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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3495-3504

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3495-3504.)
© 1997 American Heart Association, Inc.

Articles

Association of Apolipoprotein Genetic Polymorphisms With Plasma Cholesterol in a Japanese Rural Population

The Shibata Study

Mohammad Mostafa Zaman; Shinji Ikemoto; Nobuo Yoshiike; Chigusa Date; Tetsuji Yokoyama; ; Heizo Tanaka

From the Department of Epidemiology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan (M.M.Z., T.Y., H.T.); the Division of Clinical Nutrition, National Institute of Health and Nutrition, Tokyo, Japan (S.I.); the Division of Adult Health Science, National Institute of Health and Nutrition, Tokyo, Japan (N.Y.); and the Department of Public Health, Osaka City University School of Medicine, Osaka, Japan (C.D.).

Correspondence to Dr Mohammad Mostafa Zaman, Department of Epidemiology, Medical Research Institute, Tokyo Medical and Dental University, 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo, 101 Japan. E-mail zaman.epi{at}mri.tmd.ac.jp


*    Abstract
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 *Abstract
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Abstract The association between apolipoprotein (apo) genetic polymorphisms and lipid phenotypes remains to be determined because such studies have reported contradictory results. We have measured plasma total cholesterol (TC) and HDL cholesterol (HDL-C) in a cross-sectional sample of 1328 (462 men and 866 women) Japanese (aged 40 to 80 years) and analyzed their DNA for the following genotypes: apoA1-C3 Msp I and Sst I sites; apoB signal peptide insertion/deletion, Xba I site and 3' variable number of tandem repeats (VNTR); and apoE. Using multivariate analyses (ANOVA) we found that (1) the polymorphisms of apoA1-C3 and apoB genes are not significantly associated with TC or HDL-C and (2) the polymorphism of the apoE gene is significantly related with TC and the TC:HDL-C ratio. The {epsilon}2 carriers have lower levels of TC and a lower TC:HDL-C ratio, {epsilon}3 carriers have intermediate levels, and {epsilon}4 carriers have higher levels. These findings held whether sexes were analyzed separately or together. Although an opposite trend in HDL-C levels was observed, it did not reach the level of statistical significance. Multiple regression analyses have shown that apoE polymorphism accounts for about 2.3% of the variation in TC and TC:HDL-C ratio irrespective of sex. When the subjects are divided into two groups according to their age (<=59 and >=60 years old), the effect of apoE alleles on serum cholesterols appears to be diluted in the younger age group whereas it appears to be accentuated in the older group for both sexes. Our findings suggest that among the apo genetic polymorphisms examined, the apoE gene may be considered as a possible candidate for the "high-risk" strategy of atherosclerosis prevention in the Japanese population.


Key Words: apolipoprotein genes • plasma cholesterol • Japanese population


*    Introduction
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 *Introduction
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The apolipoprotein (apo) genes play a central and pervasive role in lipid metabolism by maintaining structural integrity of lipoproteins, acting as cofactors for lipid-processing enzymes, and serving as ligands for lipoprotein receptors.1 2 3 Polymorphisms of these genes may alter lipid levels in individuals, which may predispose to atherosclerosis.4 If dysfunctional alleles that predispose to atherogenic lipid alterations can be identified, screening for the presence of these alleles may identify a substantial proportion of high-risk individuals.1 5 Appropriate monitoring of these individuals, in conjunction with targeted intervention, could then delay or avert the onset of atherosclerosis.5

Thus, one of the important topics of research is to identify apo genes for significant variation in lipid phenotypes in the general population.1 The findings of studies in "healthy" subjects to test the assumption that "variations in the apo loci are associated with variation in lipid phenotypes" have been contradictory. Some studies have concluded that there is a significant association between lipid phenotypes and an apo genetic polymorphism,6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 whereas others have found no association.21 22 23 24 25 26 27 28 29 30 These studies, however, might have been complicated by nonmendelian inheritance, genetic heterogeneity, late onset, and incomplete penetrance and environmental interactions.4 As yet, we do not have a clear understanding of how many different apo genes contribute to plasma lipid phenotypes and the nature of the interactions involved: among gene products and between gene products and environmental factors.3 4 Thus, it is difficult to formulate an appropriate study that considers these interactions in human populations.3

The contradictory results of studies among or within ethnic groups indicate that the association of specific apo genetic polymorphisms with lipid phenotypes remains to be established. We have designed this study to evaluate the feasibility of identifying individuals at high-risk for atherosclerosis in a general population. On the basis of reported putative roles of the apo genes in cholesterol metabolism,4 we have selected six polymorphisms at three genes for this study: apoA1-C3 Msp I and Sst I sites; apoB signal peptide insertion/deletion, Xba I site, and 3' VNTR; and apoE. The association of the polymorphisms of these loci with plasma TC and HDL-C was determined in a large free-living healthy Japanese rural population, which is homogeneous in ethnic background.


