Apolipoprotein E (apoE) polymorphism: A risk factor for fatal coronary sclerosis?

Forensic Science International 192 (2009) 62–66

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Apolipoprotein E (apoE) polymorphism: A risk factor for fatal coronary sclerosis? Steffen Heide a,*, Kleiber Manfred a, Christiane Gla¨ser b, Susanne Schulz c a

Institute for Forensic Medicine; Halle University, Franzosenweg 1, D-06112 Halle, Germany Institute for Human Genetics and Medical Biology; Halle University, Magdeburger Str. 2, D-06097 Halle, Germany c University School of Dental Medicine, Department of Operative Dentistry and Periodontology; Halle University, Harz 42a, D-06108 Halle, Germany b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 17 December 2008 Received in revised form 7 May 2009 Accepted 28 July 2009 Available online 3 September 2009

Objective: The association of apoE polymorphism and coronary heart disease has been examined in numerous clinical studies. However, there are only very few autopsy studies on this topic, there having been none in Germany for instance. Methods: In a retrospective study, we examined 121 cases where the autopsy revealed premature coronary sclerosis. The collected data included the maximum degree of stenosis of the coronary arteries, the body mass index and a potential left heart hypertrophy. Coronary sclerosis was the cause of death in 47 and a relevant secondary finding in 74 of the cases. The control group consisted of 253 healthy blood donors and 79 patients who had undergone cardiac catheterization and did not show coronary macroangiopathy. Genotyping of apoE polymorphism was performed by using blood. Results: When assessing the influence of apoE genotype or allele no significant influence on premature death was found. However, a subdivision of deceased according to their coronary state showed that the carriers of apoE e4-allele had a higher risk of coronary artery sclerosis with a significantly higher degree of stenosis. Conclusion: In cases of fatal premature coronary sclerosis and proven apoE e4-allele, blood relatives should receive advice. ß 2009 Elsevier Ireland Ltd. All rights reserved.

Keywords: Premature coronary sclerosis Autopsy ApoE polymorphism

1. Introduction

2. Material and methods 2.1. Material

Apolipoprotein E (apoE) polymorphism is one of the genetic determinants of the lipoprotein metabolism and acquires therefore importance for the morbidity and mortality of arteriosclerosis [1]. ApoE 3/4 and apoE 4/4 carriers show reduced LDL receptor activity which causes an increased concentration of total cholesterol and LDL cholesterol [2]. Without treatment, such patients have statistically a higher risk of developing coronary heart disease [3]. The association of apoE polymorphism and coronary heart disease has been proven by numerous clinical and coronary angiographic studies. However, there are only very few autopsy studies on the association of apolipoprotein E genotypes and coronary arteriosclerosis or aortic sclerosis [1,4,5]. In Germany, no proven data on a potential association of apoE polymorphism and coronary sclerosis and coronary risk factors have been stated as yet.

* Corresponding author. Tel.: +49 345 557 1588; fax: +49 345 557 1587. E-mail address: [email protected] (S. Heide). 0379-0738/$ – see front matter ß 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2009.07.020

