© 1995 American Heart Association, Inc.
Articles |
Polymorphisms of the Apolipoprotein E Gene and Severity of Coronary Artery Disease Defined by Angiography
From the Department of Cardiovascular Medicine, University of New South Wales, Prince Henry/Prince of Wales Hospitals, Sydney, Australia.
Correspondence to Prof David Wilcken, Department of Cardiovascular Medicine, Clinical Sciences Bldg, Prince Henry Hospital, Little Bay, NSW 2036, Australia.
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Abstract |
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Abstract In a recent study, we could account for only about 50% of the variance in angiographically determined severity of coronary artery disease (CAD) with use of lipid and clinical variables as predictors. To explore the possible contribution of the apolipoprotein (apo) E polymorphisms to the severity of CAD (rather than to its occurrence), we studied 424 white patients aged 65 years or less consecutively referred for coronary angiography. Among the 304 male and 120 female patients, there were 110 with no significant CAD and 118 with one, 96 with two, and 100 with three significantly diseased major coronary arteries (>50% luminal obstruction). The allele frequencies were 0.068 for E2, 0.759 for E3, and 0.172 for E4. The E2 frequency was slightly lower and E4 higher than the frequencies reported for healthy white populations (E2: 0.072 to 0.130; E4: 0.136 to 0.160). There was a clear association between the apo E genotype and the number of significantly diseased vessels (regression coefficient=.12, P=.008). The frequency of the E4 allele increased linearly with the increase in CAD severity in both sexes (for none, one, two, and three significantly diseased vessels; female patients: 0.136, 0.161, 0.200, and 0.324; male patients: 0.136, 0.167, 0.132, and 0.229, respectively, P<.01). The frequencies of the E2 allele, on the other hand, decreased with increasing severity (for none, one, two, and three significantly diseased vessels; female patients: 0.091, 0.018, 0.050, and 0.029; male patients: 0.073, 0.089, 0.072, and 0.054, respectively, P<.05). While much of the correlation was mediated through apo E–related changes in circulating levels of apo B and apo B–containing lipoproteins, after controlling for these lipid variables, there was an independent association between apo E genotype and CAD severity (P<.05). Among the 311 CAD patients who were not receiving lipid-lowering drugs at the time of testing, the E2 allele was significantly associated with low apo B levels (-21% for male patients, P<.01, and -9% for female patients, P<.05) compared with E3E3 genotypes, whereas the E4 allele was associated with high apo B levels (+12.4% for male patients, P<.05, and +19.8% for female patients, P<.01). We conclude that among patients with coronary disease, the apo E4 allele is associated with more severe and the E2 allele with less severe disease. While the apo E genotype had an important effect on the level of circulating apo B–containing lipoprotein levels, these associations with severity were mediated not only by changes in circulating apo B but also by mechanisms unrelated to circulating lipids in this population of CAD patients.
Key Words: apolipoprotein E • polymorphisms • coronary artery disease • lipoprotein profiles
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Introduction |
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In a recent study, we identified a hierarchy of circulating lipid variables and other clinical factors that were predictive of the severity of coronary artery disease (CAD) defined angiographically.1 Our hypothesis was that factors initiating coronary atherogenesis may not be the same as those responsible for its progression. While this proved to be the case,1 we were only able to account for about 50% of the variance in severity. In the present study, we considered the possibility that variations in arterial wall responses to local injury could partly account for the unexplained variability in CAD severity and that these may not be related to circulating variables. While it is difficult to explore local vessel wall function in vivo and to relate it to CAD severity, measuring DNA variants at relevant genes may provide a way to assess this relation. Apolipoprotein (apo) E is one of the genes that could provide this link because of its reported effect on endothelial cell proliferation.2
The gene for apo E is located on chromosome 19. Three common polymorphisms designated as E2, E3, and E4 code for the three major apo E protein isoforms in plasma.3 The three isoforms differ by amino acid substitution at one or both of two sites (residues 112 and 158) on the 299–amino-acid chain of the mature apo E molecule. E4 has arginine (DNA sequence for the site: GCGC) and E2 has cysteine (GTGC) at both sites, whereas E3 has cysteine at site 112 (GTGC) and arginine at site 158 (GCGC). These base pair differences create or eliminate the restriction site for the HhaI enzyme, and this enzyme has been used for apo E genotyping.4
Many studies have shown that apo E polymorphism may enhance atherogenesis indirectly by a strong effect on circulating levels of LDL cholesterol and apo B.3 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 The apo E2 allele is associated with low LDL and may be antiatherogenic, whereas the E4 allele, which is associated with high LDL, may be atherogenic.3 However, lipoproteins are only one of many contributors to atherogenesis, and the response of the arterial wall to local and other changes is also clearly important. Genes that influence this response could contribute to or inhibit the development of CAD. Recent reports that apo E is involved in immune reactions,21 22 23 tissue regeneration, and endothelial cell proliferation2 24 25 suggest that the apo E gene also could have a direct effect on the response of the arterial wall to injury. Thus, apo E polymorphism could be associated not only with the occurrence of CAD3 14 15 16 18 19 26 27 but also with CAD severity by mechanisms related to both circulating lipids and the arterial wall response to local injury.