*    Methods
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 *Methods
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Subjects
The 1990s health examination of the Shibata Study covered four agricultural areas—Akadani, Ijimino, Matsuura, and Yonekura—of Shibata city, which is located in the northern part of the Niigata prefecture (Figure 1Down). Housewives, farmers, and small shop owners 40 years of age or older are considered for the Shibata Study. However, for our study unrelated subjects aged 40 to 80 years were considered eligible. On the basis of these eligibility criteria, 2172 subjects (840 men and 1332 women) were invited to participate in this study. They represent 38% (32% men and 44% women) of the total population for their age group. Of the 2172 subjects, 1353 (62%) participated in the survey, and apo genotypes could be determined for 1340 (overall response rate, 62%; men, 55%; women, 66%) subjects. However, this analysis was restricted to 1328 subjects (462 men and 866 women) after exclusion of 12 subjects (2 men and 10 women), who were receiving pharmacological treatment for hyperlipidemia.



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  		Figure 1. Location of the study area in Shibata City, Nügata Prefecture, Japan.

All participants were examined at several local community halls, where history of smoking, alcohol consumption, and lipid-reducing drugs was elicited. Height and weight were measured, and body mass index was calculated (weight in kilograms divided by the square of height in meters). This study was approved by the ethics review committee of the Medical Research Institute, Tokyo Medical and Dental University. Informed consent was obtained from all subjects.

Laboratory Methods
Nonfasting blood samples were drawn into tubes containing EDTA. Plasma was separated for measurement of lipids, and blood cells were stored at -80°C until used. TC and HDL-C values were determined with an AutoAnalyzer. Standardization of lipid measurements was achieved by participation in the Lipid Standardization Program of the US Centers for Disease Control and Prevention through the Osaka Prefectural Center for Adult Disease, Japan (presently, the Osaka Medical Center for Cancer and Cardiovascular Diseases).31 The ratio of TC to HDL-C was used to create a dependent variable32 that has been shown to be a better index of coronary heart disease compared with TC or LDL-C.33

Leukocytes were isolated from stored blood cells. DNA was extracted by using an IsoQuick® kit (Microprobe Corp). Genotypes were determined by using polymerase chain reaction protocols. Established methods were used to obtain genotypes of apoA1-C3 Msp I21 and Sst I21 sites; apoB signal peptide insertion/deletion,34 Xba I site,35 and 3' VNTR;36 and apoE.37

Statistical Methods
All analyses were done by using the SAS statistical package (Release 6.11, SAS Institute Inc). Both separate and combined analyses for men and women were done. The balanced-gene estimates38 were used to calculate frequency of the alleles. For example, apo {epsilon}2(balanced-gene)=apo E2/2+1/2(apo E2/3+E2/4). Allele frequencies among the age groups and between sexes were compared by {chi}2 test. This statistical test was also used to examine whether the genotype frequencies were in Hardy-Weinberg equilibrium.39

The normality in the distribution of TC, HDL-C, and TC:HDL-C ratio was confirmed by using normal probability plots. TC and HDL-C values were subjected to a linear regression procedure to obtain the adjusted values30 for variation in age, sex (for combined analysis), alcohol consumption, smoking, and body mass index. Then, ANOVA was performed using the general linear model procedure to determine genetic sources of variation for biochemical traits, with F tests computed from type III sum of squares.40 This form of sum of squares applies to unbalanced study designs and reports the effect of an independent variable after adjustment for all other variables included in the model.40 In a model, for instance, TC was the dependent variable, and the six genetic markers were independent variables.32 The ANOVA testing procedure was initially used to detect the overall difference due to the independent variables on a dependent variable. If this statistical test was significant at the corrected {alpha} level (see below), the Tukey posthoc test was performed to identify which genotypic class(es) differed significantly from the other(s).

In the next step, multiple regression analysis32 was performed, and partial R2 values were obtained. The partial R2 for a given independent variable indicates the percentage of variance in a dependent variable accounted for by that independent variable, beyond the variance accounted for by other independent variables included in the model.40 Genotypic variables were coded with interval scales before they were used in the regression analysis, and the modeling procedure was similar to that for ANOVA.

To reduce the experiment-wise error rate that would otherwise accrue by carrying out independent tests on each of three ANOVAs or regression analyses in either sex ({alpha}=1-[1-0.05]3=0.14),5 we corrected the resulting {alpha} by multiplying by 3 to suggest any association, as defined by the Bonferroni method.5 32 41


*    Results
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 *Results
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Genotype and Allele Frequencies
Table 1Down lists age and risk factor levels of the participants. We have observed homogeneous distribution of the alleles in two age groups (<=59 and >=60 years) both in men and women (data not shown). As the alleles do not differ between the age groups, we present sex-specific genotype and allele frequencies (Table 2Down). The observed genotype frequencies for both men and women and the sexes combined are in Hardy-Weinberg equilibrium. They also do not differ between men and women. The P values obtained by {chi}2 test for all the comparisons mentioned above are >=0.10. There are no alleles of apoB VNTR locus with 41 and 43 repeats (Figure 2Down). For the analyses presented here, alleles <41 repeat units are considered small alleles, and alleles >43 repeat units are considered big.35


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  		Table 1. Characteristic of the Subjects, Shibata Study 1990


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  		Table 2. Distribution of Apo Genotypes and Relative Frequency of Alleles1



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  		Figure 2. Frequency distribution of 3'-apoB VNTR alleles in a Japanese rural population.

