Introduction

Pathogenesis of coronary artery disease (CAD) is associated with multiple genetic and environmental risk factors. Several candidate genes related with lipid and lipoprotein metabolism have been studied for their association with the CAD. There is a wealth of literature on the ApoE and ApoAI polymorphism particularly for their effects on lipid profiles and CAD risk. Multiple genetic variations in apolipoprotein E and apolipoprotein A1 have been shown to influence lipoprotein metabolism and the plasma concentration of total cholesterol (TC), LDL cholesterol, apo B, and apo E, and confer risk for CAD. Apolipoprotein E is involved in cholesterol transport and is encoded by a polymorphic gene. Three common alleles designated E2, E3, and E4 code for three apo E isoforms E2, E3, and E4. ApoE4 has been found to be associated with higher plasma cholesterol levels, increased risk for CAD and subjects with E4/4 genotype showed an earlier onset of the disease [1–4]. However, association between CAD and ApoE gene variants has been found to be inconsistent and vary in different ethnic populations [5, 6].

Two polymorphisms of ApoA1 gene namely G to A substitution at −75 bp of transcription start site and C to T substitution at +83 bp have been implicated in susceptibility to CAD and shown to influence plasma lipid levels [7, 8]. Individuals carrying the A1 allele have been shown to have higher concentrations of ApoA1 and HDL-C [9–14]; however, the results have been inconsistent and inconclusive, with few studies reporting either no association [15–17] or negative association [18] between Apo1 alleles and plasma lipids.

Participation of single genes in a pathogenesis of complex diseases such as atherosclerosis and CAD is relatively small, but coexistence of several polymorphic variants of candidate genes may have higher impact on disease etiology. Several recent studies have shown that combinatorial effects of candidate genes may be better predictors of disease risk than the individual genes [19–21]. Although, ApoE and ApoAI gene variants are known to modulate plasma lipid levels and have been found to be associated with CAD, the co-occurrence of ApoE and ApoA1 gene polymorphisms on risk of CAD and lipid levels has not been evaluated. The present study was carried out: (a) to study the independent influence of ApoE and ApoA1 genotypes on lipids and apolipoprotein E and apolipoprotein A1 levels among Asian Indian patients of North Indian origin with premature CAD and (b) to determine combined impact of co occurrence of ApoE and ApoA1 polymorphisms on the risk of CAD and on serum lipid levels.

Materials and methods

Study population

The study was carried out on North Indian Punjabi population. The study subjects comprised of 140 clinically confirmed CAD patients (age <50 years), attending the cardiology Out Patients Department of Nehru Hospital at PGIMER, Chandigarh. One hundred age- and sex-matched subjects with normal clinical examination, normal ECG, negative stress test, and with no family history of CAD or diabetes were recruited as controls. The study was approved by the Institute Ethics Committee and an informed consent was obtained from all the subjects enrolled in the study. An overnight fasting venous blood sample was obtained and processed for biochemical estimations and genomic DNA isolation.

Biochemical analysis

Serum TC, triglycerides (TG), and high-density cholesterol (HDL) were measured using commercial kits (Accurex, India). The value of low-density lipoprotein was determined using Friedwald’s equation LDL − C = TC − (HDL + TG/5). Serum ApoA1 and ApoE were determined using their respective kits (Spinreact, Spain and Diiachi, Tokyo).

ApoE genotyping

ApoE genotyping was done by the method of Wenham et al. [22]. PCR was performed in a 25 μl reaction mixture containing 100 ng DNA, 2.0 mM Mg2+, 0.2 mM dNTP, and 1 U Taq DNA polymerase. The amplification protocol consisted of an initial denaturation at 95°C for 5 min, followed by 28 cycles of denaturation, primer annealing, and extension at 94, 60, and 72°C for 1 min each and final extension at 72°C for 5 min. An aliquot of 20 μl of amplified product was digested with 2 units Hha1 at 37°C for 15 h. The digested products were visualized on 12% polyacrylamide gel following silver staining (Fig. 1).

Fig. 1
figure 1

Genotyping of ApoE on 12% polyacrylamide gel electrophoresis

Full size image

ApoA1 genotyping

ApoA1 genotyping was done as described by Paul et al. [12]. PCR was performed in 25 μl containing 100 ng DNA, 2.5 mM Mg2+, 0.2 mM dNTP, and 1 U Taq DNA polymerase. The amplification protocol consisted of an initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation, primer annealing, and extension at 94, 62, and 72°C for 1 min each and final extension at 72°C for 5 min. An aliquot of 20 μl of amplified product was digested with 2.5 units Msp1 at 37°C for 15 h. The digested products were visualized on 12% polyacrylamide gel following silver staining (Fig. 2).

Fig. 2
figure 2

Genotyping of ApoA1 on 12% polyacrylamide gel electrophoresis

Full size image

Statistical analysis

Allelic frequencies were estimated by a gene count method and were tested for Hardy-Weinberg equilibrium. Differences of allele and genotype frequencies were compared using the χ2 test. The student t-test was used to compare means, and ANOVA was used with Tukey post hoc comparison. In order to estimate the risk of CAD, odd ratios were calculated by multiple logistic regression analysis after age and sex adjustments. When the number of subjects in the group was less than ten, the Fisher’s correction was used. Statistical significance was set at P < 0.05. For two- and three-way interactions, Bonferroni correction was applied and P < 0.016 was considered significant. All statistical analyses were performed using SPSS (Statistical Package for Social Sciences) for windows (Version 10).