To test this, we first explored relations between the apo E polymorphisms and the severity of CAD defined by coronary angiography and assessed the findings in relation to the changes in circulating lipoprotein levels associated with individual apo E genotypes. In a second approach, we assessed the effect of apo E polymorphisms on circulating levels of lipoproteins in the CAD population as a whole, irrespective of CAD severity.
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Methods |
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Patient Population
We studied white patients aged 65 years or less, both men and women, consecutively referred to the Eastern Heart Clinic at Prince Henry Hospital for coronary angiography over a 10-month period in 1994. We excluded only patients shown to have significant left main disease (>50% luminal obstruction) because it was difficult to categorize this small proportion of the total (4%) within the classification system we used (see below). Written consent was obtained from every patient after a full explanation of the study, which was approved by the Ethics Committee of the University of New South Wales.
A 4-mL venous blood sample was drawn into an EDTA sample tube before the angiogram after at least a 6-hour fast. The blood sample was centrifuged within 2 hours, and plasma and cellular components were stored separately at -70°C in aliquots until analysis.
DNA Analysis for the Detection of Apo E
Genotypes
DNA was extracted from the frozen cellular blood component by a
salting-out method adapted from that described by Miller et
al28 for whole frozen blood. The extracted DNA was stored
at 4°C until analysis. Apo E genotypes were
determined in a modified method as described by Hixson and
Vernier.4 In brief, a section of apo E DNA that contains
the genotype differentiating sites was amplified by polymerase
chain reaction (PCR). The sequences of the primers are
5'-TAAGCTTGGCACGGCTGTCCAAGGA-3' for upstream and
5'-AGAGAATTCGCCCCGGCCTGGTACAC-3' for downstream primers. The DNA
samples were preheated at 98°C for 30 minutes in a 25-µL PCR
reaction buffer without dNTPs and Taq polymerase. At the end
of the preheating, a mixture of Taq polymerase and dNTP in
25 µL PCR buffer was added and followed by 35 cycles of 95°C for 45
seconds and 67°C for 1.5 minutes. The PCR product (244-bp) was
then subjected to HhaI digestion for 2 hours at 37°C and
visualized by silver staining after electrophoresis in 8.0%
polyacrylamide gel.
Lipoprotein Analysis
Total cholesterol (TC), HDL cholesterol,
and triglyceride levels were measured by the hospital's
clinical chemistry department with use of standard enzymatic methods.