Analysis of Variance
For ANOVAs, we have added genotypes with very small frequencies to other genotypes in accordance with the method of single gene estimates38 (Table 3Down). Each model for ANOVA considers a biochemical trait as the dependent variable and six genetic markers as independent qualitative variables. Of the six genotypes examined, in either sex only apoE genetic polymorphism is found to be significantly associated with TC and the TC:HDL-C ratio when Bonferroni correction is applied for the {alpha} levels obtained. Subjects with the E3/4 genotype have higher least-square mean levels and those with the E2/3 genotype have lower levels than those with the E3/3 genotype. For the apoE gene system, consistent gene-dosage effects for least-square means of all dependent variables are seen. However, the variation in HDL-C levels across genotypes does not reach the corrected level of significance.


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  		Table 3. Least-Square Means of TC1 , HDL-C,1 and TC:HDL-C by Apo Genotypes

Multiple Regression Analysis
The results of regression analyses are displayed in Table 4Down. To estimate the percentages of phenotypic variations that are determined by genetic variations, biochemical variables are regressed on the linear combination of the six gene markers tested.32 The regression equation containing these six variables accounts for 3.3%, 1.5%, and 3.6% in the variance in TC, HDL-C and TC:HDL-C, respectively, in men. The corresponding values in women are 2.7%, 1.5%, and 3.3%. The partial R2 values show the relative importance of the six genetic markers in the prediction of the traits. These values show that only apoE genetic polymorphism is the significant determinant of TC and TC:HDL-C variation in both men and women when the Bonferroni correction is applied for {alpha} levels. The apoE genetic marker accounts for 2.4% of the variance in TC and 2.3% in TC:HDL-C in men. In women, these values are similar: 2.3% and 2.4%. These percentages for other genes are subtle, ranging from 0.0% to 0.5%, in both sexes.


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  		Table 4. Results of Multiple Regression Analysis Showing the Percentage of Variation in the Dependent Variable Accounted for by Independent Variables

Major Determinant of Genetic Variation in Cholesterol Levels
This analysis fails to find any significant contribution of apoA1-C3 and apoB genetic polymorphisms to the traits examined. The results of two separate analyses, ANOVA (Table 3Up) and multiple regression analysis (Table 4Up), revel a remarkable conformity: among the six genetic markers examined, apoE genetic variation is the only significant determinant of variation in TC and TC:HDL-C. In general, apoE allele {epsilon}2 has TC and TC:HDL-C lowering effects while the {epsilon}4 allele has a trait-raising effect. After confirming the equality of allele frequencies between the sexes, we have also done analyses for sexes combined. These analyses also have shown similar results (Tables 3Up and 4Up), except the finding that the variation in HDL-C levels appears statistically significant for the apoE gene in regression analysis (Table 4Up). However, the magnitude of variation remains small (partial R2 0.5%, P=.01).


*    Discussion
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 up arrowAbstract
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 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
Our study is the first to describe the association of apoB signal peptide and apoB VNTR with serum cholesterols in a Japanese general population. We have examined apoE polymorphism among Japanese women in expansion of a previous study,17 which recruited apparently healthy male subjects in a hospital. This study has confirmed that there is no association between apoB signal peptide, apoB VNTR, and cholesterol levels for Japanese men and women, and we found a significant association between apoE polymorphism and serum TC for Japanese women as well as men in a general population.

Contradictory Results of Apo Genetic Studies
The contradictory results of the association studies (Table 5Down) are probably a result of small sample sizes, ethnicity, variations including covariates such as sex and environmental factors, and failure to correct the probability levels for multiple comparisons.3 4 5 To overcome the constraint of small sample size, adequately powered studies with large sample sizes are required.5 Our study is the largest of its kind in the Japanese population and one of the largest among all ethnic groups (Table 5Down). This analysis, however, fails to reveal a significant association between five of six polymorphisms and lipid phenotypes examined. These findings held whether sexes were analyzed separately or together.


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  		Table 5. A Review of the Allele Frequencies of Six Apo Genetic Polymorphisms and Their Associations With TC and HDL-C in Healthy Subjects of Various Races1

We have compared allele frequencies of apo genes examined in this study and their effects on TC and HDL-C in "normal" subjects of different racial groups (Table 5Up) to have an overview of the current understanding. One important point to note is that the individual studies differed by settings (eg, control subjects recruited in hospitals, healthy volunteers, and cross-sectional or cohort samples), statistical procedures (univariate or multivariate approach and correction or no correction for multiple comparisons), sample sizes ranging from very small (45) to very large (2258), and covariates considered (eg, sex, body mass index, and known environmental factors such as smoking and alcohol consumption). The subjects who attended a hospital for medical consultation and subsequently were found to have no particular disease (and were recruited as the control subjects) may not be representative of normal individuals in the population.5 The studies that considered the same recruitment (such as healthy population samples) and analytic procedures (such as adjustment for the confounding by covariates) across racial groups found similar trends or association of the polymorphisms studied.29 42 This may mean that although allele distributions are heterogeneous, the relation of alleles with plasma cholesterol may be similar among populations.42 However, the amplitude of the effect of alleles may differ among populations. An impression of the conflicting association among or within ethnic groups by a simple inspection of Table 5Up may be misleading if the methodological differences across studies are not taken into the account.