From 1997 to 2004, the Halle Institute of Forensics performed 3641 autopsies. These mainly involved cases where especially younger individuals had died suddenly and by causes unknown. This often leads to suspicion of accident, homicide or suicide. In a retrospective study, 121 of these 3641 cases (3.3%) showed premature coronary arteriosclerosis. A dried blood sample was taken from these bodies when the autopsy was performed. Inclusion criteria were defined as follows: age of up to 40 years for males (100 cases, average age: 33.7  5.8 years), age of up to 45 years for females (21 cases, average age: 39.1  5.4 years) and coronary arteriosclerosis with a minimum stenosis of one third (33%). In 47 of the 121 cases (38.8%) coronary sclerosis was the cause of death. Causes of death were classified in four groups in accordance to our macroscopic and histological findings: acute myocardial infarction, recurring myocardial infarction, stenosing coronary arteriosclerosis, acute coronary thrombosis and sclerosis (Table 1). In 74 cases (61.2%) of other natural and unnatural causes of death, coronary sclerosis was a considerable secondary finding (Table 2). The maximum degree of stenosis (in %) of the atherosclerotic changes was taken from the descriptions pertaining to the macroscopic and histological examinations of the coronary arteries as recorded in the autopsy records. Further risk factors for coronary heart disease were ascertained as far as the nature of a retrospective study allows. In 119 cases (98.3%) autopsy records included body size and weight, thus enabling us to determine the body mass index (BMI). Presence of left heart hypertrophy as a morphological indicator for hypertonia was also ascertainable for nearly the whole group. However, information on other atherosclerotic risk factors such as nicotine abuse or diabetes mellitus was not always recorded. The control group consisted of 253 healthy, medically monitored long time blood donors (125 men, average age: 31.8  6.7 years) who had no health complaints and

S. Heide et al. / Forensic Science International 192 (2009) 62–66 Table 1 47 cases with coronary sclerosis as cause of death.

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Table 2 74 cases with coronary sclerosis as secondary finding.

Cause of death

Number of cases

In percent

Cause of death

Number of cases

In percent

Acute myocardial infarction Recurring myocardial infarction Stenosing coronary arteriosclerosis Acute coronary thrombosis and sclerosis

17 11 11 8

36.2 23.4 23.4 17.0

Traumata Internal diseases Intoxications Other

34 24 9 7

45.9 32.4 12.2 9.5

did not suffer from any known heart disease. Inclusion criteria were again an age of up to 40 years for men and of up to 45 years for women. Besides frequent serological and medical monitoring necessary for blood donations, atherosclerotic risk factors (e.g. smoking, hypertonia, diabetes mellitus) were ascertained by oral questioning and were excluded. We further included 79 patients (47 men, average age: 50.3  9.3 years) who were all examined by diagnostic cardiac catheterization with selective catheterization and contrast media radiography of the coronary arteries. This group of patients had been subjected to the above examination because of suspected coronary heart disease either for a differential diagnostic reason or to prepare for surgery. These patients were European Caucasians of central German origin. They too came from the Halle/ Merseburg/Bitterfeld region. Examination and evaluation was performed by experienced cardiologists. The study only included patients who did not suffer from coronary macro-angiopathy (angiographic testing).

2.2. DNA extraction and apoE genotyping Genomic DNA was extracted from a dried blood sample (autopsy cases) by using ‘‘QIAamp DNA Blood Mini Kit’’ (Quiagen, Hilden, Germany), extraction was performed in accordance to the manufacturer’s instructions, or from venous EDTA blood (blood donor, patients with coronary angiography). The quality of the prepared DNA was tested electrophoretically (separation in 1.2% (w/v) of an ethidium bromide-containing agarose gel, 0.5 mg/ml ethidium bromide, UVstimulation at 254 nm) and spectrometrically (absorption detection at 260 nm). DNA samples were stored at 20 8C. ApoE polymorphism was genotyped in accordance to Wenham et al. [6] inside the Mastercycler1 gradient (Eppendorf, Hamburg, Germany). Restriction separation through HhaI was followed by electrophoretic separation of fragments in polyacrylamide gel (T = 10.4%, C = 3.7%). Fragments were visualised by silver staining.

3. Results 3.1. Distribution of genotypes ApoE 2/2 received no further consideration in the now following investigation since it appeared in only three cases of the blood donor group. When investigating apoE polymorphism we found that apoE genotype 4/4 was a very rare event in the group comprising all deceased (2.5%), in the group of patients with coronary angiography (0%), which was even less than in the blood donor group (0.8%). Furthermore, the e4-allele was nearly as frequent in every group (all deceased: 12.0%; patients with coronary angiography: 10.1%; blood donors: 11.5%). However, a subdividing of the deceased into a group with premature coronary sclerosis as cause of death and a second with coronary sclerosis as secondary finding revealed differences in apoE allele distribution. The group of deceased with coronary sclerosis comprised of twice as much carriers of the e4-allele (18.1%) than deceased with coronary sclerosis as secondary finding (8.1%) (Table 3). For further evaluation, we subdivided the group comprising all deceased into a subgroup where coronary sclerosis had been the cause of death and into a second subgroup where arteriosclerosis of the coronary vessels had been merely a relevant secondary finding. 3.2. Maximum degree of stenosis