Results

The demographic characteristics of the patients and control subjects are given in Table 1. HDL-Cholesterol, ApoE, and ApoA1 were significantly decreased (P < 0.05), and TG levels were significantly increased in patients as compared to controls (P < 0.05). Multiple logistic regression analysis of known predictors of CAD confirmed the independent role of age (OR 1.36, 95% CI 1.03–1.80, P < 0.05), male gender (OR 0.001, 95% CI 0.00–0.29, P < 0.05), plasma ApoA1 (OR 0.001, 95% CI 0.00–0.03, P < 0.05) and plasma ApoE (OR 0.02, 95% CI 0.00–0.24, P < 0.05) as risk factors for CAD in our population. Smoking was also found to be significantly associated with risk of CAD (P < 0.05).

Table 1 Demographic profile
Full size table

Genotypes

The percentage prevalence of genotypes and allele frequencies of ApoE and ApoA1 are given in Tables 2 and 3. Prevalence of ApoE 4/4 genotype, ApoA1-75 AA, and ApoA+83 TT genotypes was significantly higher in patients as compared to controls (P < 0.05). ApoE 4/4 genotype was associated with higher risk of the disease (OR 3.11, 95% CI, P < 0.05). Percentage prevalence of E2 (21.07%) and E4 allele (49.29%) was also significantly higher in patients as compared to controls (11.50%, 31%). ApoA1-75 ‘AA’ and ApoA+83 ‘TT’ genotypes showed higher odds ratio (Table 2), associated with increased risk of CAD. For ApoA1−75 and +83 polymorphisms, the prevalence of ‘A’ and ‘T’ alleles was significantly higher in patients (52.14%, 42.14%) as compared to controls (29.50%, 8%) (P < 0.05). An analysis of ApoA1 diplotypes showed that individuals with AA/TT (OR 8.44), GA/CT (OR 30.53), and AA/CT (OR 14.60) diplotypes had higher risk of CAD (Table 4).

Table 2 Percentage prevalence of genotypes
Full size table
Table 3 Percentage prevalence of alleles
Full size table
Table 4 Percentage prevalence of ApoA1 diplotypes
Full size table

Apolipoprotein E, apolipoprotein AI genotypes, and lipid levels

Associations of ApoA1 and ApoE genotypes with lipid profile and apolipoprotein levels are shown in Table 5. Patients with ApoE 3/3 and ApoE 3/4 genotypes had significantly lower HDL-C levels than controls. HDL-C levels were also significantly lower in patients with ApoA1 AA and CC genotypes (P < 0.05). Patients with ApoA1−75 GG and +83 CC genotypes had higher TG levels than controls. Cholesterol levels were found to be higher in patients with E4/4 genotype as compared to patients with E2/2 genotype. LDL levels were also significantly higher in patients with E4/4 genotype compared to patients with E3/3 genotype. Apolipoprotein A1 was found to be significantly associated with ‘A’ allele among patients but not in controls.

Table 5 ApoA1 and ApoE genotypes and biochemical characteristics patient versus controls
Full size table

Synergistic effect of ApoE and ApoA1 gene polymorphisms

Subgroups were formed according to the ApoE and ApoA1 polymorphisms. Both two- and three-way interactions were analyzed. Genotype combinations conferring protection and risk to CAD were identified. Only those combinations which were significantly associated with either risk or protection of CAD are shown (Tables 6 and 7). ApoE 2/4 genotype in combination with heterozygous Msp1 −75 (GA) or +83 bp (CT) ApoA1 genotypes showed odd ratio of 5.88 and 19.46, respectively, indicating association with significantly increased risk of CAD (P < 0.05). This significance was abolished for E2/4 and GA genotypes after Bonferroni correction. ApoE 3/4 genotype in combination with heterozygous C/T genotype of ApoA1 also showed increased risk of CAD (OR 6.02, P < 0.001).

Table 6 Genotype combinations conferring protection
Full size table
Table 7 Genotype combinations conferring risk
Full size table

Three-way interaction between ApoE genotypes in combination with ApoA1 diplotypes showed that ApoE3/4 genotype in combination with ApoA1 GG/CC diplotype was most prevalent in controls (OR 0.06, CI 95%, P < 0.001). E3 allele in homozygous or heterozygous state in combination with ApoA1+83 CC genotype conferred highest protection (P < 0.05).

