Published online before print October 15, 2008, doi: 10.1161/CIRCGENETICS.108.791764
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Original Articles |
Interactions of Functional Apolipoprotein E Gene Promoter Polymorphisms With Smoking on Aortic Atherosclerosis
From the Department of Forensic Medicine (L.E.V., E.I., P.J.K.), University of Tampere Medical School and Centre for Laboratory Medicine, Tampere University Hospital, Tampere, Finland; Paediatric Research Centre (K.M.V., M.M.), University of Tampere Medical School and Tampere University Hospital, Tampere, Finland; Heart Center (E.I.), Department of Cardiology, Pirkanmaa Hospital District, Tampere, Finland; Tampere School of Public Health (H.H.), University of Tampere, Tampere, Finland; Department of Neurology (P.J.T.), Helsinki University Central Hospital, University of Helsinki, Biomedicum Helsinki, Molecular Neurology Programme C522b, Helsinki, Finland; National Public Health Institute (M.P.), Department of Molecular Medicine, Helsinki, Finland; and Laboratory of Atherosclerosis Genetics (T.L.), Department of Clinical Chemistry, Centre for Laboratory Medicine, University Hospital of Tampere and Tampere University Medical School, Department of Clinical Chemistry, Tampere, Finland.
Correspondence to Leena E. Viiri, Department of Forensic Medicine, School of Medicine, 33014 University of Tampere, Tampere, Finland. E-mail Leena.Viiri{at}uta.fi
Received May 13, 2008; accepted September 29, 2008.
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Abstract |
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Background— Apolipoprotein E gene (APOE) interacts with environmental factors in defining risk for atherosclerosis. We studied whether the APOE
2/3/4 genotype or APOE promoter polymorphisms –219G/T and +113G/C might interact with smoking on the development of fatty streaks. We also studied the previously unknown effects of +113G/C on transcriptional activity.
Methods and Results— The fatty streak areas of aorta were measured morphometrically in subjects of the Helsinki Sudden Death Study. Within APOE 3/3 subjects, there was a strong interaction between smoking and both –219G/T (P=0.009) and +113G/C (P=0.003) promoter polymorphisms on abdominal aorta fatty streak area: the –219T- and +113C-allele carriers had larger lesion areas compared with G/G (12.7% versus 5.9%, P=0.007; 12.9% versus 6.3%, P=0.010, respectively) within nonsmokers. Within smokers, the associations were inverse. Moreover, smoking increased the fatty streak area within –219G/G or +113G/G genotypes and –219G/+113G/3 haplotype carriers. Functional studies in reporter assay showed that in comparison with the +113G allele, the +113C allele had higher transcriptional activity and bound transcription factors from liver cell nuclear extract with significantly lower affinity.
Conclusions— In middle-aged Finnish men with APOE 3/3 genotype, the APOE promoter polymorphisms –219G/T and +113G/C interact with smoking in modulating aortic atherosclerosis. The +113G/C polymorphism has an effect on transcriptional activity.
Key Words: apolipoprotein • death, sudden • genetic transcription • genetics • lesion • smoking
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Introduction |
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Atherosclerosis begins early in childhood and progresses throughout life.1,2 Risk factors for atherosclerosis include a combination of environmental and genetic factors, such as high-serum low-density lipoprotein cholesterol, hypertension, diabetes, smoking (for review, see reference 3) and the
4 allele of the apolipoprotein E gene (APOE).4 The common APOE 2/3/4 polymorphism,5,6 as well as 2 promoter polymorphisms of the APOE gene, –219G/T and +113G/C, and their haplotype are known to have impact on serum lipid levels.7,8 In addition to single nucleotide polymorphisms (SNPs), the APOE gene haplotype has been shown to have clinical impact. The APOE –219G/+113G/3 haplotype has previously been associated with increased risk of neuropathologically verified Alzheimer disease.9 Moreover, the –219G/3 haplotype homozygosity has been associated with incident of dementia, in combination with low education and herpes seropositivity, in a cohort of elderly subjects with cardiovascular disorders.10
Clinical Perspective see p 107
In 1999, Stengård et al11 proposed that individuals with different APOE genotypes respond differently to environmental exposure such as smoking. Currently, gene–environment interactions are considered to have a crucial impact on the risk for coronary heart disease (CHD).12 In fact, several studies have explored the APOE–smoking interaction on CHD risk showing statistically significant results.13–15 However, to our knowledge, interactions of APOE promoter polymorphisms –219G/T and +113G/C with environmental factors in defining atherosclerotic lesions areas have not been studied before.