2.3. Statistical evaluation SPSS 17.0 was used for statistical evaluation. Metric data were checked for normal distribution by the Kolmogorov–Smirnov test. For analysis of independent stratified samples, we used the t-test according to Student and single factor analysis of variance (‘‘One-Way-Anova’’) for normal distribution, and the U-test according to Mann–Whitney and Kruskal–Wallis test for not normally distributed variables. For determination of bi-variat correlations of metric values we calculated the productmoment correlation according to Pearson (normal distribution) and the rank correlation according to Spearman (not normal distribution). After testing variance homogeneity (Levene test) the connection between metric variables within groups was determined by post hoc tests (LSD). Categorial data were analysed in contingency tables by making use of the chi-square test according to Pearson and the Exact Fisher test. All values p < 0.05 were assessed as statistically relevant values.

Our next step was to investigate if these two subgroups differed in the degree of coronary sclerosis. We found that in the subgroup where coronary sclerosis had been the cause of death the maximum degree of stenosis showed a mean of 69.7 (standard deviation  15.35) whereas it was considerably lower in the subgroup where coronary sclerosis had been a secondary finding (48.62; standard deviation  12.98). This difference proved to be highly significant (p < 0.001, 95% confindence interval: 15.71–26.45). Regarding the correlation between maximum degree of stenosis and apoE polymorphism (Table 4) it was conspicuous that the carriers of apoE 3/4 and 4/4 as well as apoE e4-allele of all

Table 3 ApoE polymorphism in the groups: blood donors, patients with coronary angiography and deceased. ApoE genotype

2/3 3/3 2/4 3/4 4/4

(%) (%) (%) (%) (%)

1. Deceased (total)

2. Deceased (CS = death)

3. Deceased (CS = secondary)

4. Blood donors

5. Patients (coronary angiography)

n = 121

n = 47

n = 74

n = 250

n = 79

5.8 72.7 3.3 15.7 2.5

4.3 61.7 2.1 29.8 2.1

6.8 79.7 4.0 6.8 2.7

12.4 65.2 2.0 19.6 0.8

10.1 69.6 2.6 17.7 0

p-values

1 vs. 4: p = 0.130; 1 vs. 5: p = 0.487; 2 vs. 3: p = 0.020, pcorr. = 0.100; 2 vs. 4: p = 0.281; 2 vs. 5: p = 0.268; 3 vs. 4: p = 0.020, pcorr. = 0.100; 3 vs. 5: p = 0.129

ApoE allele

n = 242

n = 94

n = 148

n = 500

n = 158

e2-allele (%) e3-allele (%) e4-allele (%)

4.5 83.5 12.0

3.2 78.7 18.1

5.4 86.5 8.1

8.3 80.2 11.5

6.3 83.5 10.1

p-values

1 vs. 4: p = 0.173; 1 vs. 5: p = 0.647; 2 vs. 3: p = 0.055; 2 vs. 4: p = 0.062; 2 vs. 5: p = 0.126; 3 vs. 4: p = 0.222; 3 vs. 5: p = 0.768

CS, coronary sclerosis, pcorr., p-values after Bonferroni correction.

