Download Association Analysis of Restriction Fragment Length

1-203
Association Analysis of Restriction
Fragment Length Polymorphism for
o?2-Adrenergic Receptor Genes in
Essential Hypertension in Japan
Satoshi Umemura, Nobuhito Hirawa, Tamio Iwamoto, Satoshi Yamaguchi,
Yoshiyuki Toya, Shunichi Kobayashi, Izumi Takasaki, Gen Yasuda, Kouichi Tamura,
Masao Ishii, Lu Sun, William A. Pettinger
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Abstract Recently, restriction fragment length polymorphism (RFLP) of aj-adrenergic receptor gene (a2-C10) digested with Bsu361 restriction enzyme has been reported in US
populations. Therefore, we examined the association of this
RFLP with essential hypertension by comparing the frequency
of specific alleles for this gene in Japanese populations. The
distribution of this RFLP was compared with that in US
populations. Subjects were hypertensive patients with a family
history of essential hypertension (n=56) and normotensive
subjects whose parents had no history of essential hypertension (n=46). DNA was prepared from leukocytes. RFLP was
determined by use of Southern blot analysis with an a2-C10
probe and Bsu36l. The frequencies of the major (12-kb) and
minor (5.8-kb) alleles were 0.30 and 0.70 in hypertensive
patients and 0.38 and 0.62 in normotensive subjects, respectively. The difference between observed alleles in all subjects
in each group was not significant (* 2 =1.33, P>.1). The
difference between the overall allelic frequency in Japan and
that reported in US populations was significant. This study
found no evidence for an association between a2-adrenergic
receptor gene/5iu36I RFLP and essential hypertension in
Japan. However, the findings showed that the allele frequency
in Japan differed from that reported in US populations.
(Hypertension. 1994;23 [suppl IJ:I-203-1-206.)
S
human chromosomes 10,4, and 2, has been suggested to
correspond to the pharmacologic classification of a2-A,
a2-C, and a2-B adrenergic receptors, respectively. Using
cDNA, two restriction fragment length polymorphisms
(RFLPs) of the a2-adrenergic receptor gene in humans
have been reported. 910 Furthermore, Sun et al,11 using
an a2-C10 probe representing the human platelet a2adrenergic receptors, reported the frequency of a2adrenergic receptor RFLPs in normotensive subjects
and hypertensive patients in US populations.
Therefore, we examined the association of these
RFLPs with essential hypertension by comparing the
frequency of specific alleles for this gene in Japanese
populations. The distribution of these RFLPs was compared with that reported in US populations.11
everal lines of evidence suggest that the a2-adrenergic receptor may play a pathogenic role in
primary hypertension in rats as well as humans.1
The a2-adrenergic receptors in the kidney and central
nervous system have been suggested to play important
roles in the regulation of blood pressure.2-3 The density
of cerebral and renal a2-adrenergic receptors is increased
in genetically hypertensive rats even before their blood
pressure becomes elevated, but not in animal models of
acquired hypertension. In humans, platelet a2-adrenergic
receptor density is better correlated in monozygotic
twins than in dizygotic twins (in whom receptor number
correlates better than in age-matched random pairs),
suggesting that the density of a2-adrenergic receptors can
be inherited.4 This platelet a2-adrenergic receptor may
be altered in patients with essential hypertension as well
as in young normotensive subjects who have a family
history of essential hypertension.1-5-7
Recently, at least three a2-adrenergic receptor genes
have been cloned in humans.8 The molecular classification of a2-C10, a2-C4, and a2-C2, which localized in
From the Second Department of Internal Medicine, Yokohama
City (Japan) University (S.U., N.H., T.I., S.Y., Y.T., S.K., I.T.,
G.Y., K.T., M.I.), and the Midwest Hypertension Research Center, Creighton University, Omaha, Neb (L.S., WAP.).
Correspondence to Satoshi Umemura, Second Department of
Internal Medicine, Yokohama City University, Fulmura 3-9, Kanazawa-ku, Yokohama, 236, Japan.
Key Words • a2-adrenergic receptors • hypertension,
primary • restriction fragment length polymorphism •
ethnicity
Methods
Subjects
All subjects were adult Japanese of either sex. The diagnosis
of hypertension was based on the criteria of a systolic blood
pressure greater than 140 mm Hg and a diastolic pressure
greater than 90 mm Hg on at least three readings on at least
two visits. To diagnose essential hypertension and exclude
secondary hypertension, all patients were admitted to the
Yokohama City University Hospital for evaluation.