Autopsy study is currently the best way of studying, at the vessel-wall level, the very early steps of the pathogenesis of atherosclerosis, such as accumulation of lipids into the arterial wall. Only a few autopsy studies, however, have investigated atherosclerosis at the vessel-wall level in relation to different APOE genotypes, some reporting association of the APOE genotypes with lesion areas in aorta,16 coronary arteries,17 or neither.18 We have earlier shown association of the APOE 2/3/4 polymorphism with coronary and aortic atherosclerosis in middle-aged male victims of sudden death.19 Using this same autopsy data, we now studied the association of the APOE promoter polymorphisms, –219G/T and +113G/C, and the APOE haplotype with the fatty streak areas measured in coronary arteries and abdominal aorta, taking into account APOE genotype–environment interactions. Furthermore, the –219G/T is known to affect APOE transcriptional activity,20 but there are no previous reports about the possible transcriptional effects of the +113G/C polymorphism. Therefore, we also addressed this issue by studying the effects of the APOE +113G/C polymorphism on the transcriptional activity in human hepatoma cell line.
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Methods |
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Subjects
The study design of the Helsinki Sudden Death Study autopsy series, subjects, and methods have previously been described.19 In short, the Helsinki Sudden Death Study contains in total 700 Finnish men age 33 to 70 (mean age, 53 years; median, 54 years) whose causes of death were cardiac in 41%, other disease in 20%, and suicides or accidents in 39% of cases. The Ethics Committee of the Department of Forensic Medicine, University of Helsinki, approved the study. Spouse, relative, or a close friend of the deceased was interviewed to determine the possible risk factors of atherosclerosis. This was possible in 500 cases. The questionnaire included questions concerning hypertension, diabetes, as well as smoking and drinking habits. Alcohol consumption was calculated as all year average daily alcohol consumption (total amount of pure alcohol in grams used per year divided by 365). Ex-smokers were included into the smokers group in the statistical analyses. We focused part of our analyses on the APOE
3/3 carriers (n=384) only to control for the previously known effects of the APOE 2/3/4 polymorphism on the lesion areas.
DNA Extraction, Genotyping, and Haplotype Reconstruction
DNA was extracted and APOE 2/3/4 genotyping done as described earlier,19 and APOE promoter –219G/T (rs405509) and +113G/C (rs440446) polymorphisms genotyped as described previously.7 Haplotypes were reconstructed using the PHASE program (version 2.0.2).21,22
Measuring the Area of Fatty Streaks
At autopsy, the proximal parts of left anterior descending artery and right common carotid artery, and the abdominal aorta were collected for analysis. The area of atherosclerosis lesion was measured using planimetry as described in detail elsewhere.19 The fatty streak areas are expressed in percentages by dividing the lesion area by the total area of the arterial wall and multiplying by 100%.
Functional Studies
All methods for functional studies are discussed in Methods section in the online-only Data Supplement. (www.circgenetics.ahajournals. org).
Statistical Analyses
For descriptive data, continuous variables were compared with ANOVA and categorical variables with the 
2 test. Alcohol consumption was tested using nonparametric Kruskal-Wallis test. The APOE 2/3/4 genotypes were grouped as follows for statistical analysis: 2+ (2/2+2/3), 3/3, and 4+ (3/4+4/4). Genotype 2/4 carriers were excluded from the analyses because they are difficult to assign in a group. The cause of death was included as a factor in statistical models to control for its effects on the lesion areas. First, we compared the mean fatty streak areas of the left anterior descending artery, right common carotid artery, and abdominal aorta in different APOE 2/3/4 genotype groups by using ANOVA. The plaque areas were square root transformed before the analyses to gain normal distribution, but the results are expressed as crude values. Second, we used ANCOVA to test for interactions between the APOE genotype and other known atherosclerotic risk factors (age, body mass index, hypertension, diabetes, smoking, and alcohol use). We used custom model and introduced genotype, cause of death, and one risk factor at a time, as well as their interaction term into the model (continuous variables were introduced as covariates and categorized variables as factors). Third, associations between the APOE promoter polymorphisms and lesion areas were tested in groups of 2- and 4-allele carriers, as well as within the most common APOE 3/3 genotype group (n=384). Interactions between the APOE promoter genotypes and other known atherosclerotic risk factors were also tested within the 2/3/4 subgroups.
