Newman, Mark F. MD*,; Laskowitz, Daniel T. MD†,; White, William D. MPH*,; Kirchner, Jerry L. BS*,; Grocott, Hilary P. MD, FRCPC*,; Stafford-Smith, Mark MD*,; Sketch, Michael H. MD‡,; Jones, Robert H. MD§,; Reves, J. G. MD*,; Saunders, Ann M. PhD†
Coronary artery disease (CAD) results in almost 500,000 deaths each year, and remains the number one cause of death in the United States (1). Atherosclerosis, an organized, active, life-long process, involves elements of chronic inflammation followed by repair of the artery wall (2). Apolipoprotein E (apoE) is a heritable determinant of total and low-density lipoprotein (LDL) cholesterol. There are three common human apoE isoforms that differ by a single amino acid substitution, designated apoE2, apoE3, and apoE4. Epidemiological studies show that the apoE structural variation is a determinant of interindividual differences about the population means of total LDL cholesterol (3,4). In vivo and in vitro investigations have examined the mechanisms by which apoE4 genotype leads to alterations in lipids and lipoproteins (5–8). apoE modulates the catabolism of triglyceride depleted remnants of chylomicrons and very LDLs by acting as a ligand for the LDL receptor and for the chylomicron remnant receptor.
The importance of apoE-mediated increases in cholesterol level on the severity of atherosclerosis has been controversial (4,9,10). However, several studies have identified a relationship between the apoE genotype and atherosclerosis (11–16). These studies indicate that apoE4 contributes to the development of atherosclerosis and is a major factor responsible for predisposition to this disease (17,18).
The purpose of our investigation was to determine whether apoE4 genotype alters the age of presentation for coronary artery bypass graft (CABG); specifically, whether the presence of the apoE4 allele is associated with an earlier age at primary surgical intervention for coronary atherosclerosis.
After IRB approval and patient informed consent, 560 patients scheduled for CABG were entered into a continuing study assessing the impact of apoE4 genotype on perioperative neurological, neuropsychological, renal, and myocardial outcomes. Exclusion criteria included emergency surgery, history of severe renal disease (preoperative creatinine level >177 μmol/L), hepatic disease, or symptomatic cerebrovascular disease with residual neurological deficits.
Peripheral blood samples were obtained from each patient and stored at 4°C before processing. Genomic DNA from these samples was analyzed for apoE genotypes by using a published method (19), with minor modifications for fluorographic rather than autoradiographic detection of DNA. All study personnel were blinded to the apoE analysis results.
Age at first CABG, the primary endpoint in this study, was calculated as the patient’s age in days at the date of first CABG. We eliminated from the analysis four cases in which the first revascularization status was unknown.
Baseline, demographic, and clinical variables were described for categorical data by summarizing frequencies and percentages, and for continuous data as means with standard deviations and medians, 25th and 75th percentiles. Differences between allelic groups in these demographic and clinical variables were assessed univariately by using the Fisher’s exact test or a χ2 test for categorical data, and the Wilcoxon’s ranked sum test for continuous numerical data. We first assessed the association between apoE genotypes and age at first CABG by using a univariate Kruskal-Wallis test and an analysis of variance, controlling for gender. Our goal was to determine the effect of apoE4 allele. The number of patients in some individual genotype groups was too small to support group comparisons; therefore, we next compared patients with one or more copies of the apoE4 allele to those without, both univariately and again when controlling for gender. Finally, we categorized genotype by the number of apoE4 alleles (0,1,2) in an “apoE4 allele dose” analysis, performed first univariately with a ranked sum test and then multivariately by using a general linear analysis of covariance model adjusting for gender, race, diabetes, and number of previous nonsurgical revascularization and all the two-way interactions of these with the apoE4 dose group. A backward stepwise variable selection method was used to sequentially drop nonsignificant terms from the model. No main effects involved in a significant interaction were eliminated. In a confirmatory, nonparametric analysis, we also tested the same model by using rank of age at first CABG as the dependent variable. P values were reported as observed in each comparison; a conservative significance level of α = 0.017 (0.05/3) may be applied to adjust for our sequential comparisons of genotype effect.
We performed apoE genotyping on the 560 patients entered in the study. Demographics of the population are included in Table 1 (categorical variables and continuous variables).
ApoE genotypes for the study population included 28.9% (162 of 560 patients) carrying at least 1 copy of the apoE4 allele. Our sample matched the published allelic frequencies of apoE2, apoE3, and apoE4 in the population at 0.08, 0.77, and 0.15, respectively (20). The apoE4 and non-apoE4 groups were compared with respect to demographic and perioperative characteristics. No significant differences were seen on any of the numerical or categorical characteristics except race, which was included as a covariable in the multivariable model. Unadjusted comparison of age at first CABG by the six apoE genotype groups showed a marginally significant effect, Kruskal-Wallis P = 0.057 (Fig. 1), with a substantially younger age for homozygous 4/4 and for all genotypes, with at least one copy of the apoE4 allele. When we grouped the population into those patients with or without at least one copy of the apoE4 allele, univariate analysis (Wilcoxon’s ranked sum test) determined a significant difference in age at first CABG by the presence or absence of the apoE4 allele (P = 0.032). Patients with one or more copies of the apoE4 allele presented at a younger age (60.6 ± 10.3 yr vs 62.6 ± 10.2 yr). Finally, our unadjusted gene-dose analysis was also significant (P = 0.0126), despite only nine patients with the homozygous apoE4/4 genotype. The age at first CABG for patients with two copies of the apoE4 allele (mean, standard deviation) was 54.21 ± 6.95, compared with 61.01 ± 10.33 for those with a single apoE4 and 62.57 ± 10.15 for those with none. Figure 2 and Table 2 illustrate the differences in mean age by allele count and gender.