Comparison of Allele Frequencies and Effects in Different Racial Groups
ApoA1-C3 Genetic Polymorphisms and TC and HDL-C
Inspection of Tables 2Up and 5Up reveals a conformity between the allele frequencies of Msp I and Sst I that we have observed (M1=0.555, S1=0.642) and those reported previously from our laboratory for another Japanese general population (M1=0.573, S1=0.664).21 The present findings of no association of apoA1-C3 Msp I and Sst I with cholesterol levels are in agreement with our previous results.21 The frequencies of M1 (0.879)23 and S1 (0.768)7 alleles in the Chinese subjects reported by Saha et al are higher than those in the Japanese. The corresponding frequencies in whites are even higher (0.924 and 0.921) (Table 5Up). The differences among the Japanese, Chinese, and white populations are statistically significant, which may mean that genetic heterogeneity persists among populations for Msp I and Sst I alleles. In spite of heterogeneity, the nonassociations of the alleles with TC and HDL-C are consistent. The only exception to this is the significant association of the S1 allele with low HDL-C in a Chinese ethnic group in Singapore.7 That report has also reviewed studies available in the literature and found no other study that showed a positive association with HDL-C. As mentioned earlier, the S1 allele frequency in this Chinese sample falls between the Japanese and the whites. Neither the Japanese nor the white studies found such a significant association. Therefore, this isolated finding may be explained by factors other than genetic heterogeneity.

ApoB Genetic Polymorphisms and TC and HDL-C
As observed for apoA1-C3 polymorphisms, population heterogeneity persists for signal peptide (insertion/deletion), Xba I, and VNTR at apoB gene (Table 5Up). The present analysis is the first to examine apoB signal peptide and VNTR in the Japanese that found no association between polymorphisms and cholesterol levels. This finding may need further inquiry. The allele frequencies we observed (X1=0.950) for Xba I are similar to the pooled estimates derived from other Japanese studies (X1=0.966). The lack of association between apoB Xba I polymorphism and TC levels seems to be consistent. The low frequency of the X2 allele among the Japanese may be responsible for low statistical power to detect a significant association if the association exists. In contrast, the studies in the white and Chinese populations reported conflicting results. However, the positive associations reported were rather weak and could disappear in most instances had they been corrected for multiple comparisons. In the context of differences in allele frequencies, the relative positions of the Japanese are closer to the Chinese and distant from the whites. The differences between the Japanese and Chinese should be studied further.

ApoE Genetic Polymorphism and TC and HDL-C
The apoE allele frequencies we observed for sexes combined ({epsilon}2, 0.052; {epsilon}3, 0.855; {epsilon}4, 0.093) are similar to the pooled estimates derived from five other Japanese studies ({epsilon}2, 0.047; {epsilon}3, 0.855; {epsilon}4, 0.098) (Table 5Up). However, in contrast with the female predominance in our sample, the pooled estimates of the other studies represent a predominantly male population. This study confirms the finding of a previous large study17 in the Japanese population that apoE alleles are associated with the variation in serum TC. The heterogeneity of apoE alleles is also found among populations. For instance, compared with whites, an extremely high frequency of {epsilon}4 allele has been found in blacks, and a high frequency in some northern European populations such as the Finns. The studies in the Japanese, including this study, have shown lower frequencies of both {epsilon}2 and {epsilon}4.

The studies on apoE polymorphism have shown relatively consistent positive associations across populations.3 In many studies (Table 5Up), the {epsilon}4 allele is found to be associated with increased levels of TC compared with {epsilon}3 while the opposite is true for {epsilon}2. In our study, the {epsilon}2 carriers have a lower level of TC, {epsilon}3 carriers have an intermediate level, and {epsilon}4 carriers have a higher level in both sexes. A similar association is found for the TC:HDL-C ratio, which has been reported to be a superior measure of risk for coronary heart disease compared with either TC or LDL-C level.33

The nonassociation of apoE alleles with HDL-C has been consistent among studies in healthy populations (Table 5Up). However, a meta-analysis43 of 27 studies in various racial groups including the Japanese, which had sufficient statistical power, showed that HDL-C level is influenced by apoE genetic polymorphism. Although such an analysis can be very informative, it remains open to criticism44 because of the failure to include effects of different settings and covariates3 that differ among samples studied. Our finding of nonassociation cannot be considered as a ß error as the sample size is relatively large.