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S. Heide et al. / Forensic Science International 192 (2009) 62–66

Table 4 Relation between maximum degree of stenosis and apoE polymorphism in the total group of deceased and its subgroups. ApoE genotype

Deceased (total, n = 121)

Deceased (coronary sclerosis as cause of death, n = 47)

Deceased (coronary sclerosis as secondary finding, n = 74)

n

Stenosis in (%) mean  SD

n

Stenosis in (%) mean  SD

n

Stenosis in (%) mean  SD

7 88 4 19 3

56.43  9.45 54.47  19.09 56.25  12.50 66.89  18.90 63.33  23.09

2 29 1 14 1

67.5  10.61 68.21  15.3 75 71.29  16.69 90

5 59 3 5 2

52  4.47 47.71  13.23 50.00  0 50.00  0 54.60  21.11

ApoE allele

n

Mean  SD

n

Mean  SD

n

Mean  SD

e2-allele e3-allele e4-allele

11 202 29

56.36  10.02 55.70  17.18 64.69  18.34

3 74 17

70.00  8.66 68.77  15.24 73.71  16.27

8 128 12

51.25  3.54 48.15  13.30 51.92  12.95

2/3 3/3 2/4 3/4 4/4 p-values (Kruskal–Wallis)

0.087

p-values (Kruskal–Wallis)

0.575

0.003, pcorr. = 0.009

0.730

0.210

0.394

pcorr = p-values after Bonferroni correction.

groups of deceased (total group, first and second subgroup) showed the highest mean of maximum stenosis. The highest mean of all (72.69  16.24) was found for carriers of e4-allele in the subgroup with fatal coronary sclerosis.

Table 6 shows that in the group of all deceased left heart hypertrophy was most frequent for carriers of apoE 3/4 genotype and the e4-allele. This is mainly due to the fact in the subgroup with fatal coronary sclerosis one third of all deceased with apoE 3/4 genotype and e4-allele showed left heart hypertrophy.

3.3. Body mass index (BMI) and left heart hypertrophy 4. Discussion The group of deceased (total) included 42 cases (35.3% of the recorded 119 cases) of pre-obesity (BMI: 25.0–29.9). We also found 11 cases (9.2%) of class I obesity (BMI: 30.0–34.9), 6 cases (5.0%) of class II obesity (BMI: 35.0–39.9) and 2 cases (1.7%) of class III obesity (BMI: >40). This means that according to WHO classification more than half the cases (n = 60, 50.4%) had been overweight. Regarding the relation between body mass index (BMI) and apoE polymorphism (Table 5) it showed that carriers of apoE 3/4 of the group of all deceased (total) and the subgroup with fatal coronary sclerosis had the highest means. However, regarding the apoE allele no consistent trend was obviously. In the subgroup where coronary sclerosis had been the cause of death carriers of the e4-allele had the highest means whereas in the second subgroup the e3-allele was associated with the highest BMI. In the group of all deceased, autopsy revealed in 23 of the cases (19.0%) left heart hypertrophy as a morphological indicator for hypertonia. This proportion was with 27.6% twice as high in the subgroup with fatal coronary sclerosis (13 cases) as compared to the subgroup where coronary sclerosis had been a secondary finding (13.5%, 10 cases).

Germany has one of the highest mortality rates in coronary heart disease in Western Europe. It is only surpassed by Ireland, Scotland and Finland [7,8]. The apoE-4 genotype is seen as a significant risk factor for coronary heart disease [3,9,10] especially in younger and middle-aged individuals [1]. Our study aimed to investigate if apoE 3/4 and 4/4 genotypes had been a significant risk factor in a group of 121 deceased individuals from Central Germany whose autopsy had revealed premature coronary arteriosclerosis. Control groups consisted of 253 blood donors and 79 patients with coronary angiography. All control group members were selected from the same geographic region since frequency of apolipoprotein E alleles differs significantly not only on an international scale but also regionally. Hallmann et al. [11] found in his study of apoE polymorphism in nine different populations from Asia and Europe significant differences in allele frequencies. The most common genotypes varied considerably, i.e. apoE 2/3 from 7.0% to 16.9%, apoE 3/3 from 39.8% to 72.1% and apoE 3/4 from 11.3% to 35.9%. For Europe it showed [12] that the frequency of the apo E-4 genotype was higher