In total, 7 different haplotypes were identified. In the statistical analyses, we included only those 4 haplotypes with a frequency >5%. In addition to performing the haplotype analyses in the whole study population, we also performed the haplotype analyses within the 3/3 haplotype group, to focus on the independent effects of the APOE promoter polymorphisms and to standardize the effects of APOE 2/3/4 polymorphism. Within the 3/3 group, 3 different haplotypes were recognized and for purposes of the statistical analyses, the study subjects were categorized into homozygous carriers of the –219G/+113G/3 haplotype, heterozygous carriers of haplotypes –219G/+113G/3, and –219T/+113C/3, as well as homozygous carriers of the –219T/+113C/3 haplotype. There were only 13 carriers of the haplotype –219T/+113G/3, so this haplotype was excluded from all statistical analyses.
Linkage disequilibrium between the APOE 2/3/4 defining SNPs +3937T/C (rs429358) and +4075C/T (rs7412) and promoter SNPs –219G/T and +113G/C were calculated using the Stata 8.0. The transcriptional efficiency in different promoter constructs was compared using Kruskal-Wallis test and pairwise tests were performed using Mann-Whitney U test. All statistical calculations were carried out with SPSS (version 14.0, SPSS Inc, Chicago, Ill) on a personal computer.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
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Results |
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Characteristics of the Helsinki Sudden Death Study Subjects
Age, body mass index, or other measured cardiovascular risk factors (hypertension, diabetes mellitus, smoking, or alcohol consumption) did not have statistically significant differences between the –219 and the +113 genotype groups within the whole Helsinki Sudden Death Study population (data not shown) or within the APOE
3/3 carriers (Table 1). Among the whole study population (3/3 carriers), there were 345 (187) smokers, 67 (37) ex-smokers, and 88 (43) nonsmokers. Information about the alcohol consumption was available in 415 (214) cases. The distribution of classes of death (cardiac, other disease, or unnatural death) did not differ between the genotype groups (data not shown). The promoter –219G/T and +113G/C allele frequencies among the whole study population (3/3 carriers) were as follows: –219G 0.56 (0.63), –219T 0.44 (0.37), +113G 0.74 (0.65), and +113C 0.26 (0.35), and the genotype frequencies (Table 1) were similar, as in previous studies of the Finnish population.7,23 Linkage disequilibrium (D') between –219G/T and +113G/C was 1.0, between –219G/T (+113G/C) and +3937T/C was 0.69 (0.95), between –219G/T (+113G/C) and +4075C/T was 0.86 (1.0), and between +3937T/C and +4075C/T was 0.62.
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APOE Promoter Polymorphisms Interact With Smoking on Aortic Atherosclerosis
Within the whole study population, including all the APOE
2/3/4 genotypes, the APOE 2/3/4 polymorphism did not associate statistical significance with fatty streak areas in neither coronary arteries nor abdominal aorta, and no statistically significant interactions with environmental factors were found (Table 2).