In contrast with the finding of the Framingham Offspring Study,19 we found no sex differences for the associations between apoE polymorphism and traits. This study also showed that the effect of apoE alleles on lipids in women appears to be more pronounced after menopause. However, this finding is not supported by the data of the Healthy Women Study.18 Another large study45 found no interaction between apoE polymorphism and menopausal status in affecting TC levels in non-Hispanic women. However, in Hispanic women, this study found a significant effect of apoE only in premenopausal women. We have made an attempt to examine this contradictory issue. As we do not have information on menopause, we have done an analysis (Table 6Down) stratified by age (<=59 and >=60 years). The results of our age-stratified analysis in women may support the findings of the Framingham Offspring Study19 that the effect of apoE alleles on TC levels appears to be more pronounced after menopause. However, our method of stratification may not conform with menopausal status. The present analysis also shows that the effect of the apoE allele is prominent in the older group of men (Table 6Down). The prominent effect of apoE alleles in the older age group (>=60 years) in both sexes may be due to differences in some other biological or lifestyle factors such as diets, given the finding that allele frequencies are similar between these two age groups. Unfortunately, we have no data on dietary intake that appears to interact with apoE alleles to modulate plasma cholesterols.46 It is noteworthy that the mean level of TC in men, 180.3 mg/dL of plasma (equivalent to 188.8 mg/dL of serum [180.3x1.047])47 is 10 mg/dL lower while in women it is close to that observed in the concurrent national survey held in 1990.48


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  		Table 6. Variability in Cholesterol Levels Accounted for by the ApoE Genetic Polymorphism Stratified by Sex and Age1

Among the whites, apoE polymorphisms account for up to 8% of the variation in TC, slightly >6% in blacks, and <2.5% in Japanese (present study). This may mean that the contribution of the apoE gene in the variation of TC is subtle in the Japanese.

Although apoE allele distributions are heterogeneous, the relation of alleles with plasma cholesterol is found to be similar among populations.42 However, the amplitude of the effect of various apoE alleles may differ among populations. The data accumulated make it, currently, the most common genetic abnormality in hyperlipidemia or its consequence, atherosclerosis.38 49

The molecular basis of apoE genetic polymorphism and its role in lipid metabolism have extensively been described.50 51 52 In humans, three common apoE isoforms can be distinguished due to the presence of three common alleles. The three apoE alleles differ from each other by amino acid substitutions either at amino acids 112 or 158 of the 299-amino acid peptide chain. The {epsilon}3 allele has arginine at codon 112 and cysteine at codon 158, but {epsilon}2 has cysteine and {epsilon}4 has arginine at both positions. ApoE regulates lipid metabolism by serving as a ligand for the receptor-mediated uptake of remnant particles via the LDL and remnant receptors. An increased association of the apo E4 isoform, compared with E3, with chylomicrons is thought to underlie the faster removal of alimentary lipoproteins from the circulation, which results in downregulation of hepatic LDL receptor expression. As a consequence, cholesterol clearance from the circulation is decreased. The E2 isoform, on the other hand, upregulates hepatic LDL receptor synthesis that results in an overall increased rate of cholesterol clearance from plasma. These mechanisms help to explain the association of {epsilon}2 allele with lower and {epsilon}4 with higher plasma cholesterol levels compared with {epsilon}3. However, the {epsilon}2 allele may not be entirely benign, as it has a propensity for hypertriglyceridemia in homozygotes.53

Attributes and Conclusions
A feature of this study is that despite analyzing a relatively large number of polymorphisms we fail to find significant association with lipid variability except for apoE. This conforms with most of the articles included in the review (Table 5Up). The consistent fit of genotypic arrays to Hardy-Weinberg expectations confirms the genetic homogeneity of our sample of unrelated Japanese subjects. Therefore, it is unlikely that our analysis is contaminated by heterogeneity or sampling bias due to a relatively low response rate (62%). The nonassociations we observed for five of six polymorphisms are probably not the results of ß errors, given the relatively large sample size compared with previous reports (Table 5Up). This study has taken a relatively comprehensive look (and the most comprehensive among the studies in the Japanese) at variations in DNA loci in lipid variability by simultaneously assessing multiple polymorphisms. The simultaneous assessment of several polymorphisms lends strength to our conclusion.

This stringent analysis shows that the apoA1-C3 and apoB genetic polymorphisms examined in this study are not significantly associated with TC, HDL-C, or the TC:HDL-C ratio. Thus, it would not be effective to develop disease prevention strategies based on these polymorphisms. However, our results suggest that apoE polymorphism may be considered as a possible candidate for a high-risk strategy54 of atherosclerosis prevention in the Japanese population.


*    Selected Abbreviations and Acronyms
 
apo = apolipoprotein
HDL-C = HDL cholesterol
LDL-C = LDL cholesterol
TC = total cholesterol
VNTR = variable number of tandem repeats


*    Acknowledgments
 
We gratefully acknowledge the collaboration of the Shibata Study investigators, Drs Momoko Yamaguchi and Takeo Nakayama. We appreciate the assistance provided by other members of our study group, Hiroko Iwaoka, Fukue Seino, Masako Iwaya, and Dr Yasuhiro Matsumura during the phase of data collection. We offer special thanks to Drs Chen Hao and Anisul Haque Chowdhury, and Masako Shiono for their assistance in DNA analysis. Appreciation is extended to Dr Torahiko Kon (mayor of Shibata city) and executives of the local medical association for their continuous support for the Shibata Study. Dr Zaman is supported by the Ministry of Education, Science, Sports, and Culture (Monbusho), Japan. Finally we are indebted to all the subjects who participated in this study; without their help, we could have achieved nothing.


