© 1997 American Heart Association, Inc.
Articles |
Association of Apolipoprotein Genetic Polymorphisms With Plasma Cholesterol in a Japanese Rural Population
The Shibata Study
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
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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
2
carriers have lower levels of TC and a lower TC:HDL-C ratio, 3
carriers have intermediate levels, and 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
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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.
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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 1
). 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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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
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 
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 
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
(=1-[1-0.05]3=0.14),5
we corrected the resulting by multiplying by 3 to suggest any
association, as defined by the Bonferroni
method.5 32 41
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Results |
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Genotype and Allele Frequencies
Table 1
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 2). 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 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 2). 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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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 3). 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 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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Multiple Regression Analysis
The results of regression analyses are displayed in Table 4. 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 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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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 3
) and multiple regression analysis (Table 4), 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 2 has TC and TC:HDL-C
lowering effects while the 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 3 and 4),
except the finding that the variation in HDL-C levels appears
statistically significant for the apoE gene in regression
analysis (Table 4). However, the magnitude of variation remains
small (partial R2 0.5%,
P=.01).
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Discussion |
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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 5) 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 5). 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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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 5) 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 5 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 2 and 5 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 5). 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 5). 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
(2, 0.052; 3, 0.855; 4, 0.093) are similar to the pooled
estimates derived from five other Japanese studies (2, 0.047; 3,
0.855; 4, 0.098) (Table 5). 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 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 2 and 4.
The studies on apoE polymorphism have shown relatively
consistent positive associations across
populations.3 In many studies (Table 5), the 4
allele is found to be associated with increased levels of TC
compared with 3 while the opposite is true for 2. In our study,
the 2 carriers have a lower level of TC, 3 carriers have an
intermediate level, and 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 5).
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 6) 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 6). 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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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 3 allele has arginine at codon 112 and
cysteine at codon 158, but 2 has cysteine and 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 2 allele with lower and 4 with
higher plasma cholesterol levels compared with 3.
However, the 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 5). 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 5). 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.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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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.
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Footnotes |
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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.
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