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Within the
2- or 4-allele carriers or within the 3/3 group, the promoter polymorphisms –219G/T and +113G/C were not associated with fatty streak areas. However, within 3/3 carriers, there was a strong genotype–smoking interaction (–219, interaction P=0.009; +113, interaction P=0.003) on fat area in abdominal aorta (Table 2). For further studies, we divided the study population into nonsmokers and smokers (including ex-smokers) and then performed ANOVA analyses comparing the abdominal aorta fatty streak areas between different promoter genotypes. These analyses showed that within nonsmokers, the –219T- and +113C-allele carriers had larger abdominal aorta fatty streak areas compared with the G/G carriers (–219T+ versus G/G: 12.7% versus 5.9%, P=0.007; +113C+ versus G/G: 12.9% versus 6.3%, P=0.010; Figure 1). Within smokers, the situation was opposite: +113C-allele carriers associated with smaller fatty streak areas compared with the G/G carriers (+113C+ versus G/G: 10.5% versus 13.9%, P=0.010; Figure 1). Regarding the –219G/T polymorphism, the association with fatty streak area was not statistically significant within smokers (–219T+ versus G/G: 10.8% versus 13.1%, P=0.083). The –219G/G and +113G/G genotype carriers had larger abdominal aorta fatty streaks in smokers compared with nonsmokers (–219G/G: 13.1% versus 5.9%, P=0.019; +113G/G: 13.9% versus 6.3%, P=0.008; Figure 1).
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There were no statistically significant differences in the fatty streak areas of left anterior descending artery, right common carotid artery, or abdominal aorta among the 4 studied APOE haplotypes within the whole study population. When the nonsmoking –219G/+113G/
3 haplotype carriers were used as the reference group, there were 2 haplotype groups that showed statistically significantly larger abdominal aorta fatty streak areas: smoking –219G/+113G/3 haplotype carriers and smoking –219T/+113G/4 haplotype carriers (12.6% versus 8.2%, P=0.005 and 12.4% versus 8.2%, P=0.041, respectively; Figure 2).
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Within the
3/3 carriers, there was a statistically significant haplotype–smoking interaction on abdominal aorta fatty streak area (interaction P=0.003). Further analyses showed that within nonsmokers, the carriers of –219T/+113C/3 haplotype had larger abdominal aorta fatty streak areas compared with homozygous carriers of the –219G/+113G/3 haplotype (12.9% versus 5.9%, P=0.008; Figure 3). Within smokers, the association was opposite (Figure 3).
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APOE +113G/C SNP Region Can Act as an Enhancer
The effect of the APOE +113G/C SNP on the transcriptional activity of APOE was studied using the Luciferase assay system. It has earlier been shown that the region between +44 and +262 (IRE1) has enhancer activity,24 but the +113 allelic status in that study is unknown because the SNP was identified almost 10 years later.25 We studied the enhancer activity using a smaller 50-bp region (from +89 to +138) of the first intron region in the APOE gene. Our transcription experiments showed that primarily the +113C allele has an effect on the luciferase gene expression, mainly acting as a transcriptional enhancer. In more detail, most of the promoter constructs carrying the C allele possessed higher transcriptional activity than the corresponding constructs with the G allele. This was most prominent when we compared the G- and C alleles cloned in sense orientation upstream of the luciferase gene, or in antisense orientation downstream of the luciferase gene (Figure 4). The transcriptional activity for the sense C-allele construct upstream of the luciferase gene was
1.4-fold compared with the similar construct with G allele (P=0.002). Furthermore, the transcriptional activity for the antisense C-allele construct downstream of the luciferase gene and was 1.6-fold compared with the antisense G allele downstream of the luciferase gene (P=0.002).
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APOE +113G/C SNP Influences Transcription Factor Binding
As the transcriptional studies clearly showed a difference in the transcriptional activity between the APOE +113G and C alleles, we used electrophoretic mobility-shift assay (EMSA) to study whether there are differences in protein binding to the area within which the APOE +113G/C SNP is situated. Our results showed distinct shifts in the +113G and +113C EMSA assays suggesting to a difference in the composition or amount of proteins binding to the G and C alleles (Figure 5A). In more detail, proteins in the HepG2 nuclear lysate had constantly >5 times higher affinity to the G allele compared with the C allele. However, the competition experiments with cold oligonucleotides showed that the DNA-protein complexes were reciprocally competed with both G and C allele oligos (Figure 5B). This proposes that +113G/C polymorphism has an effect on transcription factor affinities to the +113 region but not on the composition of transcription factors.
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In earlier studies, NF
B has been proposed to bind to the +113G/C region.26 This was confirmed by our promoter analyses with ConSite,27 which showed that the p50 subunit of transcription factor NFB binds with different affinity to the +113G and C alleles. The affinity score for the +113G allele was 7.0 and for the +113C allele 8.7 U. However, the supershift EMSAs with antibodies for p50 and p65 (tested to be functional in Western blot; data not shown) did not give any positive results (data not shown).
