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(Hypertension. 1998;32:935-938.)
© 1998 American Heart Association, Inc.
Scientific Contributions |
G Protein ß3 Subunit Variant and Essential Hypertension in Japanese
From the Graduate School of Human and Environmental Studies, Kyoto University, Kyoto (N.K., Y. Yamori); the Institute for Adult Diseases Asahi Life Foundation (T.S.), Tokyo; and the Department of Cardiovascular Medicine, Graduate School of Medicine, Tokyo University, Tokyo (H.M., H.K., Y. Yazaki), Japan.
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Abstract |
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Abstract—Enhanced G protein activation has been implicated to underlie the increased sodium-proton transport in blood cells, an impaired characteristic observed in 30% to 50% of patients with essential hypertension. Recently, significant association between a C825T polymorphism of the gene encoding the G protein ß3 subunit and hypertension was demonstrated in a white population, together with the finding that the T825 variant might be related to alternative splicing through unidentified mechanisms. We therefore investigated the disease relevance of this candidate gene by conducting an association study in a relatively large Japanese population. Participants comprised 718 hypertensive case subjects (without diabetes mellitus), 515 normotensive control subjects, and 191 hypertensive subjects with borderline or established diabetes mellitus; all individuals were recruited at a single institution. Genotype distribution of the C825T polymorphism was compared between hypertensive subjects, with or without diabetes, and the control group with
2 statistics. No significant
association was observed in the present study. Results were still
not significant when the case group was subdivided according to more
stringent classification criteria. Allele frequencies of T825
proved to be almost concordant among the 3 study groups and higher in
Japanese (49.0% to 49.6%) compared with a reported prevalence of 25%
to 31% in whites. Our data suggest that the T825 variant of the G
protein ß3 subunit gene is unlikely to constitute major
susceptibility for essential hypertension in the Japanese population
studied. However, further investigation is required to answer the
question of whether the lack of association reflects ethnic differences
in the nature of genetic susceptibility loci.
Key Words: hypertension, essential • association • case-control studies • G proteins • genetics
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Introduction |
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Avariety of approaches have been attempted to elucidate the genetic basis of essential hypertension, which is thought to be of multifactorial origin. One of the logical and promising strategies is the investigation of candidate genes that encode key components of the physiological mechanisms impaired in (or characteristic of) hypertensive patients. Among these mechanisms, enhanced signal transduction via pertussis toxin–sensitive G proteins has been demonstrated in immortalized lymphoblasts from hypertensive patients,1 which may underlie the sodium-proton transport abnormality in blood cells of a subgroup of hypertensive subjects.2 3 In the search for structural changes in the
, ß, or 
subunit of heterotrimeric G
proteins, Siffert et al4 have recently
shown that a C825T polymorphism of the gene encoding the G protein
ß3 subunit (GNB3)5 is significantly
associated with essential hypertension in a white population; the T825
allele had a higher frequency in hypertensive subjects than in
normotensive subjects. Of note is the fact that the T825 allele was
also associated with the occurrence of alternative splicing, which
caused the loss of 41 amino acids within highly conserved repeating
units of GNB3. The splice variant appeared to be
predominantly expressed in cell lines with the TC or TT
genotype. Therefore, it has been implicated that the T825
variant (or another unidentified mutation) of the GNB3 gene
may predispose to essential hypertension. Independent replications
would help to confirm this positive association.6
In addition, some studies have shown the likely ethnic variation in the
nature of genetic susceptibility loci for hypertension—an important
issue that should be further addressed.7 8 9 We
have thus performed a case-control association study for the
GNB3 locus in a group of 1233 Japanese (718 hypertensive and
515 normotensive subjects), where all participants were recruited at
the same institute with relatively clear classification criteria.
