the risk factor for CHD and ApoE ε4 gene carrier was the risk factor


Introduction
Apolipoprotein E (apoE) is a protein constituent of plasma lipoproteins that performs several functions including a role in cholesterol metabolism and as an important ligand in lipoprotein clearance.Apolipoprotein E was fi rst identifi ed as a constituent of very low density lipoprotein (VLDL) which function to transport triglycerides from the liver to peripheral tissues (Mayes and Botham, 2006).Apolipoprotein E gene is polymorphic and exists in six different isoform proteins, designated E2/E2, E2/E3, E2/E4, E3/E3, E3/ E4, and E4/E4 which are the gene products of three ApoE alleles i.e. ε2, ε3 and ε4 respectively (Belkovets et al., 2001).Apolipoprotein E2 exhibits lower affi nity for the LDL receptor, resulting in slower clearance of ApoE and higher plasma apoE levels.Conversely, ApoE4 is cleared more effi ciently, resulting in lower ApoE levels and higher cholesterol levels.The genetic variations thus affect lipid metabolism and have been shown to alter risk of cardiovascular disease and dementia (Eichner et al., 2002).
Epidemiologic studies addressing the contribution of apo ε gene to Coronary Heart Disease (CHD), reported that 6 percent of the variation in risk for CHD in North America can be attributed to this locus.Another study of middle-aged men from nine populations estimated a 40 percent increased risk for CHD mortality for ε4 carriers compared with Hastuti et al. ε3 carriers or ε2 carriers (Stengard et al., 1998).Some studies have also suggested that ε4 carriers are particularly prone to developing disseminated coronary lesions or to have an increased risk of death from CHD (Eichner et al., 1993, Lehtinen et al., 1995;Stengard et al., 1995, Wang et al., 1995).Coronary heart disease is related to dysfunction of the E4 isoform in lipoprotein metabolism and an increased concentration of serum cholesterol and triglycerides.Studies from Finland, Scotland, and northern part of Ireland have shown that populations with higher cholesterol levels and higher CHD mortality rates also have a higher frequency of the ε4 allele.Other studies have also associated the ε2 allele with increased CHD risk (Eichner et al., 2002, Zannis et al., 1996, Mahley et al., 2006).
An association between apoE ε2/ε2 and type III hyperlipoproteinemia has been known for decades.This disorder is characterized by increased cholesterol and triglyceride levels, the presence of ß-VLDL (cholesterol-enriched remnants of intestinal chylomicrons and hepatic VLDL), xanthomas, and premature vascular disease, both CHD and peripheral artery disease (Mahley et al., 1995).Overt hyperlipoproteinemia III occurs with a frequency of 1-5 per 5,000, whereas homozygosity for E2/2 occurs with a frequency of 0.5-1.0 per 100 in Caucasian populations.Thus, this genotype contributes to the hyperlipoproteinemia III phenotype without being its sole cause (Eichner et al., 2002, Fullerton et al., 2000).It examines the relationship of this genotype in CHD patients compared to the specifi ed controls.

Materials and Methods
Blood samples were collected from CHD patients in Dr. Sardjito Hospital Yogyakarta compared with controls collected from an exersice group in Yogyakarta.
Inclusion criteria: patients were diagnosed by an Internal Medicine Specialist as CHD, Javanese male or female age 40 -75 years, BMI <30, and free from Diabetes Mellitus (DM).Exclusion criteria: CHD patients with DM and non Javanese ethnic background.Inclusion criteria for the control group: apparently healthy male or female individuals, BMI < 30, no DM, age 40 -75 years and Javanese ethnic in origin.Exclusion criteria: DM and non Javanese.
Plasma from patients and controls were separated and their lipid profi les were examined.Buffy coat from patients and controls were prepared for DNA isolation.Polymerase Chain Reaction (PCR) was performed with Zivelin et al., (1997) method to amplify exon 4 and then followed by HaeII dan Afl III digestion to identify the ε2, ε3 and ε4 allele with 195 and 23 bp for ε4; 23, 50 and 145 bp for ε3 and 50 and 168 bp for ε2 respectively.Genotype of ε2/ε2 and ε2/ε3 were grouped as ε2 carrier; genotype of ε3/ε3 was grouped as ε3 carrier and genotype of ε3/ε4 and ε4/ε4 were grouped as ε4 carrier.ApoE ε2/ε4 was not included as any apoE carrier.The result was analyzed employing t-test to compare lipid profi le in CHD patients and controls with signifi cant difference at p <0.05.Odd Ratio was used to determine the risk factors, while Chi Square test was used for frequency of apoE genotype and allele as risk factor of CHD.Another Odd Ratio was apllied to analyse apoE genotype and allele as risk factor of CHD with controlled lipid profi le.