Fifty-six patients with essential hypertension, 58.5 ±11.3
years old (range, 27 to 77 years), and 46 normotensive subjects,
47.7±15.9 years old (range, 20 to 72 years), were invited to
participate in the study. A detailed history was taken for each
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Supplement I Hypertension
Vol 23, No 1 January 1994
subject. Only patients with essential hypertension and a family
history of hypertension were enrolled in this study. Normotensive subjects were selected on the basis of both parents also
being normotensive. Patients with an uncertain family history
of hypertension were excluded from the study. All subjects
were informed about the study protocol and subsequently
provided informed consent.
-12Kb
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DNA Extraction and RFLP Analysis
Blood samples of approximately 10 mL were drawn into
heparinized tubes and used for the separation of white blood
cells. Genomic DNA was extracted from peripheral leukocytes
of the hypertensive patients and normotensive subjects as
previously described.12
Human genomic DNA (10 ^g) was digested overnight with
40 U flsu36I restriction endonuclease at 37°C. The products
were separated by electrophoresis on a 1% agarose gel and
transferred to nylon membranes. The filter membrane was
baked for 2 hours at 80°C. Prehybridization was performed in
a solution containing 6x saline/sodium phosphate/EDTA
buffer (SSPE), lOx Denhardt's solution, 1% sodium dodecyl
sulfate (SDS), and 50 /ig/mL salmon sperm DNA at 42°C for
4 hours. After prehybridization, the solution was changed to
the hybridization solution containing 6 x SSPE, 5 x Denhardt's
solution, 0.5% SDS, 100 MgAnL salmon sperm DNA, 50%
deionized formamide, 10% dextran sulfate, and ["PJdeoxycytidine triphosphate-labeled DNA probes and incubated for 18
to 24 hours at 42°C. The 950-bp Pst I restriction fragment of
the human platelet a2-adrenergic receptor gene (a2-C10) was
used as a probe as previously reported in US populations.11
This Pst I restriction fragment is from the coding region of
o2-C10 and runs from base 151 to base 1099. The filters were
washed twice in 3x standard saline citrate (SSC) and 0.1%
SDS at 55°C for 30 minutes followed by once in 0.1 x SSC/
0.1% SDS at 55°C for 10 minutes. The filters were exposed to
Kodak XAR-5 film with a Lightning Plus intensifying screen
for several days at -70°C.
-5.8Kb
Southern blot of human genomic DNA digested with Ssu36l
restriction endonuclease and hybridized with the a2-C10 probe.
have either a 12-kb or a 5.8-kb band, and heterozygotes
have both the 12-kb and 5.8-kb bands. Nondifferentiating additional bands were also seen in the Southern
blots.
Genotype and derived allele frequencies are shown in
the Table. In examining data for normotensive and
hypertensive groups, the correct data for analysis were
the observed numbers of alleles, ie, the sum of the
number of alleles on each chromosome of all of the
subjects in each group (eg, for the hypertensive group,
the 12-kb allele was [2x7]+20=34). By x1 analysis of
observed alleles, no significant difference was apparent
between the hypertensive and normotensive groups in
Japanese populations. Because blood pressure elevation
usually starts after the age of 40 years in patients with
essential hypertension, we also analyzed the data only
from subjects more than 40 years old. However, we
again found no significant difference in the frequency of
observed alleles between the hypertensive and normotensive groups (data not shown).
Statistical Analysis
Data were compiled according to genotype, and allele
frequencies were calculated. Statistical analysis required calculation of the observed number of alleles from genotype data
in each group. Differences between groups were tested by x2
analysis with one degree of freedom.
X analysis was also used for the comparison between
Japanese and US populations. A value of P<.05 was considered significant.
Results
The allele frequencies were compared between the
US11 and Japanese populations. The overall allele frequencies in Japanese differ from those reported in US
populations (0.34 versus 0.66 in Japanese and 0.49
versus 0.51 in the United States, 12-kb versus 5.8-kb,
The three possible patterns of hybridization of the
a2-C10 to a Southern blot of human genomic DNA
digested with Bsu36l restriction endonuclease are
shown in the Figure. Homozygotes for the Bsu36l RFLP
Allele Frequencies for the 12-kb and 5.8-kb Alleles of a Bsu36l RFLP of the o%-C10 Gene In
Hypertensive and Normotensive Groups in Japan
Allele
Frequencies
(kb)
Genotype
(kb)
Total Alleles on
All Chromosomes
(kb)
Group
12
12+5.8
5.8
12
5.8
12
5.8
Hypertensive (n=56)
7
20
29
0.30
0.70
34
78
Normotensive (n=46)
8
19
19
0.38
0.62
35
57
2
x »1.33 (P>.1)
RFLP indicates restriction fragment length polymorphism.