*    Footnotes
 
A portion of this work was presented at the 4th International Conference on Preventive Cardiology, Montreal, Canada, June 28-July 3, 1997, and published in abstract form (Can J Cardiol. 1997;13[suppl B]:278B).

This article encompasses the PhD dissertation of M.M.Z.

Received May 5, 1997; accepted June 30, 1997.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Humphries SE. DNA polymorphisms of the apolipoprotein genes: their use in the investigation of genetic component of hyperlipidemia and atherosclerosis. Atherosclerosis. 1988;72:89–108.[Medline] [Order article via Infotrieve]
  2. Lusis AJ. Genetic factors affecting blood lipoproteins: the candidate gene approach. J Lipid Res. 1988;29:397–429.[Medline] [Order article via Infotrieve]
  3. McCluer JW, Kammerer CM. Dissecting, the genetic contribution to coronary heart disease. Am J Hum Genet. 1991;49:1139–1144.[Medline] [Order article via Infotrieve]
  4. Mehrabian M, Lusis AJ. Genetic markers for studies of atherosclerosis and related risk factors. In: Lusis AJ, Rotter JI, Sparkes RS, eds. Molecular Genetics of Coronary Artery Disease: Candidate Genes and Processes in Artherosclerosis. Basel, Switzerland: S. Karger; 1992;14:363–418.
  5. Marshall HW, Morrison LC, Wu LL, Anderson JL, Corneli PS, Stauffer DM, Allen A, Karagounis LA, Ward RH. Apolipoprotein polymorphisms fail to define risk of coronary artery disease: results of a prospective, angiographically controlled study. Circulation. 1994;89:567–577.[Abstract/Free Full Text]
  6. Porkka KVK, Taimela S, Kontula K, Lehtimaki T, Aalto-Setala K, Akerblom HK, Viikari LSA. Variability gene effects of DNA polymorphisms at the apo B, apo AI/CIII and apo E loci on serum lipids: the Cardiovascular Risk in Young Finns Study. Clin Genet. 1994;45:113–121.[Medline] [Order article via Infotrieve]
  7. Saha N, Tay JSH, Low PS, Basair J, Hong S. Five restriction fragment length polymorphisms of the APOA1–C3 gene and their influence on lipids and apolipoproteins in healthy Chinese. Hum Hered. 1995;45:303–310.[Medline] [Order article via Infotrieve]
  8. Bohn M, Bakken A, Erikssen J, Berg K, The apolipoprotein B signal peptide insertion/deletion polymorphism is not associated with myocardial infarction in Norway. Clin Genet. 1994;45:255–259.[Medline] [Order article via Infotrieve]
  9. Turner PR, Talmud PJ, Visvikis S, Enholm C, Tiret L. DNA polymorphisms of the apoprotein B gene are associated with altered plasma lipoprotein concentrations but not with perceived risk of cardiovascular disease: European Atherosclerosis Research Study. Atherosclerosis. 1995;116:221–234.[Medline] [Order article via Infotrieve]
  10. Saha N, Tay JSH, Chew LS. Influence of apolipoprotein B signal peptide insertion/deletion polymorphism on serum lipids and apolipoproteins in a Chinese population. Clin Genet. 1992;41:152–156.[Medline] [Order article via Infotrieve]
  11. Berg K. DNA polymorphism at the apolipoprotein B locus is associated with lipoprotein level. Clin Genet. 1986;30:515–520.[Medline] [Order article via Infotrieve]
  12. Law A, Wallis SC, Powell LM, Pease RJ, Brunt H, Priestley LM, Knott TJ, Scott J, Altman DG, Miller GJ, Rajput J, Miller NE. Common DNA polymorphism within coding sequence of apolipoprotein B gene associated with altered lipid levels. Lancet. 1986;1:1301–1303.[Medline] [Order article via Infotrieve]
  13. Talmud PJ, Barni N, Kessling AM, Carlsson P, Darnfors C, Bjursell G, Galton D, Wynn V, Kirk H, Hayden MR, Humphries SE. Apolipoprotein B gene variants are involved in the determination of serum cholesterol levels: a study in normo- and hyperlipidemic individuals. Atherosclerosis. 1987;67:81–89.[Medline] [Order article via Infotrieve]
  14. Saha N, Tong MC, Tay JSH, Jeyaseelan K, Humphries SE. DNA polymorphisms of the apolipoprotein B gene in the Chinese coronary artery disease patients. Clin Genet. 1992;42:164–170.[Medline] [Order article via Infotrieve]
  15. Eto M, Watanabe K, Ishi K. Reciprocal effects of apolipoprotein E alleles ({epsilon}2 and {epsilon}4) on plasma lipid levels in normolipidemic subjects. Clin Genet. 1986;29:477–484.[Medline] [Order article via Infotrieve]