Table 5 Relation between body mass index (BMI) and apoE polymorphism in the total group of deceased and its subgroups. Deceased (total, n = 119)

Deceased (coronary sclerosis as cause of death, n = 47)

Deceased (coronary sclerosis as secondary finding, n = 72)

ApoE genotype

n

BMI in kg/m2 mean  SD

n

BMI in kg/m2 mean  SD

n

BMI in kg/m2 mean  SD

2/3 3/3 2/4 3/4 4/4

7 86 4 19 3

23.83  3.69 25.53  4.76 23.72  1.24 27.67  6.56 21.27  3.93

2 29 1 14 1

24.3  5.37 25.69  5.57 25.4 28.19  5.93 25.8

5 57 3 5 2

23.64  3.6 25.46  4.34 23.17  0.67 26.22  8.72 19.00  0.14

p-value (Anova)

0.142

ApoE allele

n

Mean  SD

n

Mean  SD

n

Mean  SD

e2-allele e3-allele e4-allele

11 198 29

23.79  2.94 25.68  4.94 25.80  6.12

3 74 17

24.67  3.85 26.12  5.62 27.74  5.44

8 124 12

23.46  2.76 25.41  4.49 23.05  6.18

p-value (Anova) BMI = body mass index.

0.666

0.469

0.278

0.484

0.139

S. Heide et al. / Forensic Science International 192 (2009) 62–66

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Table 6 Frequency of left heart hypertrophy in correlation to apoE polymorphism in the total group of deceased and its subgroups. Deceased (total, n = 121)

Deceased (coronary sclerosis as cause of death, n = 47)

Deceased (coronary sclerosis as secondary finding, n = 74)

ApoE genotype

n

LH positive in %

n

LH positive in %

n

LH positive in %

2/3 3/3 2/4 3/4 4/4

7 88 4 19 3

14.3 17.0 0 31.6 0

2 29 1 14 1

0 24.1 0 35.7 0

5 59 3 5 2

20 13.6 0 20.0 0

p-value (x2)

0.407

0.706

0.888

ApoE allele

n

%

n

%

n

%

e2-allele e3-allele e4-allele

11 202 29

9.1 18.3 20.7

3 74 17

0 25.7 29.4

8 112 12

12.5 14.1 8.3

p-value (x2)

0.692

0.559

0.854

LH = left heart hypertrophy.

in its northern parts (Scandinavia, Scotland, Germany and the Netherlands) that in its southern regions (Switzerland, Tyrol, France, Italy and Spain). Even within one country, distribution of apolipoprotein E alleles may be quite varied especially in dependency from ethnic origin [13,14]. In our study we found differences in apoE genotype distribution even between both control groups. They were, however, not significant. Distribution of the apoE 2/3 genotype varied from 12.4% (blood donors) to 10.1% (patients where angiography did not reveal and coronary sclerosis); apoE 3/3: 65.2% as compared to 69.6% and apoE 3/4 and 4/4: 20.4% vs. 17.7%. Studies of apoE polymorphism in healthy control groups showed clear differences for Germany as a whole. Different studies from different regions ascertained the frequency of apoE 2/3 to vary between 8.0% [[15]; 120 individuals from Central Germany] and 17.3% [[16]; 571 individuals from Southwest Germany]. ApoE 3/3 varied from 59.2% [16] to 65.0% [15], apoE 3/4 and 4/4 from 17.6% [[17]; 182 individuals from Southeast Germany] to 23.8% [[18]; 282 individuals from Berlin]. Nearly no differences were found for apoE 2/4 (approximately 2%) and apoE 2/2 genotypes (approximately 1%). In our group of deceased (total), apoE 2/3 genotype was with 5.8% considerably rarer and apoE 3/3 with 72.7% more frequent than in the control groups. Despite the premature coronary scleroses being proven by autopsy, distribution of apoE 3/4 was with 15.7% in the group of all deceased almost equal to the control groups. In this respect, we therefore found no significant differences between these groups. Our investigation of the group of all deceased did therefore not confirm the association of apo E-4 genotype and coronary disease as ascertained by a series of clinical and coronary angiographic studies [3,19,20]. Several studies from previous years were also unable to proof significant association [16,21]. In relation to apoE polymorphism, Volcik et al. [22] found for 12,491 individuals (black and white Americans) from different regions in the US no significant differences between the patients with coronary heart disease and the control group. The association of apoE polymorphism and coronary heart disease has been investigated in numerous clinical and coronary angiographic studies. There are, however, not many autopsy studies on this subject. In a study comprising 700 autopsy cases from Finland (Helsinki Sudden Death Study (HSDS) with cardiac deaths but also with intoxications and traumata) Ilveskoski et al. [1] confirmed for up to early middle-age (<53 years) the importance of the apoE e4-allele as a significant risk for coronary sclerosis. In respect to older men however, no significant association to atherosclerotic lesions was found. Ilveskoski et al.