The LDL cholesterol levels were calculated using the
Friedewald formula. We measured levels of apo AI, apo B, and
lipoprotein(a) [Lp(a)] using enzyme-linked immunosorbent assay
methods developed in our laboratory and previously
reported.1
Patient Histories
We obtained each patient's medical history by use of a
questionnaire with standardized choices of answers to be ticked during
the interview. We recorded the presence or absence (yes/no) of a
history of hypertension requiring treatment, of diabetes, and angina
pectoris. A "don't know" box was also included for those not
clear about aspects of their past medical history and which were
unavailable from the patient's file. Current medications were
recorded, in particular, the usage of lipid-lowering drugs. The
presence or absence of CAD among first-degree relatives (parents and
siblings) and the age of first onset were recorded for a
quantitative assessment of family history of premature CAD. We
recorded the presence and severity of angina according to whether
each patient was experiencing no angina, stable angina, or unstable
angina before and during the current hospitalization. All those
classified as having unstable angina had an increase in pain frequency
as well as rest pain. The lifetime smoking dose in pack-years was
recorded as described previously.1
Documentation of CAD Severity
The severity of CAD was determined as follows. The angiograms
were assessed by two cardiologists who were unaware that the patients
were to be included in the study. Each angiogram was classified as
revealing either no coronary lesion with >50% luminal
stenosis or as having one, two, or three major epicardial
coronary arteries with >50% luminal obstructions. In a second
approach, we used the Green Lane coronary scoring
system.29 This provides a numerical value for lesion
severity and takes account of the amount of myocardium
supplied by an affected vessel; the maximum score is 15.
Statistical Analysis
Levels of the quantitative variables are presented
as mean±SEM. We used a full factorial design of ANOVA to assess
relations between the levels of lipoproteins, apolipoproteins, and the
apo E genotype.
We used log linear analysis for associations between the number
of significantly diseased vessels and apo E genotypes. The
number of diseased vessels was regarded as an ordinal variable
(none, one, two, or three significantly diseased vessels). The log
linear model was also used to evaluate the relation between apo E
genotype and the medical history, which included categorical
variables (eg, history of hypertension requiring treatment and
diabetes). Likelihood ratio 
2 values were used
for the assessment of significance.
We also used logistic linear regression analysis to evaluate the independent contribution of apo E genotype to the severity of CAD. In the analysis, the number of significantly diseased vessels was regarded as the ordinal variable, and apo E genotypes, sex, age, smoking dose, hypertension, diabetes, and levels of apolipoproteins and lipoproteins were entered as independent variables.
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Results |
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Patient Population
Of the 424 patients studied, 110 had normal coronary arteries or mild lesions, 118 had one, 96 had two, and 100 had three significantly stenosed major coronary arteries (>50% luminal obstruction). There were 304 male and 120 female patients. At the time of testing 114 were receiving lipid-lowering drugs, of whom 13 had mild or no lesions, 28 had one, 30 had two, and 43 had three significantly diseased vessels. In our patient population, there were 8 patients (6 men, 2 women) with the E2E2 genotype, 33 patients (25 men and 8 women) with E2E3, 9 patients (7 men and 2 women) with E2E4, 254 patients (183 men and 71 women) with E3E3, 103 patients (70 men and 33 women) with E3E4, and 17 patients (13 men and 4 women) with the E4E4 genotype. The frequencies of E2, E3, and E4 alleles were 0.068, 0.759, and 0.172. The distribution patterns of apo E alleles between male and female patients and ages were not different. The female patients had less severe CAD than did the male patients (P=.0001). The numbers with none, one, two, and three significantly diseased vessels for women were 56, 27, 19, and 18 and for men were 54, 91, 77, and 82, respectively.
Among patients not receiving lipid-lowering drugs (n=311), levels of HDL cholesterol and apo AI were significantly higher in the women (n=89) than in the men (n=217) (women versus men: 1.27±0.04 mmol/L versus 0.98±0.02 mmol/L for HDL cholesterol, P=.0001; 1.09±0.04 versus 0.93±0.02 g/L for apo AI, P=.001). Female patients also had higher TC and LDL cholesterol levels than did the male patients (women versus men for TC: 5.96±0.13 mmol/L versus 5.56±0.07 mmol/L, P=.01; for LDL cholesterol: 3.86±0.12 mmol/L versus 3.57±0.06 mmol/L, P=.037). However, levels of triglycerides and TC/HDL cholesterol were lower among female patients (women versus men for triglycerides: 1.84±0.13 mmol/L versus 2.24±0.08 mmol/L, P=.01; for TC/HDL cholesterol: 4.99±0.20 versus 6.04±0.11, P=.0001).
