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(Circulation. 1995;91:265-269.)
© 1995 American Heart Association, Inc.

Articles

Apolipoprotein E Polymorphism Predicts Death From Coronary Heart Disease in a Longitudinal Study of Elderly Finnish Men

Jari H. Stengård, MD; Kim E. Zerba, PhD; Juha Pekkanen, MD; Christian Ehnholm, MD; Aulikki Nissinen, MD; Charles F. Sing, PhD

From the National Public Health Institute (J.H.S., J.P., C.E.), Helsinki, Finland; Department of Human Genetics (J.H.S., K.E.Z., C.F.S.), University of Michigan (Ann Arbor); and Department of Community Health and General Practice (A.N.), University of Kuopio, Finland.

Correspondence to Dr Jari H. Stengård, National Public Health Institute, Department of Epidemiology and Health Promotion, Mannerheimintie 166, FIN-00300, Finland, Helsinki.

	Abstract

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Background There is ample evidence from cross-sectional studies of an association between allelic variation of the gene coding for apolipoprotein E (apoE) and interindividual variation in plasma lipids, and the presence of coronary heart disease (CHD). There have been no prospective studies, however, to evaluate the usefulness of allelic variation of the apoE gene for predicting CHD.

Methods and Results Two samples of elderly Finnish men were followed for 5 years, one in the east (n=297) and the other in the southwest of Finland (n=369). At baseline, when the apoE genotypes were assessed, the men were 65 to 84 years old. At the end of the follow-up, the vital status of each man was determined, and cause of death was coded. At baseline, relative frequencies of the three alleles–2, 3, and 4–were 0.037, 0.827, and 0.136 in the eastern and 0.062, 0.763, and 0.175 in the southwestern samples, respectively (²=8.89, df=2, P<.012 for difference between the samples). During the 5-year follow-up, a total of 28 deaths from CHD were recorded in the eastern and 42 in the southwestern sample. Relative CHD mortality was not heterogeneous between the samples. Among those who died from CHD, there was a doubling of the relative 4 allele frequency in both samples (²=4.70, df=1, P<.03 for the eastern sample; ²=7.11, df=1, P<.01 for the southwestern sample).

Conclusions Allelic variation in the apoE gene is a statistically significant predictor of CHD death in these samples of elderly Finnish men.

Key Words: coronary disease • apolipoproteins • mortality

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
There is ample evidence that certain quantitative measures of biochemical and physiological traits are predictors of coronary heart disease (CHD) morbidity and mortality.^{1 2 3} Because these traits are influenced by genetic variation, they are links between interindividual genome variation and variation in risk of CHD. There is evidence from cross-sectional studies of an association between allelic variation responsible for the polymorphic apolipoprotein E (apoE) phenotypes and variation in lipid levels^{4 5 6 7 8 9} and presence of CHD.^{4 5 10 11 12 13} To date, however, there have been no longitudinal studies to evaluate whether the alleles of the gene coding for apoE are predictors of CHD.

The objective of the present study was to evaluate whether the polymorphism of three alleles of the gene coding for apoE is associated with CHD death during a 5-year follow-up of two samples of elderly Finnish men aged 65 to 84 years, one in east and the other in southwest Finland. At baseline, the relative frequencies of apoE genotypes and alleles were different for the eastern and the southwestern samples. Average serum total cholesterol level and measures of other putative CHD risk factor traits were within the ranges reported in other studies of elderly men.^{14 15 16} Approximately one third of the men died during the follow-up period, with CHD the most common cause of death in both samples. Among those who died of CHD, there was a statistically significant excess of the 4 allele in both samples. Therefore, we conclude that allelic variation in gene coding for apoE is a risk factor for CHD death in these samples of elderly Finnish men.