Allele frequencies of the T825 variant proved to be higher in
Japanese (49.0% to 49.6%) compared with a reported prevalence of 25%
to 31% in whites.4 Although the present
study does not provide any support for the GNB3 association
as previously reported,4 the lack of association
has to be carefully interpreted, which may simply reflect the ethnic
differences between the 2 ethnic groups. |
Methods |
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Study Population
This study was approved by an institutional review committee. Participants in the present study comprised a total of 1424 individuals (1233 individuals for the case-control study [718 hypertensive and 515 normotensive subjects] and 191 hypertensive subjects with either impaired glucose tolerance or non–insulin-dependent diabetes mellitus) selected from outpatients and hospital staff at the Institute for Adult Diseases Asahi Life Foundation, Tokyo. Informed consent for participation was obtained from all subjects. Clinical characteristics of each group are described in Table 1
. Two blood pressure (BP)
measurements were taken with a sphygmomanometer on separate visits and
averaged for each individual's reading. Hypertension was defined
according to the following criteria: (1) systolic BP 
160
mm Hg and/or diastolic BP 95 mm Hg on 2
consecutive visits for untreated subjects, (2) chronic antihypertensive
treatment of patients, and (3) absence of a secondary form of
hypertension through extensive workup; subjects with a history of
diabetes mellitus and renal failure were excluded from the case
(hypertensive) group. In addition, more stringent criteria were used to
subdivide the case group, ie, age of onset <60 years and BP readings
clearly documented in hospital records with systolic BP
160 mm Hg and/or diastolic BP 95 mm Hg
before establishing of medication. Here, the age onset of hypertension
was defined as the time when BP readings exceeded the above criteria on
consecutive visits before start of medication or when chronic
medication was initiated. Subjects with systolic BP <140
mm Hg and diastolic BP <90 mm Hg were categorized
into a control group, which comprised hospital staff and those
attending voluntary health checks or with noncardiac symptoms. Because
G proteins encompassing the ß3 subunit can be involved in several
signaling pathways regulating hypertension-associated
phenotypes,10 11 the presence of diabetes
may affect results for association study through some genetic
mechanisms; for example, the molecular variant of GNB3 could
be a genetic predisposition to clustering of hypertension and other
cardiovascular risk factors.12
The association was therefore separately tested between the control
group above and another group of 191 hypertensive patients who were
concomitantly diagnosed as having impaired glucose tolerance or
non–insulin-dependent diabetes mellitus without serious renal
dysfunction (serum creatinine level <115 µmol/L),
where impaired glucose tolerance was defined on the basis of an oral
glucose tolerance test.
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Genotyping of GNB3 Polymorphism
The C825T polymorphism was characterized following the
method described previously,4 except that a
restriction endonuclease, BsaJI (New England Biolabs, Inc)
was used instead of BseDI (Fermentas). Briefly, polymerase
chain reaction (PCR) was performed in PTC-100 (MJ Research Inc) in a
15-µL reaction volume containing 200 nmol/L each of the PCR primers,
10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 25 µmol/L
each of dNTPs, 0.4 U Ampli-Taq DNA polymerase (Perkin Elmer), and
1.5 mmol/L MgCl2. The initial denaturation
for 3 minutes at 95°C was followed by 35 cycles of denaturation for
20 seconds at 94°C, annealing for 30 seconds at 60°C, and extension
for 20 seconds at 72°C. The size of PCR products after
BsaJI digestion was 268 bp for the TT genotype, and
a set of 116 bp and 152 bp for the CC genotype, which were
clearly resolved on 2% agarose gel (SeaKem agarose, FMC
Bioproducts).
Statistical Analysis
The likelihood ratio 2 statistics were
calculated between genotype distribution and hypertension
status, with TT and TC genotypes being analyzed
separately and analyzed together according to the observation
by Siffert et al4; the associated cellular
phenotype did not appear to be different between TT and TC
genotypes. Differences in each of the clinical characteristics
between the case and control groups were also examined by 1-way ANOVA.
Confounding influences of age and body mass index (BMI) were assessed
in a multiple logistic regression model using the JMP statistical
package (SAS Institute Inc). Approximate 95% confidence intervals
(CIs) of the odds ratio were given by Woolf's
method.13
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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No statistically significant association was observed between the T825 variant of GNB3 and hypertension in our study (Table 2
). When the case group was subdivided
according to more stringent criteria, there was still no association.