The Matinspector analyses predicted that +113C would recruit 2 transcription factors RBP-J and RFX1 that were not recruited by the +113G allele. Core similarity and matrix similarity for RBP-J were 1.0 and 0.843 and for RFX1 were 0.881 and 0.920, respectively. With RBP-J we conducted an experiment to verify this prediction. First, we tried to study the binding of RBP-J to the +113 region by using supershift EMSA, but the antibody for RBP-J proved to be nonfunctional in Western blot with HePG2 nuclear lysate (data not shown). Second, we decided to conduct EMSA using prokaryotically produced transcription factor RBP-J. This experiment showed a 4-fold affinity of the RBP-J to the +113C allele compared with the G allele (Figure 6).
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Discussion |
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Smoking is known to associate especially with the development of atherosclerosis of the abdominal aorta28 and arteries of the lower limbs,29 leading to claudication due to obstruction of blood flow to lower extremities and eventually resulting in gangrene.30 In the present study, we show that there is a statistically significant APOE promoter genotype–smoking interaction considering the fatty streak area in abdominal aorta but not in the coronary arteries. It is evident that factors influencing the development of early atherosclerotic lesions differ in coronary arteries and aorta, which is comprehensible when considering 2 vessels that are completely different regarding, for example, their hemodynamic conditions. Regarding abdominal aorta, the –219T- or +113C-allele carriers as well as men carrying the haplotype –219T/+113C/
3 had statistically significant larger lesion areas compared with the G-allele homozygotes or homozygous carriers of haplotype –219G/+113G/3 within nonsmokers. The associations were opposite within smokers.
We did not find statistically significant APOE 2/3/4 genotype–smoking interaction in this study regarding the fatty streak areas in coronary arteries or abdominal aorta. Such interaction studies have not been performed earlier in relation to autopsy verified atherosclerosis, but there are several previous studies on the APOE–smoking interaction regarding clinical phenotypes of atherosclerosis such as MI and CHD. Considering the subject of APOE–smoking interaction in general, our negative result is in agreement with a previous study by Liu et al31 but conflicts with most previous studies exploring the APOE 2/3/4 genotype–smoking interaction on CHD risk.13–15 Liu et al31 performed a nested case-control study from the Physicians Heart Study and failed to find any association between APOE 2/3/4 and the risk of MI nor any interaction between 4 and smoking on MI risk. Humphries et al13 showed that smoking increases CHD risk particularly in 4 carrier men. Later on, a reanalysis of the Framingham Offspring Study32 showed that within nonsmokers, the cardiovascular disease risk does not differ between APOE 2/3/4 genotypes.14 In smokers, however, both 2 and 4 carriers showed increased cardiovascular disease risk compared with 3/3 carriers. Therefore, the association of 4 with cardiovascular disease was limited to smokers.14 In our study, the –219T- and +113C allele as well as –219T/+113C/3 haplotype carriers associated with larger abdominal aorta fatty streak areas only within nonsmokers. On the other hand, smoking was clearly shown to increase the fatty streak area within the –219G/G, +113G/G as well as within –219G/+113G/3 haplotype carriers.