	Methods

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Sample
The Seven Countries Study was initiated in the late 1950s to study cardiovascular disease mortality and morbidity and related risk factor levels in different countries, including Finland.^{1 17} The original Finnish samples consisted of all men born between 1900 and 1919 in two geographically defined rural areas, one in eastern and the other in southwestern Finland. There were 823 men in the eastern sample and 888 in the southwestern sample. In 1984, when baseline assays for the present study were performed, 766 of the 1711 men were 65 to 84 years old and were still alive. Of these eligible men, 716 men (93%) participated in the medical examination. The reasons for the nonparticipation of 50 individuals included long distance to travel, recent myocardial infarction, severe rheumatic fever, and refusal to participate. Knowledge about apoE genotype was available for 666 men (87%). Of the 50 nonparticipants and 50 participating subjects whose apoE genotype was not known at baseline, a total of 45 died during the follow-up period.

At the time of follow-up survey in 1989, 467 of the men whose apoE genotype was known at baseline were still alive and 199 had died during the 5-year follow-up period. The vital status of each man was determined through the Finnish Death Registry or personal contacts, except for 11 men whose vital status was determined through the Finnish Population Registry. Death certificates and hospital records were obtained for all the deceased men, and the cause of death was coded by one of the authors (J.P.) according to standardized criteria.¹⁸

Study Protocol
Two field surveys of each geographic region were conducted during the same month of the year, in October in the east and in November in the southwest. Complete details of the study protocols of the 1984 baseline and 1989 follow-up surveys are given elsewhere.^{19 20} All the men were requested to fast at least 4 hours before examination in the first survey and overnight in the second survey. At the clinic, blood samples were drawn from the antecubital vein for laboratory analyses. Body weight was measured to the nearest 0.1 kg with the participant in light undergarments. Height was measured only in 1959, when the men were first seen in connection with the Seven Countries Study. This height was used to calculate body mass index (BMI, kg/m²), an index of body size used in current analyses. Subjects with BMI 27 kg/m² were classified as being obese.

Smoking status was assessed using a standard questionnaire.¹⁸ Subjects were classified into three categories: current smokers of cigarettes, cigars, or pipes; ex-smokers; and never smokers. A subject who had been an ex-smoker for <1 year before the survey was considered to be a current smoker. Never smokers were classified as those men who had never smoked cigarettes, cigars, or pipes.

Blood pressure was measured by a trained nurse. After at least a 5-minute rest at the end of clinical examination, two successive readings were taken from the right arm using a mercury manometer with a 12x33.5-cm cuff.¹⁹ Readings were taken to the nearest 2 mm Hg, and complete disappearance of the fifth phase of Korotkoff sounds was recorded as the diastolic pressure. We used the mean value of the two readings in analyses reported here. Hypertension was defined according to the recommendation of the World Health Organization²¹ (systolic blood pressure 160 mm Hg and/or diastolic blood pressure 95 mm Hg, or receiving antihypertensive drug treatment).

Laboratory Measurements
Total and high-density lipoprotein (HDL) cholesterol concentrations were assayed using an enzymatic method (Monotest, Boehringer Mannheim) and Olli C3000 Photometer, total cholesterol was determined from fresh sera.²² HDL cholesterol was measured after precipitation of very-low- density lipoprotein and low-density lipoprotein particles with dextran-magnesium-chloride.²² ApoE phenotypes were assessed using serum samples stored at -20°C until used in 1992. We used a modification of the method of Havekes et al,^{23 24} which is based on isoelectric focusing of delipidated serum followed by immunoblotting using rabbit anti-human apoE antiserum. ApoE genotypes were inferred from the respective isoform phenotypes. The apoE allele frequencies (denoted 2, 3, and 4) were estimated by the gene-counting method, separately for the eastern and the southwestern samples.

Statistical Analysis
Differences between the two samples were tested using ANOVA for quantitative traits and a ² statistic for categorical variables. All statistical analyses were performed with the SAS statistical software package.²⁵ The level of statistical significance was taken to be P=.05 unless otherwise designated.