Apo E Genotypes and Lipid Variables
We conducted the statistical analysis of the lipoprotein
variables first among patients (n=311) not receiving lipid-lowering
drugs at the time of testing. Levels of TC, LDL
cholesterol, and apo B were significantly related to the
genotypes (Table 1
), but those of Lp(a), HDL
cholesterol, and apo AI were not. Patients who were E2
homozygotes had lowest apo B, TC, and LDL cholesterol
levels, and the higher levels were in patients with E4 alleles.
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We also assessed the effect of the apo E genotype on lipoproteins in male and female patients separately. To facilitate valid statistical comparisons, we regrouped patients into those with E2 alleles (18 men and 9 women), those with E3E3 genotypes (144 men and 51 women), and those with E4 alleles (54 men and 29 women). We excluded the 9 patients with E2E4 genotype because it was difficult to assign them to any of the three groups. Significant effects of apo E genotypes were seen on the levels of TC, LDL, and apo B. There was a linear increase in apo B levels with apo E genotypes both in male patients (E2: 0.83±0.07 g/L, E3: 1.05±0.03 g/L, E4: 1.18±0.05 g/L, P=.008) and female patients (E2: 0.88±0.09 g/L, E3: 0.96±0.03 g/L, E4: 1.15±0.06 g/L, P=.003). Patients with E2 alleles also had significantly (P<.05) lower LDL cholesterol levels (men: 2.96±0.22 mmol/L, women: 2.98±0.21 mmol/L) than those with either E3E3 genotypes (men: 3.61±0.08 mmol/L, women: 3.70±0.14 mmol/L) or E4 alleles (men: 3.61±0.13 mmol/L, women: 3.82±0.16 mmol/L). The same linear trends were seen in patients who were receiving lipid-lowering drugs, although they were not statistically significant (data not shown).
Apo E Genotypes and Number of Significantly Diseased
Vessels
We used a log linear model to assess the relation between apo E
genotypes and the number of significantly diseased vessels and
found a statistically significant association (regression
coefficient=.12, P=.008). As shown in Table 2, more patients with E4 alleles had three-vessel
disease than expected by the model of null hypothesis, and in the
patients with E2 alleles there were more than predicted with no
significantly diseased vessels. The association was further illustrated
when we compared the frequencies of the E2 and E4 alleles among
patients with differing CAD severity. As shown in Table 3, there was a near linear increase in the frequencies
of the E4 allele in patients with the increase in number of
significantly diseased vessels that was accompanied by a decrease in
the frequencies of the E2 allele. These linear changes were
particularly obvious in the 120 female patients. There was also a
nonsignificant trend for increased coronary scores among
patients with E4 alleles (E2E2: 4.93±1.05, E2E3: 4.62±0.79, E2E4:
5.93±2.1, E3E3: 4.99±0.20, E3E4: 6.25±0.47, E4E4: 6.02±1.21,
P=.14).
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In the logistic analysis, levels of TC/HDL
cholesterol, Lp(a), and apo B, age, smoking dose, sex,
history of diabetes, and hypertension remained to be significant
predictors of the severity of CAD, as we had found
previously.1 The association between apo E and the number
of significantly diseased vessels was minimized by the presence of
these independent risk factors, but it was still statistically
significant (regression coefficient=.097, P=.04). Further
analysis showed that apo B played the major role in this
attenuation. Nevertheless, after controlling for apo B in the log
linear analysis, the relation between the number of
significantly diseased vessels and apo E genotypes remained
statistically significant (regression coefficient=.117,
P=.03). As shown in Table 4, there is a
linear increase in apo B in relation to apo E genotypes, which
is not mirrored by a corresponding increase in the number of diseased
vessels.
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Apo E Genotypes and Clinical Data
We found no relations between apo E genotypes and
diabetes, hypertension, positive family history of premature CAD, or
the presence and severity of angina.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The key finding in this study is a significant association between apo E polymorphisms and CAD severity. While many studies have found a higher frequency of the E4 allele in CAD patients3 14 15 18 19 26 27 than in healthy control subjects, as far as we are aware, a significant association with severity of CAD has not been documented previously. In our study, the frequency of the E4 allele increased linearly with the increase in the number of significantly diseased vessels while there was a decline in the E2 allele with increasing severity, and these associations were particularly evident in the female patients.