Smoking is known to raise serum lipid levels33,34 and is considered as one of the major risk factors for the development of atherosclerotic disease.35,36 Moreover, the effect of APOE–smoking interaction on lipid concentrations37 and CHD risk have been studied (for review, see reference 12), but the biological basis for it is still unclear. Smoking might lower APOE transcription, for example, by somehow disrupting the binding of transcription factors, therefore, leading to higher serum lipid values and eventually resulting in larger fatty streak areas of the abdominal aorta. Unfortunately, we did not have the apoE or lipid levels measured in this autopsy study series, and the lack of lipid data could possibly bias the estimates of the effects of APOE–smoking interaction on fatty streak areas. Many studies have shown that the effects of the APOE 2/3/4 genotype on cardiovascular disease risk remain even after adjusting for lipid variables,13,14,32 therefore, suggesting to an independent effect for the APOE genotype. The effects of APOE–smoking interaction on CHD risk could be explained through low density lipoprotein oxidation. The APOE 4 carriers are in higher risk only if they smoke,14 and it is known that APOE E4 is more susceptible to oxidation than E3 or E2.38 This difference, however, does not explain our results because we focused our promoter association studies mainly on the APOE 3/3 carriers, and therefore, the susceptibility to oxidation does not differ between the study subjects in this sense. It is unknown whether the APOE promoter polymorphisms have a role in determining the APOE susceptibility to oxidation as well. It is of note that our study series only included male subjects, which can be considered a deficiency because, for example, the effects of smoking are thought to be more detrimental in women than in men.35
The second part of this article investigated the possible effects of the +113G/C SNP on transcriptional activity. It has been shown in a previous study that the region between +44 and +262 (IRE1) in the first intron of the APOE gene has promoter enhancing activity,24 but there were no preceding functional studies on APOE +113G/C SNP. Our experiments showed that the transcriptional activity of the +113C allele is constantly higher compared with the +113G allele, suggesting that the enhancer activity of this region is specifically dependent on the +113C allele. We also found the +113 region to be evolutionary conserved (>60%) between mouse and human, suggesting that it is important in the regulation of APOE transcription. In EMSA assays, both +113 alleles were able to bind proteins from the hepatic nuclear lysate suggesting that this region can recruit transcription factors in hepatic cells. Thus, it is likely that the +113 region can regulate APOE gene transcription also in vivo. The EMSA bands were constantly more intense for the G allele compared with the C allele, suggesting to an affinity difference in proteins binding to the 2 alleles. Because DNA-protein complexes were reciprocally competed in the competition experiments, they most likely contain the same proteins but possibly in different amounts. Protein affinity difference is also evident in competition experiments (Figure 5B), where an excess of cold G allele always competes more strongly than cold C allele. We suggest that the +113G allele has higher affinity to some transcriptional repressors because it has lower transcriptional activity but a stronger shift in EMSA compared with the C allele. Unfortunately, our EMSA supershift studies did not reveal any of the binding transcription factors. Our EMSA experiments with the in vitro produced RBP-J suggest that this protein could be one of the transcription factors binding to the +113 region but to verify this, more in vivo studies are admittedly warranted. Furthermore, there most probably are also other, currently unidentified, transcription factors, which bind to the +113G/C region and take part in regulating the APOE transcription.
In smokers, the +113C allele associated with smaller abdominal aorta fatty streak areas compared with G/G, which agrees with the higher transcriptional activity of the C allele in the functional analyses. In nonsmokers, the +113C-allele carriers had larger lesion areas compared with G/G, which agrees with our previous results showing higher cholesterol concentrations in C-allele carriers in a longitudinal study8 but disagrees with the results from our functional experiments. It is central to remember that based on association analyses, it is difficult to make definite conclusions about the causality of SNPs. Our functional studies, on the other hand, are in vitro studies including a 50-bp bit of the first intron of the APOE gene. In vivo, the circumstances are much more complicated when many other SNPs and regulatory regions are involved in regulating the APOE transcription.
In conclusion, we show that there is a statistically significant APOE genotype–smoking interaction on the abdominal aorta fatty streak areas of the middle-aged Finnish male victims of sudden death. This is a new finding, but further studies are needed to affirm these results and to clarify the molecular mechanisms behind these associations. We still consider smoking to be an important risk factor for CHD in all genotype groups, but nonetheless, we suggest that its effects could be different in people carrying different (APOE promoter) genotypes. Additionally, we started the functional studies regarding the APOE +113G/C polymorphism, and showed that it has an effect on the transcriptional activity and furthermore, that the +113C allele possesses transcription enhancing capabilities.
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Acknowledgments |
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Sources of Funding
This work was supported by the Kalle Kaihari Heart Research Fund (L.E.V.), Helsinki University Central Hospital, the Pirkanmaa Regional Fund of the Finnish Cultural Foundation (L.E.V.), the Emil Aaltonen Foundation (T.L.), and the Tampere University Hospital Medical Fund.
Disclosures
None.
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The online-only Data Supplement is available at http://circimaging.ahajournals.org/cgi/content/full/CIRCIMAGING.108.791764/DC1.
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