Moreover, the GNB3 association was not significant between
the control group and diabetic hypertensives, although the latter group
comprised a smaller number of individuals, producing less statistical
power. Each of the 3 study groups was consistent with
Hardy-Weinberg equilibrium. The lack of observed association was
independent of age and BMI. Allele frequencies of the T825 variant
proved to be higher in Japanese (49.0% to 49.6%) compared with a
reported prevalence of 25% to 31% in
whites.4
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The present study is the first conducted to date to assess the relevance of the GNB3 gene to essential hypertension in a nonwhite population. Our failure to reproduce the positive association originally demonstrated in whites4 has to be interpreted with considerable caution. Confounding factors such as population stratification and misclassification are known to frequently cause false-positive and false-negative results in case-control study.14 15 Although the Japanese are thought to be monoracial, some phylogenetic studies have reported that there is a moderate spectrum of genetic variation among persons living in different parts of the country.16 17 All the participants were therefore recruited at the same institute to minimize the chance of mixing populations with inherently diverse allele frequencies of a susceptibility gene. In addition, diagnosis of hypertension was basically determined after long-term clinical follow-up, which enabled us to reduce the risk of misclassification to some extent. Furthermore, our trial size involving over 1200 participants in the case-control study should have decreased the substantial sampling variation (or selection bias) that is generally unavoidable in case-control association strategy. This thereby lends us an appropriate, though far from sufficient, statistical power to evaluate a susceptibility gene exerting principal effects on BP regulation.
Three interpretations can be proposed to explain the lack of observed
association in the Japanese population. First, the contribution of
GNB3 to the pathogenesis of hypertension may be less
significant than originally implicated4 in a
given population, partly because of the population-specific combination
of genetic and environmental factors. In the assessment of this
interpretation, our data can be examined as follows. The odds ratio for
the TT+TC versus CC genotype is 1.44 (95% CI, 1.09 to 1.88) in
the reported association study,4 while the
corresponding odds ratio is 0.94 (95% CI, 0.72 to 1.22) in the
present study; both CIs partially overlap. If results for these 2
studies potentially represent the same value of the effect
measure (as is possible but may be less likely than the hypothesis of
"population-specificity"), the sample size required to confirm the
susceptibility in question will be very large. For example, it is
roughly estimated that >1000 individuals are required in each group of
cases and controls for detecting odds ratio 
1.2 with 80% power at a
5% type I error probability.18 Thus, no
conclusive claims can be made from our data because statistical power
is insufficient based on this assumption. On the other hand, the higher
frequency of T825 allele in Japanese may have hampered the
detection of the potential association, although our trial size is
larger than the previous study. With these arguments considered
together, the T825 variant at least is unlikely to constitute major
susceptibility for essential hypertension in the population
studied.
Second, detailed characterization of the pathophysiological mechanisms may help further investigate the disease association of GNB3. Given the moderate effects of GNB3 on hypertension as assumed above, an intermediate phenotype, which the molecular variant of the GNB3 gene itself represents, would allow for more appropriate evaluation of this candidate gene by dissecting the genetic heterogeneity of essential hypertension. There is also the possibility that a "true" mutation as yet unidentified, which is in linkage disequilibrium with the T825 variant, may exist in the GNB3 locus. If so, a search for other base-substitution polymorphisms, together with haplotype analysis,19 will provide a better chance to detect the potential association.
Third, the lack of association may result from an ethnic variation. Some of the phenotypic characteristics related to hypertension are known to show ethnic differences. For example, the prevalence of salt sensitivity is estimated to be higher in blacks than in whites.20 The likely presence of ethnic variation has been also indicated in the disease relevance of the angiotensinogen gene7–9; that is, despite a number of studies supporting positive linkage and/or association as originally reported by Jeunemaitre et al21, lack of linkage in Chinese9 and lack of association in a few black populations7 8 have been demonstrated. Accordingly, the issue of ethnic variation remains to be extensively explored for the GNB3 gene.
In summary, the present study does not replicate the association between the T825 variant of GNB3 and essential hypertension in the Japanese population, although there is a possibility that this particular polymorphism is uninformative in the Japanese. Use of an intermediate phenotype and/or haplotype analysis, as well as a genetic linkage analysis, would allow for further investigation of this candidate gene in relation to hypertension, and thereby the question of whether the lack of association in the Japanese population simply reflects ethnic differences can be answered.
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Acknowledgments |
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We gratefully acknowledge Chie Fujinami for assisting us in DNA preparation and data arrangement.
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Footnotes |
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Reprint requests to Norihiro Kato, MD, PhD, Otsuka Department of International Preventive Nutritional Medicine, Graduate School of Human and Environmental Studies, Kyoto University, Yoshida Nihonmatsu-cho, Sakyo-ku, Kyoto 606, Japan.
Received March 3, 1998; first decision March 10, 1998; accepted July 13, 1998.
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References |
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