These findings are different from those of Reardon et al,30 who were unable to show any significant correlation between apo E polymorphisms and CAD severity. However, the numbers in that study were comparatively small (65 male and 42 female patients), and Green Lane Coronary Scores were used to assess CAD severity. The Green Lane Score relates to the extent and site of the coronary obstruction and takes into account the amount of myocardium supplied by the affected vessel. It reflects the severity of ischemic heart disease and in our study was strongly correlated with the number of significantly stenosed arteries. However, it does represent a different end point from the number of significantly stenosed vessels, which comments more on the extent of the atherosclerotic involvement. Nevertheless, there was a trend for an increase in coronary score with the presence of the E4 allele in our study, although this did not reach statistical significance.
In a study by Hixson and the PDAY research group,15 the extent of atherosclerotic involvement of thoracic and abdominal aorta at autopsy was assessed in young male subjects aged 15 to 34 years who had died unexpectedly of external causes. The greatest involvement was in those with E4 alleles and the least in those with the E2 polymorphism. Their results indicated that the apo E genotype could account for 5.9% of the observed variation in atherosclerotic lesions in aorta and that this genotypic effect was independent of cholesterol levels. These autopsy findings are consistent with our in vivo findings in the coronary arteries of an older age group of both men and women.
An association between the E2 allele and raised triglyceride levels has been consistently reported in healthy control populations,3 7 8 and this was confirmed in our CAD patient population. Patients with the E2 genotype had relatively high triglyceride levels, although this did not offset the coronary protective effect of the E2 allele. Also in our study, there was no association between the E2 allele and circulating HDL and apo A-1 in accordance with the findings in normal populations.3 We also confirmed in our CAD patient population the significant contribution of the apo E genotype to the variation in LDL and TC levels, in particular to apo B levels.3 10 12 Compared with the E3E3 genotype, in male patients levels of apo B were increased by 12.4% with the E4 allele and were lowered by 21% with E2, whereas in female patients levels were +19.8% and -9% for E4 and E2 alleles. Similar increases and decreases were also observed for TC and LDL cholesterol. The same trend was observed among the patients receiving lipid-lowering drugs. Thus these associations are relevant to both healthy and coronary populations.
Finally, to explore the question whether or not the apo E genotype could have a more direct role in atherogenesis, when we tested whether the association between CAD severity and apo E genotypes was independent of altered circulating lipid levels, our results showed that this was indeed the case. Even though a large part of the association was mediated by elevated apo B and apo B–containing lipoprotein levels, the apo E genotype remained an independent significant predictor of CAD severity after controlling for the levels of these variables. This finding provides support for the concept of a more direct role for the apo E gene in atherogenesis.
Summary
We found a significant dose-dependent relation between apo E
polymorphisms and the number of significantly diseased
coronary arteries in that the E2 allele was associated with
mild disease and the E4 allele was associated with severe disease.
Although these associations were largely mediated by relations between
apo E polymorphisms and circulating apo B and apo B–containing
lipoproteins as predictors of CAD severity, there was nevertheless a
significant independent contribution from the apo E genotype.
This latter finding suggests that the apo E gene is involved
independently and more directly in atherogenesis, the E4 allele
being atherogenic and the E2 being antiatherogenic. We suggest that
this could be a locally mediated vessel wall effect unrelated to
circulating lipoproteins, but further studies are required to explore
this concept.
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Acknowledgments |
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This work was supported by grants from National Health and Medical Research Council of Australia. We wish to thank Dr Bridget Wilcken for reviewing the manuscript, Lily Fenech, Shelly Brown, Steven Brouwer, Dr Greg Cranny, and all nurses in the Eastern Heart Clinic for their assistance in clinical data collection, A.S. Sim for her laboratory assistance, and J. Kessey for the data entry. We are also most grateful to the cardiologists in the department for allowing us to study their patients.
Received March 25, 1995; accepted May 22, 1995.
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