To control for other variables associated with an increased risk for coronary atherosclerosis, gender, diabetes, and number of previous nonsurgical coronary interventions were added into a multivariable model assessing the impact of apoE4 on age at first CABG. We also tested ethnicity in the multivariable analysis, because of previously described ethnicity differences in the impact of apoE4 on atherosclerosis (21).
In the two-group multivariable analysis, a significant relationship persisted between the presence of the apoE4 allele and the age at first myocardial revascularization (P = 0.037). Further, the three-group gene-dose analysis was also significant (P = 0.012). Of the other variables included in the model, gender obtained statistical significance (P < 0.001), with male patients presenting at a younger age as expected. Also, the number of coronary interventions predicted a younger age for surgery, potentially identifying individuals with more severe CAD or more reactive vessels (Table 3). After adjustment for gender, the estimated mean ages by gene dose were 52.2 yr for the homozygous apoE4, 60.2 yr for the single apoE4, and 61.6 yr for those with no apoE4 (least-squares means). The nonparametric multivariable analysis on age rank confirmed these results, showing significant effects of apoE4 (P = 0.007), gender (P < 0.001), and previous coronary interventions (P = 0.011).
We report an association between apoE4 and an earlier age at first CABG. The severity of disease, number of vessels grafted, and left ventricular ejection fraction are similar among groups, despite the difference in age. There also appears to be a dose effect with homozygous 4/4 patients demonstrating the youngest age at primary surgery (Fig. 1 and 2).
apoE-ε4 has been linked to disease progression in a few organ systems. A member of our investigative group (AS) was involved in investigating apoE4 as a susceptibility gene in late and sporadic forms of Alzheimer disease (22). This has led to a substantial increase in the study of this genotype on a number of different long-term and perioperative outcomes. The apoE4 genotype plays a well characterized role in cholesterol metabolism (23). The role of apoE4 in the progression of atherosclerosis has been more controversial, but studies have linked apoE4 with the severity of coronary, carotid, and aortic atherosclerosis (17,18,21,24,25). Small studies have also indicated a substantial increase in the prevalence of the apoE4 genotypes in those patients presenting before age 40 for coronary angioplasty (26). However, this association is less clear, with some reports indicating a predilection for an increased presence of the apoE2 allele in families with early myocardial infarction (27). Our study supports the role of apoE4 in determining susceptibility for progression of CAD.
The mechanism by which apoE4 is associated with increased atherosclerosis appears to be related to increased levels of LDL cholesterol, but several studies controlling for cholesterol level have still shown an increased propensity for atherosclerosis (21). In addition to its role in lipid metabolism, one possibility is that apoE, locally secreted by the macrophages at the vessel wall, modulates the inflammatory process associated with atherosclerosis. This concept is consistent with the known immunomodulatory effects of apoE on peripheral immune tissue (28,29) and in brain tissue (30–32). Of particular relevance to the atherosclerotic process, apoE inhibits platelet-derived growth factor induced smooth muscle migration and proliferation (33). This is consistent with recent data demonstrating that the small-dose expression of human apoE transgene by macrophages in apoE-deficient mice significantly reverses their atherosclerotic genotype, with no effect on systemic hyperlipidemia (34). Mice that are apoE deficient have increased levels of autoantibodies (35,36). The possibility that apoE may protect against oxidation insults and lipid peroxidation is supported by observations that plasma lipoproteins from apoE-deficient mice are more susceptible to in vitro oxidation than those of wild-type mice (36), and that apoE-deficient animals express higher titers of autoantibodies directed against oxidized lipids than control animals (35).
The possibility that apoE modulates the vascular response to injury and atherosclerosis is also consistent with two clinical studies demonstrating an increased susceptibility for restenosis after angioplasty in patients with the apoE4 allele (37,38); however, this is neither universally accepted nor well demonstrated (39).
The primary limitation of our study is our exclusion of some patients (see Methods) to allow for the assessment of outcome in our continuing trials. Limiting enrollment makes our results less generalizable and could have introduced bias. The most obvious potential bias is the exclusion of patients with chronic renal insufficiency and residual neurological deficit after stroke. apoE-ε2 has been associated with the development of chronic renal disease (40,41), which could have reduced the percentage of apoE2 patients in our population. In some studies, but not others, the apoE4 allele has been associated with an increased incidence of stroke (42). If this association does exist, then we may have excluded more apoE4 patients by excluding patients with residual neurological deficits. The other confounding issue in our analysis is the possible change in allelic frequency that has been identified with increasing age by some groups (43,44). Decline in relative frequency of apoE4 with age is thought to occur because of differential survival based on the excess of Alzheimer disease and CAD; one study has confirmed this differential survival (45). Determining that the decline in frequency of apoE4 is caused by CAD would strengthen our hypothesis that apoE4 is related to a propensity for premature development of CAD. Our population is unique in that it was selected for surgical coronary revascularization and does not represent a cross-section of the population as a whole. Numerous factors may alter the decision to refer for coronary intervention, and we can only speculate on what these factors may be, because we have not followed a complete cross-sectional community. However, the fact remains that in the population studied, there was a distinct association between the presence of the apoE4 allele and earlier presentation for coronary revascularization.
We have demonstrated that the presence of the apoE4 allele is associated with an earlier presentation for surgical coronary revascularization. Our understanding of the genetics of coronary atherosclerosis is preliminary, and the relationship of this information to the efficacy of treatment strategies is currently insufficient. Prospective trials are necessary to assess the progression of patients who are relatively young at first cardiac surgery. In the future, the most appropriate intervention may be indicated by a patient’s genetic profile.
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