	Results

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The eastern sample included 297 men, and the southwestern sample included 369 men. At baseline, the eastern sample tended to be younger and leaner and have lower systolic and diastolic blood pressures than the southwestern sample (Table 1). In accordance with these findings, prevalence of hypertension was lower in the eastern than in the southwestern sample, whereas the prevalence of obesity and the use of antihypertensive drugs did not vary significantly between these two samples. The proportions of current and ex-smokers were higher in the eastern than in the southwestern sample.

View this table: [in this window] [in a new window]   Table 1. Baseline Characteristics Population by Sample

At baseline, the 3/3 genotype was the most frequent genotype in both samples; 2/2 was not recorded in either sample; and 4/4 was recorded only in three men in the eastern sample (Table 2). Therefore, 4/4 was the least frequent genotype in the eastern sample, and 2/4 was the least frequent in the southwestern sample. Allele frequencies were heterogeneous between the two samples (²=8.890, df=2, P=.012). Significantly lower relative frequencies of the 2 and 4 alleles were observed in the eastern sample (²=4.346, df=1, P<.05 for the 2 allele; ²=3.661, df=1, P=.056 for the 4 allele), whereas the relative frequency of the 3 allele was significantly higher (²=8.093, df=1, P<.01).

View this table: [in this window] [in a new window]   Table 2. Common Genotypes and Distribution of the Three Common Alleles for the Gene Coding Apolipoprotein in E at Baseline by Sample

During the 5-year follow-up period, a total of 88 deaths was recorded in the eastern sample, and 111 in the southwestern sample (Table 3). CHD was the most common cause of death in both samples; it accounted for 34.5% of all deaths in the eastern sample and 37.8% in the southwestern sample. There was no statistically significant difference in either all-cause or CHD mortality rates between the two samples.

View this table: [in this window] [in a new window]   Table 3. Five-Year Mortality by Sample

The distribution of the 2, 3, and 4 alleles varied significantly between survivors and those who died from CHD in both the eastern and the southwestern samples (Table 4). There was a doubling of the relative 4 allele frequency among those who died of CHD in both samples compared with the relative 4 frequency among survivors (²=4.70, df=1, P<.03 for the eastern sample; ²=7.11, df=1, P<.01 for the southwestern sample). In the eastern sample, the relative 2 allele frequency also tended to be higher in those who died of CHD, whereas in the southwestern sample, the relative 2 frequency in those who died of CHD tended to be lower than among survivors. This difference in the relative 2 frequency in those who died of CHD was not different between the samples. There was no second-order interaction among the relative apoE frequency, CHD mortality, and the sample (²=1.67, df=2, P=.433). In the eastern sample, but not in the southwestern sample, increases in the relative frequencies of the 2 and 4 alleles were also associated with non-CHD death and all-cause mortality.

View this table: [in this window] [in a new window]   Table 4. Distribution of the Common Alleles for the Gene Coding for the Apolipoprotein E Polymorphism Among Survivors and Nonsurvivors by Sample

	Discussion

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An ultimate goal for genetic studies of CHD is to provide information that will improve our ability to identify individuals, families, and populations at increased risk. Such information is expected to be useful in developing public health programs and to predict the response of patients with CHD to a proposed treatment. Because the 4 allele is associated with atherogenic changes in measures of lipid metabolism, the presence of this allele is hypothesized to confer an increased susceptibility to CHD.^{4 5 10 11 12 13} This hypothesis is supported in many cross-sectional studies,^{4 5 10 11 12 13} although conflicting results also exist.^{4 26 27 28} In our study of elderly Finnish men, there was an excess of the 4 allele in the men who died of CHD during a 5-year follow-up. This finding provides the first prospective evidence that allelic variation in the gene coding apoE may act as a statistically significant predictor of a CHD outcome.

