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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:91-94.)
© 1997 American Heart Association, Inc.

Articles

Polymorphism of the Apolipoprotein E Gene and Early Carotid Atherosclerosis Defined by Ultrasonography in Asymptomatic Adults

L. Cattin; M. Fisicaro; M. Tonizzo; M. Valenti; G.M. Danek; M. Fonda; P.G. Da Col; S. Casagrande; E. Pincetti; M. Bovenzi; F. Baralle

the Atherosclerosis Research Center, Institute of Internal Medicine (L.M., M. Fisicaro, M.T., M.V., M. Fonda, P.G. Da C., S.C., E.P.), and the Institute of Occupational Health (M.B.), University of Trieste; and the International Centre for Genetic Engineering and Biotechnology (G.M.D., F.B.), Trieste, Italy.

Correspondence to Prof Dr L. Cattin, Clinica Medica Generale, Ospedale di Cattinara, Strada di Fiume 447, 34149-Trieste, Italy. E-mail luigic@clmed.univ.trieste.it.

	Abstract

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Clinical and autoptical studies have suggested a predisposing role of the allele E4 of apolipoprotein E (apoE) in the development of atherosclerosis and cardiovascular disease. To investigate the possible contribution of apoE allele polymorphism to the carotid intima-media thickness (IMT) as assessed by ultrasound, we studied 260 asymptomatic nondiabetic subjects (121 men, 139 women; mean±SD age, 53±7 years), randomly selected from the population register of the inhabitants of Trieste, Italy. B-mode ultrasound was used to quantify the maximum IMT at 12 sites on the near and far wall of the common, bifurcation, and internal carotid arteries. ApoE genotypes were determined from amplified apoE sequences by restriction isotyping. The frequencies of E2, E3, and E4 alleles were 0.073, 0.827, and 0.100, respectively. As expected, subjects with E4 allele had the highest levels of total serum cholesterol and LDL cholesterol, subjects with E2 allele had the lowest levels, and those with E3 genotype had intermediate levels. The echographic measurements of carotid IMT showed increasing values from E2 to E4 carriers. After adjustment for total and LDL cholesterol serum levels, triglycerides, ratio of LDL to HDL cholesterol, age, sex, and body mass index, ANCOVA showed that the common carotid IMT was significantly greater (P=.029) in subjects with E4 allele compared with E3 carriers. Our data confirm the influence of apoE4 on cholesterol levels and clearly show that apoE genotype affects carotid atherosclerosis in its early stages in middle-aged asymptomatic subjects.

Key Words: apolipoprotein E • polymorphism • carotid atherosclerosis • ultrasonography

	Introduction

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Apolipoprotein E is one of the major proteins involved in catabolism of triglyceride-rich lipoproteins (VLDL and remnants). ApoE acts as ligand for two receptors: the "remnants" receptor and the apoB/E receptor. In humans, the gene locus for apoE is polymorphic: three common alleles, E2, E3, and E4, code for three major apoE isoforms in plasma. ApoE3 is the predominant isoform; the other two isoforms differ by an amino acid substitution: apoE4 differs at position 112 (CysArg) and apoE2 at position 158 (ArgCys). These substitutions affect ligand binding of triglyceride-rich lipoproteins to the remnants and apoB/E receptors, thus affecting cholesterol serum levels.^{1 2} E2 allele is associated with lower LDL-C levels, and E4 allele with higher LDL-C levels, compared with E3 allele.^{1 3} Population studies have demonstrated that the different ethnic and geographic distributions of apoE isoforms are associated with a different prevalence of dyslipidemia and CAD.^{4 5 6} In Europe, there is a clear-cut gradient for the allele E4 frequency that increases from the south to the north, and it is associated with an increased incidence of CAD.⁷

Clinical and autoptical studies have suggested a predisposing role for E4 and a protective role for E2 in the development of atherosclerosis and cardiovascular disease.^{8 9 10 11} Up to now, no data are available on the role of apoE isoforms in early asymptomatic carotid atherosclerotic lesions in vivo. Atherosclerosis is a disease of the arterial wall, with increasing wall thickness representing an early event in the progression of the disease. In the past decade, ultrasound assessment of the arterial wall allowed in vivo study of atherosclerosis even in its early stages.^{12 13 14 15 16} The purpose of this study was to investigate the relationship between polymorphism of the apoE gene and carotid IMT as assessed by ultrasound in a group of middle-aged asymptomatic subjects.

