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b1-adreno-ceptor polymorphisms may help us to explain intraindividual
differences in drug response and disease susceptibility.
Therefore, in the present study we investigated the
frequencies of the main mutants of the
b1-adrenoceptor gene in Han and Dai Chinese populations. Because we consider that
polymerase chain reaction-restriction fragment length
polymorphism (PCR-RFLP) analysis is currently one of the most
reliable methods for population genotyping, PCR-RFLP
methods for Ser49Gly and Gly389Arg genotyping of the
b1-adrenoceptor gene were used.
Materials and methods
Subjects The study protocol was approved by the
Ethics Committee of Xiang-Ya School of Medicine, Central South
University. A total of 225 Han Chinese (123 men, and 102
women) living in Hu-nan province and 175 Dai Chinese
living in Yunnan province (89 men, and 86 women) were
recruited for the present study. The subjects were unrelated
healthy volunteers aged 18_21 years, and all participated
after giving their written informed consent. All of the Han
subjects were students from Xiang-Ya School of Medicine,
Central South University, and the Dai subjects were
students from the Dali Nationality School, Yunnan province.
None of the subjects had any abnormalities according to
their medical records, routine laboratory tests, physical
examinations, or electrocardiography. Samples (5 mL per
subject) of peripheral vein blood were collected for
genotyping analysis (Table 1).
Genotyping procedures for b1-adrenergic
receptor Genomic DNA was extracted from peripheral lymphocytes with
phenol-chloroform followed by ethanol
precipitation[14]. Genotyping analysis was carried out by using the PCR-RFLP
assay. The PCR procedure for the b1-adrenergic receptor
gene was performed as described previously with minor
modifications[15]. For the Ser49Gly locus, we used the
following primer pairs: the sense primer P1, 5กฏ-CCGGGCTTCTGG-
GGTGTTCC-3กฏ, and the antisense primer P2,
5กฏ-GGCGAGGTG-ATGGCGAGGTAGC-3กฏ. The Gly389Arg polymorphic locus
was amplified by using the sense primer P3,
5กฏ-CATCATGGG-CGTCTTCACGC-3กฏ, and the antisense primer P4,
5กฏ-TGGGC-TTCGAGTTCACCTGC-3กฏ. The amplified DNA fragments
including the Ser49Gly or Gly389Arg polymorphic sites were
separately digested with Eco0109I (TaKaRa Biotech, China)
or BcgI (England Biolabs, Beverly, USA) at 37 °C for 8 h.
The different patterns produced by the digested fragments
were visualized on 2% agarose gels stained with ethidium
bromide. Following the amplifications, some of the PCR
product was purified and sequenced to confirm the accuracy of
the RFLP assay.
Ser49Gly and Gly389Arg point mutations The Ser49Gly
point mutation gave rise to an Eco0109I cleavage site. The
PCR product of the Gly49 allele contained a unique site for
restriction by Eco0109I. The PCR product of the Gly49 allele
contained a unique site for restriction by
Eco0109I. The Ser49Gly mutant allele had 3 bands, 564 bp, 345 bp, and 219
bp, which became apparent when digested fragments were
detected with an ultraviolet detector (Figure 1). The
Gly389Arg substitution gave rise to a BcgI cleavage site. The Gly389Arg
mutant had 3 bands, 530 bp, 342 bp, and 154 bp.
BcgI cleaves twice, excising its recognition site, which accounts for the 34
bp discrepancy in the fragments generated (Figure 2). When
the PCR-RFLP genotyping assay was repeated for randomly
selected samples, the result of the second genotyping of
each sample was identical to the first in every case, thus
verifying the reproducibility of the genotyping results.
Statistical analysis Data analysis was performed using
SPSS (version 10.0 for Windows, SPSS, Chicago, IL, USA).
A two-tailed value of P<0.05 was considered statistically
significant. The frequencies of each allele polymorphism
were calculated based on the observed number of different
alleles (49Ser or 49Gly, or 389Gly or 389Arg) in each ethnic
group. Hardy-Weinberg equilibrium and possible differences
in allele frequencies between the 2 populations were tested
by using the c2-test.