The presence of the 2 allele is associated with both anti-atherogenic and atherogenic changes in the content and composition of plasma lipids.^{4 29} The atherogenic potential of the 2 allele is mainly associated with 2/2 homozygotes and/or presence of other factors such as obesity, diabetes, and aging.⁴ In accordance with the hypothesized anti-atherogenic potential of the 2 allele, in cross-sectional studies a higher relative frequency of the 2 allele has been observed in samples of subjects free of CHD compared with subjects with CHD.^{4 30} We found no evidence for a statistically significant association between the 2 allele and CHD death in either sample of the elderly Finnish men. A finding that there were no 2 homozygotes in either sample might indicate that the 2 homozygotes were deceased before the baseline study. However, only one or two individuals could be expected to be homozygous for the 2 allele based on Hardy-Weinberg expectations. Therefore, the lack of the 2 homozygotes in the current sample may be entirely attributable to chance.

Age of onset, rate of progression, and severity of atherosclerosis are emergent properties of interactions between genetic effects with exposures to many environmental factors acting through numerous intermediate physiological and biochemical processes, including lipid metabolism.³¹ The relative importance of different combinations of factors to this cascade is, however, context (eg, sex and age) dependent. Elderly subjects, for example, appear to be free of many of CHD risk factors because the disease already has taken its greatest toll among high-risk subjects in their early adulthood.³² In accordance with the latter findings, the elderly men of this study had lower mean levels of serum total cholesterol and measures of other CHD risk factor traits at baseline than they had 20 to 25 years earlier.¹ However, elevated serum cholesterol level remains a predictor of CHD death among elderly subjects.¹⁴ Similarly, the apoE polymorphism is an important determinant of interindividual variation in lipid metabolism among elderly men (Haviland et al, unpublished observations, 1994) and susceptibility for CHD death as seen in the present study. Therefore, we believe that the results of the present study are applicable not only to elderly male populations but also to middle-aged male populations.

The association between the apoE polymorphism and CHD in the present study measures only marginal effects of the apoE polymorphism. The contribution of apoE polymorphism to variation in quantitative measures of lipid and lipoprotein metabolism depends on the levels of other risk factor traits, such as body size, sex, and smoking status.³³ We believe that this also is the case when considering the contribution of apoE polymorphism, or any other measure of genetic variability, to susceptibility to CHD. Therefore, knowledge about apoE genotype combined with a knowledge about other risk factor levels can provide additional information for CHD risk assessment that is not obtained from the apoE polymorphism alone. Studies that address the predictive value of the apoE polymorphism among elderly Finnish men, when variation in other risk factor traits are considered simultaneously, are in progress.

The atherogenic potential of the 4 allele is hypothesized to result in a selective mortality against this allele and subsequent reduction of the 4 allele frequency among elderly subjects.^{4 32} Conversely, the proposed anti-atherogenic potential of the 2 allele is expected to result in an excess of this allele among the elderly subjects. Recent observations among octogenarians and centenarians have supported this hypothesis.^{32 34} In accordance with the selective mortality hypothesis, the baseline 4 allele frequency in the current samples of elderly Finnish men was lower than that obtained from a sample of younger Finns (the estimates of relative 2, 3, and 4 frequencies of Finnish children are 0.035, 0.781, and 0.184 in East Finland and 0.045, 0.753, and 0.202 in West Finland, respectively).³⁵ The deficiency was statistically significant in the eastern sample (²=4.591, df=1, P=.032) but not in the southwestern sample. This uneven decline in the 4 allele frequency across the area of residence, together with a finding that the baseline 4 allele frequency was lower in East than in Southwest Finland in the current samples of elderly Finnish men but not in a sample of younger Finns,³⁵ could be a reflection of excess of CHD mortality in the eastern sample in the past.¹ A finding that the averages of many risk factor levels in this study were higher in the southwestern sample than in the eastern sample, a situation that is opposite to previous findings,^{1 18} provides further support for the selection hypothesis. In this context, we emphasize that during the follow-up the excess of the 4 allele in men who died from CHD resulted in a decrease of the 4 allele in the eastern sample but not in the southwestern sample because in the southwestern sample there was deficiency of the 4 allele among men who died from non-CHD cause. This result suggests strong caution in making inferences about the cause(s) of a heterogeneity of allele frequencies between younger and elderly subjects from cross-sectional data.