	Methods

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Subjects
The study subjects were randomly selected from the population register of the inhabitants of Trieste, northeastern Italy. A total of 260 subjects (121 men, 139 women) aged 45 to 65 years (mean±SD, 53±7 years) who were nondiabetic and asymptomatic for cardiovascular disease were invited to participate in the study. To exclude cardiovascular diseases, all subjects underwent a clinical examination and interview that included the Rose Cardiovascular Questionnaire.¹⁷ Diabetic patients were excluded on the basis of a previous history and/or fasting serum glucose levels 7.8 mmol/L. Current medications were recorded, and subjects taking lipid-lowering drugs were excluded. The presence or absence of premature CAD among first-degree relatives (parents and siblings) was recorded. A smoking habit was defined as current smoking. Subjects were considered to have hypertension if a history of hypertension requiring treatment was recorded.

During the screening visit, weight and height were measured with subjects in light clothing without shoes. BMI was calculated as weight divided by height squared. Waist circumference was measured at the level of the umbilicus and hip circumference at the level of the greatest hip girth, with the subject standing and breathing normally. Body fat distribution was measured as waist-to-hip ratio. Informed consent was obtained from each subject. None of the invited subjects refused to participate in the study.

Lipid Analysis
Venous blood obtained from subjects was collected after an overnight fast in tubes containing EDTA. Total serum cholesterol and triglyceride levels were assayed enzymatically (Boehringer-Biochemia). HDL-C was measured enzymatically after precipitation of apoB containing lipoproteins with Mg-phosphotungstate. LDL-C level was then computed with the Friedewald formula.¹⁸

DNA Analysis for ApoE Genotypes
DNA was extracted from the frozen cellular blood component, and apoE genotypes were determined with a modified method as described by Hixson and Vernier.¹⁹ A section of apoE DNA that contains the genotype-differentiating sites was amplified by PCR. The DNA samples were preheated at 96°C for 5 minutes in a 50-µL PCR reaction buffer including dNTPs and Taq polymerase. The preheating was followed by 30 cycles of 96°C for 1 minute, 63°C for 1 minute, and 70°C for 45 seconds. The PCR product (244 bp) was subjected to Hha I digestion for 2 hours at 37°C, and the reaction mixture was loaded onto 11% polyacrylamide nondenaturing gel and electrophoresed. The gel was then treated with ethidium bromide, and the DNA fragments were visualized by ultraviolet illumination. The PCR isoform typing results were checked in 10% of the samples by the direct sequencing of amplified DNA.²⁰ No difference between the enzymatic and DNA sequencing methods was found.

Ultrasound Assessment of the Carotid Arteries
The ultrasound evaluation of the extracranial carotid arteries was performed using the Biosound 2000 II B-Mode ultrasound system with an 8-MHz probe. The scanning protocol used in this study was detailed elsewhere²¹ and is similar to that described by Crouse et al.²² The protocol entails the longitudinal scanning of the near and far wall of the left and right carotid arteries at the distal common carotid artery, carotid bifurcation, and proximal internal carotid artery. The common carotid was defined as the portion 10 mm below the dilatation of the bulb; the bifurcation was defined proximally by this landmark and distally by the tip of the flow divider; and the internal carotid artery was defined as the portion 10 mm above the tip of the flow divider. In summary, 12 carotid walls in each subject were scanned and analyzed; the measurements were performed three times in real time with an electronic caliper to the nearest 0.1 mm. The mean of these measurements was used in the analysis. Each of the 12 sites was examined with a circumferential scan; of the multiple longitudinal scans, those showing the maximum IMT were selected. An aggregate score was calculated on the basis of the extent of IMT in the 12 carotid walls: the mean maximum IMT represents the mean of the 12 individual maximum thicknesses. All the examinations were performed by two trained physicians certified by the Division of Vascular Ultrasound Research of the Bowman Gray School of Medicine (Wake Forest University, Winston-Salem, NC) during the CAIUS trial.²³ Examinations in which the artery wall was not visualized or showed poor imaging or extensive shadowing were excluded from the statistical analysis.