[1] conclude that apoE polymorphism mainly influences the first stages of arteriosclerosis whereas, at an older age, the accumulation of other known risk factors becomes more important. Within the scope of the PDAY study (Pathobiochemical Determinants of Atherosclerosis in Youth), Hixson [4] examined 720 accidental deaths of young male US Americans (15–34 years). He found that apoE polymorphism and the severity of the atherosclerotic lesions in the aorta were significantly associated. This association was weakest in apoE 2/3 carriers and strongest in carriers of the apoE4allele. In their examination of 130 deaths in Alaska (males and females; 9–85 years), Scheer [5] was able to prove association between the apoE4-allele and arteriosclerosis in the coronary arteries (Ramus interventricularis anterior of A. coronaria sinistra and right coronary artery). The autopsy of 98 Greenland Inuit (males and females) revealed no such association [23]. In recent years, autopsy examinations have increasingly focussed on the association of apoE polymorphism with neuropathological changes or with atherosclerotic findings on brain vessels in Alzheimer disease [24–29]. Systematic examinations of deceased individuals with premature coronary sclerosis in association with apoE polymorphism are still lacking in Germany. In our study, we formed a separate subgroup of those deaths that were due to coronary sclerosis in order to distinguish them clearly from other cases. The hitherto conducted studies on the association of apoE polymorphism with coronary heart disease did not make this distinction. We found that there was a significant distinction between the two subgroups’ genotype distribution (p = 0.020). However, after Bonferroni correction this association remained not significantly (p = 0.100). Regarding the allele distribution a distinct increase in e4-allele carriers could be estimated in the subgroup with fatal coronary sclerosis (p = 0.055, pcorr. = 0.275). In the subgroup with fatal coronary sclerosis, the rate of apoE 3/4 and e4-allele was with 29.8% and 18.1% more than four and two times, respectively, higher than the rate found in the subgroup where arteriosclerosis of the coronary arteries had only been a relevant second finding. Compared to the control groups was the rate of apoE 3/4 clearly higher in the subgroup with fatal coronary sclerosis. We found that the rate of nearly one third of apoE 3/4 carriers in the group with fatal coronary sclerosis was considerable higher than the rate found in the healthy control groups but was also higher than the rate found in the groups of patients suffering from coronary heart disease (up to 27.9, respectively, 25.9%) in other German regions [16,21]. Our investigation included other risk factors of arteriosclerosis as much as the possibilities of our retrospective study of autopsy cases allowed since there is a great number of different factors that