Migration is a possible confounding factor when interpreting the cause for unequal allele frequencies between younger and elderly subjects, particularly in the present study. In the early 1960s, there was considerable migration from rural areas to large cities within Finland, and emigration from Finland to Sweden as well. The children and young subjects included in the study of younger Finns³⁵ were born after the migration period. Therefore, the allele frequency in the samples of younger Finns does not necessarily represent the allele frequencies of these areas when the elderly men of the current samples were in their childhood or youth.

In conclusion, the results of the present study provide the first prospective evidence that allelic variation in gene coding for apoE may be of value in predicting CHD mortality. This is the only gene identified thus far to have common allele (ie, 4 relative frequency >0.10) that is a predictor of future CHD when other risk factors are not considered. Because the influence of allelic variation in the apoE gene on the phenotypic distributions of intermediate CHD risk factor traits, and thus interindividual variation in disease risk, varies among subdivision of population defined by sex, age, body size, and smoking status,^{31 33} the use of the gene as a predictor of future CHD may also vary among such subdivisions. The next step is to determine the extent to which the findings of the present study are applicable to subdivisions of the Finnish population or to other populations with different risk factor profiles.

	Acknowledgments

 
This study was supported by Academy of Finland, Medical Research Council, and by National Institutes of Health grants NIH EDC-1, 1-RO1AG08762-01A1, and NIH HL-39107.

Received July 5, 1994; accepted August 11, 1994.

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				K. M. Mann, F. E. Thorngate, Y. Katoh-Fukui, H. Hamanaka, D. L. Williams, S. Fujita, and B. T. Lamb Independent effects of APOE on cholesterol metabolism and brain A{beta} levels in an Alzheimer disease mouse model Hum. Mol. Genet., September 1, 2004; 13(17): 1959 - 1968. [Abstract] [Full Text] [PDF]

				T. M. Doherty, L. A. Fitzpatrick, D. Inoue, J.-H. Qiao, M. C. Fishbein, R. C. Detrano, P. K. Shah, and T. B. Rajavashisth Molecular, Endocrine, and Genetic Mechanisms of Arterial Calcification Endocr. Rev., August 1, 2004; 25(4): 629 - 672. [Abstract] [Full Text] [PDF]

				Y. Song, M. J. Stampfer, and S. Liu Meta-Analysis: Apolipoprotein E Genotypes and Risk for Coronary Heart Disease Ann Intern Med, July 20, 2004; 141(2): 137 - 147. [Abstract] [Full Text] [PDF]

				G. D. Kolovou, D. Ch. Daskalova, M. Hatzivassiliou, N. Yiannakouris, N. D. Pilatis, M. Elisaf, D. P. Mikhailidis, M. A. Cariolou, and D. V. Cokkinos The Epsilon 2 and 4 Alleles of Apolipoprotein E and Ischemic Vascular Events in the Greek Population -- Implications for the Interpretation of Similar Studies Angiology, January 1, 2003; 54(1): 51 - 58. [Abstract] [PDF]

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				T. Lehtimaki, P. Dastidar, H. Jokela, T. Koivula, S. Lehtinen, C. Ehnholm, and R. Punnonen Effect of Long-Term Hormone Replacement Therapy on Atherosclerosis Progression in Postmenopausal Women Relates to Functional Apolipoprotein E Genotype J. Clin. Endocrinol. Metab., September 1, 2002; 87(9): 4147 - 4153. [Abstract] [Full Text] [PDF]

				J. E. Eichner, S. T. Dunn, G. Perveen, D. M. Thompson, K. E. Stewart, and B. C. Stroehla Apolipoprotein E Polymorphism and Cardiovascular Disease: A HuGE Review Am. J. Epidemiol., March 15, 2002; 155(6): 487 - 495. [Abstract] [Full Text] [PDF]