Umemura et al
RFLP of or2-Adrenergic Receptor Gene in the Japanese
respectively; * 2 =9.40, / > <.01). The same probe and
restriction enzyme were used to examine the frequency
of distribution of allelic polymorphisms of the a2-adrenergic receptor gene in normotensive (n=47) and hypertensive (n=60) humans in the US study.11
Discussion
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In the present study, we found no association of an
a2-adrenergic receptor RFLP with essential hypertension in Japan using an a2-C10 probe and Bsu56\ digestion. However, we found that the allele frequency in
Japanese differed from that reported in US populations.
Because essential hypertension is an inherited disease, many investigators are trying to find a gene
responsible for this disease. The angiotensinogen,13
insulin receptor,14 and angiotensin converting enzyme15
genes have been reported to be candidates using techniques similar to those used in the present study.
However, results are not yet consistent.16
Altered a2-adrenergic receptors have also been suggested as a possible pathogenic factor in the genetic
predisposition to hypertension.1 In humans, the density
of a2-adrenergic receptors in platelets can be inherited,4
and this density has been reported to be altered in
patients with essential hypertension as well as in young
normotensive subjects with a family history of essential
hypertension.1-57 Thus, we compared the a2-adrenergic
receptor genes in patients with essential hypertension
and normotensive control subjects by use of RFLP.
Recently, the RFLPs of human a2-adrenergic receptors
have been reported. 910 With these RFLPs, association
studies have been performed by others.11'17 However,
both prior studies demonstrated no difference in the
frequencies of a2-adrenergic receptor RFLPs between
patients with essential hypertension and normotensive
subjects. One study11 used RFLPs shown by Bsu7>6\
digestion and an a2-C10 probe (950 bp) in a US
population, and the other17 used Dra I digestion and the
platelet a2-adrenergic receptor gene (1500 bp) in an
Australian population. Our results also revealed no
association between a2-adrenergic receptor RFLPs and
essential hypertension in a Japanese population.
This lack of association indicates that the particular
DNA changes causing these polymorphisms are not
themselves responsible for hypertension. However, we
cannot rule out the possibility that polymorphism of this
gene might be associated with essential hypertension if
other detection methods could be used. The distribution
of a2-adrenergic receptor alleles did not differ between
the hypertensive patients and the normotensive subjects. We calculated that approximately 270 participants
would be needed to see significant differences in gene
frequency based on this study.
There are additional nondifferentiating bands in the
Southern blots (Figure). The actual reason for the
existence of these bands is not known. One possibility is
the cross-hybridization between a2-C4 and a2-C10, because a previous report showed the same bands and
further demonstrated that the 3' end probes (corresponding to the third cytoplasmic loop, which shares
less sequence homology) from both a2-C10 and a2-C4
genes identified the RFLP.11 Additionally, after hybridization of the a2-C2 probe, they failed to identify the
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RFLP as seen with the other two probes.11 Therefore,
these results might suggest that the observed polymorphism could exist in a2-C10 or «2-C4 adrenergic receptor genes.
There was a significant association between allelic
frequencies and ethnic group for these RFLPs. Similar
ethnic differences have been reported in renin gene
RFLP between Afro-Caribbeans and Europeans.18 The
actual meaning of these ethnic differences is unknown.
However, these differences are interesting because the
reports from the United States1 show increased a2adrenergic receptor density compared with normotensive control subjects with no patient manipulation,
which is different from reports from Japan5"7'19 showing
that an alteration of a2-adrenergic receptor density in
patients with essential hypertension can be demonstrated by manipulations such as salt restriction5'6 and
standing stress.7
In summary, the present study could find no evidence
for an association of essential hypertension with a2adrenergic receptor RFLP in Japanese. However, the
allele frequency in Japan was different from that reported in US populations.
Acknowledgments
This study was supported in part by Grants-in-Aid 0104412
and 02670404 for Scientific Research from the Ministry of
Education, Science and Culture, Japan. We thank Dr John
Regan for kindly providing the a2-C10 probe and C. Hayashi
for secretarial help.
References
1. Michel MC, Insel PA, Brodde OE. Renal o-adrenergic receptor
alterations: a cause of essential hypertension? FASEB 3. 1989;3:
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aj-adrenoceptors and the adenylate cyclase-cAMP system: biochemical and physiological interactions. Am J Physioi. 1987;252:
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Vol 23, No 1 January 1994
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Association analysis of restriction fragment length polymorphism for alpha 2-adrenergic
receptor genes in essential hypertension in Japan.
S Umemura, N Hirawa, T Iwamoto, S Yamaguchi, Y Toya, S Kobayashi, I Takasaki, G Yasuda, K
Tamura and M Ishii
Hypertension. 1994;23:I203
doi: 10.1161/01.HYP.23.1_Suppl.I203
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