Results
Apolipoprotein E polymorphism was examined in 33 CHD patients and 38 controls.There were no signifi cant difference between patients and controls in the body weight, height, BMI, blood pressure and blood glucose with t-test p>0.05 (Table 1).
There were no significant difference between CHD patients and controls in their lipid profi le (p>0.05)despite higher levels of triglyceride, cholesterol, and LDL-C and lower level of HDL-C in CHD patients (Table 2 and Figure 1).
When Chi square analysis was employed, higher frequency of dyslipidemia in CHD patients was observed with    signifi cant difference (p<0.05) in cholesterol concentration.Applying Odd Ratio calculation, it was showed that dyslipidemia was a strong risk factor for CHD with 1.4 -3.93 times higher (Table 3) The result of PCR (Figure 2) was digested by Afl III and Hae II to determine the apoE ε2, apoE ε3, and apoE ε4 genotypes (Figure 3).
On the other hand, despite p>0.05 for ε4 allele, OR 2.05 indicates its role as risk factor for CHD.It can be summarized that apoE ε3/ ε4 genotype and ε4 allele were risk factors for CHD, whereas apoE ε2/ε2 genotype and ε2 allele were protective factors for CHD.
B e i n g a s r i s k f a c t o r f o r C H D , dyslipidemic condition especially the increase of cholesterol and LDL-C as well as the decrease of HDL-C levels were risk factors for CHD for all of apoE genotypes (Tabel 5, 6, and 7).In contrast, the increase of triglyceride level in ε2 carrier gene was not the risk factor for CHD (Table 8).
Table 5 showed the relation of apoE polymorphism with hypercholesterolemia which is 5 and 7.88 times higher to have CHD in high concentration of cholesterol but in the case of ε4 carrier the OR of this gene carrier could not be calculated due to low level of cholesterol in the control group.This result summarized that hypercholesterolemia caused CHD in all of apoE gene carrier.Table 6 showed that high level of LDL-C was risk factor for CHD with ε2 and ε4 alleles, but not for ε3 allele.In low HDL-C level, individual with ε2 and ε3 alleles were at risk for CHD (Table 7).8 showed that high level of triglyceride was the risk factor for CHD with ε3 and ε4 alleles, but not for ε2 allele.
The overall OR of lipid profi le showed ε2 carrier of apoE gene was the risk factor for CHD provided that the respective individuals have high cholesterol and low HDL-C level.Similarly, carrier of ε3 and ε4   .197 -14.165 apoE gene were also at risk of CHD in the presense of dyslipidemia with high levels of triglycerides, total cholesterol and LDL-C and low level of HDL-C.This study was designed as a case control study without special treatment to control lipid profi le in both groups which was later controlled by multifactorial analysis.Table 9 showed even in controlled lipid profi le, ε4 gene carrier has the risk factor for CHD 3.94 times higher except in triglyceride level, while the ε2 gene carrier was not the risk factor for CHD with OR <1.