  16. Kobori S, Nakamura N, Uzawa H, Shichiri M. Influence of apolipoprotein E polymorphism on plasma lipid and apolipoprotein levels, and clinical characteristics of type III hyperlipoproteinemia due to apolipoprotein E phenotype E2/2 in Japan. Atherosclerosis. 1988;69:81–88.[Medline] [Order article via Infotrieve]
  17. Yamanouchi Y, Arinami T, Tsuchiya S, Miyazaki R, Takaki H, Takano T, Hamaguchi H. Apolipoprotein E5 and E7 in apparently healthy Japanese males: frequencies and relation to plasma lipid levels. Jpn J Hum Genet. 1994;39:315–325.[Medline] [Order article via Infotrieve]
  18. Eichner JE, Kuller LH, Ferrell RE, Meilahn EN, Kamboh MI. Phenotypic effects of apolipoprotein structural variation on lipid profiles: III. Contribution of apolipoprotein E phenoytpe to prediction of total cholesterol, apolipoprotein B, and low density lipoprotein cholesterol in the Healthy Women Study. Arteriosclerosis. 1990;10:379–385.[Abstract/Free Full Text]
  19. Schaefer EJ, Lamon-Fava S, Johnson S, Ordovas JM, Schaefer MM, Castelli WP, Wilson PWF. Effects of gender and menopausal status on the association of apolipoprotein E phenotype with plasma lipoprotein levels: results from the Framingham Offspring Study. Arterioscler Thromb. 1994;14:1105–1113.[Abstract/Free Full Text]
  20. Kamboh MI, Evans RW, Aston CE. Genetic effect of apolipoprotein(a) and apolipoprotein E polymorphisms on plasma quantitative risk factors for coronary heart disease in American black women. Atherosclerosis. 1995;117:73–81.[Medline] [Order article via Infotrieve]
  21. Chen H, Date C, Ikemoto S, Nakayama T, Yoshiike N, Yokoyama T, Seino F, Zaman MM, Iwaoka H, Matsumura Y, Iwaya M, Sugiyama M, Tsuchida M, Yamaguchi M, Itakura H, Tanaka H. Allele frequency of apolipoprotein gene polymorphisms and association between genotype and serum lipids and lipoprotein levels. J Epidemiol. 1995;5:141–151.
  22. Ordovas JM, Civeira F, Genest JJ, Craig S, Robbins AH, Meade T, Pocovi M, Frossard PM, Masharani U, Wilson PWF, Salem DN, Ward RH, Schaefer EJ. Restriction fragment length polymorphism of the apolipoprotein A-I, C-III, A-IV gene locus: relationship with lipids, apolipoproteins, and premature coronary artery disease. Atherosclerosis. 1991;87:75–86.[Medline] [Order article via Infotrieve]
  23. Saha N, Tay JSH, Low PS, Humphries SE. Guanidine to adenine (G/A) substitution in the promoter region of the apolipoprotein AI gene is associated with elevated serum apolipoprotein AI levels in Chinese non-smokers. Genet Epidemiol. 1994;11:255–264.[Medline] [Order article via Infotrieve]
  24. Ferns GAA, Stocks J, Ritchie C, Galton DJ. Genetic polymorphisms of apolipoprotein C-III and insulin in survivors of myocardial infarction. Lancet. 1985;2:300–303.[Medline] [Order article via Infotrieve]
  25. Aburatani H, Matsumoto A, Itoh H, Yamada N, Murase T, Takaku F, Itakura H. A study of DNA polymorphism in the apolipoprotein B gene in a Japanese population. Atherosclerosis. 1988;72:71–76.[Medline] [Order article via Infotrieve]
  26. Hegele RA, Huang LS, Herbert PN, Blum CB, Buring JE, Hennekens CH, Breslow JL. Apolipoprotein B gene DNA polymorphisms associated with myocardial infarction. N Engl J Med. 1986;315:1509–1515.[Abstract]
  27. Eto M, Watanabe K, Iwashima Y, Morikawa A, Oshima E, Sekiuchi M, Ishi K. Apolipoprotein E phenotypes and plasma lipids in young and middle-aged subjects. Tohoku J Exp Med. 1986;148:25–34.[Medline] [Order article via Infotrieve]
  28. Imari Y, Koga S, Ibayashi H. Phenotypes of apolipoprotein E and abnormalities in lipid metabolism in patients with non-insulin-dependent diabetes mellitus. Metabolism. 1988;12:1134–1138.
  29. Sanghera DK, Ferrell RE, Aston CE, McAllister AE, Kamboh MI, Kimm SYS. Quantitative effects of the apolipoprotein E polymorphism in a biracial sample of 9–10-year-old girls. Atherosclerosis. 1996;126:35–42.[Medline] [Order article via Infotrieve]
  30. Sing CF, Davignon J. Role of the apolipoprotein E polymorphism in determining normal plasma lipid and lipoprotein variation. Am J Hum Genet. 1985;37:268–285.[Medline] [Order article via Infotrieve]
  31. Baba T, Tanaka H, Date C, Hayashi M, Tanaka Y, Okazaki K, Nakamura M, Iida M, Komachi Y, Yoshikawa K, Horimoto T, Shimada T, Yamamoto H, Owada K. The standardization of measurement of lipids for epidemiologic study of stroke. Jpn J Hyg. 1983;38:606–613.