Statistical Analysis
Data analysis was performed with BMDP/Dynamic (release 7.0) software. Continuous data were summarized with the mean as a measure of central tendency and standard deviation as a measure of dispersion. The relationship between variables was tested by the least-squares method. Bivariate correlation was tested by the Pearson product-moment correlation coefficient. Group means were compared by one-way ANOVA. A multiple comparison test (Scheffe's method) was used to test the difference between pairs of means. To adjust for the influence of covariates on the outcome variable, ANCOVA was also used. The difference between categorical data tabulated in 2xk contingency tables was tested by ² statistic. Because triglyceride concentration and IMT of the arterial wall were not normally distributed, they were logarithmically transformed in statistical analysis. A value of P<.05 (two-sided) was chosen as the limit of statistical significance.

	Results

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The frequencies of E2, E3, and E4 alleles in the population included in this analysis were 0.073, 0.827, and 0.100, respectively. We regrouped subjects into those with E2 alleles, those with E3/3 genotype, and those with E4 alleles. We excluded 6 subjects (1.9%) with E2/4 genotype because it was difficult to assign them to any of the three groups. Therefore, data analysis was performed on 32 subjects with E2 allele, 177 with E3 genotype, and 45 with E4 allele. As shown in Table 1, the distribution of apoE alleles between men and women was not different. Age was similar in the three groups. There were no significant differences in BMI, waist-to-hip ratio, smoking habit, hypertension, and positive family history of premature CAD between apoE allele groups. The mean values of lipids in the apoE genotypes are reported in Table 2. After adjustment for age, sex, and BMI, levels of total cholesterol, LDL-C, and LDL-C to HDL-C ratio were significantly higher in subjects with E4 alleles than in those with E2 and E3 alleles (.005<P<.05). In the overall sample, IMT at the common carotid and bifurcation arteries was found to be positively correlated with age, sex, LDL-C, and LDL-C to HDL-C ratio (.001<P<.05). IMT at the same sites was inversely correlated to HDL-C (P<.02). Table 3 gives the sum of the maximum IMT of the near and far walls of the left and right carotid arteries at the three identified sites. Subjects who were E2 carriers had the lowest carotid IMT at each of the measured sites, whereas the highest values were observed in those with E4 alleles. One-way ANOVA showed that IMT at the common carotid artery was significantly greater in the E4 group than in both E2 and E3 groups (P<.05). After controlling for age, sex, BMI, total cholesterol, triglycerides, and LDL-C to HDL-C ratio, IMT at the common carotid artery remained significantly different in the three allele groups (P=.029). However, a multiple comparison test showed that the significant F ratio of ANCOVA was due to the difference between the E3 and the E4 groups. After adjustment for age, the estimated percent variance for IMT explained by apoE genotype was approximately 6%. After adjustment for total cholesterol and LDL-C serum levels, fasting triglycerides, and LDL-C to HDL-C ratio, as well as age, apoE genotype still explained approximately 3% of the IMT variance.

View this table: [in this window] [in a new window]   Table 1. Age, Sex, Anthropometric Data, and Main Cardiovascular Risk Factors According to Apo E Genotypes

View this table: [in this window] [in a new window]   Table 2. Plasma Lipids According to Apo E Genotypes

View this table: [in this window] [in a new window]   Table 3. Sum of Maximum IMT of Left and Right Carotid Arteries According to Apo E Genotypes