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influence and hasten both origin and progress of the atherosclerotic process. In our study, we found that the risk factors BMI and left heart hypertrophy were most pronounced in the subgroup with fatal coronary sclerosis. In this subgroup, it was shown for the first time that carriers of apoE 3/4 and e4-allele had the highest BMI as well as the highest rate of left heart hypertrophy. When examining the maximum degree of stenosis we found as we had expected a highly significant difference between the two subgroups. This was due to the fact that the group of deceased who had died from other causes of death also included cases with a relatively low degree of stenosis (minimum: one third). The carriers of apoE 3/4 genotype and e4-allele showed the highest degrees of stenosis in the subgroup with fatal coronary sclerosis. Our findings did not ascertain any significant association of apo E-4 genotype or allele with premature coronary sclerosis for the group of all deceased comparing with blood donors as well as patients who had angiographically proven no coronary angiography. However, e4-allele carriers have shown to be at a significantly higher risk of a higher degree of sclerosis in the group of deceased with coronary sclerosis as cause of death. Several clinical studies have described a relation between apoE polymorphism and risk of myocardial infarction. Approximately 8% of myocardial infarctions can be explained by an underlying apoE polymorphism [30]. Luc et al. [31] found a relative myocardial infarction risk of 0.73 for carriers of the apoE2-allele whereas this amounted to 1.33 for apoE4-allele carriers. The results of the EARS study (European Atherosclerosis Research Study) indicate that in populations with a high apoE4 frequency an increased incidence of myocardial infarction is to be expected [32]. Our investigation leads to the conclusion that relatives should receive advice if there was a case of premature fatal coronary sclerosis accompanied by the proven presence of apoE 3/4 genotype or e4-allele. Especially the deceased’s children and siblings should be tested for dyslipoproteinaemia and, if required, apoE isoforms should be analysed. For primary prevention of coronary heart disease in apoE4 allele carriers, lipometabolism and other risk factors should be monitored and in case of increased concentrations of LDL-cholesterol treatment should be given accordingly [33,34]. Further autopsy studies on premature coronary sclerosis should aim to include a larger number of cases as well as prospective collection of further candidate genes for arteriosclerosis and coronary risk factors. References [1] E. Ilveskoski, M. Perola, T. Lethimaki, P. Laippala, V. Savolainen, J. Pajarinen, A. Penttila, K.H. Lalu, A. Mannikko, K.K. Liesto, T. Koivula, P.J. Karhunen, Agedependent association of apolipoprotein E genotype with coronary and aortic atherosclerosis in middle-aged men: an autopsy study, Circulation 100 (1999) 608–613. [2] J. Dallongeville, S. Lussier, J. Davignon, Modulation of plasma triglyceride levels by apoE phenotype: a meta-analysis, J. Lipid Res. 33 (1992) 447–454. [3] P.W. Wilson, E.J. Schaefer, M.G. Larson, J.M. Ordovas, Apolipoprotein E alleles and risk of coronary disease: a meta-analysis, Arterioscler. Thromb. Vasc. Biol. 16 (1996) 1250–1255. [4] J.E. Hixson and PDAY Research Group, Apolipoprotein E polymorphisms affect atherosclerosis in young males. Arterioscler. Thromb. 11 (1991) 1237–1244. [5] W.D. Scheer, D.A. Boudreau, G.T. Malcom, J.P. Middaugh, Apolipoprotein E and atherosclerosis in Alaska Natives, Atherosclerosis 114 (1995) 197–202. [6] P.R. Wenham, W.H. Pric, G. Blundell, Apolipoprotein E genotyping by one-stage PCR, Lancet 337 (1991) 1158–1159. [7] S. Sans, H. Kesteloot, D. Kromhout, The burden of cardiovascular diseases mortality in Europe. Task Force of the European Society of Cardiology on Cardiovascular Mortality and Morbidity Statistics in Europe, Eur. Heart J. 18 (8) (1997) 1231–1248. [8] A. Kay, W. Ma¨rz, M.M. Hoffmann, Q. Zhang, L.I. Masana, J. Cavanna, M.G. Baroni, B. Shine, D.J. Galton, Coronary artery disease and dyslipidemia within Europe: Genetic variants in lipid transport gene loci in German subjekts with premature coronary artery disease, Atheroscler. Suppl. 3 (1) (2002) 27–33.

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