  32. Hegele RA, Brunt JH, Connelly PW. Multiple genetic determinants of variation of plasma lipoproteins in Arberta Hutterites. Arterioscler Thromb Vasc Biol. 1995;15:861–871.[Abstract/Free Full Text]
  33. Kinosian B, Glick H, Garland G. Cholesterol and coronary heart disease: predicting risk by levels and ratios. Ann Intern Med. 1994;121:641–647.[Abstract/Free Full Text]
  34. Boerwinkle E, Chan L. A three codon insertion/deletion polymorphism in the signal peptide region of the human apolipoprotein B (APOB) gene directly typed by the polymerase chain reaction. Nucleic Acids Res. 1989;17:4003.[Free Full Text]
  35. Boerwinkle E, Lee SS, Butler R, Schumaker VN, Chan L. Rapid typing of apo B DNA polymorphisms by DNA amplification. Atherosclerosis. 1990;81:225–232.[Medline] [Order article via Infotrieve]
  36. Boerwinkle E, Xiong W, Fourest E, Chan L. Rapid typing of tandemly repeated hypervariable loci by the polymerase chain reaction: application to the apolipoprotein B 3' hypervariable region. Proc Natl Acad Sci U S A. 1989;86:212–216.[Abstract/Free Full Text]
  37. Hixson JE, Vernier DT. Restriction isotyping of human apolipoprotein E by gene amplification and cleavage with HhaI. J Lipid Res. 1990;31:545–548.[Abstract]
  38. Wilson PW, Myers RH, Larson MG, Ordovas JM, Wolf PA, Schaefer EJ. Apolipoprotein E alleles, dyslipidemia, and coronary heart disease: the Framingham Offspring Study. JAMA. 1994;272:1666–1671.[Abstract]
  39. Hartl DL, Clark AG. Principles of Population Genetics. 2nd ed. Sunderland, Mass: Sinauer Associates, Inc; 1989.
  40. Hatcher L, Stephanski EJ. A Step by Step Approach to Using the SAS System for Univariate and Multivariate Statistics. Cary, NC: SAS Institute Inc; 1994.
  41. Bland JM, Altman DG. Multiple significance tests: the Bonferroni method. Br Med J. 1995;310:170.[Free Full Text]
  42. Hallman DM, Boerwinkle E, Saha N, Sandholzer C, Menzel HJ, Csazar A, Utermann G. The apolipoprotein E polymorphism: a comparison of allele frequencies and effects in nine populations. Am J Hum Genet. 1991;49:338–349.[Medline] [Order article via Infotrieve]
  43. Dallongeville J, Lussier-Cacan S, Davignon J. Modulation of plasma triglycerides levels by apoE phenotype: a meta-analysis. J Lipid Res. 1992;33:447–454.[Abstract]
  44. Jenicek M. Epidemiology: the Logic of Modern Medicine. Montreal, Canada: Epimed International; 1995.
  45. Kamboh MI, Aston CE, Hamman RF. The relation of APOE polymorphism and cholesterol levels in normoglycemic and diabeticsubjects in a biethnic population from the San Luis Valley, Colorado. Atherosclerosis. 1995;112:145–159.[Medline] [Order article via Infotrieve]
  46. Cobb MM, Teitlebaum H, Risch N, Jekel J, Ostfeld A. Influence of dietary fat, apolipoprotein E phenotype, and sex on plasma lipoprotein levels. Circulation. 1992;86:849–857.[Abstract/Free Full Text]
  47. Cloey T, Bachorik PS, Becker D, Finney C, Lowry D, Sigmund W. Reevaluation of serum-plasma differences in total cholesterol concentration. JAMA. 1990;263:2788–2789.[Abstract]
  48. Ministry of Health, and Welfare. Summary of National Survey of Circulatory Disorders 1990. Tokyo, Japan: Japan Heart Foundation; 1993.
  49. Castelli W. Lipids, risk factors and ischaemic heart disease. Atherosclerosis. 1996;124(suppl):S1–S9.
  50. Utermann G. Apolipoprotein E polymorphism in health and disease. Am Heart J. 1987;113:433–440.[Medline] [Order article via Infotrieve]
  51. Mahley RW. Apolipoprotein E: cholesterol transport protein with expanding role in cell biology. Science. 1988;240:622–630.[Abstract/Free Full Text]
  52. Surguchov AP, Boerwinkle E, Sharett AR, Patsch WP. Apolipoprotein E genotype and lipid transport: insight into the role of {epsilon}4 allele. Atherosclerosis. 1994;106:119–121.[Medline] [Order article via Infotrieve]
  53. Wilson PWF, Schaefer EJ, Larson MG, Ordovas JM. Apolipoprotein E alleles and risk of coronary heart disease: a meta-analysis. Arterioscler Thromb Vasc Biol. 1996;16:1250–1255.[Abstract/Free Full Text]
  54. Rose G. High-risk and population strategies of prevention: ethical considerations. Ann Med. 1989;21:409–413.[Medline] [Order article via Infotrieve]



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