	Discussion

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Because the classic risk factors do not explain all cardiovascular morbidity and mortality, new risk factors have been sought. In recent years, apolipoproteins have been one of the main targets of interest.^{24 25 26} ApoE modulates lipoprotein transport and metabolism, and its polymorphism explains about 7% of cholesterol variation at the population level.¹ Accordingly, in our study, E4 allele has been found to be associated with higher cholesterol levels and a higher LDL-C to HDL-C ratio compared with E2 and E3 alleles. However, the key finding in this study is a significant relationship between apoE polymorphism and early carotid atherosclerosis in a sample of a middle-aged population, as indicated by highest values of carotid IMT in E4 carriers. Although many studies have found a higher frequency of the E4 allele in CAD patients than in healthy subjects^{10 11 27 28 29 30} and a more severe atherosclerosis compared with other phenotypes, as far as we are aware, a significant association with early carotid atherosclerosis in asymptomatic subjects has not been documented previously. Our results for E4 allele disagree with those of the recent cross-sectional study by de Andrade et al,³¹ who found an unexpected association between the E2 allele and carotid artery atherosclerotic disease identified by B-mode ultrasonography. The most likely causes of the discrepancy with our results are the study design and the statistical methods used for data analysis. Furthermore, the 95% confidence intervals for the odds ratios estimated by the authors were rather wide, suggesting that the association between carotid atherosclerosis and different apoE alleles was weak, even for E2/3 allele. Nevertheless, these data raised the question of whether carotid atherosclerosis depends not only on the direct effect of the apoE polymorphism on lipid metabolism and the atherosclerotic process but also on the heterogeneity of the study population with respect to cardiovascular risk factors. In our study, the echographic measurement of carotid IMT showed increasing values from E2 to E4 carriers, and this association was statistically significant at the common carotid artery. In agreement with other studies,^{21 32} our asymptomatic subjects showed the greatest IMT at the carotid bifurcation, with increasing values from E2 to E4 carriers; however, the differences among the three allele groups did not reach statistical significance because of the wide IMT range measured at the bifurcation due to ultrasonographic complexity of this area. These findings are consistent with those of the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) Study,⁸ in which the extent of atherosclerotic involvement of thoracic and abdominal aorta was assessed in young male subjects who died unexpectedly of unrelated causes. The greatest involvement was in those with the E4 allele and the least in those with the E2 polymorphism; this genotypic effect was independent of cholesterol levels. Considering the fact that apoE polymorphism modulates LDL-C levels and that the prevalence of early carotid atherosclerosis was higher in hypercholesterolemic subjects than in control subjects,^{21 33 34 35} it remains questionable as to whether LDL-C levels represent a confounding factor when the risk for carotid atherosclerosis is assessed according to apoE polymorphism. As expected, in the present study, carotid IMT was significantly related to the aging process, LDL-C levels, and LDL-C to HDL-C ratio. Nevertheless, after adjustment for these covariates and the other potential confounding factors, IMT at the common carotid artery remained significantly greater in the E4 allele group than in the E3 genotype group. The explained variance for IMT in the common carotid artery due to apoE genotype was approximately 6% after adjustment for age, quite similar to values for apoE effects on serum cholesterol concentration found at the population level.¹ After adjustment for total serum cholesterol and LDL-C levels, apoE genotype still explained approximately 3% of the variance for IMT in the common carotid artery, consistent with the variance for total lesions in the thoracic and abdominal aorta found by PDAY investigators in young males.⁸ However, Bergeron and Havel³⁶ have recently found that the postprandial increases in triglyceride-rich lipoproteins are prolonged in persons with an apoE4/3 phenotype compared with an apoE 3/3 phenotype. In our subjects, lipid levels were determined after an overnight fast. Therefore, we do not have information on serum levels of chylomicron and VLDL remnants, which are known to be atherogenic³⁷ and may well account for the 3% variance in carotid IMT that we observed in apoE4 carriers. These data support the view that apoE genotype affects both cholesterol levels and carotid atherosclerosis in its early stages in middle-aged asymptomatic subjects. More information is needed to determine whether apoE genotype affects atherogenesis independently of serum lipid profile.

	Selected Abbreviations and Acronyms

 

apo = apolipoprotein BMI = body mass index CAD = coronary artery disease HDL-C = HDL cholesterol IMT = intima-media thickness LDL-C = LDL cholesterol PCR = polymerase chain reaction

	Acknowledgments

 
This work was partly supported by the European Union Human Capital and Mobility Project "Biological History of European Populations," contract number ERBCHRXCT 920032, and partly by a research grant from the Regional Project for Prevention of Cardiovascular Diseases of Friuli-Venezia Giulia, Italy.

Received January 23, 1996; revision received May 14, 1996;

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