Discussion
To demonstrate the role of ApoE polymorphism as the risk factor for CHD, the frequency of ApoE gene in CHD patients was compared with controls.It was shown that ε2 allele was the protective factor for CHD and ε4 allele was the risk factor for CHD.This risk factor was not different in other world populations, in which ε4 gene was the risk factor for CHD than those ε2 and ε3 genes (Eichner et al., 2002;Elousa et al., 2004;Mahley et al., 2006;Mc. Neale et al., 2000;Pirim et al., 2001).This ε4 gene was also the risk factor for myocard infarc, atherosclerosis, stroke, and neurodegeneration (Elousa et al., 2004;Frikke-Schmidt et al., 2000 (a); Guera et al., 2003;Leshinsky-Silver et al., 2006;Mahley et al., 2006;Masemola et al., 2007;Moghadasian et al., 2001;Sheehan et al., 2000;Sima et al., 2006;Yang et al., 2004).Population with high frequency of ε4 allele have high incidence of ischemic heart disease and this genetic determinant was related to mortality in isolated populations (Garces et al., 2004).Different result were found in China, where polymorphism of ε4 gene was not the risk factor for CHD (Liu et al., 2003), as well as in coronary artery disease.This ε4 gene was not the risk factor in Oman, Greek and Brazalian populations (Al-Yahyaee et al., 2007;De Franca et al., 2004;Kolovou et al., 2002;Souza et al., 2007) The role of apoE polymorphism in causing dyslipidemia was studied in CHD patients and controls.Apolipoprotein ε2 allele has protective effect for CHD, but ε3 and ε4 alleles were the risk factor for CHD especially in individuals with dyslipidemia.The role of apoE polymorphism on dyslipidemia in Indonesian populations seems to be almost similar to that in the world's populations in which the ε2 allele was the protective factor for CHD despite suffering from dyslipidemia (Chaaba et al., 2009;Chanprasetyothin et al., 2000;Eichner et al., 2002;Elousa et al., 2004;Mahley et al., 2006;Masemola et al., 2007;Moghadasian et al., 2001;Rodsari et al., 2005;Sheehan et al., 2000;Yang et al., 2004;Zannis et al., 1996).Other studies reported that ε2 allele was related with high triglyceride level and the incidence of type III hyperlipoproteinemia (Batal et al., 2000;Bennet et al., 2007;Eichner et al., 2002;Letonja et al., 2004;Liberopoulos et al., 2004;Pallaud et al., 2001).
The role of apoE polymorphism in causing dyslipidemia is due to the ability of apoE3 to accept more cholesterol from fi broblast than apoE2 and apoE4 (Huang et al., 2009).In HDL 3 , apoE3 binds cholesterol better than apoE4, because the structure of carbon end domain of apoE4 was irregular and more exposed to the water; these differences causes the pathologically of cardiovascular and neurodegenerative disease (Sakamoto et al., 2008).These conditions showed that genetic factors and lipid profi le varies with age, sex, and the differences of environmental factors (Pallaud et al., 2001).Studies by Eichner et al., (2002), giving hypolipidemic drugs to block HMG-CoA reductase or drugs to reduce lipid profi les, was effective for apoE ε2 and apoE ε3 gene carriers, but apoE ε4 gene carrier was difficult to be influenced by medical intervention.Low lipid and cholesterol diets induced the decrease of cholesterol and LDL levels higher in apoE ε4 gene carrier than in apoE ε2 and apoE ε3 gene carriers.It was shown that response to hypolipidemic drugs was different in apoE polymorphism.Treatment with phenofi brate showed that ε2 allele reduce lower in triglyceride level than others (Irvin et al., 2010).The defect in E4 protein, causes effi cacy to bind and transport of lipid decrease.Statin drug to decrease lipid profi les was not responded by some individuals because of high response variability of hypolipidemic drugs.It can be summarized that the detection of genetic variability that infl uence lipoprotein levels in the plasma supports to predict therapeutic response (Morrison, 2007).The ε4 allele is consistently lower in reduced cholesterol level after hypolipidemic therapeutic in Portugese (Withers, 2011).Regarding the effect of therapeutic intervention to modify the disease related with apoE polymorphism, apoE ε4 gene carrier had the worst effect (Cacabelos et al., 2010) As conlusion, dyslipidemia was the risk factor for CHD.Apolipoprotein E ε3/ε4 genotype and ε4 allele were the risk factor for CHD whereas apoE ε2/ε2 genotype and ε2 allele were protective factor for CHD.Polymorphism of apoE led to variability of triglyceride level but not causes variability of other lipid profi le.Apolipoprotein E ε3/ε4 genotype and ε4 allele were the risk factor for CHD.

Figure 1 .
Figure 1.Lipid profi les in CHD and control

Table 2 .
Lipid profi le in CHD patients and controls

Table 1 .
Sex, age, body weight, height, blood pressure, and blood glucose level in CHD patients and control

Table 3 .
Odd Ratio of lipid profi le between CHD and controls

Table 6 .
Risk factor of ApoE polymorphism in LDL-C level in CHD patients and Control

Table 7 .
Risk factor for apoE polymorphism in HDL-C level in CHD patients and control

Table 5 .
Risk factor of apoE polymorphism to high level of cholesterol in CHD patients and control

Table 8 .
Risk factor for ApoE polymorphism in triglyceride level in CHD patients and control

Table 9 .
The role of ApoE polymorphism as the risk factor for CHD with controlled lipid profi le.