Results
In the present study, we found that the 2 single
nucleotide polymorphisms causing the Ser49Gly and Gly389Arg
mutants of the b1-adrenoceptor existed in both the healthy
Han and Dai Chinese populations.The allele and genotype frequencies for the
b1-adreno-ceptor Ser49Gly and Gly389Arg polymorphisms in Han and
Dai populations are summarized in Table 2. The allelic
frequencies for Gly49 and Arg389 were 16.2% and 76.4% in the
Han population and 14.6% and 75.7% in the Dai population,
respectively. The allele and genotype frequencies for the 2
different populations were in Hardy-Weinberg equilibrium
as tested by comparisons of the frequencies found versus
the frequencies expected on the basis of the proportion
represented by the more frequently occurring allele. There was
no difference between the Han and Dai populations with
respect to either of the allele frequencies. Compared with
data in the literature regarding the 2 polymorphisms in healthy
subjects from different ethnic groups, significant interethnic
differences in the incidence of the Arg389 polymorphism
were found between a Chinese and an African American
population; however, the frequencies of the Ser49Gly
polymorphism were not significantly different in these 2
populations[11].
Discussion
The b1-adrenoceptor is an important target for many
drugs and endogenous substances[16]. In the present study,
we carried out the first investigation of the frequencies of
the Ser49Gly and Gly389Arg genetic polymorphisms of
b1-adrenoceptor in Han and Dai Chinese populations and found
the frequencies for both polymorphisms were not
significantly different in the 2 populations; however, the frequency
of the Gly389 variant appeared to be significantly lower in
the Han and Dai Chinese populations than in an
African-American population.
In the present study, we found that the allelic
frequencies of Gly49 and Arg389 were approximately 14.6%_16.2%
and 75.7%_76.4% in the combined Han and Dai populations,
respectively. Gly389 is considered to be the wild type, and
was first cloned by Frielle et al as a Gly389-containing
receptor[1].
The Dai ethnic group is one of many minority groups in
China who have unique characteristics with respect to
genetic make-up, culture, diet and
environment[12]. Previous studies carried out by our laboratory have found that
interethnic differences with respect to polymorphisms in drug
metabolizing enzymes such as CYP2C19 exist between Han
and Dai populations. He et al reported that the frequency of
the CYP2C19*3 allele in a Dai population was significantly
lower than that in a Han population[13]. In the present study,
we found that there was no significant difference in the
frequencies of the b1-adrenoceptor Ser49Gly and Gly389Arg
polymorphisms between the two ethnic groups. An
explanation for the difference between the 2 enzymes with respect
to the level of polymorphism in different populations is that
b1-adrenoceptor may well be more conserved throughout
different populations than drug metabolizing enzymes. Many
drugs that have effects mediated by b1-adrenoceptor have
obvious interethnic differences in their
effects[17]. If such a difference was also found between Han and Dai populations,
the differences would be unlikely to be due to
b1-adreno-ceptor polymorphisms, but would rather be due to genetic
variations in drug-metabolizing enzymes and the effects of
environmental factors.
A previous study reported that the incidence of the
Ser49Gly amino acid substitution was 16.2% in patients with
IDCM, but zero in a healthy
population[6]. In contrast, we found that the frequency of the Gly49 allele was
approximately 14.6%_16.2% in healthy subjects, which is
consistent with the results of other studies, in which the frequency
of the Gly49 allele was found to be 2%_15% in healthy
subjects[8,15,18]. Because one study in which the Gly49 allele was
only detected in patients with IDCM used only a small sample
size (37 patients), the resulting in low statistical power might
have produced a false result. However, several other
studies that used a large sample size have also found that there
was a higher frequency of the Gly49 allele in patients with
ICDM relative to healthy
subjects[6,8,15].
An in vitro mutagenesis study of the codon 389
polymorphism revealed that the Arg389 receptor form had nearly
two-fold greater basal and 3-fold greater agonist-mediated
adenylyl cyclase activity[9], which suggests that the Gly389
mutation produces a hypoactive b1-adrenoceptor, and that a
higher frequency of the Gly389 allele could contribute to
decreased sensitivity to b-blockers in African-Americans.
However, we found no ethnic differences in the frequency
of the b1-adrenoceptor Gly389Arg polymorphism between
Han and Dai Chinese populations in the present study,
indicating that this polymorphism is unlikely to account for any
ethnic differences in sensitivity to b-blockers between these
two populations.
In conclusion, we found that the b1-adrenoceptor
Ser49Gly and Gly389Arg mutations both existed in both Han
and Dai Chinese populations, and that the frequencies of
the two polymorphisms were not significantly different in
these two ethnic groups.
References
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