				A.J.C. Slooter, M. L. Bots, L. M. Havekes, A. I. del Sol, M. Cruts, D. E. Grobbee, A. Hofman, C. Van Broeckhoven, J.C.M. Witteman, and C. M. van Duijn Apolipoprotein E and Carotid Artery Atherosclerosis: The Rotterdam Study Stroke, September 1, 2001; 32(9): 1947 - 1952. [Abstract] [Full Text] [PDF]

				K. L. Klos, S. L. R. Kardia, R. E. Ferrell, S. T. Turner, E. Boerwinkle, and C. F. Sing Genome-Wide Linkage Analysis Reveals Evidence of Multiple Regions That Influence Variation in Plasma Lipid and Apolipoprotein Levels Associated With Risk of Coronary Heart Disease Arterioscler. Thromb. Vasc. Biol., June 1, 2001; 21(6): 971 - 978. [Abstract] [Full Text] [PDF]

				A. Batalla, R. Alvarez, J. R. Reguero, S. Hevia, G. Iglesias-Cubero, V. Alvarez, A. Cortina, P. Gonzalez, M. M. Celada, A. Medina, et al. Synergistic Effect between Apolipoprotein E and Angiotensinogen Gene Polymorphisms in the Risk for Early Myocardial Infarction Clin. Chem., December 1, 2000; 46(12): 1910 - 1915. [Abstract] [Full Text] [PDF]

				R. Frikke-Schmidt, B. G. Nordestgaard, B. Agerholm-Larsen, P. Schnohr, and A. Tybjærg-Hansen Context-dependent and invariant associations between lipids, lipoproteins, and apolipoproteins and apolipoprotein E genotype J. Lipid Res., November 1, 2000; 41(11): 1812 - 1822. [Abstract] [Full Text]

				D. A. Nickerson, S. L. Taylor, S. M. Fullerton, K. M. Weiss, A. G. Clark, J. H. Stengård, V. Salomaa, E. Boerwinkle, and C. F. Sing Sequence Diversity and Large-Scale Typing of SNPs in the Human Apolipoprotein E Gene Genome Res., October 1, 2000; 10(10): 1532 - 1545. [Abstract] [Full Text]

				C. R. Isasi, S. Shea, R. J. Deckelbaum, S. C. Couch, T. J. Starc, J. D. Otvos, and L. Berglund Apolipoprotein epsilon 2 Allele Is Associated With an Anti-atherogenic Lipoprotein Profile in Children: The Columbia University BioMarkers Study Pediatrics, September 1, 2000; 106(3): 568 - 575. [Abstract] [Full Text]

				J. M. Olichney, L. A. Hansen, C. R. Hofstetter, J.-H. Lee, R. Katzman, and L. J. Thal Association Between Severe Cerebral Amyloid Angiopathy and Cerebrovascular Lesions in Alzheimer Disease Is Not a Spurious One Attributable to Apolipoprotein E4 Arch Neurol, June 1, 2000; 57(6): 869 - 874. [Abstract] [Full Text] [PDF]

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				A. M. Dart and B. Cooper Independent Effects of Apo E Phenotype and Plasma Triglyceride on Lipoprotein Particle Sizes in the Fasting and Postprandial States Arterioscler. Thromb. Vasc. Biol., October 1, 1999; 19(10): 2465 - 2473. [Abstract] [Full Text] [PDF]

				S. T. Bogardus Jr, J. Concato, and A. R. Feinstein Clinical Epidemiological Quality in Molecular Genetic Research: The Need for Methodological Standards JAMA, May 26, 1999; 281(20): 1919 - 1926. [Abstract] [Full Text] [PDF]

				S. L. R. Kardia, M. B. Haviland, R. E. Ferrell, and C. F. Sing The Relationship Between Risk Factor Levels and Presence of Coronary Artery Calcification is Dependent on Apolipoprotein E Genotype Arterioscler. Thromb. Vasc. Biol., February 1, 1999; 19(2): 427 - 435. [Abstract] [Full Text] [PDF]