Discussion

The prevalence and mortality rates of CAD have been known to be higher in Indians than the Western population. Most data on lipid levels in Indians have been obtained from studies on migrant Asian Indians. There are insufficient data on lipid profile and other conventional risk factors in Indian patients living within India. We observed normal cholesterol, LDL-C, decreased HDL-C, and raised TG levels in our patient group with premature CAD. Our results are in agreement with those reported earlier for Asian Indians [23] indicating that CAD occurs at much lower levels of TC and low-density lipoprotein cholesterol in northern Indians than other populations, and high triglyceride and low high-density lipoprotein levels are more of a universal phenomenon in this population. The absence of traditional dyslipidemia among Indian patients has also prompted studies on role of non-traditional risk factors in CAD in Asian Indians. Low serum levels of ApoAI and ApoE have been proposed to be independent risk factors for CAD and may initiate atherosclerosis, especially in patients with no major risk factors [24]. We too observed significantly lower levels of ApoAI and ApoE in our patients and found that higher ApoA1 and ApoE levels were protective against CAD in our subjects (ApoA1 OR 0.001, 95% CI 0.00–0.03, P < 0.05; ApoE OR 0.02, 95% CI 0.00–0.24, P < 0.05). Our results suggest that quantitation of apolipoproteins could be another useful parameter to identify subjects at risk of CAD and may assume importance in the definite subset of Indian patients with normal levels of cholesterol and LDL-C and an early-onset of CAD.

Genetic variations at the ApoE locus have been found to influence the risk of CAD and myocardial infarction (MI) in different ethnic populations. In India, data on association between ApoE polymorphism and CAD are limited and inconclusive. Conflicting studies indicating an increased prevalence of ApoE4 allele in patients [25] and a lack of association between genotype prevalence and CAD have been reported [26]. These conflicting reports could be due to regional variations in genotype frequencies among the populations studied or small sample size in these studies [27, 28]. We found a significant higher prevalence of ApoE4/4 genotype compared to controls and E4 allele to be associated with higher risk of the disease in our patients who were all from northern India and were selected on the basis of rigid clinical phenotype of early onset of disease. Our study confirms a positive association of E4/4 genotype with higher risk of premature CAD in our patients similar to those observed in other ethnic groups.

Studies in several different populations have shown varied association of ApoAI polymorphisms and risk of CAD [29–32]. A higher but non-significant prevalence of ApoA1-75 ‘A’ allele has been reported in CAD patients of north Indian origin [19]. We observed significantly higher frequency of ApoA1-75 AA and ApoA+83 TT genotypes in CAD patients and co-occurrence of these genotypes further increased the risk of CAD as indicated by higher odd ratio (OR 8.44). Our results are similar to those reported by Wang et al. [17] who also found homozygous AA and TT genotypes to be associated with CAD in Australian Caucasian population.

ApoE and ApoAI gene variants may influence serum lipid and lipoprotein metabolism; for example, ApoE4 isoform, which has a higher affinity for LDL-receptor, could lead to increased TC, LDL-C, and apo B levels by down regulating these receptors. We observed higher cholesterol and LDL-C levels in patients with ApoE4/4 genotypes. These results are similar to those reported earlier in other populations [33–36] and confirm the strong influence of ApoE4 allele on increased cholesterol levels. However, none of the ApoE genotypes was found to be associated with the raised TG in the present study, whereas a strong association between TG and ApoE4 genotype has been reported in Caucasian population. It appears that genes such as lipoprotein lipase and ApoC3 that are also known to modulate TG levels may be playing a more dominant role in modulating TG levels in our population [37].

Besides ApoE, ApoAI polymorphisms were also seen to influence plasma ApoAI concentrations in the present study. We observed higher serum ApoAI levels in patients carrying ‘A’ allele. An association between ApoAI ‘A’ allele and higher concentrations of ApoA1 and HDL-C has been reported in Caucasian populations [9, 10, 32]. However, we did not observe any association between HDL-C and ApoAI genotypes, which could be either due to ethnic or environmental differences as Asian Indians tend to have lower HDL-C levels [38].

Since ApoE and ApoAI alleles showed significant association with the risk of CAD and were found to influence plasma lipid levels, we also examined the combined effects of genetic polymorphisms in these two genes on the risk of CAD. We observed that individuals with ApoE4 allele in association with ApoA1 ‘A’ and ‘T’ alleles were more susceptible to CAD. Moreover we also observed that ApoE3 allele in combination with ApoA1 ‘G’ allele was protective against CAD. Although ApoA1 ‘A’ and ApoE4 alleles have been shown to be associated with increased susceptibility to CAD, ours is the first study showing the additive effects of these genotypes on susceptibility to CAD. A synergistic effect between ApoE and AGT polymorphisms and MI has been also observed [39] which has been attributed to higher cholesterol levels in patients with AGT-TT + ApoE4 allele carriers. Zak et al. [21] have also reported a considerably higher frequency of contemporaneous carriers of two or three proatherosclerotic variants of candidate genes in CAD patients. Thus, it appears that co-occurrence of polymorphic alleles of genes involved in the disease process may increase the risk of the disease.

Conclusions

Our results indicate that aggregate effects of candidate gene loci such as ApoE and ApoAI could significantly contribute to increased risk of CAD even though individual effect of these gene variations on the disease may be small. If studies based on other populations confirm our results, simultaneous genotyping of ApoE and ApoA1 polymorphisms could identify individuals at a higher risk of developing CAD.